Development and Optimization of In-House Made Indirect Elisa Kit for the Detection of Antibodies Against Pasteurella Multocida in Chicken

Development and Optimization of In-House Made Indirect Elisa Kit for the Detection of Antibodies Against Pasteurella Multocida in Chicken | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and Optimization of In-House Made Indirect Elisa Kit for the Detection of Antibodies Against Pasteurella Multocida in Chicken Dubale Beyene This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8122359/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Feb, 2026 Read the published version in BMC Immunology → Version 1 posted 10 You are reading this latest preprint version Abstract Fowl cholera is caused by P. multocida. The currently available commercial kit is relatively expensive for Ethiopian laboratories. To address this issue indirect ELISA kit to detect antibodies against P. multocida in chicken was developed. In-house made indirect ELISA kit was developed and optimized. Re-constituted local avian P. multocida biotype A was used for the preparation of coating antigen. The purity of the P. multocida biotype A inoculum was checked by gram staining and centrifugation at 4500 rpm for 30 minutes \(\:.\) The assay was developed and evaluated using 128 samples of chicken serum. Optimizations of various components were carried out against both anti-P. multocida hyper immune and pre immune serum. To develop an indirect ELISA test, washing buffer concentration of 500µl of T-20, blocking buffer concentration of 200µl, antigen concentration of 0.1µg/ml, serum samples with a final dilution of 1:500 and optimal dilution of Rabbit anti-sheep IgG HRP-conjugate of 1: 1000 µg/ml are determined by checker board titration (CBT) method. The cut-off value was determined to be 0.23. Sensitivity, specificity and accuracy of in-house made indirect ELISA were 96%, 90% and 93%, respectively. This test result indicated good correlation with that of the commercial kit with a Cohen's κ agreement of 0.86 with a 95% confidence interval of 0.77 to 0.95. We found that in-house made indirect ELISA was more sensitive and convenient to perform. Consequently, in-house made indirect ELISA was as good as the commercial indirect ELISA in screening chicken serum samples for detection of antibodies against P. multocida. Antibodies Cut-off value In house made indirect ELISA Sensitivity Specificity Figures Figure 1 1. INTRODUCTION 1.1. Background of the study The poultry sector continues to grow and industrialize in several parts of the world. An increasing population, bigger getting power, and urbanization are robust drivers of growth (FAO, 2014 ). In Ethiopia, raising poultry could be an important business. The entire numbers of chickens in Ethiopia were 56 Million and are currently reached to be 65.87 Million with an expected increase within the future (Abdo et al., 2016 ). The backyard poultry represents an important part of the national economy and provides about 98·5% and 99·2% of national egg and poultry meat production, respectively (Tadelle et al., 2003 ). The poultry trade is growing quick in Ethiopia and is facing several constraints particularly infectious diseases inflicting a significant setback to the development of this sector (Dana et al., 2008 ). Colibacillosis, salmonellosis, mycoplasmosis, and fowl cholera are among the numerous bacterial diseases that threaten the poultry trade worldwide, including Ethiopia. An important infectious agent that causes fowl cholera in domestic and wild birds is P. multocida (Xiao et al., 2015 ). In poultry, P. multocida is often associated with severe economic loss due to the loss of cattle or poultry species (Marza et al., 2015 ). One of the most dangerous infectious illnesses of poultry is fowl cholera (FC). Most of Ethiopia is endemic to illness, which causes major economic losses due to decreased productivity and mortality worldwide (Molalegne et al., 2009 ). Controlling the disease is crucial due to its enormous influence on chicken productivity. The use of vaccinations is the most effective control method in places where the disease is endemic from both a practical and financial standpoint (OIE, 2012 ). ELISAs with various technical solutions are continually being developed, which enhances and broadens the scope of application. ELISAs have been around for 50 years. Engvall and Perlmann first reported the test in 1971 (Engval and Perlmann, 1972), building on Avrameas' work in which he employed enzyme-linked antibodies in histochemistry (Avrameas, 1969 ; Avrameas, 1970 ). Since its discovery, ELISA has been utilized in a wide variety of applications to identify both antigens and antibodies. An enzyme-linked immunosorbent assay was developed by Marshall et al., ( 1981 ) for the detection of antibodies to P. multocida in turkeys. They found ELISA to be superior to microtiter agglutination tests for measuring serum antibodies against P. Multocida and ELISA was more sensitive and convenient to perform than conventional serologic tests (Marshall et al., 1981 ). The sensitivities of those ELISA tests were higher than both and MA tests (Marshall et al., 1981 ; Solano et al ., 1983). ‘Due to its simplicity in preparation, type specificity, and ability to distinguish effectively between protected and unprotected animals, the heat extract antigen was preferred to other antigens for use in ELISA (Afzal et al., 1992 ; Tankaew et al., 2017 ). 1.2. Statement of the problem and justification To identify and assess the chickens' antibody response to P. multocida biotype A, a quick, sensitive, specific, and reproducible in-house made indirect ELISA kit which has a strong agreement with commercial indirect ELISA kit is required here in Ethiopia. There were no previous studies conducted in such areas in Ethiopia. The use of the ELISA with other infectious agents of poultry has given researchers and diagnostic workers a rapid, relatively inexpensive to set up, and sensitive method to detect and quantitate antibodies (Smith et al ., 1983). The ELISA is appropriate for testing on a wide scale and is rapid and simple to use (Tod et al ., 1990b). The cost of antigen preparation may be an advantage of the ELISA test (Lamichhane et al ., 1990). Although there are numerous commercial ELISA kits available, they are all imported, which drives up the cost and makes them unaffordable for large-scale P. multocida antibody screening in chickens. 1.3. Objectives 1.3.1. General objective To develop and optimize an in-house made indirect ELISA kit for the detection of antibodies against P. multocida in chicken. 1.3.2. Specific objectives To develop and standardize an in-house made indirect ELISA kit. To determine the analytical parameters of an in-house made indirect ELISA kit (Cut off value, Specificity, Sensitivity, Accuracy, Positive predictive value, and Negative predictive value). To determine in-house made and commercial indirect ELISA kit agreement. 1.4. Significance of the study In- house made indirect Enzyme-Linked Immunosorbent Assay (ELISA) is a multi-well plate-based immunoassay which will offer rapid, quantitative and sensitive antibody detection against P. multocida in chicken at relatively low cost. Furthermore, the ease of adapting an ELISA kit to a higher throughput screening method empowers National Veterinary Institute (NVI) researchers and other institutes to test large sample numbers in a single run. This study is also convenient for blood screening to detect markers of infection. This newly developed ELISA kit will be useful in the detection of antibodies for the diagnosis of fowl cholera and for sero-surveillance during vaccination campaigns. 2. MATERIALS AND METHODS 2.1. Experimental site From Jan to July 2022, this study was conducted at the National Veterinary Institute (NVI), Bishoftu, Ethiopia. At an elevation of 1850 meters on top of water level, Bishoftu is located within the Oromiya Regional State about forty five kilometers southeast of Addis Ababa, within the geographic coordinates of 9°N latitude and 40°E (NMSA, 2010). The National Veterinary Institute could be a center for the development, research, and production of livestock vaccines. It currently produces over twenty two vaccines against the most common bacterial and viral diseases that have an effect on livestock and poultry. 2.2. Preparation of purified antigen (whole bacterial) For the manufacture of the coated antigen for an in-house made indirect ELISA kit for the detection of antibodies against P. multocida in chicken, working seeds of avian P. multocida biotype A (MK802880, NVI-01-2017, Ethiopia) were used. P. multocida biotype A that had been locally freeze-dried was diluted and allowed to grow on the tryptose agar plate (SIGMA-ALDRICH, Co., 3050 Spruce Street, St. louis Mo 63103, USA 314-771-5765). Once being totally homogenized with two ml of Tryptose soya Broth (REF M177-500G, lot # 0000216822, India), the freeze-dried P. multocida biotype A was inoculated into sterile twenty ml of Tryptic soya agar was added on a petri dish. Then 10% horse serum was added in 2ml of Tryptic soya broth at hemolytic tube and incubated at 37ºC overnight. Following gram staining, the colonies were inspected visually and microscopically for Cocco-bacilli organisms, gram-negative bacteria, and purity. The purity was again verified by Gram staining once one colony was transferred to a two ml haemolytic tube containing P. multocida biotype A inoculums media and incubated for seven hours at 37°C. Once checking the purity of the P. multocida biotype A inoculums by gram-staining, 0.5 ml of the broth culture was transferred into a hundred ml of P. multocida A inoculums media and inoculated into P. multocida biotype A production media at the ratio of seven ml of P. multocida biotype A inoculums, seven ml of glucose, and three ml of serum per 800 ml of P. multocida production media then incubated for forty eight hour with slow agitation at eighty rpm (NVI/SOP, 2017). The culture was harvested at a pH of 5.5 to 6.2, with a desirable titer of 1 CFU/ml as measured by the plate count technique, once 2 days, throughout that the purity was assessed by Gram staining. By centrifuging many equal volumes of two ml haemolytic tubes containing P. multocida biotype A inoculum media at 4500 rpm for half-hour and putting them in numerous directions during a centrifuge (REMI ELECTRO Technic LTD, VASAI-401208, made in India), the purity of the P. multocida biotype A inoculum was another time examined. The supernatant was removed and washed 3 times with PBS. The washed sediment (antigen) was reconstituted with PBS. The re-constituted antigen served as the coating antigen rather than the supernatant. 2.3. Sample selection and preparation The assay was developed and evaluated using groups of serum samples. A total of fifty-seven negative sera samples collected from non-vaccinated chickens and 71 sera samples collected from experimentally vaccinated chicken with 0.5ml formalin-inactivated fowl cholera vaccine from National Veterinary Institute poultry experimental room were used for the assay development. Serum samples were tested to be classified as positive or negative for P. multocida by commercial ID Screen® Pasteurella multocida Chicken and Turkey Indirect (ID Vet, 310, Rue gladiator Pasteur-Grabels-France, Innovative Diagnostic). Four positive controls (C1, D1, C2, and D2) and four-negative controls (A1, B1 and A2, and B2) were chosen for the event of this in-house made indirect ELISA kit. The indirect ELISA was carried out in accordance with the directions provided within the ID Screen® Pasteurella multocida Chicken and Turkey Indirect. Proper sample collection procedures, serum harvest, and serum sample storage (4℃ for up to four days or -20℃ for extended periods) were wont to offer reliable test results. A 96-well plate containing the test and control specimens was ready before being transferred into an ELISA micro plate employing a multichannel pipette so as to prevent variations in incubation periods between specimens. 2.4. Preparation of reagents The carbonate-bicarbonate buffer (C-3041, SIGMA Lot # 65H8931) was dissolved in 1liter of distilled water to make the coating buffer, which was then placed on a stirrer (Stuart ®, STIR, Undergrad, CAT No US152, Designed in the UK, built in PRC, Serial no. R630008616). A magnetic stirrer was added and set on the stirrer to assist saturate the buffer. It was utilized as a coating buffer in the formulation of this test after being saturated. P. multocida biotype A antigen with 24 mixed with an equal volume of carbonate-bicarbonate buffer to prepare of in-house-made indirect ELISA plates. On Vortex (IKA®MS3 basic, model = MS3B, 03.377259, USA), it was blended and placed. Antigens were adsorbed to flat-bottom 96-well microtiter plates (Nunc MaxiSorp™ ELISA plates, Nunc, Roskilde, Denmark) for 18 hr at 4°C. Phosphate Buffered Solution was made by dissolving a number of 1 Phosphate Buffered Solution tablet (Lot # 163607, Medicago AB, Sweden) in 1000ml of distilled water. Magnet beads were then added and placed on a stirrer machine. To create 0.05 percent Tween 20, 50 ml of PBS and 25of Tween-20 (Lot # 038K009, SIGMA-ALDRICH) were combined. The washing buffer was then made by combining 0.05of Tween-20 with 1 lit of PBS. The mixture was then gently shaken or gently stirred until homogeneous. It was then put on a stirrer machine to prepare it for usage. Using a measuring balance, 2.5g of skim milk powder (SIGMA-ALDRICH Lot#BCBD9552) was combined with 50ml of PBS in bottles equipped with magnetic bead stirrers to create a blocking buffer. After that, it was put on a stirrer machine that receives electricity until homogenized. The substrate solution 3, 3′, 5, 5′-Tetramethylbenzidine (TMB), the stop solution 0.16M sulfuric acid, and the conjugate (Rabbit anti-sheep IgG HRP-conjugate, lot #: 30520, Alpha Diagnostics) were all utilized in this test. They were homogenized by placing them on a vortex before adding them to ELISA plates. 2.5. Antigen coating and blocking of indirect ELISA plates By combining 21,600µl of PBS diluents with 2400µl of bacterial stock solution, a concentration of dilution at 1: 10(10%) was produced. The coating buffer containing prepared antigen solution ( P. multocida biotype A-PBS) was applied to the NUNC micro plate’s flat bottom plates for 24 hours at room temperature. After that, the plates were washed three times in a prepared washing solution that contained no additional proteins that could have challenged the target antigen's ability to adhere to the solid phase of plastic. An amount of 300µl PBS was used to wash the coated antigen. At the last washing, the plates were blotted against clean paper towels to remove the remaining buffer. The plates were blocked for two hours at room temperature using 200µl of homemade blocking buffer. Blocking buffer was removed from the plates after two hours of incubation and rinsed three times with 300µl of washing solution. Before usage, the plates were sealed and kept at 4°C. 2.6. Indirect-Enzyme Linked Immunosorbent Assay Procedure Before use, the reagents were brought to room temperature (21°C). All the reagents were homogenized by the vortex. Commercial indirect ELISA was given ready-to-use negative and positive controls. The control wells A1, B1, C1, and D1 controls, which were evaluated undiluted, did not receive dilution buffer 14. In the case of in-house made indirect ELISA, dilution buffer that was prepared by adding 1g of skimmed milk in 100ml of PBS was added to the control wells. Samples, however, were tested at a final dilution of 1:500 in Dilution buffer 14(1:50 pre-dilution, followed by 1:10 dilution in the microplate). Each sample, which weighed 5µl, was added to one of the two pre-dilution plates (Ref: 6.44/1614, Supertek = Micro plate U-shape, PK OF 50) to create a 1:50 pre-dilution. All the wells other than the control wells of the commercial ELISA kit's dilution buffer 14 and in-house made dilution buffer for in-house made indirect ELISA kit of 245µl were added. The ELISA micro plate's wells A1 and B1 received 100µl of the negative control, 100µlof the positive control, and 90µl of dilution buffer 14 in order to prepare a final dilution of 1:500. The wells C1 and D1 received 100µl of the positive control. Additionally, 10µlpre-diluted samples were created. The controls for an in-house made indirect ELISA kit was chosen from wells with good color development from a commercial ID Screen® Pasteurella multocida Chicken and Turkey Indirect to prepare a final dilution of 1:500 in dilution buffer. Vortex homogenized both the positive and negative controls. In the NUNC ELISA microplate, wells A1 and B1 received the 5µl negative control, wells C1 and D1 received the 5µl positive control, and the 128 wells containing the test samples received the 90µl dilution buffer. Additionally, the 5µl pre-diluted samples were made. The plates were covered and let to sit for 30 minutes at 21°C in the case of both commercial and in-house made indirect ELISA. For 160 wells, a total volume of 300ml of wash solution was made. By diluting the wash concentrate (20 the wash solution of 15ml was prepared. The concentrated conjugate was diluted (10to 1:10 in 15 ml of dilution buffer to prepare the conjugate, which has a volume of 1.6 ml. The dilution buffer and conjugate were both homogenized by a vortex. The wells had been drained. With around 300µl of wash solution 1 time each well was washed three times. Between washes, the wells were not allowed to dry out. For commercial ELISA kits, an anti-chicken horseradish peroxidase conjugate was added to each well, and a 100µl rabbit anti-sheep IgG HRP conjugate was used for the in-house made ELISA kits. The plates were sealed with a plate sealer and left to sit at 21°C for 30 minutes. The wells were cleaned out. With around 300 µl of wash solution 1 time, each well was washed three times. Between washes, the wells were not allowed to dry out. Each well received 100µl of the substrate solution (TMB). At 21°C in the dark, the plate was covered and incubated for 15 minutes. To stop the reaction, 100µl of the stop solution was applied to each well. Using a Multiplex microplate ELISA reader, the O.D was read and recorded at 450 nm. 2.7. Optimization of the indirect ELISA The Checkerboard titration method was used to optimize the reagents as described by Crowther, (2001). The P. multocida biotype A antigen was coated on a plate with 1:10; 1:100 and 1:1000 dilutions. To optimize antigen dilution a number of fifteen negative sera and twenty positive sera were used. A number of twenty positive samples with 1:100: 1:500; 1:1000 and 1:2000 different dilutions were done to determine the detection limit of antibodies. Conjugate with 1:10; 1:100; 1:1000 and 1:10,000 different concentrations were done to optimize it. 2.8. Determination of the cut-off value The cut-off value was obtained by determining the optical density (OD) at a wavelength of 450 nm (OD450) calculated from the mean of negative sera plus three standard deviations, as described previously (Crowther, 2001; Tankaew et al., 2017 ). A sample was considered seropositive when the sample OD value at 450 nm exceeds the mean OD value of negative controls plus three times the standard deviation (SD) defined as the cut-off value. For a sample to be seronegative the value should be equal to or less than the cut-off OD. Cut-off OD = Mean OD of negative sera + (3X Standard Deviation). 2.9. Determination of sensitivity and specificity The serum samples (n = 128) were collected from NVI, poultry experimental room for the determination of sensitivity and specificity of in-house made indirect ELISA. Seventy- one chickens were vaccinated against formalin-inactivated Fowl cholera vaccine and fifty-seven chickens were non-vaccinated. For diagnostic validation, positive and negative serum was assayed to determine the sensitivity, specificity, and accuracy of the in-house made indirect ELISA. The sensitivity, specificity, accuracy, Positive predictive value and negative predictive value were calculated according to Chansiripornchai ( 2007 ). \(\:\text{S}\text{e}\text{n}\text{s}\text{i}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:=\frac{True\:positive}{True\:positive\:+False\:negative}\times\:100;\:\text{S}\text{p}\text{e}\text{c}\text{i}\text{f}\text{i}\text{c}\text{i}\text{t}\text{y}\:=\frac{True\:negative}{True\:negative+False\:positive}\times\:\) 100 Accuracy \(\:=\frac{number\:of\:positives\:in\:both\:tests\:+number\:of\:negatives\:in\:both\:tests}{total\:number\:of\:samples}\) Positive and negative predictive value was calculated by described formula: PPV= \(\:\frac{True\:positive}{True\:positive\:+False\:negative}\times\:\) 100; NPV= \(\:\frac{True\:negative}{True\:negative+False\:positive}\times\:\) 100. 3.10. Determination of in-house made and commercial indirect PM-ELISA kit agreement: Qualitative agreement between in-house made and commercial indirect ELISA kits was assessed by calculating percent agreement and Cohen’s kappa coefficient (k) statistic to assess the degree of (inter-rater) agreement for serum antibody status (Ubersax, 2010). Calculation of Cohen’s kappa was performed according to the following formula: K \(\:=\frac{\text{P}\text{r}\left(a\right)-\text{P}\text{r}\left(e\right)}{1-\text{P}\text{r}\left(e\right)}\) Where Pr (a) represents the actually observed agreement and Pr (e) represents chance agreement. Note that the sample size consists of the number of observations made across which in-house made and commercial indirect PM-ELISA were compared. The kappa is based on the chi-square table, and the Pr (e) is obtained through the following formula: Expected (Chance)Agreement \(\:=\frac{\frac{{\:\:\:cm}^{1}\times\:{rm}^{1}}{n}+\frac{{\:\:\:cm}^{2}\times\:{rm}^{2}}{n}}{n}\:\:\:\) Where: represents column 1 marginal, represents column 2 marginal, represents row 1 marginal, represents row 2 marginal, and n represents the number of observations (not the number of raters).Confidence interval was determined according to the formula: κ – 1.96SEκ to κ + 1.96SEκ. The standard error of kappa (SEκ) was calculated according to the following formula \(\:\sqrt{\frac{p(1-p}{{n(1-pe)}^{2}}}\) 3.11. Ethical clearance The research ethical committee of the National Veterinary Institute (NVI) reviewed and discussed this research project entitled ‘ Development and optimization of an in-house made indirect ELISA kit for the detection of antibodies against Pasteurella multocida in chicken’ on Sep19, 2021. After discussion and review of this project proposal, it was found scientifically and ethically sound from relevance, originality and technical competence point of view. 3.12. Statistical Analysis. A number of true positive and negative sera and false positive and negative sera were used to determine the sensitivity, specificity, accuracy, positive, and negative predictive value. Cut-off value, sensitivity, specificity, accuracy, positive, and negative predictive value of the in-house made indirect ELISA was calculated according to Chansiripornchai ( 2007 ). A Cohen’s Kappa test was also applied for agreement between in-house made and commercial indirect ELISA test (Marston, 2010 ). The intensity of color development was measured in the form of optical density at 450 nm wavelength using a microtitre plate reader. 4. RESULTS Characterization of seropositive and seronegative samples by commercial indirect ELISA kit An in-house made and commercial indirect ELISA antibody titers of positive sera were greater than 396 and titers of negative sera were less than or equal to 396. A total of 65 out of 128 (51%) sera derived from chicken were classified as true seropositive to fowl cholera and 54 sera out of 128 (42%) were classified as true seronegative. A total of 3 out of 128 (2%) sera derived from chicken were classified as false seronegative and 6 out of 128 (5%) sera derived from chicken were classified as false seropositive. The results analyzed by the Graph pad prism and ID Soft data analysis program indicated that all tested positive samples were reactive and the negative samples were non-reactive. Optimization of in-house made indirect ELISA kit : To optimize antigen dilution fifteen negative sera and twenty positive sera were used. As indicated in Figure 2, 1:10antigen dilution was selected as optimal dilution to develop an assay. To define the working optimal dilution of Rabbit anti-sheep IgG HRP-conjugate, four dilutions were used for in house made indirect ELISA kit. As shown in Table 3and, the optimal dilution, in which the ratio between the absorbance of negative control and the positive one has the lowest value, was defined at the dilution of 1:1000. Table 3: Optical densities from four different dilutions to optimize conjugate at 450 nm. OD at 450nm Dilutions of conjugate 1/10000 1/1000 1/100 1/10 Negative 0.071 0.201 2.705 0.337 Positive 0.076 0.841 2.958 1.306 Ratio of the absorbance 0.9 0.24* 0.91 0.26 Abbreviation: OD, optical density; NC, negative control; PC, positive control. Optimal dilution of conjugate was 1/1000. As indicated in Table 4, the optimal detection limit was at a dilution of 1:500.A 1:100 dilution was not selected because we lose a lot of sample. The left two dilutions (1:1000 and 1:2000) also were not selected because of lowest OD Values. Table 4: Checker board titration method (CBT) that indicates detection limit of antibodies: The row C and D in red color indicated the ideal OD450 values of antibodies. 1 2 3 4 5 6 7 8 9 10 11 12 A 0.057 2.925 2.97 2.939 3.059 2.834 2.91 3.059 2.955 2.822 2.646 1.974 1:100 B 0.069 2.764 2.831 2.841 2.949 2.683 2.791 2.874 2.773 1.252 0.969 1.447 C 0.694 1.688 1.691 2.361 2.323 1.658 2.368 2.216 2.003 1.286 0.647 0.436 1:500 D 0.738 1.853 1.633 2.437 2.66 1.839 2.3 2.371 1.899 0.783 0.403 0.416 E 0.064 1.04 0.868 1.943 1.312 0.888 1.719 1.175 1.047 0.746 0.294 0.156 1:1000 F 0.06 1.21 0.343 1.558 2.008 0.331 1.988 1.668 0.407 0.129 0.115 0.303 G 0.06 0.97 0.655 1.66 0.99 0.517 1.507 0.945 0.809 0.601 0.171 0.063 1:2000 H 0.101 0.701 0.221 0.763 1.536 0.221 0.754 0.554 0.266 0.086 0.096 0.034 Cut-off value of in-house made indirect ELISA : Cut-off value was calculated by mean of negative sera samples at OD450nm were 0.1286, and the value of three standard deviation of mean of negative sera were 0.099. Then, the set calculated cut-off value was 0.23. On the base of cut-off value, seropositive and seronegative samples were decided. If the OD 450nm value of sample sera is more than 0.23 that is to be considered as seropositive and the sample with less than 0.23 OD450nm value is considered as seronegative. Sensitivity and specificity of in-house made indirect ELISA: According to the two by two tables 5 true positive (TP) and true negative samples were 65 and 54 respectively while false positive and false negative samples were 6 and 3, respectively. Table 5: Two by two table which indicate sensitivity and specificity of in-house indirect ELISA. Commercial ELISA kit positive Commercial ELISA kit negative Sum In-house made ELISA kit positive 65(51%) 6(5%) 71(56%) True positive(TP) False positive(FP) In-house made ELISA kit negative 3(2%) 54(42%) 57(44%) False negative(FN) True negative(TN) Sum 68(53%)) 60(47%)) 128(100%) According to the formula the calculated sensitivity, specificity and accuracy was 96%, 90% and 93% respectively. Positive and negative predictive value was 92% and 95% respectively. Test agreement between in-house made and commercial indirect ELISA: According to the formula, the actual observed agreement (Pr (a)) and the chance agreement (Pr (e)) was 0.93 and 0.5 respectively. Therefore the value of Cohen’s kappa (K) was calculated as 0.86 with a 95% confidence interval of 0.77 to 0.95 and standard error of kappa (SEk) was 0.045. Table 6: In-house made and commercial indirect ELISA test agreement result. Commercial Indirect ELISA kit In-house made indirect ELISA kit Positive Negative Sum Positive 65 3 68 Negative 6 54 60 Sum 71 57 128 Interpretation: The value of K (K) was between 0.80-0.90. Therefore, it was interpreted as there is strong level of agreement and 64%-80% of data were reliable b/n both commercial and in-house made indirect PM-ELISA. 5. DISCUSSION Fowl cholera may be a contagious avian diseases caused by P. multocida . The disease affects commercial poultry like chickens and ducks (Glisson et al ., 2003). Early detection will scale back morbidity and mortality, however none of in-house made highly sensitive serological tests for the antibody against the bacteria in avian sera are available at the moment. Since the event of assorted commercially accessible assay kits for the detection and measurement of antibodies to P. multocida , enhancements to the present technique are carried out. These ELISA test kits are widely used by poultry diagnostic laboratories and analysis institutes. Conflicting results are reportable by various researchers regarding the utilization of gel-precipitin and varied agglutination for the detection of antibodies to P. multocida in poultry. The utilization of the gel-precipitin takes a look at by Heddleston et al ., (1972) demonstrated that there wasn't perpetually a correlation between the immune reaction in chickens and therefore the serologic reaction. In a very comparison study of the microtiter agglutination test (MAT) and enzyme-linked-immunosorbent serologic assay done by Marshall et al ., (1981), a poor correlation was found between agglutination titers of P. multocida antibody in turkey sera and protection from challenge, whereas enzyme-linked-immunosorbent serologic assay demonstrated an honest correlation between titer and challenge Marshall et al ., (1981). One reason that agglutination tests aren't as dependable as enzyme-linked-immunosorbent serologic assay is that there's some discrepancy in determinative actual antibody titer, particularly at high titers, as a result of their supported two-fold serial dilutions and therefore the actual endpoint is troublesome to see. Enzyme-linked-immunosorbent serologic assay is a lot of advantageous for reporting exact antibody titer in that it is based on a continuous linear scale addicted to a color modification that is directly proportional to the number of antibody present in serum samples. ELISA has advantages over the IHA test in that it is less labor intensive and less time consuming. A lot of experiments were permitted to develop an in-house made indirect-ELISA kit, for detection of fowl cholera disease. In this study the ratio of the mean values of the positive and negative controls was 8.36 and that of commercial one was 5.93. The mean values of in-house made indirect ELISA kit were greater than commercial one. This was because of locally isolated P. multocida biotype A strain that we have used. The optimal concentration of whole-antigens for binding to the surface of the microtiter plate was determined. The optimal concentration refers to one that provides the minimum possible amount of antigen that completely covers the bottom of the well of the microtiter plate. Further increase of antigen concentration does not lead to signal amplification (absorbance), but could form layers of antigens that may be unstable, leading to erroneous measurements. Determination of the cut-off point is usually a troublesome step within the standardization of an ELISA, as a result of a continual variable like OD should be transformed into a qualitative response (positive or negative). Previous studies have used multiple criteria to work out the cut-off value of ELISA, like the mean of the sample to positive ratio value and a pair of standard deviations of the control sera. The calculated cut-off value during this study used the mean OD450 of non-vaccinated serum (negative) and three times standard deviations due to the convenience of calculation and effectiveness in practice (Tankaew et al ., 2017). It was absolutely determined as 0.23. In addition, this technique was accustomed calculate the cut-off point of elephant antibody against P. multocida in a very previous study (Tankaew et al ., 2017). The diagnostic performance of the in-house made indirect ELISA in terms of its diagnostic sensitivity and specificity was also evaluated in comparison with commercial kit which is the gold standard. The sensitivity of in-house made indirect ELISA kit was 96% slightly lower than that of the IHA (97.6%); in contrast, the specificity of this ELISA kit was 90% higher than that of the IHA test (76.5%). In poultry, a high specificity of serological tests is more important than high sensitivity since low sensitivity can be compensated for by using a greater number of chickens’ sera (De Wit et al ., 1997). Thus this in-house made indirect ELISA appears to be 93% accurate for detection of antibodies against P. multocida in chicken. The positive predictive value was 92%. That means in-house made indirect ELISA kit has performance to identify 92% of chickens which have probability of positive test result actually having antibodies against P. multocida. The negative predictive value was 95%. That means in-house made indirect ELISA kit have performance to identify 95% of chickens which have probability of negative test result truly do not have antibodies against P. multocida. At present, the strong agreement (Kappa value =0.86) between an in-house made indirect ELISA kit and the commercial indirect ELISA means that it is possible to use this in-house made indirect ELISA to screen chicken serum samples for detection of antibodies against P. multocida. On the basis of the analysis of 128 serum samples, the results obtained by in-house made indirect ELISA showed strong agreement with those of commercial kit. This means that the obtained data from in-house made kit results are in the same range with the commercial indirect ELISA kit. We suggest that the agreement of results is due to the high purity of produced re-constituted P. multocida biotype A antigen, optimization of factors affecting reactions of the test, including antigen, primary antibody, and Rabbit anti-sheep IgG HRP-conjugate concentrations, incubation time and temperature. There was no in-house made indirect ELISA kit available in Ethiopia to detect the immune response against P. multocida of vaccinated and non-vaccinated chicken. Before that commercial indirect ELISA test was used to monitor the immune status of chicken against Fowl cholera. In-house made indirect ELISA would be able to monitor the immune status of chicken and decrease the economic losses. The ELISA is easy to perform, can be completed within a workday, can be used to screen many samples at a time, easy to interpret; produces objective results, and is economical. This in-house made indirect ELISA kit gives reliable antibody titer with less labor than other assays; it can strongly support vaccine development. Thus, in-house made indirect ELISA has advantages as a tool for disease surveillance in chicken. There are some limitations in our study. First of all, all reagents of indirect ELISA kit were not in-house made. In this study, we used commercial enzyme labeled secondary conjugated antibody, substrate and stop solution. Second, in case of standardization and optimization of in-house made indirect ELISA kit, only antigen, primary antibody and conjugate were optimized. Third, we have challenged to test replicates or triplicate of all samples in conjunction with a known standard to ensure the accuracy of results and for quantitation due to resource shortage. 6. CONCLUSION AND RECOMMENDATION It was possible to develop and optimize in-house made indirect ELISA kit that has strong agreement with commercial indirect ELISA kit by sensitivity, specificity, accuracy and other analysis ways. During this study, in-house made indirect ELISA kit was effectively developed and optimized by locally freeze-dried P. multocida biotype A whole antigen for detection of antibody. Most of the elements like whole antigen, primary antibody and secondary conjugated antibody were optimized at totally different dilutions. It absolutely was found that in-house made indirect ELISA kit was sensitive and reproducible to be used in detecting antibody responses to P. multocida in chickens. There was strong agreement between in-house made and commercial indirect ELISA kit consistent with kappa value. This in-house made indirect ELISA kit has the advantage of constant basic principle having the ability to be used for detecting antibodies against different chicken pathogens using constant reagent except the coating antigen. Based on the above conclusion the following recommendations are forwarded: Institutes and organizations should try and expand this assay, perform a series of tests and replicates of samples and cooperative studies with different laboratories. Researchers and organizations of institutes additionally should to focus in such novel technology that is extremely economical and speedy test. Institutes and organizations should try and develop conjugate, substrate and stop solution. Abbreviations ABTS Azino-Benzthiazoline ALP Alkaline Phosphatase BSA Bovine Serum Albumin CFT Complement Fixation Test COD Coefficient of determination CSA Central statistical agency CV Coefficient of Variation CVMA College of Veterinary Medicine and Agriculture ELISA Enzyme Linked Immunosorbent Assay GDPT Gel Diffusion Precipitation Test HRP Horse Radish Peroxidase IHA IndirectHemagglutination LOD Lower Limit of Detection LPS Lipopolysaccharide MA Microtiter Agglutination NAD Nucleic Acid Detection NMSA National Meteorological Services Agency NPV Negative Predictive Value NVI National Veterinary Institute OD Optical Density OIE Office International Des Epizooties OPD O-PhenylenediamineDihydrochloride PBS Phosphate buffered saline PCR Polymerase Chain Reaction P-NPP P-nitrophenylphosphate PPV Positive Predictive Value QA Quality Assurance QC Quality Control RIA Radio Immuno Assay S/P Sample/Positive Ratio TMB Tetra Methyl Benzidine VF Virulence Factor VNT Virus Neutralization Test Declarations Acknowledgements We thank the National Veterinary Institute, Research and Development Directorate, Bishoftu, Ethiopia, for their invaluable technical support. Authors' contributions Anmaw Shite and Belaynah Getachew conceptualized and supervised the study. Anmaw Shite, Belaynah Getachew and Dubale Beyene were responsible for sample preparation, data collection, and statistical analyses. Belaynah Getachew provided technical support. Dubale Beyene prepared the manuscript. All authors reviewed the manuscript. Funding We thank the National Veterinary institute, Research and Development directorate, Bishoftu, Ethiopia for their invaluable technical support. This work was not supported by any funding from different organizations. Data availability All data needed to evaluate the conclusions are provided in the supplemental material. All other raw data can be obtained from the corresponding author upon reasonable request. Ethics approval All experimental animal procedures were approved by the research ethical committee of the National Veterinary Institute (NVI). The experimental procedures conducted in this study received approval from the Ethical Committee of National Veterinary Institute. Consent for publication All the contributors have given their consent (verbally) for the data to be published. Competing interests The authors declare no competing interests. Author details 1Jinka University, College of Agriculture and Natural Resources, Department of Veterinary Science, Ethiopia 2University of Gondar, College of Veterinary Medicine and Animal Sciences, Department of Veterinary Pathobiology 3Research and Development directorate of National Veterinary institute, Bishoftu, Ethiopia References Abdo M., Samson H., Gebremedhin G. and Kefyalew G. (2016): Challenges and opportunities of small scale poultry production system in Jigjiga zone, Somalia regional state, Ethiopia, Poultry, Fisheries and Wildlife Sciences, 4 :1 Aberra, M. and Tegene, N. (2007): Study on the characterization of local chicken in Southern Ethiopia, Proceeding shield in Awassa, March 16-17. Adler, B., Bulach, D., Chung, J., Doughty, S., Hunt, M., Rajakumar, K., Serrano, M., van Zanden, A., Zhang, Y. and Ruffolo C. (1999): Candidate vaccine antigens and genes in Pasteurella multocida, Journal of Biotechnology , 73 : 83-90. Afzal, M., Muneer, R., and Akhtar, S. (1992): Serological evaluation of Pasteurella multocida antigens associated with protection in buffalo calves. 11 : 917-923. Rev Sci Tech OffInt 'Epiz. Akhtar, M. (2013): Isolation, identification, and characterization of Pasteurella multocida from chickens and development of an oil-based vaccine. Ph.D. Thesis, Department of Microbiology and Hygiene, Bangladesh Agricultural University. Avrameas, S. (1969): Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies.Immunochemistry. Jan; 6(1):43-52. DOI: 10.1016/0019- 2791(69)90177-3 PMID: 4975324. Avrameas, S., T, Ternynck. and J. L. Guesdon. (1978): Coupling of enzymes to antibodies and antigens in quantitative enzyme immunoassays. Scand J ImmunolSuppl , 8 . 7:7-24. Avrameas, S. (1970): Immunoenzyme techniques: Enzymes as markers for the localization of antigens and antibodies. International Review of Cytology , 27 :349385. DOI: 10.1016/s0074- 7696(08)61250-4 PMID: 4190535. Aye, P. P., Angrick, E. J., Morishita, T. Y. and Harr B. S. (2001): Prevalence and characteristics of Pasteurella multocida in commercial turkeys. Avian Disease , 45 : 182-190. Baba, T. (1977): Immunogenic activity of a ribosomal fraction obtained from Pasteurella multocida . Infect Immun , 15 :1-6. Bauer, A.W., Kirby, W.M., Sherris, J.C. & Turck, A.M. (1966): Antibiotic susceptibility test by a standardized single disk method. American Journal of Clinical Pathology , 45 , 493 /496. Bierer, B. W., Derieux, W. T. (1972): Immunologic response of turkeys to a virulent Pasteurella multocida vaccine in the drinking water. Poultry Sci , 51 :408-416. Boyce, J. D., Chung J. Y. and Adler B. (2000): Pasteurella multocida capsule composition, function and genetics, Journal of Biotechnology , 83 :153-160. Boyce, J. D., Adler, B. (2000): The capsule is a virulence determinant in the pathogenesis of Pasteurella multocida M1404 (B: 2). Infect Immun , 68 , 3463–3468. Briggs, D.J., Skeeles, J.K. (1984): An enzyme-linked immunosorbent assay for detecting antibodies to Pasteurella multocida in chickens. Avian Dis . 28 , 208–215. Brumpt, E. (1910): Précis de parasitologie (1st edn). Paris: Masson. Bullen, J. J., Wilson, A. B., Cushnie, G. H. (1968): The abolition of the protective effect of Pasteurella septica antiserum by iron compounds. Immunology , 14 :889-898. Bulletin, (1929): Poultry Diseases, Their Prevention and Control; Agricultural Experiment Station Kansas State Agricultural College Manhattan, Kansas. Carter, G. R. (1955): Studies on Pasteurella multocida . I. A hemagglutination test for the identification of serological types. Am J Vet Res , 16 , 481–484. Carver, C., Emery, D., Straub, D. and Wileman, B. (2013): Evidence of Serotype Cross Protection against Fowl Cholera by Vaccination with Siderophore Receptors and Porins. In: Proceedings of Western Poultry Disease Conference, Sacramento. California. Chansiripornchai, N. (2007): Screening and diagnostic tests. In: Veterinary Epidemiology and Preventive Medicine. Triranasarn Publishing, Bangkok, Thailand: 93-111. Christensen, J. P. and Bisgaard, M. (2000): Fowl cholera review, Scientific Technique, 19 : 626–637. Chung, J.Y., Wilkie, I., Boyce, J. D., Townsend, K.M., Frost, A.J., Ghoddusi, M., Adler, B. (2001): Role the of the capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A. Infect Immun , 69 , 2487–2492. Clinical and Laboratory Standard Institute (CLSI). (2016): Performance standards for antimicrobial susceptibility testing. M100S, 26th ed. Wayne. Coates, S. R., M. M. Jensen. and E. D. Brown. (1977): The response of turkeys to varying doses of live oral Pasteurella multocida vaccine. Poult Sci , 56 :273-2. Cowan, S. T. and steel. K. L. (2004): Manual for Identification of Medical Bacteria. 2nd Edition, Cambridge UniversiPressess, Cambridge, Pp 28-106. CSA (2014): Federal Democratic Republic of Ethiopia Central Statistical Authority; Report on livestock and livestock characteristics, Volume II. Addis Ababa. Dana, N., Dessie, T., Vander, W.L.H. and Van Arendonk J.A.M. (2008): Morphological features of indigenous chicken populations of Ethiopia, Animal Genetics Research, 46 : 11-23. De Ley, J., Mannheim, W., Mutters, R., Piechulla, K., Tytgat, R., Segers, P., Bisgaard, M., Frederiksen, W., Hinz, K-H.&Vanhoucke, M. (1990): Inter- and intrafamilial similarities of rRNAcistrons of the Pasteurellaceae. International Journal of Systematic Bacteriology , 40 , 126-13. Dewhirst, F. E., Paster, B.J., Fraser, G.J. & Olsen, I. (1995): Taxonomy of the Pasteurellaceae: a difficult problem even when the phylogeny is known. In Donachie, W., Lainson, F.A. & Hodgson, J.C. (Eds) Haemophilus, Actinobacillus, and Pasteurella (p. 199) New York: Plenum Press. De Wit, JJ. (1997): Sensitivity and specificity of serological tests for infectious bronchitis virus antibodies in broilers. Avian Pathol, 26 : 105-118. Dick, J. W., Johnson, J. W. (1985): Fowl cholera immunity in broiler breeder chickens determined by the enzyme-linked immunosorbent assay. Avian Dis , 29 :706-714. Dick, J.W. and Avakian, A. P. (1991): Response of broiler chickens to fowl cholera vaccination at 1 to 6 weeks of age. Avian Diseases , 35 : 761-766. Engvall E, Perlmann P. (1972): Enzymelinked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled nti-immunoglobulin in antigen-coated tubes. Journal of Immunology . Jul; 109(1):129-135 PMID: 4113792. Ethiopia Animal Health Yearbook (2011): Veterinary Epidemiology, Disease Prevention, Control and Eradication, Ethiopia, Pp22-42. Ewers, C., Lubker, B A., Bethe, A., Kiebling, S., Filter M. and Wieler, L. H. (2006): Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status, Veterinary Microbiology , 114 : 304-317. FAO (2014): Emergency Centre for Trans-boundary Animal Diseases (ECTAD). Annual Report. Jakarta, FAO. Frederiksen, W. (1993): Ecology and significance of Pasteurellaceae in man—an update. ZentralblattfürBakteriologie, 279 , 27-34. Fuller, T. E., Kennedy, M. J. and Lowery D. E. (2000): Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis, Micro, biology and Pathology , 29 : 25-38. Ganfield, D. J., Rebers, PA., Heddleston, K. L. (1976): Immunogenic and toxic properties of a purified lipopolysaccharide-protein complex from Pasteurella multocida . Infect Immun , 14 :990-999. Gaunt, G., Moffat, R., Mukkur, T. K. S. (1977): Fowl cholera: immunization of chickens with potassium thiocyanate (KSCN) extract of Pasteurella multocida serotype 3. Avian Dis , 21 :543-548. Gebre-gziabher, M. (2007): Characterization of smallholder poultry production and marketing system of Dale, Wonsho and Loka Abaya Weredas of southern Ethiopia. M.Sc Hawassa University, Hawassa, Ethiopia.Thesis, https://cgspace.cgiar.org/bitstream/handle/10568/701/Thesis Gebre-gziabher Characterization .pdf? Sequence=1 Glisson, J. R. (2008): Pasteurellosis and other respiratory bacterial infection. In: Diseases of poultry, ed. Saif, Y. M, Fadly A. M., Glisson J. R, 12th ed., Blackwell Publishing, Ames, IA. Pp. 739-758. Glisson, J. R., Hofacre, C. L. and Christensen J. P. (2013): Pasteurellosis and Other Respiratory Bacterial Infections, In Diseases of Poultry 13th Edition, Swayne, D.E., Editor in Chief, Glisson, J. R., McDougald, L. R., Nolan, L. K., Suarez, D. L. and Nair, V. Wiley-Blackwell, Ames, Iowa, USA and Oxford, UK, Pp. 807–823. Glisson, J.R., Hofacre, C.L., Christensen, J.P. (2003): Fowl cholera. In: Saif, Y.M., Barnes , H., Glisson, J.R., Fadly, A.M., McDougald, L.R., Swayne, D.E. (Eds.), Diseases of Poultry. Iowa State University Press, Ames, Iowa, USA, pp. 657–690. Glisson, J.R., Sandhu, T.S. & Hofacre, C.L. (2008): Pasteurellosis, Avibacteriosis, Gallibacteriosis, Riemerellosis, and Pseudo tuberculosis. In: A Laboratory Manual for the Isolation, Identification, and Characterization of Avian Pathogens, Fifth Edition, Dufour-Zavala, L., Swayne, D.E., Glisson, J.R., Pearson, J.E., Reed, W.M., Jackwood, M.W. & Woolcock P.R., eds. American Association of Avian Pathologists, Athens, Georgia, USA, 12–14. Goodwin, M., Lamichhane, C. M., Brown, J., Smeltzer, M. A., Miller, S. L., Girshick, T., Snyder, D. B, and Dickson, T. G. (1992): Relationship of the enzymelinked immunofluorescent antibody test for the detection of so-called chicken anemia agent antibodies in serum from broiler breeders. Avian Dis . 36 : 512-514. Greiner, M., Pfeiffer, D., Smith, R.D. (2000): Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Prev. Vet. Med . 45 , 23–41. Harper, M., Boyce, JD., Adler, B. (2006): Pasteurella multocida pathogenesis: 125 years after Pasteur. FEMS Microbiology Letters, 265: 1-10. Harper, M., Cox, A.D., Michael, F.S., Parnas, H., Wilkie, I., Blackall, P.J.; Adler, B.; Boyce, J.D. (2007): Decoration of Pasteurella multocida LPS with phosphocholine is important for virulence. J Bacteriol , 189 , 7384–7391. Harper, M., John, M., Edmunds, M., Wright, A., Ford, M., Turni, C., Blackall, P., Cox, A., Adler, B., Boyce, J.D. (2016): Protective efficacy afforded by live Pasteurella multocida vaccines in chickens is independent of lipopolysaccharide structure. Vaccine, in press. Harper, M., John, M., Turni, C., Edmunds, M., Michael, F.S., Blackall, P.J., Cox, A.D., Boyce, J.D. (2015): Development of a rapid multiplex PCR to genotype Pasteurella multocida strains using the lipopolysaccharide outer core biosynthesis locus. J Clin Microbiol , 53 , 477–485. Harper, M., Cox, A.D., Michael, F.S., Wilkie, I.W., Boyce, J.D., Adler, B. (2004): A heptosyltransferase mutant of Pasteurella multocida produces a truncated lipopolysaccharide structure and is attenuated in virulence. Infect Immun , 72 , 3436–3443. Heddleston, K. L., Rebers, P. A., Ritchie, A. E. (1966): Immunizing and toxic properties of particulate antigens from two immunogenic types of Pasteurella multocida of avian origin. J. Immunol , 96 :124-133. Heddleston, K. L., Watko, L. P., Rebers, P. A. (1964): Dissociation of a fowl cholera strain of Pasteurella multocida . Avian Dis , 8 : 649-657. HU, I. S. L. & Walter S. D. (1980): Estimating the error rates of diagnostic tests. Biometrics, 36, 167–171. Hunt, M. L., Adler, B., and Townsend, K. M. (2000): The molecular biology of Pasteurella multocida , Veterinary Microbiology , 72 : 3-25. Ievy, S., Khan, M. R. F., Islam, M. A. and Rahman M. B. (2013): Isolation and identification of Pasteurella multocida from chicken for the preparation of oil adjuvanted vaccine Bangladesh, Journal of Veterinary Medicine , 2 : 1-4. Jean, E. Sander. (2019): Fowl Cholera, MSD, and the MSD Veterinary Manual. Jens, P. K. C. (2016): Citing Websites. In: Etiology and Transmission. Retrieved June 11, 2018, from http://www.merckvetmanual.com/poultry/fowlcholera/overview-of fowl-cholera/. Jenssen, H.A., Dolberg, F. (2003): A conceptual framework for using poultry as a tool in poverty alleviation. Livestock Research for Rural Development 15(5):1-7. K, Snehaa., Abhay, Kumar., Amit, Banik., Sanjeev, H., Manoj, Joshi. (2018): Melioidosis in Andaman’s: A report of two cases and a short review DOI: 10.18231/2455-6807.0010. Kajikawa, O, Matsumoto M. (1984/85): A protective antigen for turkeys purified from a type 1 strain of Pasteurella multocida . Vet Microbiol , 10 :43-55. Kawamoto, E., Sawada, T., Suzuki, K., Maruyama, T. (1990b): Serotypes of Pasteurella multocida isolates from rabbits and their environment in Japan. Japanese Journal of Veterinary Science 52 , 1277-1279. Lamichhane, C. M., Snyder, D. B., Girschick, T., Goodwin, M. A, and Miller, S. L. (1992): Development and comparison of serologic methods for diagnosis chicken anemia virus infection. Avian Dis . 36 : 725-729. Lamont, J. D., Wichman, R. (1977): Observations on the use of CU strain Pasteurella multocida drinking water vaccine in California. Proceedings of the 26th Western Poultry Disease Conference, Davis, California. Pp. 17-20. Lowry, O. N., Rosebrough, A., Farr. and R, Randall. (1951): Protein measurement with the folin phenol reagent. J Biol Chem , 193 :265-275. Marshall, M. S., Robison R. A., Jensen, M. M. (1981): Use of an enzyme-linked immunosorbent assay to measure antibody responses in turkeys against Pasteurella multocida , Avian Disease , 25 : 964–971. Marston, L. (2010): Introductory Statistics for Health and Nursing Using SPSS. Thousand Oaks, California: Sage Publications, Ltd. Marza, A. D., Abdullah, F. F. J., Ahmed, I. M., Chung, E. L. T., Ibrahim, H. H., Zamri-Saad, M., Omar, A. R., Bakar, M. Z. A., Saharee, A. A., Haron, A. W., Lila, M. A. M. (2015): Involvement of nervous system in cattle and buffaloes due to Pasteurella multocida B:2 infection: A review of clinic-pathological and pathophysiological changes, Journal of Advanced Veterinary and Animal Research , 2 : 252-26. Molalegne, B., Kelay, B., Berhe, G. E., Kyule, M. (1974): Development and Efficacy Trial of Inactivated Fowl Cholera Vaccine Using Local Isolates of Pasteurella multocida . Ethiopian Veterinary J , 13(2):81–98. Molalegne, Bitew., Kelay, Belihu., Berhe, GebreEgziabher. And Kyule, M. (2009): Development and efficacy trial of inactivated Fowl cholera vaccine using local isolates of Pasteurella multocida , Ethiopian Veterinary Journal , 13 : 81-98 Nathanson, R. M., M. S. Hofstad. and E. L., Jeska. (1981): Cyclophosphamide-induced immunosuppression demonstrated in Pasteurella multocida -vaccinated chickens. Avian Dis , 25 :218-221. National Veterinary Institute/SOP (2017): Fowl Cholera Vaccine Production NVI Standard Operational Procedure. Debrezeit, Ethiopia 1–7. OIE (2008): Tests for sterility and freedom from contamination of biological materials OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Office International des Epizooties (OIE), Chapter 1.1.9. OIE (2009): Manual of Diagnostic Tests for Aquatic Animals, principles and methods of validation of diagnostic assays for infectious diseases, c h a p t e r 1 .1 .2. OIE (2012): World Organization for Animal Health, OIE manual of diagnostic tests and vaccines for terrestrial animals, mammals, birds and bees, Office International des Epizooties (OIE), Chapter 1.1.6. OIE (2015): Fowl Cholera In Manual of principles of veterinary vaccine production Terrestrial manual, Office International des Epizooties (OIE), Chapter 2.3.9. OIE (2018): Terrestrial Manual, Principles and methods of validation of diagnostic assays for infectious diseases , Chapter 1.1.6. Phillips, M., Rimler, RB., Rebers, PA. (1981): Failure of ribosomes from non encapsulated Pasteurella multocida to protect CF-1 mice. Am J Vet Res , 42 :1769-1774. Pohl, S. (1979): Reklassifizierung der GattungenActinobacillusBrumpt 1910, Haemophilus Winslow et al ., 1917 und PasteurellaTrevisan, 1887 anhandphänotypischer und molecular Daten, insbesondere der DNS-Verwandtschaftenbei DNS: DNS-Hybridisierung in vitro und VorschlageinerneuenFamilie, Pasteurellaceae. Thesis, FachbereichBiologie der Philipps-Universität Marburg/Lahn. Pohl, S. (1981): DNA relatedness among members of Haemophilus, Pasteurella and Actinobacillus. In: Kilian, M., Frederiksen, W. &Biberstein, E.L. (Eds) Haemophilus, Pasteurella and Actinobacillus (pp. 245-253) London: Academic Press. Rana, M., Hossain, M.T., Islam, M.A., Rahman, M.M., Alam, M.K. and Dutta, U.K. (2010): Comparative immunogenicity study in ducks of different breeds available at coastal regions of Bangladesh against duck plague and duck cholera vaccines, International Journal Biological Research , 2 : 23-27. Rhoades, K. R., Rimler, R. B. (1984): Avian Pasteurellosis. In: Hofstad, k MS., Barnes, HJ.,Calnek, BW.eds. Diseases of Poultry. 8th ed. Iowa State University Press, Ames, Iowa, Pp. 141-156. Rimler, R. B., and Glisson, J. R. (1997): Fowl cholera. In B.W. Calnek, H.J., Barnes, C.W., Beard, L.R., Mcdougald. And Y.M. Saif (Eds.), Diseases of Poultry 10th eds (Pp. 143 /159). Ames, IA: State University Press. Samuel, M. D., Botzler, R. G., Wobeser, G. A. (2007): Avian cholera Infectious diseases of wild birds. Ames: Blackwell Publishing, pp. 239-269. Sawada, T., Rimler, R.B., Rhoades, K.R. (1982): Indirect hemagglutination test that uses glutaraldehyde-fixed sheep erythrocytes sensitized with extract antigens for detection of Pasteurella antibody. J Clin Microbiol , 15 , 752–756. Schlink, G. T. and L. D. Olson. (1979): A microtiter agglutination test for anti-Pasteurella multocida antibodies in turkeys. Avian Dis , 23 :1066-1071. Schlink, G. T. and Olson L. D. (1987): Effects of Bursectomy, irradiation, and cyclophosphamide on turkeys vaccinated with CU cholera Strain. Avian Dis, 31 :13-21. Smith, E. J. and R. L. Witter. (1983): Detection of antibodies against reticuloendotheliosis viruses by an enzyme-linked immunosorbent assay. Avian Dis , 27 :225-234. Syuto, B., Matsumoto. and M. (1982): Purification of a protective antigen from a saline extract of Pasteurella multocida . Infect Immun , 37 :1218-1226. Tadelle, D., Million, T., Alemu, Y., Peters, K. (2003): Village chicken production system in Ethiopia: use patterns and performance evaluation and chicken products and socio-economic functions of chicken. DebreZeit agricultural research Centre: DebreZeit, Ethiopia. Livestock research and rural development, 15. http://www.lrrd.org/lrrd15/1/tadet151.htm. Tankaew, P., Singh-La, T., Titaram, C., Punyapornwittaya, V., Vongchan, P., Sawada, T., Sthitmatee, N. (2017): Evaluation of an in-house indirect ELISA for detection of antibody against haemorrhagic septicemia in Asian elephants. J Microbiol Methods, 134 : 30–34. Tatum, F.M., Yersin, A.G., Briggs, R.E. (2005): Construction and virulence of a Pasteurella multocida fhaB2 mutant in turkeys. Microb Pathog , 39 : 9–17. Todd, D., Mackie, D. P., Mawhinney, K. A., Connor, T. J., McNeilly, Fand McNulty, MS (1990b): Development of an enzymelinked immunosorbent assay to detect serum antibody to chicken anemia agent. Avian Dis . 34 : 359-363. Townsend, K.M., Boyce, J.D., Chung, J.Y., Frost, A.J. and Adler, B. (2001): Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system, Journal of clinical microbiology , 39 :924–929. Trevisan, V. (1887): Sulmicrococcodellarabbia e sullapossibilità di riconoscereduranteilperioded'incubazione, dall'esamedelsanguedella persona moricata, se ha contrattal'infezionerabbica. Rend. 1st. Lombardo (Ser. 2), 20 , 88-105 Van Weemen, B. K., Schuurs, A. H. (1971): Immunoassay using antigen-enzyme conjugates. FEBS Lett ; 15 (3):232–236. [PubMed: 11945853]. Weigend, S., Mielenz, N. and Lamont, J. (1997): Application of a nonlinear regression function to evaluate the kinetics of antibody response to vaccines in chicken lines divergently selected for multitrait immune response. Poultry Science , 76 : 1248-1255. Doi: https://dx.doi.org/10.1093/ps/76.9.1248. White, B. J., Goehl, DR., Amrine, DE. (2016): Bayesian evaluation of clinical diagnostic test characteristics of visual observations and remote monitoring to diagnose bovine respiratory disease in beef calves. Prev Vet Med, 126 :74-80. Winslow, C-E.A., Broadhurst, J., Buchanan, R.E., Krumwiede, C. Jr, Rogers, L.A. & Smith, G.H. (1917): The families and genera of the bacteria. Preliminary report of the committee of the society of American bacteriologists on characterization and classification of bacterial types. Journal of Bacteriology , 2 , 506-566. Xiao, K., Liu, Q., Liu, X., Hu, Y., Zhao, X., Kong, Q. (2015): Identification of the avian Pasteurella multocida phoP gene and evaluation of the effects of phoP deletion on virulence and immunogenicity. International Journal of Molecular Science , 217 : PII: E12. DOI: 10.3390/ijms17010012. Yap, H.Y., Ghazali, K.L., Nazarie, W.F.W.M., Isa M.N.M., Zakaria, Z. and Omara, A.R. (2013): Draft Genome Sequence of Pasteurella multocida subsp. multocida Strain PMTB, Isolated from a Buffalo, Genome Announcements, 1 : 1-3. Additional Declarations No competing interests reported. Supplementary Files Supplementaldata.pdf Cite Share Download PDF Status: Published Journal Publication published 23 Feb, 2026 Read the published version in BMC Immunology → Version 1 posted Editorial decision: Revision requested 24 Dec, 2025 Reviews received at journal 16 Dec, 2025 Reviews received at journal 11 Dec, 2025 Reviewers agreed at journal 10 Dec, 2025 Reviewers agreed at journal 07 Dec, 2025 Reviewers invited by journal 02 Dec, 2025 Editor invited by journal 21 Nov, 2025 Editor assigned by journal 18 Nov, 2025 Submission checks completed at journal 18 Nov, 2025 First submitted to journal 15 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8122359","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":554298461,"identity":"58b2b235-65bb-49d8-bc62-81e5aec5f3fa","order_by":0,"name":"Dubale Beyene","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYDCCAzwMzCCaH0QkFJCiRbIBpMWAFC0GB8AkETr4buQefFxQcVjO+PzqxA8PDBjk+cUO4NcieSMv2XjGmcPGZjfebpYAOsxw5uwE/FoMbuSYSfO2HU7cduPsBpCWBIPbhLWY/+b9d7h+84yzm38Qq8WMmbfhcIIBf+824myRPPMuWXrGsXTDGTd4t1kkGEgQ9gvf8dyDnwtqrOX5+89uvvmjwkaeX5qAFihoZmCQAKuUIEo5CNQBU8wBolWPglEwCkbBCAMAAN9JNuK0LPEAAAAASUVORK5CYII=","orcid":"","institution":"Jinka University, College of Agriculture and Natural Resources, Department of Veterinary Science","correspondingAuthor":true,"prefix":"","firstName":"Dubale","middleName":"","lastName":"Beyene","suffix":""}],"badges":[],"createdAt":"2025-11-15 13:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8122359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8122359/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12865-026-00818-8","type":"published","date":"2026-02-23T15:58:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":97952944,"identity":"f42b89dc-216c-48ff-b04b-398c18b7f96c","added_by":"auto","created_at":"2025-12-11 07:24:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118678,"visible":true,"origin":"","legend":"","description":"","filename":"FulllengthresearchpaperofDrDubaleBeyeneedited.docx","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/90ddfcddec576085a65f93ca.docx"},{"id":98423740,"identity":"df600c92-6c47-4440-91b8-cfd280a1caf5","added_by":"auto","created_at":"2025-12-17 16:32:34","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4030,"visible":true,"origin":"","legend":"","description":"","filename":"18cc90509dab4c298293ec8c24460f55.json","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/d3b12e4ee666ec22be3951e4.json"},{"id":97952933,"identity":"b34841c4-01e9-4450-b98e-f941acd56638","added_by":"auto","created_at":"2025-12-11 07:24:31","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":444558,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/0626f690c5ec4be574b85801.pdf"},{"id":97952942,"identity":"4a08d04a-03cf-4698-ad14-1826091eb36d","added_by":"auto","created_at":"2025-12-11 07:24:34","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":208001,"visible":true,"origin":"","legend":"","description":"","filename":"18cc90509dab4c298293ec8c24460f551enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/68a19bf49733d8e0ef43a2ea.xml"},{"id":97952928,"identity":"1550b1de-289a-4623-9549-e8f456aa1125","added_by":"auto","created_at":"2025-12-11 07:24:29","extension":"emf","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":28656,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.emf","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/5b4f24280737112049f4748f.emf"},{"id":97952931,"identity":"c021442c-d112-4305-a156-e90f5973c5af","added_by":"auto","created_at":"2025-12-11 07:24:30","extension":"emf","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":264,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.emf","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/3cf1a79fe9cac8c724afc34d.emf"},{"id":97952930,"identity":"7bf55039-cdc7-4b61-9a06-4a9d335c03e4","added_by":"auto","created_at":"2025-12-11 07:24:30","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6841,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/fe882e760c49f899550e861e.png"},{"id":97952943,"identity":"990d6bce-0bec-48d4-a224-9e96f5fc5910","added_by":"auto","created_at":"2025-12-11 07:24:34","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":317,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/7d1320e7960beb28d40fe19d.png"},{"id":97952929,"identity":"f60af2f8-ebc0-4d91-9d2a-b03853868937","added_by":"auto","created_at":"2025-12-11 07:24:30","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":193138,"visible":true,"origin":"","legend":"","description":"","filename":"18cc90509dab4c298293ec8c24460f551structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/4379b3f3c52d8d6b46921bb8.xml"},{"id":97952934,"identity":"fa4e0e48-169d-4cb9-a893-652d6c65cfa4","added_by":"auto","created_at":"2025-12-11 07:24:31","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":223950,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/25b3ff0f6e3c5cb27b62eee1.html"},{"id":97952932,"identity":"155bd84b-4044-44c1-aa2e-32c7a9c82874","added_by":"auto","created_at":"2025-12-11 07:24:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"placeholderimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/d91c81094f6af989235f9463.png"},{"id":103765578,"identity":"b12deaf1-aea6-4800-89cd-307d5e3c3257","added_by":"auto","created_at":"2026-03-02 16:05:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1478309,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/f328bc3a-1602-4343-a64d-bab5da24cd8a.pdf"},{"id":97952941,"identity":"20042cad-d99f-4e02-b0e9-978fb55101f7","added_by":"auto","created_at":"2025-12-11 07:24:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":444558,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8122359/v1/8b072acc03f3d795c9877186.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDevelopment and Optimization of In-House Made Indirect Elisa Kit for the Detection of Antibodies Against Pasteurella Multocida in Chicken\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e1.1. Background of the study\u003c/h2\u003e\u003cp\u003eThe poultry sector continues to grow and industrialize in several parts of the world. An increasing population, bigger getting power, and urbanization are robust drivers of growth (FAO, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In Ethiopia, raising poultry could be an important business. The entire numbers of chickens in Ethiopia were 56\u0026nbsp;Million and are currently reached to be 65.87\u0026nbsp;Million with an expected increase within the future (Abdo et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The backyard poultry represents an important part of the national economy and provides about 98\u0026middot;5% and 99\u0026middot;2% of national egg and poultry meat production, respectively (Tadelle et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The poultry trade is growing quick in Ethiopia and is facing several constraints particularly infectious diseases inflicting a significant setback to the development of this sector (Dana et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Colibacillosis, salmonellosis, mycoplasmosis, and fowl cholera are among the numerous bacterial diseases that threaten the poultry trade worldwide, including Ethiopia.\u003c/p\u003e\u003cp\u003eAn important infectious agent that causes fowl cholera in domestic and wild birds is \u003cem\u003eP. multocida\u003c/em\u003e (Xiao et al., \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In poultry, \u003cem\u003eP. multocida\u003c/em\u003e is often associated with severe economic loss due to the loss of cattle or poultry species (Marza et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). One of the most dangerous infectious illnesses of poultry is fowl cholera (FC). Most of Ethiopia is endemic to illness, which causes major economic losses due to decreased productivity and mortality worldwide (Molalegne et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eControlling the disease is crucial due to its enormous influence on chicken productivity. The use of vaccinations is the most effective control method in places where the disease is endemic from both a practical and financial standpoint (OIE, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). ELISAs with various technical solutions are continually being developed, which enhances and broadens the scope of application. ELISAs have been around for 50 years. Engvall and Perlmann first reported the test in 1971 (Engval and Perlmann, 1972), building on Avrameas' work in which he employed enzyme-linked antibodies in histochemistry (Avrameas, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Avrameas, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). Since its discovery, ELISA has been utilized in a wide variety of applications to identify both antigens and antibodies.\u003c/p\u003e\u003cp\u003eAn enzyme-linked immunosorbent assay was developed by Marshall et al., (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) for the detection of antibodies to \u003cem\u003eP. multocida\u003c/em\u003e in turkeys. They found ELISA to be superior to microtiter agglutination tests for measuring serum antibodies against \u003cem\u003eP. Multocida\u003c/em\u003e and ELISA was more sensitive and convenient to perform than conventional serologic tests (Marshall et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). The sensitivities of those ELISA tests were higher than both and MA tests (Marshall et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Solano \u003cem\u003eet al\u003c/em\u003e., 1983). \u0026lsquo;Due to its simplicity in preparation, type specificity, and ability to distinguish effectively between protected and unprotected animals, the heat extract antigen was preferred to other antigens for use in ELISA (Afzal et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Tankaew et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.2. Statement of the problem and justification\u003c/h2\u003e\u003cp\u003eTo identify and assess the chickens' antibody response to \u003cem\u003eP. multocida\u003c/em\u003e biotype A, a quick, sensitive, specific, and reproducible in-house made indirect ELISA kit which has a strong agreement with commercial indirect ELISA kit is required here in Ethiopia. There were no previous studies conducted in such areas in Ethiopia. The use of the ELISA with other infectious agents of poultry has given researchers and diagnostic workers a rapid, relatively inexpensive to set up, and sensitive method to detect and quantitate antibodies (Smith \u003cem\u003eet al\u003c/em\u003e., 1983). The ELISA is appropriate for testing on a wide scale and is rapid and simple to use (Tod \u003cem\u003eet al\u003c/em\u003e., 1990b). The cost of antigen preparation may be an advantage of the ELISA test (Lamichhane \u003cem\u003eet al\u003c/em\u003e., 1990). Although there are numerous commercial ELISA kits available, they are all imported, which drives up the cost and makes them unaffordable for large-scale \u003cem\u003eP. multocida\u003c/em\u003e antibody screening in chickens.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.3. Objectives\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e1.3.1. General objective\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eTo develop and optimize an in-house made indirect ELISA kit for the detection of antibodies against \u003cem\u003eP. multocida\u003c/em\u003e in chicken.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e1.3.2. Specific objectives\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eTo develop and standardize an in-house made indirect ELISA kit.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eTo determine the analytical parameters of an in-house made indirect ELISA kit (Cut off value, Specificity, Sensitivity, Accuracy, Positive predictive value, and Negative predictive value).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eTo determine in-house made and commercial indirect ELISA kit agreement.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e1.4. Significance of the study\u003c/h2\u003e\u003cp\u003eIn- house made indirect Enzyme-Linked Immunosorbent Assay (ELISA) is a multi-well plate-based immunoassay which will offer rapid, quantitative and sensitive antibody detection against \u003cem\u003eP. multocida\u003c/em\u003e in chicken at relatively low cost. Furthermore, the ease of adapting an ELISA kit to a higher throughput screening method empowers National Veterinary Institute (NVI) researchers and other institutes to test large sample numbers in a single run. This study is also convenient for blood screening to detect markers of infection. This newly developed ELISA kit will be useful in the detection of antibodies for the diagnosis of fowl cholera and for sero-surveillance during vaccination campaigns.\u003c/p\u003e\u003c/div\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Experimental site\u003c/h2\u003e\u003cp\u003eFrom Jan to July 2022, this study was conducted at the National Veterinary Institute (NVI), Bishoftu, Ethiopia. At an elevation of 1850 meters on top of water level, Bishoftu is located within the Oromiya Regional State about forty five kilometers southeast of Addis Ababa, within the geographic coordinates of 9\u0026deg;N latitude and 40\u0026deg;E (NMSA, 2010). The National Veterinary Institute could be a center for the development, research, and production of livestock vaccines. It currently produces over twenty two vaccines against the most common bacterial and viral diseases that have an effect on livestock and poultry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Preparation of purified antigen (whole bacterial)\u003c/h2\u003e\u003cp\u003eFor the manufacture of the coated antigen for an in-house made indirect ELISA kit for the detection of antibodies against \u003cem\u003eP. multocida\u003c/em\u003e in chicken, working seeds of avian \u003cem\u003eP. multocida\u003c/em\u003e biotype A (MK802880, NVI-01-2017, Ethiopia) were used. \u003cem\u003eP. multocida\u003c/em\u003e biotype A that had been locally freeze-dried was diluted and allowed to grow on the tryptose agar plate (SIGMA-ALDRICH, Co., 3050 Spruce Street, St. louis Mo 63103, USA 314-771-5765). Once being totally homogenized with two ml of Tryptose soya Broth (REF M177-500G, lot # 0000216822, India), the freeze-dried \u003cem\u003eP. multocida\u003c/em\u003e biotype A was inoculated into sterile twenty ml of Tryptic soya agar was added on a petri dish. Then 10% horse serum was added in 2ml of Tryptic soya broth at hemolytic tube and incubated at 37\u0026ordm;C overnight. Following gram staining, the colonies were inspected visually and microscopically for Cocco-bacilli organisms, gram-negative bacteria, and purity. The purity was again verified by Gram staining once one colony was transferred to a two ml haemolytic tube containing \u003cem\u003eP. multocida\u003c/em\u003e biotype A inoculums media and incubated for seven hours at 37\u0026deg;C. Once checking the purity of the \u003cem\u003eP. multocida\u003c/em\u003e biotype A inoculums by gram-staining, 0.5 ml of the broth culture was transferred into a hundred ml of \u003cem\u003eP. multocida\u003c/em\u003e A inoculums media and inoculated into \u003cem\u003eP. multocida\u003c/em\u003e biotype A production media at the ratio of seven ml of \u003cem\u003eP. multocida\u003c/em\u003e biotype A inoculums, seven ml of glucose, and three ml of serum per 800 ml of \u003cem\u003eP. multocida\u003c/em\u003e production media then incubated for forty eight hour with slow agitation at eighty rpm (NVI/SOP, 2017).\u003c/p\u003e\u003cp\u003eThe culture was harvested at a pH of 5.5 to 6.2, with a desirable titer of 1 CFU/ml as measured by the plate count technique, once 2 days, throughout that the purity was assessed by Gram staining. By centrifuging many equal volumes of two ml haemolytic tubes containing \u003cem\u003eP. multocida\u003c/em\u003e biotype A inoculum media at 4500 rpm for half-hour and putting them in numerous directions during a centrifuge (REMI ELECTRO Technic LTD, VASAI-401208, made in India), the purity of the \u003cem\u003eP. multocida\u003c/em\u003e biotype A inoculum was another time examined. The supernatant was removed and washed 3 times with PBS. The washed sediment (antigen) was reconstituted with PBS. The re-constituted antigen served as the coating antigen rather than the supernatant.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Sample selection and preparation\u003c/h2\u003e\u003cp\u003eThe assay was developed and evaluated using groups of serum samples. A total of fifty-seven negative sera samples collected from non-vaccinated chickens and 71 sera samples collected from experimentally vaccinated chicken with 0.5ml formalin-inactivated fowl cholera vaccine from National Veterinary Institute poultry experimental room were used for the assay development. Serum samples were tested to be classified as positive or negative for \u003cem\u003eP. multocida\u003c/em\u003e by commercial ID Screen\u0026reg; \u003cem\u003ePasteurella multocida\u003c/em\u003e Chicken and Turkey Indirect (ID Vet, 310, Rue gladiator Pasteur-Grabels-France, Innovative Diagnostic). Four positive controls (C1, D1, C2, and D2) and four-negative controls (A1, B1 and A2, and B2) were chosen for the event of this in-house made indirect ELISA kit. The indirect ELISA was carried out in accordance with the directions provided within the ID Screen\u0026reg; \u003cem\u003ePasteurella multocida\u003c/em\u003e Chicken and Turkey Indirect. Proper sample collection procedures, serum harvest, and serum sample storage (4℃ for up to four days or -20℃ for extended periods) were wont to offer reliable test results. A 96-well plate containing the test and control specimens was ready before being transferred into an ELISA micro plate employing a multichannel pipette so as to prevent variations in incubation periods between specimens.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Preparation of reagents\u003c/h2\u003e\u003cp\u003eThe carbonate-bicarbonate buffer (C-3041, SIGMA Lot # 65H8931) was dissolved in 1liter of distilled water to make the coating buffer, which was then placed on a stirrer (Stuart \u0026reg;, STIR, Undergrad, CAT No US152, Designed in the UK, built in PRC, Serial no. R630008616). A magnetic stirrer was added and set on the stirrer to assist saturate the buffer. It was utilized as a coating buffer in the formulation of this test after being saturated. \u003cem\u003eP. multocida\u003c/em\u003e biotype A antigen with 24 mixed with an equal volume of carbonate-bicarbonate buffer to prepare of in-house-made indirect ELISA plates. On Vortex (IKA\u0026reg;MS3 basic, model\u0026thinsp;=\u0026thinsp;MS3B, 03.377259, USA), it was blended and placed. Antigens were adsorbed to flat-bottom 96-well microtiter plates (Nunc MaxiSorp\u0026trade; ELISA plates, Nunc, Roskilde, Denmark) for 18 hr at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003ePhosphate Buffered Solution was made by dissolving a number of 1 Phosphate Buffered Solution tablet (Lot # 163607, Medicago AB, Sweden) in 1000ml of distilled water. Magnet beads were then added and placed on a stirrer machine. To create 0.05 percent Tween 20, 50 ml of PBS and 25of Tween-20 (Lot # 038K009, SIGMA-ALDRICH) were combined. The washing buffer was then made by combining 0.05of Tween-20 with 1 lit of PBS. The mixture was then gently shaken or gently stirred until homogeneous. It was then put on a stirrer machine to prepare it for usage. Using a measuring balance, 2.5g of skim milk powder (SIGMA-ALDRICH Lot#BCBD9552) was combined with 50ml of PBS in bottles equipped with magnetic bead stirrers to create a blocking buffer. After that, it was put on a stirrer machine that receives electricity until homogenized. The substrate solution 3, 3\u0026prime;, 5, 5\u0026prime;-Tetramethylbenzidine (TMB), the stop solution 0.16M sulfuric acid, and the conjugate (Rabbit anti-sheep IgG HRP-conjugate, lot #: 30520, Alpha Diagnostics) were all utilized in this test. They were homogenized by placing them on a vortex before adding them to ELISA plates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Antigen coating and blocking of indirect ELISA plates\u003c/h2\u003e\u003cp\u003eBy combining 21,600\u0026micro;l of PBS diluents with 2400\u0026micro;l of bacterial stock solution, a concentration of dilution at 1: 10(10%) was produced. The coating buffer containing prepared antigen solution (\u003cem\u003eP. multocida\u003c/em\u003e biotype A-PBS) was applied to the NUNC micro plate\u0026rsquo;s flat bottom plates for 24 hours at room temperature. After that, the plates were washed three times in a prepared washing solution that contained no additional proteins that could have challenged the target antigen's ability to adhere to the solid phase of plastic. An amount of 300\u0026micro;l PBS was used to wash the coated antigen. At the last washing, the plates were blotted against clean paper towels to remove the remaining buffer. The plates were blocked for two hours at room temperature using 200\u0026micro;l of homemade blocking buffer. Blocking buffer was removed from the plates after two hours of incubation and rinsed three times with 300\u0026micro;l of washing solution. Before usage, the plates were sealed and kept at 4\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Indirect-Enzyme Linked Immunosorbent Assay Procedure\u003c/h2\u003e\u003cp\u003eBefore use, the reagents were brought to room temperature (21\u0026deg;C). All the reagents were homogenized by the vortex. Commercial indirect ELISA was given ready-to-use negative and positive controls. The control wells A1, B1, C1, and D1 controls, which were evaluated undiluted, did not receive dilution buffer 14. In the case of in-house made indirect ELISA, dilution buffer that was prepared by adding 1g of skimmed milk in 100ml of PBS was added to the control wells. Samples, however, were tested at a final dilution of 1:500 in Dilution buffer 14(1:50 pre-dilution, followed by 1:10 dilution in the microplate).\u003c/p\u003e\u003cp\u003eEach sample, which weighed 5\u0026micro;l, was added to one of the two pre-dilution plates (Ref: 6.44/1614, Supertek\u0026thinsp;=\u0026thinsp;Micro plate U-shape, PK OF 50) to create a 1:50 pre-dilution. All the wells other than the control wells of the commercial ELISA kit's dilution buffer 14 and in-house made dilution buffer for in-house made indirect ELISA kit of 245\u0026micro;l were added. The ELISA micro plate's wells A1 and B1 received 100\u0026micro;l of the negative control, 100\u0026micro;lof the positive control, and 90\u0026micro;l of dilution buffer 14 in order to prepare a final dilution of 1:500. The wells C1 and D1 received 100\u0026micro;l of the positive control. Additionally, 10\u0026micro;lpre-diluted samples were created.\u003c/p\u003e\u003cp\u003eThe controls for an in-house made indirect ELISA kit was chosen from wells with good color development from a commercial ID Screen\u0026reg; \u003cem\u003ePasteurella multocida\u003c/em\u003e Chicken and Turkey Indirect to prepare a final dilution of 1:500 in dilution buffer. Vortex homogenized both the positive and negative controls. In the NUNC ELISA microplate, wells A1 and B1 received the 5\u0026micro;l negative control, wells C1 and D1 received the 5\u0026micro;l positive control, and the 128 wells containing the test samples received the 90\u0026micro;l dilution buffer. Additionally, the 5\u0026micro;l pre-diluted samples were made.\u003c/p\u003e\u003cp\u003eThe plates were covered and let to sit for 30 minutes at 21\u0026deg;C in the case of both commercial and in-house made indirect ELISA. For 160 wells, a total volume of 300ml of wash solution was made. By diluting the wash concentrate (20 the wash solution of 15ml was prepared. The concentrated conjugate was diluted (10to 1:10 in 15 ml of dilution buffer to prepare the conjugate, which has a volume of 1.6 ml. The dilution buffer and conjugate were both homogenized by a vortex. The wells had been drained. With around 300\u0026micro;l of wash solution 1 time each well was washed three times. Between washes, the wells were not allowed to dry out.\u003c/p\u003e\u003cp\u003eFor commercial ELISA kits, an anti-chicken horseradish peroxidase conjugate was added to each well, and a 100\u0026micro;l rabbit anti-sheep IgG HRP conjugate was used for the in-house made ELISA kits. The plates were sealed with a plate sealer and left to sit at 21\u0026deg;C for 30 minutes. The wells were cleaned out. With around 300 \u0026micro;l of wash solution 1 time, each well was washed three times. Between washes, the wells were not allowed to dry out. Each well received 100\u0026micro;l of the substrate solution (TMB). At 21\u0026deg;C in the dark, the plate was covered and incubated for 15 minutes. To stop the reaction, 100\u0026micro;l of the stop solution was applied to each well. Using a Multiplex microplate ELISA reader, the O.D was read and recorded at 450 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Optimization of the indirect ELISA\u003c/h2\u003e\u003cp\u003eThe Checkerboard titration method was used to optimize the reagents as described by Crowther, (2001). The \u003cem\u003eP. multocida\u003c/em\u003e biotype A antigen was coated on a plate with 1:10; 1:100 and 1:1000 dilutions. To optimize antigen dilution a number of fifteen negative sera and twenty positive sera were used. A number of twenty positive samples with 1:100: 1:500; 1:1000 and 1:2000 different dilutions were done to determine the detection limit of antibodies. Conjugate with 1:10; 1:100; 1:1000 and 1:10,000 different concentrations were done to optimize it.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Determination of the cut-off value\u003c/h2\u003e\u003cp\u003eThe cut-off value was obtained by determining the optical density (OD) at a wavelength of 450 nm (OD450) calculated from the mean of negative sera plus three standard deviations, as described previously (Crowther, 2001; Tankaew et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A sample was considered seropositive when the sample OD value at 450 nm exceeds the mean OD value of negative controls plus three times the standard deviation (SD) defined as the cut-off value. For a sample to be seronegative the value should be equal to or less than the cut-off OD.\u003c/p\u003e\u003cp\u003eCut-off OD\u0026thinsp;=\u0026thinsp;Mean OD of negative sera + (3X Standard Deviation).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Determination of sensitivity and specificity\u003c/h2\u003e\u003cp\u003eThe serum samples (n\u0026thinsp;=\u0026thinsp;128) were collected from NVI, poultry experimental room for the determination of sensitivity and specificity of in-house made indirect ELISA. Seventy- one chickens were vaccinated against formalin-inactivated Fowl cholera vaccine and fifty-seven chickens were non-vaccinated. For diagnostic validation, positive and negative serum was assayed to determine the sensitivity, specificity, and accuracy of the in-house made indirect ELISA. The sensitivity, specificity, accuracy, Positive predictive value and negative predictive value were calculated according to Chansiripornchai (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\text{S}\\text{e}\\text{n}\\text{s}\\text{i}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}\\:=\\frac{True\\:positive}{True\\:positive\\:+False\\:negative}\\times\\:100;\\:\\text{S}\\text{p}\\text{e}\\text{c}\\text{i}\\text{f}\\text{i}\\text{c}\\text{i}\\text{t}\\text{y}\\:=\\frac{True\\:negative}{True\\:negative+False\\:positive}\\times\\:\\)\u003c/span\u003e\u003c/span\u003e100\u003c/p\u003e\u003cp\u003eAccuracy \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:=\\frac{number\\:of\\:positives\\:in\\:both\\:tests\\:+number\\:of\\:negatives\\:in\\:both\\:tests}{total\\:number\\:of\\:samples}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003ePositive and negative predictive value was calculated by described formula:\u003c/p\u003e\u003cp\u003ePPV= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{True\\:positive}{True\\:positive\\:+False\\:negative}\\times\\:\\)\u003c/span\u003e\u003c/span\u003e100; NPV=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{True\\:negative}{True\\:negative+False\\:positive}\\times\\:\\)\u003c/span\u003e\u003c/span\u003e100.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.10. Determination of in-house made and commercial indirect PM-ELISA kit agreement:\u003c/h2\u003e\u003cp\u003eQualitative agreement between in-house made and commercial indirect ELISA kits was assessed by calculating percent agreement and Cohen\u0026rsquo;s kappa coefficient (k) statistic to assess the degree of (inter-rater) agreement for serum antibody status (Ubersax, 2010). Calculation of Cohen\u0026rsquo;s kappa was performed according to the following formula: \u003cem\u003eK\u003c/em\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:=\\frac{\\text{P}\\text{r}\\left(a\\right)-\\text{P}\\text{r}\\left(e\\right)}{1-\\text{P}\\text{r}\\left(e\\right)}\\)\u003c/span\u003e\u003c/span\u003e Where Pr (a) represents the actually observed agreement and Pr (e) represents chance agreement. Note that the sample size consists of the number of observations made across which in-house made and commercial indirect PM-ELISA were compared. The kappa is based on the chi-square table, and the Pr (e) is obtained through the following formula:\u003c/p\u003e\u003cp\u003eExpected (Chance)Agreement\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:=\\frac{\\frac{{\\:\\:\\:cm}^{1}\\times\\:{rm}^{1}}{n}+\\frac{{\\:\\:\\:cm}^{2}\\times\\:{rm}^{2}}{n}}{n}\\:\\:\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eWhere: represents column 1 marginal, represents column 2 marginal, represents row 1 marginal, represents row 2 marginal, and n represents the number of observations (not the number of raters).Confidence interval was determined according to the formula: κ \u0026ndash; 1.96SEκ to κ\u0026thinsp;+\u0026thinsp;1.96SEκ. The standard error of kappa (SEκ) was calculated according to the following formula \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sqrt{\\frac{p(1-p}{{n(1-pe)}^{2}}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.11. Ethical clearance\u003c/h2\u003e\u003cp\u003eThe research ethical committee of the National Veterinary Institute (NVI) reviewed and discussed this research project entitled \u0026lsquo;\u003cb\u003eDevelopment and optimization of an in-house made indirect ELISA kit for the detection of antibodies against\u003c/b\u003e \u003cb\u003ePasteurella multocida\u003c/b\u003e \u003cb\u003ein chicken\u0026rsquo;\u003c/b\u003e on Sep19, 2021. After discussion and review of this project proposal, it was found scientifically and ethically sound from relevance, originality and technical competence point of view.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.12. Statistical Analysis.\u003c/h2\u003e\u003cp\u003eA number of true positive and negative sera and false positive and negative sera were used to determine the sensitivity, specificity, accuracy, positive, and negative predictive value. Cut-off value, sensitivity, specificity, accuracy, positive, and negative predictive value of the in-house made indirect ELISA was calculated according to Chansiripornchai (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). A Cohen\u0026rsquo;s Kappa test was also applied for agreement between in-house made and commercial indirect ELISA test (Marston, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The intensity of color development was measured in the form of optical density at 450 nm wavelength using a microtitre plate reader.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. RESULTS","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of seropositive and seronegative samples by commercial indirect ELISA kit\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn in-house made and commercial indirect ELISA antibody titers of positive sera were greater than 396 and titers of negative sera were less than or equal to 396. A total of 65 out of 128 (51%) sera derived from chicken were classified as true seropositive to fowl cholera and 54 sera out of 128 (42%) were classified as true seronegative. A total of 3 out of 128 (2%) sera derived from chicken were classified as false seronegative and 6 out of 128 (5%) sera derived from chicken were classified as false seropositive. The results analyzed by the Graph pad prism and ID Soft data analysis program indicated that all tested positive samples were reactive and the negative samples were non-reactive.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization of in-house made\u0026nbsp;indirect ELISA\u0026nbsp;kit\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eTo optimize antigen dilution fifteen negative sera and twenty positive sera were used. As indicated in Figure 2, 1:10antigen dilution was selected as optimal dilution to develop an assay.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan id=\"_Toc238236253\"\u003eTo define the working\u0026nbsp;optimal dilution of Rabbit anti-sheep IgG HRP-conjugate, four dilutions were used for in house made indirect ELISA\u0026nbsp;kit.\u0026nbsp;As\u0026nbsp;shown in Table 3and, the optimal dilution, in which the\u0026nbsp;ratio between the absorbance of negative control and the\u0026nbsp;positive one has the lowest value, was defined at the dilution of 1:1000.\u003c/span\u003e\u003c/p\u003e\n\u003cp id=\"_Toc238236254\"\u003eTable 3:\u0026nbsp;Optical densities from four different dilutions to optimize conjugate at 450 nm.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 415px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; OD at 450nm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003eDilutions of conjugate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e1/10000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e1/1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e1/100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e1/10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.071\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e0.201\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e2.705\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0.337\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e0.841\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e2.958\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e1.306\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 175px;\"\u003e\n \u003cp\u003eRatio of the absorbance\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e0.24*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviation: OD, optical density;\u0026nbsp;NC, negative control; PC, positive control.\u003cbr\u003e\u0026nbsp;Optimal dilution of conjugate was 1/1000.\u003c/p\u003e\n\u003cp\u003eAs indicated in Table 4, the optimal detection limit was at a dilution of 1:500.A 1:100 dilution was not selected because we lose a lot of sample. The left two dilutions (1:1000 and 1:2000) also were not selected because of lowest OD Values.\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003eTable 4:\u003c/span\u003e\u003c/strong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;Checker board titration method (CBT) that indicates detection limit of antibodies: The row C and D in red color indicated the ideal OD450 values of antibodies.\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"width:444.5pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.6pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 38.1pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.6pt;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height: 150%;font-family:\"Times New Roman\",serif;'\u003eA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e0.057\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.925\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.97\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.939\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e3.059\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.834\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.91\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e3.059\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.955\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.822\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.646\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e1.974\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 38.1pt;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e1:100\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.6pt;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eB\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e0.069\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.764\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.831\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.841\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.949\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.683\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.791\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.874\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e2.773\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e1.252\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e0.969\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#00B050;'\u003e1.447\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.6pt;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height: 150%;font-family:\"Times New Roman\",serif;'\u003eC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e0.694\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e1.688\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e1.691\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e2.361\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e2.323\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e1.658\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e2.368\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e2.216\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e2.003\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e1.286\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e0.647\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e0.436\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 38.1pt;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e1:500\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.6pt;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eD\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e0.738\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e1.853\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e1.633\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e2.437\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e2.66\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e1.839\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e2.3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e2.371\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e1.899\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e0.783\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e0.403\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:red;'\u003e0.416\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.6pt;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height: 150%;font-family:\"Times New Roman\",serif;'\u003eE\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.064\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.04\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.868\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.943\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.312\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.888\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.719\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.175\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.047\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.746\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.294\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.156\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 38.1pt;border: none;padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e1:1000\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.6pt;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eF\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.06\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.21\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.343\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.558\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e2.008\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.331\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.988\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e1.668\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.407\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.129\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.115\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 16.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:#7030A0;'\u003e0.303\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.6pt;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height: 150%;font-family:\"Times New Roman\",serif;'\u003eG\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.06\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.97\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.655\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e1.66\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.99\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.517\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e1.507\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.945\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.809\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.601\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.171\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border: none;padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e0.063\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 38.1pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);padding: 0in 5.4pt;height: 15.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e1:2000\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 17.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003eH\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.101\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.701\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.221\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.763\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e1.536\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.221\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.754\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.554\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.266\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.086\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.096\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.45in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid rgb(91, 155, 213);background: rgb(214, 230, 244);padding: 0in 5.4pt;height: 3.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;color:black;'\u003e0.034\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCut-off\u0026nbsp;value of in-house made indirect ELISA\u003c/strong\u003e: Cut-off value was calculated by mean of negative sera samples at OD450nm were 0.1286, and the value of three standard deviation of mean of negative sera were 0.099. Then, the set calculated cut-off value was 0.23. On the base of cut-off value, seropositive and seronegative samples were decided. If the OD 450nm value of sample sera is more than 0.23 that is to be considered as seropositive and the sample with less than 0.23 OD450nm value is considered as seronegative.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSensitivity and specificity of in-house made indirect ELISA:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the two by two\u0026nbsp;tables 5\u0026nbsp;true positive (TP) and true negative samples were 65 and\u0026nbsp;54 respectively while false positive and false negative\u0026nbsp;samples were 6 and 3, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cspan id=\"_Toc238236256\"\u003eTable 5: Two by two table which indicate sensitivity and specificity of in-house indirect ELISA.\u003c/span\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCommercial ELISA kit positive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCommercial ELISA kit negative \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIn-house made ELISA kit positive\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e65(51%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e6(5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e71(56%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003eTrue positive(TP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eFalse positive(FP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIn-house made ELISA kit negative\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e3(2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e54(42%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e57(44%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003eFalse negative(FN)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eTrue negative(TN)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 182px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSum\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e68(53%))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e60(47%))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e128(100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAccording to the formula the calculated sensitivity, specificity and accuracy was 96%, 90% and 93% respectively. Positive and negative predictive value was 92% and 95% respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTest agreement between in-house made and commercial indirect ELISA:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the formula, the actual observed agreement\u0026nbsp;(Pr\u0026nbsp;(a)) and the chance agreement (Pr\u0026nbsp;(e)) was 0.93 and 0.5 respectively. Therefore the value\u0026nbsp;of Cohen\u0026rsquo;s kappa (K) was calculated as 0.86 with a 95% confidence interval of 0.77 to 0.95 and standard error of kappa (SEk) was 0.045.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6:\u0026nbsp;\u003c/strong\u003eIn-house made\u0026nbsp;and commercial indirect ELISA test agreement result.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eCommercial Indirect ELISA kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 443px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;In-house made indirect ELISA kit \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003eNegative \u0026nbsp; \u0026nbsp; \u0026nbsp; Sum\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e3 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e54 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003eSum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 148px;\"\u003e\n \u003cp\u003e57 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eInterpretation: The value of K (K) was between 0.80-0.90. Therefore, it was interpreted as there is strong level of agreement and 64%-80% of data were reliable b/n both commercial and in-house made indirect PM-ELISA.\u003c/p\u003e"},{"header":"5. DISCUSSION","content":"\u003cp\u003eFowl cholera may be a contagious avian diseases caused by \u003cem\u003eP. multocida\u003c/em\u003e. The disease affects commercial poultry like chickens and ducks (Glisson \u003cem\u003eet al\u003c/em\u003e., 2003). Early detection will scale back morbidity and mortality, however none of in-house made highly sensitive serological tests for the antibody against the bacteria in avian sera are available at the moment. Since the event of assorted commercially accessible assay kits for the detection and measurement of antibodies to \u003cem\u003eP. multocida\u003c/em\u003e, enhancements to the present technique are carried out. These ELISA test kits are widely used by poultry diagnostic laboratories and analysis institutes.\u003c/p\u003e\n\u003cp\u003eConflicting results are reportable by various researchers regarding the utilization of gel-precipitin and varied agglutination for the detection of antibodies to \u003cem\u003eP. multocida\u003c/em\u003e in poultry. The utilization of the gel-precipitin takes a look at by Heddleston \u003cem\u003eet al\u003c/em\u003e., (1972) demonstrated that there wasn\u0026apos;t perpetually a correlation between the immune reaction in chickens and therefore the serologic reaction. In a very comparison study of the microtiter agglutination test (MAT) and enzyme-linked-immunosorbent serologic assay done by Marshall \u003cem\u003eet al\u003c/em\u003e., (1981), a poor correlation was found between agglutination titers of \u003cem\u003eP. multocida\u0026nbsp;\u003c/em\u003eantibody in turkey sera and protection from challenge, whereas enzyme-linked-immunosorbent serologic assay demonstrated an honest correlation between titer and challenge Marshall \u003cem\u003eet al\u003c/em\u003e., (1981). One reason that agglutination tests aren\u0026apos;t as dependable as enzyme-linked-immunosorbent serologic assay is that there\u0026apos;s some discrepancy in determinative actual antibody titer, particularly at high titers, as a result of their supported two-fold serial dilutions and therefore the actual endpoint is troublesome to see. Enzyme-linked-immunosorbent serologic assay is a lot of advantageous for reporting exact antibody titer in that it is based on a continuous linear scale addicted to a color modification that is directly proportional to the number of antibody present in serum samples.\u003c/p\u003e\n\u003cp\u003eELISA has advantages over the IHA test in that it is less labor intensive and less time consuming. A lot of experiments were permitted to develop an in-house made indirect-ELISA kit, for detection of fowl cholera disease.\u003c/p\u003e\n\u003cp\u003eIn this study the ratio of the mean values of the positive and negative controls was 8.36 and that of commercial one was 5.93. The mean values of in-house made indirect ELISA kit were greater than commercial one. This was because of locally isolated \u003cem\u003eP. multocida\u003c/em\u003e biotype A strain that we have used.\u003c/p\u003e\n\u003cp\u003eThe optimal concentration of\u0026nbsp;whole-antigens for binding to the surface of the\u0026nbsp;microtiter plate was determined. The optimal concentration refers\u0026nbsp;to one that provides the minimum possible amount\u0026nbsp;of antigen that completely covers the bottom of the well\u0026nbsp;of the microtiter plate. Further increase of antigen concentration\u0026nbsp;does not lead to signal amplification (absorbance),\u0026nbsp;but could form layers of antigens that may be\u0026nbsp;unstable, leading to erroneous measurements.\u003c/p\u003e\n\u003cp\u003eDetermination of the cut-off point is usually a troublesome step within the standardization of an ELISA, as a result of a continual variable like OD should be transformed into a qualitative response (positive or negative). Previous studies have used multiple criteria to work out the cut-off value of ELISA, like the mean of the sample to positive ratio value and a pair of standard deviations of the control sera. The calculated cut-off value during this study used the mean OD450 of non-vaccinated serum (negative) and three times standard deviations due to the convenience of calculation and effectiveness in practice (Tankaew \u003cem\u003eet al\u003c/em\u003e., 2017). It was absolutely determined as 0.23. In addition, this technique was accustomed calculate the cut-off point of elephant antibody against \u003cem\u003eP. multocida\u003c/em\u003e in a very previous study (Tankaew \u003cem\u003eet al\u003c/em\u003e., 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe diagnostic performance of the in-house made indirect ELISA in terms of its diagnostic sensitivity and specificity was also evaluated in comparison with commercial kit which is the gold standard. The sensitivity of in-house made indirect ELISA kit was 96% slightly lower than that of the IHA (97.6%); in contrast, the specificity of this ELISA kit was 90% higher than that of the IHA test (76.5%). In poultry, a high specificity of serological tests is more important than high sensitivity since low sensitivity can be compensated for by using a greater number of chickens\u0026rsquo; sera (De Wit \u003cem\u003eet al\u003c/em\u003e., 1997). Thus this in-house made indirect ELISA appears to be 93% accurate for detection of antibodies against \u003cem\u003eP.\u0026nbsp;multocida\u003c/em\u003e in chicken.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe positive predictive value was 92%. That means in-house made indirect ELISA kit has performance to identify 92% of chickens which have probability of positive test result actually having antibodies against \u003cem\u003eP. multocida.\u0026nbsp;\u003c/em\u003eThe negative predictive value was 95%. That means in-house made indirect ELISA kit have performance to identify 95% of chickens which have probability of negative test result truly do not have antibodies against \u003cem\u003eP. multocida.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAt present, the strong \u0026nbsp;agreement (Kappa value =0.86) between an in-house made indirect ELISA kit and the commercial indirect \u0026nbsp;ELISA means that it is possible to use this in-house made indirect ELISA to screen chicken serum \u0026nbsp;samples for detection of antibodies against \u003cem\u003eP. multocida.\u003c/em\u003e On the basis of the analysis of 128 serum samples, the results obtained by in-house made indirect ELISA showed strong agreement with those of commercial kit. This means that the obtained data from in-house made kit results are in the same range with the commercial indirect ELISA kit. We suggest that the agreement of results is due to the high purity of produced re-constituted \u003cem\u003eP. multocida\u0026nbsp;\u003c/em\u003ebiotype A antigen, optimization of factors affecting reactions of the test, including antigen, primary antibody, and Rabbit anti-sheep IgG HRP-conjugate concentrations, incubation time and temperature.\u003c/p\u003e\n\u003cp\u003eThere was no in-house made indirect ELISA kit available in Ethiopia to detect the immune response against \u003cem\u003eP. multocida\u003c/em\u003e of vaccinated and non-vaccinated chicken. Before that commercial indirect ELISA test was used to monitor the immune status of chicken against Fowl cholera. In-house made indirect ELISA would be able to monitor the immune status of chicken and decrease the economic losses. The ELISA is easy to perform, can be completed within a workday, can be used to screen many samples at a time, easy to interpret; produces objective results, and is economical.\u0026nbsp;This in-house made indirect ELISA kit gives reliable antibody titer with less labor than other assays; it can strongly support vaccine development. Thus, in-house made indirect ELISA has advantages as a tool for\u0026nbsp;disease surveillance in chicken.\u003c/p\u003e\n\u003cp\u003eThere are some limitations in our study. First of all, all reagents of indirect ELISA kit were not in-house made. In this study, we used commercial enzyme labeled secondary conjugated antibody, substrate and stop solution. Second, in case of standardization and optimization of in-house made indirect ELISA kit, only antigen, primary antibody and conjugate were optimized. Third, we have challenged to test replicates or triplicate of all samples in conjunction with a known standard to ensure the accuracy of results and for quantitation due to resource shortage.\u003c/p\u003e"},{"header":"6. CONCLUSION AND RECOMMENDATION","content":"\u003cp\u003eIt was possible to develop and optimize in-house made indirect ELISA kit that has strong agreement with commercial indirect ELISA kit by sensitivity, specificity, accuracy and other analysis ways. During this study, in-house made indirect ELISA kit was effectively developed and optimized by locally freeze-dried \u003cem\u003eP. multocida\u003c/em\u003e biotype A whole antigen for detection of antibody. Most of the elements like whole antigen, primary antibody and secondary conjugated antibody were optimized at totally different dilutions. It absolutely was found that in-house made indirect ELISA kit was sensitive and reproducible to be used in detecting antibody responses to \u003cem\u003eP. multocida\u003c/em\u003e in chickens. There was strong agreement between in-house made and commercial indirect ELISA kit consistent with kappa value. This in-house made indirect ELISA kit has the advantage of constant basic principle having the ability to be used for detecting antibodies against different chicken pathogens using constant reagent except the coating antigen.\u003c/p\u003e\n\u003cp\u003eBased on the above conclusion the following recommendations are forwarded:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eInstitutes and organizations should try and expand this assay, perform a series of tests and replicates of samples and cooperative studies with different laboratories.\u003c/li\u003e\n \u003cli\u003eResearchers and organizations of institutes additionally should to focus in such novel technology that is extremely economical and speedy test.\u003c/li\u003e\n \u003cli\u003eInstitutes and organizations should try and develop conjugate, substrate and stop solution.\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eABTS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Azino-Benzthiazoline\u003c/p\u003e\n\u003cp id=\"_Toc238236139\"\u003eALP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Alkaline Phosphatase\u003c/p\u003e\n\u003cp id=\"_Toc238236140\"\u003eBSA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Bovine Serum Albumin\u003c/p\u003e\n\u003cp\u003e\u003cspan id=\"_Toc238236141\"\u003eCFT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Complement Fixation Test\u003c/span\u003e\u003c/p\u003e\n\u003cp id=\"_Toc238236142\"\u003eCOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Coefficient of determination\u003c/p\u003e\n\u003cp id=\"_Toc238236143\"\u003eCSA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Central statistical agency\u003c/p\u003e\n\u003cp id=\"_Toc238236144\"\u003eCV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Coefficient of Variation\u003c/p\u003e\n\u003cp id=\"_Toc238236145\"\u003eCVMA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;College of Veterinary Medicine and Agriculture\u003c/p\u003e\n\u003cp id=\"_Toc238236146\"\u003eELISA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Enzyme Linked Immunosorbent Assay\u003c/p\u003e\n\u003cp id=\"_Toc238236147\"\u003eGDPT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Gel Diffusion Precipitation Test\u003c/p\u003e\n\u003cp id=\"_Toc238236148\"\u003eHRP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Horse Radish Peroxidase\u003c/p\u003e\n\u003cp id=\"_Toc108133651\"\u003eIHA IndirectHemagglutination\u003c/p\u003e\n\u003cp id=\"_Toc108133652\"\u003eLOD Lower Limit of Detection\u003c/p\u003e\n\u003cp id=\"_Toc238236151\"\u003eLPS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Lipopolysaccharide\u003c/p\u003e\n\u003cp id=\"_Toc238236152\"\u003eMA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Microtiter Agglutination\u003c/p\u003e\n\u003cp id=\"_Toc238236153\"\u003eNAD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Nucleic Acid Detection\u003c/p\u003e\n\u003cp id=\"_Toc238236154\"\u003eNMSA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; National Meteorological Services Agency\u003c/p\u003e\n\u003cp\u003eNPV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Negative Predictive Value\u003c/p\u003e\n\u003cp id=\"_Toc238236155\"\u003eNVI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;National Veterinary Institute\u003c/p\u003e\n\u003cp id=\"_Toc238236156\"\u003eOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Optical Density\u003c/p\u003e\n\u003cp id=\"_Toc238236157\"\u003eOIE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Office International Des Epizooties\u003c/p\u003e\n\u003cp id=\"_Toc238236158\"\u003eOPD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; O-PhenylenediamineDihydrochloride\u003c/p\u003e\n\u003cp id=\"_Toc238236159\"\u003ePBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Phosphate buffered saline\u003c/p\u003e\n\u003cp id=\"_Toc238236160\"\u003ePCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Polymerase Chain Reaction\u003c/p\u003e\n\u003cp id=\"_Toc238236161\"\u003eP-NPP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; P-nitrophenylphosphate\u003c/p\u003e\n\u003cp id=\"_Toc238236162\"\u003ePPV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Positive Predictive Value\u003c/p\u003e\n\u003cp id=\"_Toc238236163\"\u003eQA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Quality Assurance\u003c/p\u003e\n\u003cp id=\"_Toc238236164\"\u003eQC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Quality Control\u003c/p\u003e\n\u003cp id=\"_Toc238236165\"\u003eRIA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Radio Immuno Assay\u003c/p\u003e\n\u003cp id=\"_Toc238236166\"\u003eS/P \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sample/Positive Ratio\u003c/p\u003e\n\u003cp id=\"_Toc238236167\"\u003eTMB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Tetra Methyl Benzidine\u003c/p\u003e\n\u003cp id=\"_Toc238236168\"\u003eVF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Virulence Factor\u003c/p\u003e\n\u003cp id=\"_Toc111792339\"\u003eVNT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Virus Neutralization Test\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the National Veterinary Institute, Research and Development Directorate, Bishoftu, Ethiopia, for their invaluable technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnmaw Shite and Belaynah Getachew conceptualized and supervised the study. Anmaw Shite, Belaynah Getachew and Dubale Beyene were responsible for sample preparation, data collection, and statistical analyses. Belaynah Getachew provided technical support. Dubale Beyene prepared the manuscript. All authors reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the National Veterinary institute, Research and Development directorate, Bishoftu, Ethiopia for their invaluable technical support. This work was not supported by any funding from different organizations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions are provided in the supplemental material. All other raw data can be obtained from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental animal procedures were approved by\u0026nbsp;the research ethical committee of the National Veterinary Institute (NVI). The experimental procedures conducted in this study received approval from the Ethical Committee of National Veterinary Institute.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the contributors have given their consent (verbally) for the data to be published.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1Jinka University, College of Agriculture and Natural Resources, Department of Veterinary Science, Ethiopia\u003c/p\u003e\n\u003cp\u003e2University of Gondar, College of Veterinary Medicine and Animal Sciences, Department of Veterinary Pathobiology\u003c/p\u003e\n\u003cp\u003e3Research and Development directorate of National Veterinary institute, Bishoftu, Ethiopia\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdo M., Samson H., Gebremedhin G. and Kefyalew G. (2016): Challenges and\u003cbr\u003e opportunities of small scale poultry production system in Jigjiga zone, Somalia\u003cbr\u003eregional state, Ethiopia, \u003cem\u003ePoultry, Fisheries and Wildlife Sciences, \u003c/em\u003e\u003cstrong\u003e4\u003c/strong\u003e:1\u003c/li\u003e\n\u003cli\u003eAberra, M. and Tegene, N. (2007): Study on the characterization of local chicken in Southern Ethiopia, Proceeding shield in Awassa, March 16-17.\u003c/li\u003e\n\u003cli\u003eAdler, B., Bulach, D., Chung, J., Doughty, S., Hunt, M., Rajakumar, K., Serrano, M., van Zanden, A., Zhang, Y. and Ruffolo C. (1999): Candidate vaccine antigens and genes in \u003cem\u003ePasteurella multocida, Journal of Biotechnology\u003c/em\u003e, \u003cstrong\u003e73\u003c/strong\u003e: 83-90.\u003c/li\u003e\n\u003cli\u003eAfzal, M., Muneer, R., and Akhtar, S. (1992): Serological evaluation of \u003cem\u003ePasteurella multocida\u003c/em\u003e antigens associated with protection in buffalo calves. \u003cstrong\u003e11\u003c/strong\u003e\u003cem\u003e: 917-923. Rev Sci Tech OffInt \u0026apos;Epiz.\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eAkhtar, M. (2013): Isolation, identification, and characterization of Pasteurella multocida from chickens and development of an oil-based vaccine. Ph.D. Thesis, Department of Microbiology and Hygiene, Bangladesh Agricultural University.\u003c/li\u003e\n\u003cli\u003eAvrameas, S. (1969): Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies.Immunochemistry. Jan; 6(1):43-52. DOI: 10.1016/0019- 2791(69)90177-3 PMID: 4975324.\u003c/li\u003e\n\u003cli\u003eAvrameas, S., T, Ternynck. and J. L. Guesdon. (1978): Coupling of enzymes to antibodies and antigens in quantitative enzyme immunoassays. \u003cem\u003eScand J ImmunolSuppl\u003c/em\u003e, \u003cstrong\u003e8\u003c/strong\u003e. 7:7-24. \u003c/li\u003e\n\u003cli\u003eAvrameas, S. (1970): Immunoenzyme techniques: Enzymes as markers for the localization of antigens and antibodies. \u003cem\u003eInternational Review of Cytology\u003c/em\u003e, \u003cstrong\u003e27\u003c/strong\u003e:349385. DOI: 10.1016/s0074- 7696(08)61250-4 PMID: 4190535.\u003c/li\u003e\n\u003cli\u003eAye, P. P., Angrick, E. J., Morishita, T. Y. and Harr B. S. (2001): Prevalence and characteristics of \u003cem\u003ePasteurella multocida\u003c/em\u003e in commercial turkeys.\u003cem\u003eAvian Disease\u003c/em\u003e, \u003cstrong\u003e45\u003c/strong\u003e: 182-190.\u003c/li\u003e\n\u003cli\u003eBaba, T. (1977): Immunogenic activity of a ribosomal fraction obtained from \u003cem\u003ePasteurella multocida\u003c/em\u003e. \u003cem\u003eInfect Immun\u003c/em\u003e, \u003cstrong\u003e15\u003c/strong\u003e:1-6.\u003c/li\u003e\n\u003cli\u003eBauer, A.W., Kirby, W.M., Sherris, J.C. \u0026amp; Turck, A.M. (1966): Antibiotic susceptibility test by a standardized single disk method. \u003cem\u003eAmerican Journal of Clinical Pathology\u003c/em\u003e, \u003cstrong\u003e45\u003c/strong\u003e, 493 /496.\u003c/li\u003e\n\u003cli\u003eBierer, B. W., Derieux, W. T. (1972): Immunologic response of turkeys to a virulent\u003cem\u003ePasteurella multocida\u003c/em\u003e vaccine in the drinking water. \u003cem\u003ePoultry Sci\u003c/em\u003e, \u003cstrong\u003e51\u003c/strong\u003e:408-416.\u003c/li\u003e\n\u003cli\u003eBoyce, J. D., Chung J. Y. and Adler B. (2000): \u003cem\u003ePasteurella multocida\u003c/em\u003e capsule composition, function and genetics, \u003cem\u003eJournal of Biotechnology\u003c/em\u003e, \u003cstrong\u003e83\u003c/strong\u003e:153-160.\u003c/li\u003e\n\u003cli\u003eBoyce, J. D., Adler, B. (2000): The capsule is a virulence determinant in the pathogenesis of \u003cem\u003ePasteurella multocida\u003c/em\u003e M1404 (B: 2). \u003cem\u003eInfect Immun\u003c/em\u003e, \u003cstrong\u003e68\u003c/strong\u003e, 3463\u0026ndash;3468. \u003c/li\u003e\n\u003cli\u003eBriggs, D.J., Skeeles, J.K. (1984): An enzyme-linked immunosorbent assay for detecting antibodies to \u003cem\u003ePasteurella multocida\u003c/em\u003e in chickens. \u003cem\u003eAvian Dis\u003c/em\u003e. \u003cstrong\u003e28\u003c/strong\u003e, 208\u0026ndash;215.\u003c/li\u003e\n\u003cli\u003eBrumpt, E. (1910): Pr\u0026eacute;cis de parasitologie (1st edn). Paris: Masson.\u003c/li\u003e\n\u003cli\u003eBullen, J. J., Wilson, A. B., Cushnie, G. H. (1968): The abolition of the protective effect of Pasteurella septica antiserum by iron compounds. \u003cem\u003eImmunology\u003c/em\u003e, \u003cstrong\u003e14\u003c/strong\u003e:889-898.\u003c/li\u003e\n\u003cli\u003eBulletin, (1929): Poultry Diseases, Their Prevention and Control; Agricultural Experiment Station Kansas State Agricultural College Manhattan, Kansas.\u003c/li\u003e\n\u003cli\u003eCarter, G. R. (1955): Studies on \u003cem\u003ePasteurella multocida\u003c/em\u003e. I. A hemagglutination test for the identification of serological types. \u003cem\u003eAm J Vet Res\u003c/em\u003e, \u003cstrong\u003e16\u003c/strong\u003e, 481\u0026ndash;484.\u003c/li\u003e\n\u003cli\u003eCarver, C., Emery, D., Straub, D. and Wileman, B. (2013): Evidence of Serotype Cross Protection against Fowl Cholera by Vaccination with Siderophore Receptors and Porins. In: Proceedings of Western Poultry Disease Conference, Sacramento. California.\u003c/li\u003e\n\u003cli\u003eChansiripornchai, N. (2007): Screening and diagnostic tests. In: Veterinary Epidemiology and Preventive Medicine. Triranasarn Publishing, Bangkok, Thailand: 93-111.\u003c/li\u003e\n\u003cli\u003eChristensen, J. P. and Bisgaard, M. (2000): Fowl cholera review, Scientific Technique, \u003cstrong\u003e19\u003c/strong\u003e: 626\u0026ndash;637.\u003c/li\u003e\n\u003cli\u003eChung, J.Y., Wilkie, I., Boyce, J. D., Townsend, K.M., Frost, A.J., Ghoddusi, M., Adler, B. (2001): Role the of the capsule in the pathogenesis of fowl cholera caused by \u003cem\u003ePasteurella multocida\u003c/em\u003eserogroup A. \u003cem\u003eInfect Immun\u003c/em\u003e, \u003cstrong\u003e69\u003c/strong\u003e, 2487\u0026ndash;2492. \u003c/li\u003e\n\u003cli\u003eClinical and Laboratory Standard Institute (CLSI). (2016): Performance standards for antimicrobial susceptibility testing. M100S, 26th ed. Wayne.\u003c/li\u003e\n\u003cli\u003eCoates, S. R., M. M. Jensen. and E. D. Brown. (1977): The response of turkeys to varying doses of live oral \u003cem\u003ePasteurella multocida\u003c/em\u003e vaccine. \u003cem\u003ePoult Sci\u003c/em\u003e, \u003cstrong\u003e56\u003c/strong\u003e:273-2.\u003c/li\u003e\n\u003cli\u003eCowan, S. T. and steel. K. L. (2004): Manual for Identification of Medical Bacteria. 2nd Edition, Cambridge UniversiPressess, Cambridge, Pp 28-106.\u003c/li\u003e\n\u003cli\u003eCSA (2014): Federal Democratic Republic of Ethiopia Central Statistical Authority; Report on livestock and livestock characteristics, Volume II. Addis Ababa.\u003c/li\u003e\n\u003cli\u003eDana, N., Dessie, T., Vander, W.L.H. and Van Arendonk J.A.M. (2008): Morphological features of indigenous chicken populations of Ethiopia, Animal Genetics Research, \u003cstrong\u003e46\u003c/strong\u003e: 11-23.\u003c/li\u003e\n\u003cli\u003eDe Ley, J., Mannheim, W., Mutters, R., Piechulla, K., Tytgat, R., Segers, P., Bisgaard, M., Frederiksen, W., Hinz, K-H.\u0026amp;Vanhoucke, M. (1990): Inter- and intrafamilial similarities of rRNAcistrons of the Pasteurellaceae. \u003cem\u003eInternational Journal of Systematic Bacteriology\u003c/em\u003e, \u003cstrong\u003e40\u003c/strong\u003e, 126-13.\u003c/li\u003e\n\u003cli\u003eDewhirst, F. E., Paster, B.J., Fraser, G.J. \u0026amp; Olsen, I. (1995): Taxonomy of the Pasteurellaceae: a difficult problem even when the phylogeny is known. In Donachie, W., Lainson, F.A. \u0026amp; Hodgson, J.C. (Eds) Haemophilus, Actinobacillus, and Pasteurella (p. 199) New York: Plenum Press.\u003c/li\u003e\n\u003cli\u003eDe Wit, JJ. (1997): Sensitivity and specificity of serological tests for infectious bronchitis virus antibodies in broilers. \u003cem\u003eAvian Pathol, \u003c/em\u003e\u003cstrong\u003e26\u003c/strong\u003e: 105-118.\u003c/li\u003e\n\u003cli\u003eDick, J. W., Johnson, J. W. (1985): Fowl cholera immunity in broiler breeder chickens determined by the enzyme-linked immunosorbent assay. \u003cem\u003eAvian Dis\u003c/em\u003e, \u003cstrong\u003e29\u003c/strong\u003e:706-714.\u003c/li\u003e\n\u003cli\u003eDick, J.W. and Avakian, A. P. (1991): Response of broiler chickens to fowl cholera vaccination at 1 to 6 weeks of age. \u003cem\u003eAvian Diseases\u003c/em\u003e, \u003cstrong\u003e35\u003c/strong\u003e: 761-766.\u003c/li\u003e\n\u003cli\u003eEngvall E, Perlmann P. (1972): Enzymelinked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled nti-immunoglobulin in antigen-coated tubes. \u003cem\u003eJournal of Immunology\u003c/em\u003e. Jul; 109(1):129-135 PMID: 4113792.\u003c/li\u003e\n\u003cli\u003eEthiopia Animal Health Yearbook (2011): Veterinary Epidemiology, Disease Prevention, Control and Eradication, Ethiopia, Pp22-42.\u003c/li\u003e\n\u003cli\u003eEwers, C., Lubker, B A., Bethe, A., Kiebling, S., Filter M. and Wieler, L. H. (2006): Virulence genotype of \u003cem\u003ePasteurella multocida\u003c/em\u003e strains isolated from different hosts with various disease status, \u003cem\u003eVeterinary Microbiology\u003c/em\u003e, \u003cstrong\u003e114\u003c/strong\u003e: 304-317.\u003c/li\u003e\n\u003cli\u003eFAO (2014): Emergency Centre for Trans-boundary Animal Diseases (ECTAD). Annual Report. Jakarta, FAO.\u003c/li\u003e\n\u003cli\u003eFrederiksen, W. (1993): Ecology and significance of Pasteurellaceae in man\u0026mdash;an update. Zentralblattf\u0026uuml;rBakteriologie, \u003cstrong\u003e279\u003c/strong\u003e, 27-34.\u003c/li\u003e\n\u003cli\u003eFuller, T. E., Kennedy, M. J. and Lowery D. E. (2000): Identification of \u003cem\u003ePasteurella multocida\u003c/em\u003e virulence genes in a septicemic mouse model using signature-tagged mutagenesis, \u003cem\u003eMicro, biology and Pathology\u003c/em\u003e, \u003cstrong\u003e29\u003c/strong\u003e: 25-38.\u003c/li\u003e\n\u003cli\u003eGanfield, D. J., Rebers, PA., Heddleston, K. L. (1976): Immunogenic and toxic properties of a purified lipopolysaccharide-protein complex from \u003cem\u003ePasteurella multocida\u003c/em\u003e. \u003cem\u003eInfect Immun\u003c/em\u003e, \u003cstrong\u003e14\u003c/strong\u003e:990-999.\u003c/li\u003e\n\u003cli\u003eGaunt, G., Moffat, R., Mukkur, T. K. S. (1977): Fowl cholera: immunization of chickens with potassium thiocyanate (KSCN) extract of \u003cem\u003ePasteurella multocida\u003c/em\u003e serotype 3. \u003cem\u003eAvian Dis\u003c/em\u003e, \u003cstrong\u003e21\u003c/strong\u003e:543-548.\u003c/li\u003e\n\u003cli\u003eGebre-gziabher, M. (2007): Characterization of smallholder poultry production and marketing system of Dale, Wonsho and Loka Abaya Weredas of southern Ethiopia. M.Sc Hawassa University, Hawassa, Ethiopia.Thesis, https://cgspace.cgiar.org/bitstream/handle/10568/701/Thesis Gebre-gziabher Characterization .pdf? Sequence=1\u003c/li\u003e\n\u003cli\u003eGlisson, J. R. (2008): Pasteurellosis and other respiratory bacterial infection. In: Diseases of poultry, ed. Saif, Y. M, Fadly A. M., Glisson J. R, 12th ed., Blackwell Publishing, Ames, IA. Pp. 739-758. \u003c/li\u003e\n\u003cli\u003eGlisson, J. R., Hofacre, C. L. and Christensen J. P. (2013): Pasteurellosis and Other Respiratory Bacterial Infections, In Diseases of Poultry 13th Edition, Swayne, D.E., Editor in Chief, Glisson, J. R., McDougald, L. R., Nolan, L. K., Suarez, D. L. and Nair, V. Wiley-Blackwell, Ames, Iowa, USA and Oxford, UK, Pp. 807\u0026ndash;823.\u003c/li\u003e\n\u003cli\u003eGlisson, J.R., Hofacre, C.L., Christensen, J.P. (2003): Fowl cholera. In: Saif, Y.M., Barnes , H., Glisson, J.R., Fadly, A.M., McDougald, L.R., Swayne, D.E. (Eds.), Diseases of Poultry. Iowa State University Press, Ames, Iowa, USA, pp. 657\u0026ndash;690.\u003c/li\u003e\n\u003cli\u003eGlisson, J.R., Sandhu, T.S. \u0026amp; Hofacre, C.L. (2008): Pasteurellosis, Avibacteriosis, Gallibacteriosis, Riemerellosis, and Pseudo tuberculosis. In: A Laboratory Manual for the Isolation, Identification, and Characterization of Avian Pathogens, Fifth Edition, Dufour-Zavala, L., Swayne, D.E., Glisson, J.R., Pearson, J.E., Reed, W.M., Jackwood, M.W. \u0026amp; Woolcock P.R., eds. American Association of Avian Pathologists, Athens, Georgia, USA, 12\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eGoodwin, M., Lamichhane, C. M., Brown, J., Smeltzer, M. A., Miller, S. L., Girshick, T., Snyder, D. B, and Dickson, T. G. (1992): Relationship of the enzymelinked immunofluorescent antibody test for the detection of so-called chicken anemia agent antibodies in serum from broiler breeders. \u003cem\u003eAvian Dis\u003c/em\u003e. \u003cstrong\u003e36\u003c/strong\u003e: 512-514.\u003c/li\u003e\n\u003cli\u003eGreiner, M., Pfeiffer, D., Smith, R.D. (2000): Principles and practical application of the\u003cbr\u003ereceiver-operating characteristic analysis for diagnostic tests. \u003cem\u003ePrev. Vet. Med\u003c/em\u003e. \u003cstrong\u003e45\u003c/strong\u003e,\u003cbr\u003e 23\u0026ndash;41.\u003c/li\u003e\n\u003cli\u003eHarper, M., Boyce, JD., Adler, B. (2006): \u003cem\u003ePasteurella multocida\u003c/em\u003e pathogenesis: 125 years after Pasteur. FEMS Microbiology Letters, 265: 1-10.\u003c/li\u003e\n\u003cli\u003eHarper, M., Cox, A.D., Michael, F.S., Parnas, H., Wilkie, I., Blackall, P.J.; Adler, B.; Boyce, J.D. (2007): Decoration of \u003cem\u003ePasteurella multocida\u003c/em\u003e LPS with phosphocholine is important for virulence. \u003cem\u003eJ Bacteriol\u003c/em\u003e, \u003cstrong\u003e189\u003c/strong\u003e, 7384\u0026ndash;7391.\u003c/li\u003e\n\u003cli\u003eHarper, M., John, M., Edmunds, M., Wright, A., Ford, M., Turni, C., Blackall, P., Cox, A., Adler, B., Boyce, J.D. (2016): Protective efficacy afforded by live \u003cem\u003ePasteurella multocida\u003c/em\u003e vaccines in chickens is independent of lipopolysaccharide structure. Vaccine, in press.\u003c/li\u003e\n\u003cli\u003eHarper, M., John, M., Turni, C., Edmunds, M., Michael, F.S., Blackall, P.J., Cox, A.D., Boyce, J.D. (2015): Development of a rapid multiplex PCR to genotype \u003cem\u003ePasteurella multocida\u003c/em\u003e strains using the lipopolysaccharide outer core biosynthesis locus. \u003cem\u003eJ Clin Microbiol\u003c/em\u003e, \u003cstrong\u003e53\u003c/strong\u003e, 477\u0026ndash;485.\u003c/li\u003e\n\u003cli\u003eHarper, M., Cox, A.D., Michael, F.S., Wilkie, I.W., Boyce, J.D., Adler, B. (2004): A heptosyltransferase mutant of Pasteurella multocida produces a truncated lipopolysaccharide structure and is attenuated in virulence. \u003cem\u003eInfect Immun\u003c/em\u003e, \u003cstrong\u003e72\u003c/strong\u003e, 3436\u0026ndash;3443. \u003c/li\u003e\n\u003cli\u003eHeddleston, K. L., Rebers, P. A., Ritchie, A. E. (1966): Immunizing and toxic properties of particulate antigens from two immunogenic types of \u003cem\u003ePasteurella multocida\u003c/em\u003e of avian origin. \u003cem\u003eJ. Immunol\u003c/em\u003e, \u003cstrong\u003e96\u003c/strong\u003e:124-133. \u003c/li\u003e\n\u003cli\u003eHeddleston, K. L., Watko, L. P., Rebers, P. A. (1964): Dissociation of a fowl cholera strain of \u003cem\u003ePasteurella multocida\u003c/em\u003e. \u003cem\u003eAvian Dis\u003c/em\u003e, \u003cstrong\u003e8\u003c/strong\u003e: 649-657.\u003c/li\u003e\n\u003cli\u003eHU, I. S. L. \u0026amp; Walter S. D. (1980): Estimating the error rates of diagnostic tests. Biometrics, 36, 167\u0026ndash;171.\u003c/li\u003e\n\u003cli\u003eHunt, M. L., Adler, B., and Townsend, K. M. (2000): The molecular biology of \u003cem\u003ePasteurella multocida\u003c/em\u003e, \u003cem\u003eVeterinary Microbiology\u003c/em\u003e, \u003cstrong\u003e72\u003c/strong\u003e: 3-25.\u003c/li\u003e\n\u003cli\u003eIevy, S., Khan, M. R. F., Islam, M. A. and Rahman M. B. (2013): Isolation and identification of \u003cem\u003ePasteurella multocida\u003c/em\u003e from chicken for the preparation of oil adjuvanted vaccine Bangladesh, \u003cem\u003eJournal of Veterinary Medicine\u003c/em\u003e, \u003cstrong\u003e2\u003c/strong\u003e: 1-4.\u003c/li\u003e\n\u003cli\u003eJean, E. Sander. (2019): Fowl Cholera, MSD, and the MSD Veterinary Manual.\u003c/li\u003e\n\u003cli\u003eJens, P. K. C. (2016): Citing Websites. In: Etiology and Transmission. Retrieved June 11, 2018, from http://www.merckvetmanual.com/poultry/fowlcholera/overview-of fowl-cholera/.\u003c/li\u003e\n\u003cli\u003eJenssen, H.A., Dolberg, F. (2003): A conceptual framework for using poultry as a tool in poverty alleviation. Livestock Research for Rural Development 15(5):1-7.\u003c/li\u003e\n\u003cli\u003eK, Snehaa., Abhay, Kumar., Amit, Banik., Sanjeev, H., Manoj, Joshi. (2018): Melioidosis in Andaman\u0026rsquo;s: A report of two cases and a short review DOI: 10.18231/2455-6807.0010.\u003c/li\u003e\n\u003cli\u003eKajikawa, O, Matsumoto M. (1984/85): A protective antigen for turkeys purified from a type 1 strain of \u003cem\u003ePasteurella multocida\u003c/em\u003e. \u003cem\u003eVet Microbiol\u003c/em\u003e, \u003cstrong\u003e10\u003c/strong\u003e:43-55.\u003c/li\u003e\n\u003cli\u003eKawamoto, E., Sawada, T., Suzuki, K., Maruyama, T. (1990b): Serotypes of \u003cem\u003ePasteurella multocida\u003c/em\u003e isolates from rabbits and their environment in Japan. \u003cem\u003eJapanese Journal of Veterinary Science\u003c/em\u003e\u003cstrong\u003e52\u003c/strong\u003e, 1277-1279.\u003c/li\u003e\n\u003cli\u003eLamichhane, C. M., Snyder, D. B., Girschick, T., Goodwin, M. A, and Miller, S. L. (1992): Development and comparison of serologic methods for diagnosis chicken anemia virus infection. \u003cem\u003eAvian Dis\u003c/em\u003e. \u003cstrong\u003e36\u003c/strong\u003e: 725-729.\u003c/li\u003e\n\u003cli\u003eLamont, J. D., Wichman, R. (1977): Observations on the use of CU strain \u003cem\u003ePasteurella multocida\u003c/em\u003e drinking water vaccine in California. Proceedings of the 26th Western Poultry Disease Conference, Davis, California. Pp. 17-20.\u003c/li\u003e\n\u003cli\u003eLowry, O. N., Rosebrough, A., Farr. and R, Randall. (1951): Protein measurement with the folin phenol reagent. \u003cem\u003eJ Biol Chem\u003c/em\u003e, \u003cstrong\u003e193\u003c/strong\u003e:265-275. \u003c/li\u003e\n\u003cli\u003eMarshall, M. S., Robison R. A., Jensen, M. M. (1981): Use of an enzyme-linked immunosorbent assay to measure antibody responses in turkeys against \u003cem\u003ePasteurella multocida\u003c/em\u003e, \u003cem\u003eAvian Disease\u003c/em\u003e, \u003cstrong\u003e25\u003c/strong\u003e: 964\u0026ndash;971.\u003c/li\u003e\n\u003cli\u003eMarston, L. (2010): Introductory Statistics for Health and Nursing Using SPSS. Thousand Oaks, California: Sage Publications, Ltd. \u003c/li\u003e\n\u003cli\u003eMarza, A. D., Abdullah, F. F. J., Ahmed, I. M., Chung, E. L. T., Ibrahim, H. H., Zamri-Saad, M., Omar, A. R., Bakar, M. Z. A., Saharee, A. A., Haron, A. W., Lila, M. A. M. (2015): Involvement of nervous system in cattle and buffaloes due to \u003cem\u003ePasteurella multocida\u003c/em\u003e B:2 infection: A review of clinic-pathological and pathophysiological changes, \u003cem\u003eJournal of Advanced Veterinary and Animal Research\u003c/em\u003e, \u003cstrong\u003e2\u003c/strong\u003e: 252-26.\u003c/li\u003e\n\u003cli\u003eMolalegne, B., Kelay, B., Berhe, G. E., Kyule, M. (1974): Development and Efficacy Trial of Inactivated Fowl Cholera Vaccine Using Local Isolates of \u003cem\u003ePasteurella multocida\u003c/em\u003e. \u003cem\u003eEthiopian Veterinary J\u003c/em\u003e, 13(2):81\u0026ndash;98.\u003c/li\u003e\n\u003cli\u003eMolalegne, Bitew., Kelay, Belihu., Berhe, GebreEgziabher. And Kyule, M. (2009): Development and efficacy trial of inactivated Fowl cholera vaccine using local isolates of \u003cem\u003ePasteurella multocida\u003c/em\u003e, \u003cem\u003eEthiopian Veterinary Journal\u003c/em\u003e, \u003cstrong\u003e13\u003c/strong\u003e: 81-98\u003c/li\u003e\n\u003cli\u003eNathanson, R. M., M. S. Hofstad. and E. L., Jeska. (1981): Cyclophosphamide-induced immunosuppression demonstrated in \u003cem\u003ePasteurella multocida\u003c/em\u003e-vaccinated chickens. \u003cem\u003eAvian Dis\u003c/em\u003e, \u003cstrong\u003e25\u003c/strong\u003e:218-221. \u003c/li\u003e\n\u003cli\u003eNational Veterinary Institute/SOP (2017): Fowl Cholera Vaccine Production NVI Standard Operational Procedure. Debrezeit, Ethiopia 1\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eOIE (2008): Tests for sterility and freedom from contamination of biological materials OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Office International des Epizooties (OIE), Chapter 1.1.9.\u003c/li\u003e\n\u003cli\u003eOIE (2009): Manual of Diagnostic Tests for Aquatic Animals, principles and methods of validation of diagnostic assays for infectious diseases, c h a p t e r 1 .1 .2.\u003c/li\u003e\n\u003cli\u003eOIE (2012): World Organization for Animal Health, OIE manual of diagnostic tests and vaccines for terrestrial animals, mammals, birds and bees, Office International des Epizooties (OIE), Chapter 1.1.6.\u003c/li\u003e\n\u003cli\u003eOIE (2015): Fowl Cholera In Manual of principles of veterinary vaccine production Terrestrial manual, Office International des Epizooties (OIE), Chapter 2.3.9.\u003c/li\u003e\n\u003cli\u003eOIE (2018): Terrestrial Manual, Principles and methods of validation of diagnostic assays for infectious diseases\u003cimg width=\"3\" height=\"1\" src=\"data:image/emf;base64,R0lGODlhBQABAHcAMSH+GlNvZnR3YXJlOiBNaWNyb3NvZnQgT2ZmaWNlACH5BAEAAAAALAAAAAABAAEAgAAAAAECAwICRAEAOw==\" alt=\"image\"\u003e, Chapter 1.1.6.\u003c/li\u003e\n\u003cli\u003ePhillips, M., Rimler, RB., Rebers, PA. (1981): Failure of ribosomes from non encapsulated Pasteurella multocida to protect CF-1 mice. \u003cem\u003eAm J Vet Res\u003c/em\u003e, \u003cstrong\u003e42\u003c/strong\u003e:1769-1774.\u003c/li\u003e\n\u003cli\u003ePohl, S. (1979): Reklassifizierung der GattungenActinobacillusBrumpt 1910, Haemophilus Winslow \u003cem\u003eet al\u003c/em\u003e., 1917 und PasteurellaTrevisan, 1887 anhandph\u0026auml;notypischer und molecular Daten, insbesondere der DNS-Verwandtschaftenbei DNS: DNS-Hybridisierung in vitro und VorschlageinerneuenFamilie, Pasteurellaceae. Thesis, FachbereichBiologie der Philipps-Universit\u0026auml;t Marburg/Lahn.\u003c/li\u003e\n\u003cli\u003ePohl, S. (1981): DNA relatedness among members of Haemophilus, Pasteurella and Actinobacillus. In: Kilian, M., Frederiksen, W. \u0026amp;Biberstein, E.L. (Eds) Haemophilus, Pasteurella and Actinobacillus (pp. 245-253) London: Academic Press.\u003c/li\u003e\n\u003cli\u003eRana, M., Hossain, M.T., Islam, M.A., Rahman, M.M., Alam, M.K. and Dutta, U.K. (2010): Comparative immunogenicity study in ducks of different breeds available at coastal regions of Bangladesh against duck plague and duck cholera vaccines, \u003cem\u003eInternational Journal Biological Research\u003c/em\u003e, \u003cstrong\u003e2\u003c/strong\u003e: 23-27. \u003c/li\u003e\n\u003cli\u003eRhoades, K. R., Rimler, R. B. (1984): Avian Pasteurellosis. In: Hofstad, k MS., Barnes, HJ.,Calnek, BW.eds. Diseases of Poultry. 8th ed. Iowa State University Press, Ames, Iowa, Pp. 141-156.\u003c/li\u003e\n\u003cli\u003eRimler, R. B., and Glisson, J. R. (1997): Fowl cholera. In B.W. Calnek, H.J., Barnes, C.W., Beard, L.R., Mcdougald. And Y.M. Saif (Eds.), Diseases of Poultry 10th eds (Pp. 143 /159). Ames, IA: State University Press.\u003c/li\u003e\n\u003cli\u003eSamuel, M. D., Botzler, R. G., Wobeser, G. A. (2007): Avian cholera Infectious diseases of wild birds. Ames: Blackwell Publishing, pp. 239-269.\u003c/li\u003e\n\u003cli\u003eSawada, T., Rimler, R.B., Rhoades, K.R. (1982): Indirect hemagglutination test that uses glutaraldehyde-fixed sheep erythrocytes sensitized with extract antigens for detection of Pasteurella antibody. \u003cem\u003eJ Clin Microbiol\u003c/em\u003e, \u003cstrong\u003e15\u003c/strong\u003e, 752\u0026ndash;756.\u003c/li\u003e\n\u003cli\u003eSchlink, G. T. and L. D. Olson. (1979): A microtiter agglutination test for anti-Pasteurella multocida antibodies in turkeys. \u003cem\u003eAvian Dis\u003c/em\u003e, \u003cstrong\u003e23\u003c/strong\u003e:1066-1071.\u003c/li\u003e\n\u003cli\u003eSchlink, G. T. and Olson L. D. (1987): Effects of Bursectomy, irradiation, and cyclophosphamide on turkeys vaccinated with CU cholera Strain. \u003cem\u003eAvian Dis, \u003c/em\u003e\u003cstrong\u003e31\u003c/strong\u003e:13-21.\u003c/li\u003e\n\u003cli\u003eSmith, E. J. and R. L. Witter. (1983): Detection of antibodies against reticuloendotheliosis viruses by an enzyme-linked immunosorbent assay. \u003cem\u003eAvian Dis\u003c/em\u003e, \u003cstrong\u003e27\u003c/strong\u003e:225-234.\u003c/li\u003e\n\u003cli\u003eSyuto, B., Matsumoto. and M. (1982): Purification of a protective antigen from a saline extract of \u003cem\u003ePasteurella multocida\u003c/em\u003e. \u003cem\u003eInfect Immun\u003c/em\u003e, \u003cstrong\u003e37\u003c/strong\u003e:1218-1226.\u003c/li\u003e\n\u003cli\u003eTadelle, D., Million, T., Alemu, Y., Peters, K. (2003): Village chicken production system in Ethiopia: use patterns and performance evaluation and chicken products and socio-economic functions of chicken. DebreZeit agricultural research Centre: DebreZeit, Ethiopia. Livestock research and rural development, 15. http://www.lrrd.org/lrrd15/1/tadet151.htm.\u003c/li\u003e\n\u003cli\u003eTankaew, P., Singh-La, T., Titaram, C., Punyapornwittaya, V., Vongchan, P., Sawada, T., Sthitmatee, N. (2017): Evaluation of an in-house indirect ELISA for detection of antibody against haemorrhagic septicemia in Asian elephants. \u003cem\u003eJ Microbiol Methods,\u003c/em\u003e \u003cstrong\u003e134\u003c/strong\u003e: 30\u0026ndash;34.\u003c/li\u003e\n\u003cli\u003eTatum, F.M., Yersin, A.G., Briggs, R.E. (2005): Construction and virulence of a \u003cem\u003ePasteurella multocida\u003c/em\u003e fhaB2 mutant in turkeys. \u003cem\u003eMicrob Pathog\u003c/em\u003e, \u003cstrong\u003e39\u003c/strong\u003e: 9\u0026ndash;17.\u003c/li\u003e\n\u003cli\u003eTodd, D., Mackie, D. P., Mawhinney, K. A., Connor, T. J., McNeilly, Fand McNulty, MS (1990b): Development of an enzymelinked immunosorbent assay to detect serum antibody to chicken anemia agent. \u003cem\u003eAvian Dis\u003c/em\u003e. \u003cstrong\u003e34\u003c/strong\u003e: 359-363.\u003c/li\u003e\n\u003cli\u003eTownsend, K.M., Boyce, J.D., Chung, J.Y., Frost, A.J. and Adler, B. (2001): Genetic organization of \u003cem\u003ePasteurella multocida\u003c/em\u003e cap Loci and development of a multiplex capsular PCR typing system, \u003cem\u003eJournal of clinical microbiology\u003c/em\u003e, \u003cstrong\u003e39\u003c/strong\u003e:924\u0026ndash;929.\u003c/li\u003e\n\u003cli\u003eTrevisan, V. (1887): Sulmicrococcodellarabbia e sullapossibilit\u0026agrave; di riconoscereduranteilperioded\u0026apos;incubazione, dall\u0026apos;esamedelsanguedella persona moricata, se ha contrattal\u0026apos;infezionerabbica. \u003cem\u003eRend. 1st. Lombardo\u003c/em\u003e (Ser. 2), \u003cstrong\u003e20\u003c/strong\u003e, 88-105\u003c/li\u003e\n\u003cli\u003eVan Weemen, B. K., Schuurs, A. H. (1971): Immunoassay using antigen-enzyme conjugates. \u003cem\u003eFEBS Lett\u003c/em\u003e; 15 (3):232\u0026ndash;236. [PubMed: 11945853].\u003c/li\u003e\n\u003cli\u003eWeigend, S., Mielenz, N. and Lamont, J. (1997): Application of a nonlinear regression function to evaluate the kinetics of antibody response to vaccines in chicken lines divergently selected for multitrait immune response. \u003cem\u003ePoultry Science\u003c/em\u003e, \u003cstrong\u003e76\u003c/strong\u003e: 1248-1255. Doi: https://dx.doi.org/10.1093/ps/76.9.1248.\u003c/li\u003e\n\u003cli\u003eWhite, B. J., Goehl, DR., Amrine, DE. (2016): Bayesian evaluation of clinical diagnostic test characteristics of visual observations and remote monitoring to diagnose bovine respiratory disease in beef calves. \u003cem\u003ePrev Vet Med,\u003c/em\u003e\u003cstrong\u003e126\u003c/strong\u003e:74-80.\u003c/li\u003e\n\u003cli\u003eWinslow, C-E.A., Broadhurst, J., Buchanan, R.E., Krumwiede, C. Jr, Rogers, L.A. \u0026amp; Smith, G.H. (1917): The families and genera of the bacteria. Preliminary report of the committee of the society of American bacteriologists on characterization and classification of bacterial types.\u003cem\u003eJournal of Bacteriology\u003c/em\u003e, \u003cstrong\u003e2\u003c/strong\u003e, 506-566.\u003c/li\u003e\n\u003cli\u003eXiao, K., Liu, Q., Liu, X., Hu, Y., Zhao, X., Kong, Q. (2015): Identification of the avian \u003cem\u003ePasteurella multocida\u003c/em\u003ephoP gene and evaluation of the effects of phoP deletion on virulence and immunogenicity. \u003cem\u003eInternational Journal of Molecular Science\u003c/em\u003e, \u003cstrong\u003e217\u003c/strong\u003e: PII: E12. DOI: 10.3390/ijms17010012.\u003c/li\u003e\n\u003cli\u003eYap, H.Y., Ghazali, K.L., Nazarie, W.F.W.M., Isa M.N.M., Zakaria, Z. and Omara, A.R. (2013): Draft Genome Sequence of \u003cem\u003ePasteurella multocida\u003c/em\u003e subsp. multocida Strain PMTB, Isolated from a Buffalo, Genome Announcements, \u003cstrong\u003e1\u003c/strong\u003e: 1-3.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"imno","sideBox":"Learn more about [BMC Immunology](http://bmcimmunol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/imno/default.aspx","title":"BMC Immunology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antibodies, Cut-off value, In house made indirect ELISA, Sensitivity, Specificity","lastPublishedDoi":"10.21203/rs.3.rs-8122359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8122359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFowl cholera is caused by P. multocida. The currently available commercial kit is relatively expensive for Ethiopian laboratories. To address this issue indirect ELISA kit to detect antibodies against P. multocida in chicken was developed. In-house made indirect ELISA kit was developed and optimized. Re-constituted local avian P. multocida biotype A was used for the preparation of coating antigen. The purity of the P. multocida biotype A inoculum was checked by gram staining and centrifugation at 4500 rpm for 30 minutes\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:.\\)\u003c/span\u003e\u003c/span\u003e The assay was developed and evaluated using 128 samples of chicken serum. Optimizations of various components were carried out against both anti-P. multocida hyper immune and pre immune serum. To develop an indirect ELISA test, washing buffer concentration of 500\u0026micro;l of T-20, blocking buffer concentration of 200\u0026micro;l, antigen concentration of 0.1\u0026micro;g/ml, serum samples with a final dilution of 1:500 and optimal dilution of Rabbit anti-sheep IgG HRP-conjugate of 1: 1000 \u0026micro;g/ml are determined by checker board titration (CBT) method. The cut-off value was determined to be 0.23. Sensitivity, specificity and accuracy of in-house made indirect ELISA were 96%, 90% and 93%, respectively. This test result indicated good correlation with that of the commercial kit with a Cohen's κ agreement of 0.86 with a 95% confidence interval of 0.77 to 0.95. We found that in-house made indirect ELISA was more sensitive and convenient to perform. Consequently, in-house made indirect ELISA was as good as the commercial indirect ELISA in screening chicken serum samples for detection of antibodies against P. multocida.\u003c/p\u003e","manuscriptTitle":"Development and Optimization of In-House Made Indirect Elisa Kit for the Detection of Antibodies Against Pasteurella Multocida in Chicken","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 07:24:17","doi":"10.21203/rs.3.rs-8122359/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-24T07:12:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T20:57:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-11T17:53:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296119984053760779220882385993096612195","date":"2025-12-10T13:12:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268721323848422474969972152578713015965","date":"2025-12-07T20:27:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T20:57:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-21T21:01:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-18T09:30:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-18T09:29:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Immunology","date":"2025-11-15T13:24:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"imno","sideBox":"Learn more about [BMC Immunology](http://bmcimmunol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/imno/default.aspx","title":"BMC Immunology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"146f3254-b023-445d-b647-02b28c81a781","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:01:51+00:00","versionOfRecord":{"articleIdentity":"rs-8122359","link":"https://doi.org/10.1186/s12865-026-00818-8","journal":{"identity":"bmc-immunology","isVorOnly":false,"title":"BMC Immunology"},"publishedOn":"2026-02-23 15:58:40","publishedOnDateReadable":"February 23rd, 2026"},"versionCreatedAt":"2025-12-11 07:24:17","video":"","vorDoi":"10.1186/s12865-026-00818-8","vorDoiUrl":"https://doi.org/10.1186/s12865-026-00818-8","workflowStages":[]},"version":"v1","identity":"rs-8122359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8122359","identity":"rs-8122359","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}