Sero-prevalence and an Outbreak Investigation of Foot and Mouth Disease with Isolation and Identification of Circulating Virus from Cattle in Selected Districts of Oromia and Amhara Regional States, Ethiopia

Sero-prevalence and an Outbreak Investigation of Foot and Mouth Disease with Isolation and Identification of Circulating Virus from Cattle in Selected Districts of Oromia and Amhara Regional States, Ethiopia | 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 Sero-prevalence and an Outbreak Investigation of Foot and Mouth Disease with Isolation and Identification of Circulating Virus from Cattle in Selected Districts of Oromia and Amhara Regional States, Ethiopia Tsegaye Mitiku¹, Sisay Alemu², Ayelech Muluneh³, Shihun Shimelis², and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9365906/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Foot and Mouth Disease (FMD)is a highly contagious transboundary viral disease of cloven-hoofed animals. It is one of the most economically important animal diseases in developing countries, including Ethiopia. A cross-sectional study was conducted from March 2022 to February 2023to estimate sero-prevalence, isolate, and molecular detection of foot and mouth disease virus (FMDV) circulating in selected districts of Oromia and Amhara regional states, Ethiopia. Multistage sampling was conducted to select the required sample size of cattle for the sero-prevalence study, and purposive sampling was implemented for collecting field epithelial tissues and oro-pharyngeal fluid samples for FMDV isolation and molecular detection. A total of 654 cattle sera samples were collected and tested for antibodies against FMDV using 3ABC-ELISA. The result showed an overall sero-prevalence of FMDVof 27.2%(CI = 24.0%-30.0%). The multivariable logistic regression analysis revealed that FMDV sero-positivity was significantly (P < 0.05) affected by factors such as districts, production system, herd size, and age. Cattle that were kept in large herd sizes were 2.2 times more likely to be infected with FMDV than those in small herds (OR = 2.2, 95% CI = 1.3–3.8). The odds of being positive for FMDV were also 4 times higher in cattle in the extensive production systems than those in intensive production systems. From 25 epithelial tissue and oro-pharyngeal samples, which were inoculated into monolayer BHK-21 cell lines, 19 (76%) samples exhibited foot and mouth disease virus cytopathic effect, and the viruses were isolated. In this study, three serotypes: serotype O (50%), SAT2 (42.86%), and serotype A (7.14%) were identified using antigen detection ELISA. Out of 29 samples tested by rRT-PCR, 17(58.6%) were positive for FMDV genome with Ct values ranging from 18-34.1. The current study findings revealed that FMD was prevalent in the study districts. Thus, the control strategy should be designed in such a way that early reporting of the disease outbreaks, regular sero-surveillance, and effective targeted vaccination. Further study on sequencing and characterizing the circulating virus to a lineage level is also recommended. Ethiopia Cattle FMD Virus Molecular Detection Sero-prevalence Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Background Ethiopia is one of the countries that possesses a huge number of livestock populations in the African. According to the Central Statistical Agency (2021), the country has approximately 70 million head of cattle, 42.9 million head of sheep, 52.5 million head of goats, 2.15 million head of horses, 10.80 million head of donkeys, 0.38 million head of mules, 8.1 million head of camels, and 56.87 million head of poultry at the country level. Even though the country is endowed with huge livestock populations, the contribution of livestock to the national economy is minimal compared to its potential. The main constraint is the spread of many infectious diseases, including foot and mouth disease (FMD), which drastically reduces the production and productivity of livestock (Belege et al ., 2019). Foot and mouth disease is an important contagious viral disease caused by foot and mouth disease virus (FMDV), which is a positive-sense, single-stranded, small non-enveloped RNA virus belonging to genus Aphthovirus of family Picornaviridae (Knowles and Samuel, 2003 ; OIE,2012). According to immunological classification, there are seven distinct serotypes, namely, O, A, C, Southern African Territories (SAT)1, SAT2, SAT3, and Asia 1, as well as over 60 subtypes (OIE, 2016 ). These serotypes do not provide any cross-immunity among each other after immunization or infection, so there is no universal vaccine that can confer protection against all serotypes (Knight-Jones et al., 2016 ). The disease spreads directly through contact with an infected host or mechanically by indirect contact with contaminated fomites such as clothing, shoes, vehicles, and veterinary instruments. In addition to contact, viruses can be transmitted orally or through the respiratory tract to a new susceptible animal. Aerosol transmission is the most common method of spreading within a herd. The reasons for the rapidity of disease spread are due to the presence of susceptible populations, the production of high titers in respiratory secretions, the large volumes of droplets, aerosols of virus shed by infected animals, and the rapid replication cycle with very high virus yields (Mesfin et al ., 2004; Belegio et al ., 2019). Foot and mouth disease is widely distributed in most developing countries, particularly in large areas of Africa, Asia, and South America. It has shown an extraordinary ability to cross international boundaries and cause epidemics in previously free areas (Knowles et al., 2001 ). FMDV serotypes are not distributed evenly around the world. Serotype O is the most common serotype in most parts of the world, including Ethiopia (Endris et al. , 2020). In most of the sub-Saharan African countries, serotypes O, A, SAT1, and SAT2 are still in circulation. According to Vosloo et al. ( 2002 ), serotype SAT3 has been recorded only in Uganda. The introduction of FMD in disease-free countries is mainly due to the import of live animals, animal products, and feed of animal origin (Tolosa et al., 2015 ; Knight-Jones et al., 2016 ). Foot and mouth disease is the major endemic disease in Ethiopia with abundant socio-economic importance because of reduced production, deaths in newborn animals, the huge cost of veterinary services, and restricted animal and meat movement locally and between countries (Knight-Jones and Rushton, 2013). According to the report of Jemberu et al. ( 2016 ), the total annual costs of FMD under the official control program were estimated at 1354 (864–2042) million ETB. The major cost (94%) was due to production losses, the rest were export losses, and control costs (Gezahegn et al ., 2014). Studies undertaken in Ethiopia revealed that the disease is still endemic and occurs in different parts of the country, mainly due to a lack of effective vaccines, the absence of animal movement control, and a lack of systematic disease surveillance and reliable epidemiological data (Shiferaw et al ., 2010). The disease was first recorded in Ethiopia in 1957; ever since, it has remained endemic in the country, causing several outbreaks each year (Ayelet et al ., 2012). According to the report of Gelagay et al . (2009), 4 of the 7 serotypes, namely A, O, SAT1, and SAT2, were circulated in all regions of the country. Generally, studies undertaken on FMD revealed the existence of a disease with sero-prevalence varying from 5.66% to 85.1% (Kalkidan et al ., 2023; Betelihem et al ., 2023), which indicated the status of FMD in different parts of Ethiopia. Several sero-prevalence studies (Gezahegn et al ., 2014; Yasmin et al ., 2023) and a few molecular detection studies (Sentayehu et al ., 2014; Tesfaye et al ., 2020) have been done on FMD in different parts of the country. The study areas were known for their abundance of cattle, and there was high cattle movement towards central Ethiopia for trading purposes and across different borders in the surrounding districts in search of food and water. This leads to an increase in the distribution of the disease in the area. Moreover, the absence of updated epidemiological studies and the detection of circulating serotypes in the current study areas aggravate the problems. However, the constraints of disease might be solved with appropriate monitoring, surveillance, and regular vaccination strategies. Hence, by considering the above problems, the current study was planned to conduct an epidemiological study and serotyping of circulating FMDV isolates from suspected cattle, which are useful for vaccine strain selection, tracing the source of outbreaks, the implementation of good control programs, and the eradication of the disease in the country. General Objective To estimate the sero-prevalence, isolation, and molecular detection of foot and mouth disease virus from cattle circulating in selected districts of Oromia and Amhara regional states from March 2022 to February 2023. Specific Objectives To estimate the sero-prevalence and determine risk factors associated with FMD in the study areas. To identify FMD serotypes responsible for FMD outbreaks in the selected areas To isolate and molecularly detect FMDV from lesions of diseased cattle in the study areas from March 2022 to February 2023 MATERIALS AND METHODS Study Areas The study was carried out from March 2022 to February 2023 in the selected districts. The selected districts were Shashemene district from West Arsi zone, Adami Tullu and Jido Kombolcha district from East Shewa zone of Oromia, and Moretinajiru district from North Shewa Zone of Amhara regional state (Fig. 1 ). AdamiTullu and JidoKombolchadistrict is located in the Great Rift Valley between 38°25′E and 38° 55′E and 7°35′N and 8°05′N. It is located about 160 km from Addis Ababa, Southwest of Lake Ziway at an altitude of 1500–2300 meters above sea level. The annual rainfall varies from 600 to 800mm, and it is characterized by bimodal rainfall (Martha et al ., 2020). The area is characterized by arid and semiarid climatic conditions, and it has a rapidly growing population. This district has a livestock population of 345356 cattle, 78976 sheep, 157598 goats, 19645 horses, 49687 donkeys, and 51764 poultry (AJLFRO, 2022). Other agricultural production systems in this area comprise small-scale irrigated vegetable and fruit production as well as livestock farming systems. In some areas, agro-pastoral livelihoods are also present. Feed availability and quality, especially during the dry season, are important constraints in livestock production, and it determines the physical performance of the livestock sector (Holden and Bekele, 2004 ). This district has substandard veterinary practices, and unqualified animal health experts provide services, so that animals do not receive the essential preventive care and timely treatment they need. There were FMD vaccination services in the area where the disease outbreak occurred, and unfortunately, they gave the vaccine even to diseased animals. Shashemene district is located at 250 km south of Addis Ababa. It is situated at 7 o 05′ to 7 o 19′N and 38 o 23′ to 38 o 41′E. Its climate is characterized by an annual temperature ranging from 12°C to 27°C. It has an altitude range from 1,685 to 2,722 meters above sea level. The district has an estimated 267234 cattle, 87345 sheep, 111156 goats, 43245 equine, and 56264 poultry (SDLFRO, 2022). The agro-climatic conditions of the district are favorable for agriculture with two rainy seasons. It has an annual rainfall ranging from 700 mm to 950 mm (SWADO, 2015). Animals in this district did not receive timely, appropriate, and high-quality treatment when they fell ill or were injured. This leads to unnecessary animal suffering and economic losses for owners. Veterinary services in this district were limited, especially since the problems were serious outside Sheshemene town due to the limited number of licensed veterinarians, especially in rural and remote areas, and uneven distribution of services (number of clinics). Vaccination service was given in the study areas, particularly for FMD, but insufficient animal coverage was given regardless of the serotypes circulating in the area. The third study area was the Moretina Jiru district. The coordinates for the study area are 39° 19’ 24’’ E and 10° 6’ 2’’N with an altitude range from 1,500 to 2694 m above sea level. The area is located at 195 km North East of Addis Ababa and receives an annual rainfall of 850 mm, while the temperature varies from 5.2°C in November to 28.8°C (Atinkugn, 2020). The livestock populations in the district are composed of 172536 goats, 51043 sheep, 124959 cattle, and 64868 equine (MWLFRO, 2022). The livestock farming system in this area is mainly a mixed farming system for a source of meat, milk, manure, hide, and skin, in addition to crop production. Cattle fattening is an effective system for poverty alleviation and has become an important business for smallholder farmers in this study area. Mainly, oxen used for draught power are put into a feedlot period to improve body condition under traditional feedlot conditions, as a source of income. The animal management system, particularly health care services in this district, focused on preventive measures like vaccination and early disease reporting. However, it was challenged with a shortage of licensed veterinarians, particularly in rural areas, uneven distribution of services, lack of infrastructure, and high costs of services. Many livestock owners, especially smallholder farmers, lack awareness about the importance of regular animal vaccination. This contributes to poor uptake of vaccination services. These systemic issues result in many animals not receiving necessary care. Study population The study populations were cattle with various sexes, age groups, and breeds with different management and production systems selected from two districts of the West Arsi and North Shewa Zone of ORS and ARS, respectively. In this study, the age of cattle was categorized as < 2 years, 2–4 years, and 4 years old according to Bereket et al . (2010). The herd size of the study cattle was categorized as large (30 animals), medium (15–30 animals), and small (< 15 animals) according to Amare et al. (2019). The body condition of cattle was also categorized as poor, medium, and good (Ibrahim, 2015). Inclusion and Exclusion Criteria For the sero-prevalence study, due to the presence of maternal antibodies in cattle below six months, only those older than six months were included. For isolation and molecular detection of FMDV, only those that manifested clinical signs of FMD irrespective of age group, sex, and breed were considered, while cattle without any suspected FMD clinical signs or not experienced the disease were excluded from the study on purposive sampling. Study Variables In this study, the variables were considered as independent and dependent variables, in which the independent variable is hypothesized to affect the dependent variable (outcome). The outcome variable was the sero-positivity of cattle tested by the 3ABC non-structural competitive ELISA test. Whereas the explanatory variables were animal-related factors such as sex, age, breed, and body condition, and mixed factors including study area, herd size, and production systems, which were assessed to determine the influence on FMDV sero-positivity. Study Design and Duration A cross-sectional study was conducted from March 2022 to February 2023 in the selected districts of Oromia and Amhara regional states to estimate sero-prevalence, isolate, and molecularly detect FMDV from cattle. For the isolation and molecular detection of the virus, field tissue and oropharyngeal lesions were collected from active cases of disease outbreak based on reports. Sampling Technique A multistage sampling method was used to select the sampling unit. The study districts (Moretinajiru and Shashemene) were selected based on livestock population, agro-ecologies, and route of trade, accessibility, and relative security. A total of 14 kebeles were selected; 9 kebeles were selected in Shashemene district from 36 kebeles, and 5 kebeles were selected in MoretinaJiru district from 17 kebeles. According to the livestock proportion to households in Shashemene and Moretinajiru, the average cattle size per household was 5 for Shashemene and 4 for Moretinajiru district. Then, household selection for each respective kebele was carried out by a simple random method. Hence, 53 and 97 households were selected from each of the selected kebeles of Shashemene and Moretinajiru districts, respectively. So, a total of 150 households were selected, and in each household, 5 animals in Shashemene and 4 animals in Moretinajiru were sampled. Then, if the randomly selected household owned fewer than 5 and 4 cattle for Shashemene and Moretinajiru, respectively, the household was rejected, and the next randomized household was sampled. Finally, sampling of individual animals was carried out by using a simple random sampling method from Shashemene and Moretinajiru districts (Fig. 2 ). During sampling, risk factors related to cattle, such as age, sex, breed, body condition, herd size, and production system were also recorded. Following a report of the disease outbreak, an investigation was carried out purposively in and around specific outbreak areas. A thorough physical examination was conducted on clinically sick animals to record clinical signs and disease conditions. Those animals that had obvious clinical signs and symptoms suggestive of FMD were sampled. Sample Size Determination The sample size for this study was estimated based on the formula described by Thrusfield(2018) using 95% confidence interval and 5% desired absolute precision with previous sero prevalence findings of 44% (Molla et al ., 2020) and 22.2% (Getnet et al ., 2016) in Moretinajiru and Shashemene districts, respectively, using the formula: N= \(\:\frac{\left(1.96\right)2\:\text{x}\:\text{P}\text{e}\text{x}\text{p}\:(1-\text{P}\text{e}\text{x}\text{p})}{\text{d}2}\) Where N = required sample size, Pexp = expected prevalence, and d 2 =desired absolute precision. Thus, substituting the respective values in the formula, a total of 654 (389 from Moretinajiru and 265 from Shashemene) cattle were considered for the sero-prevalence part of this study. Study Methods Clinical examination Soon after arrival at the specific outbreak site, based on outbreak reports received, the animals were examined from a distance for evidence of salivation and lameness in each animal owner’s homestead, since there was no crush in the area. In each outbreak, animals that have manifested signs of disease, such as vesicles on the tongue or ruptured vesicles in the oral cavity, on the feet and teat, as well as excessive salivation, lameness, anorexia, and a temperature rise, were considered clinically sick animals. Animals with salivation and lameness were restrained for thorough examination and specimen collection. The oral cavities of salivating animals were examined for evidence of any intact and ruptured vesicles, erosions, and ulcers on the tongue, dental pad, hard palate, gum, and mucosa of the mouth cavity. The hooves of lamed animals were thoroughly washed with clean water and carefully examined for the presence of lesions, and the coronary bands and inter-digital spaces of the hooves were examined. Animals were also examined their teat and external genitalia for the presence of any vesicular lesion (OIE, 2012 ). Sample collection and transportation Blood samples were collected from the jugular vein using plain vacutainer tubes and venoject needles for serological analysis, and tissue and oro-pharyngeal (OP) samples were aseptically collected from clinically suspected cases for molecular detection. Briefly, the jugular vein area was disinfected using 70% alcohol, and approximately 3–5 mL of blood was collected using a venoject needle and vacutainer tubes. Sampling was conducted as part of routine veterinary surveillance and outbreak investigation activities using standard minimally invasive procedures. All animals were handled in accordance with accepted animal welfare practices, and informed verbal consent was obtained from animal owners prior to sample collection. In such a way, the collected blood sample was labeled with date of collection, specific identification number, and species of the animals and transported to DebreBerhan Agricultural Research Center and Hawassa University Veterinary Medicine Microbiology Laboratory with a cool ice box for temporary storage. In the laboratory, the blood sample collected with plain vacutainer tubes was allowed to stand in a slant position for 24 hours at room temperature to collect serum. After 24 hours, samples from which serum was not clearly separated were centrifuged at 5000 rpm for three minutes to remove the remaining red blood cells and collect clear serum. Then, the serum was harvested using a sterile micropipette into cryogenic vials and stored at -20°C in aliquots. Finally, the samples were transported to AHI, being chilled in a cool icebox for serological analysis. For epithelial tissue sample collection, epithelial tissues that were freshly ruptured or unruptured vesicles were collected from FMD-suspected cattle, usually from the tongue, buccal mucosa, or feet, by using sterile forceps and scissors. The samples were collected and stored in cryovials containing virus transport medium composed of equal amounts of glycerol and 0.04-M phosphate-buffered saline (PBS) solution (pH 7.2–7.6) with some antibiotics and antifungal (OIE, 2004 ). Oro-pharyngeal samples were collected from previously FMD-suspected, infected, and asymptomatic cattle that were found near the affected herd. Samples were collected in advance or convalescent cases by a probang cup and poured into a 20 ml bottle. The fluid was added to a 5 ml tube containing about 2ml of transport medium (OIE, 2004 ). Both tissue and OP fluid samples were properly labeled and immediately transported at 4°C using an ice pack box from the collection site to Animal Health Institute (AHI), Sebeta, Ethiopia, for sample processing and other laboratory investigations. All the samples were coded by laboratory code at the reception room and transferred to the laboratory upon arrival. Tissue samples were immediately stored at -80ºC until processed. Serological diagnostic tests Serum samples were screened for antibodies against conserved non-structural protein (NSP) of the FMDV using FMD non-structural protein ELISA (3ABC-ELISA) to identify FMDV sero-positive or sero-negative animals. A commercially available test kit (ID Screen ® FMD NSP Competition, ID.VetInnovative Diagnostics, Louis Pasteur, Grables, France) was used. The test plates of the kit contain FMDV NSP captured by the coated 3ABC-specific monoclonal antibodies (MAbs). The assay was performed according to the manufacturer’s protocol. In brief, all reagents were allowed to come to room temperature(21 o C±5 o C) and were homogenized by vortex before use. The test was carried out in 3ABC antigen-coated 96 wells of micro plates. Then, the procedure commences as follows: 50µl of dilution buffer 18 was added to each microplate well. A 30µl of positive control (to A1 and B1 wells)and 30µlof negative control (to C1 and D1 wells) were added to respective plate wells. Subsequently, 30µl of each sample was added to the remaining wells using a multichannel pipette, sealed with adhesive plastic plate sealants to prevent cross-contamination and evaporation of the samples during incubation. Then, it was incubated at 37°C for 2 hours. The sera were discarded from the plates after incubation and washed 5 times by adding 300µl of wash solution immediately to avoid drying between washes. Then, 100µl of the conjugate 1X was added to each well after washing and incubated for 30minutes at 21°C. After incubation, the contents were discarded from the plates, and each well was washed 5 times by adding 300µl of wash solution. Then, 100µl of the substrate solution (Tetramethylbenzidine)was added to each plate well and again incubated for 15 minutes at 21°C in a dark place. Finally, after a final incubation, the substrate reaction was stopped by adding 100µl stop solution, and the color reactions were quantified by measuring the optical density (OD) of each well at 450 nm wavelength using an ELISA reader. The test was validated if the mean value of the negative control OD (OD NC ) was greater than 0.7 and the mean value of the positive control OD (OD PC ) was less than 30% of the negative control OD. The interpretation for each sample was based on the competition percentage (S/N%): S/N%= OD sample x100 OD NC Samples presenting S/N% less than or equal to 50% were considered positive, and those greater than 50% were considered negative. Virus isolation Virus isolation (VI) was conducted in a laminar air flow class II biosafety cabinet. The collected samples were processed and cultured on a BHK-21 cell monolayer with three subsequent passages as follows: The tissue epithelium samples were first taken from the transport media and blotted dry on absorbent paper to reduce the glycerol content that is toxic to cell culture. About 1gram of epithelial tissue was ground with sterile sand by a sterile pestle and mortar with a small volume of tissue culture medium and antibiotics (penicillin, streptomycin, and Amphotericin B solution). Dulbecco's Modified Eagle Medium (DMEM) was added until a final volume of nine times that of the added epithelial sample was reached, giving a 10% suspension (OIE, 2012 ). The suspension was clarified on a bench centrifuge at 3000 rpm for 10 minutes. The supernatant of the suspension was collected in sterile cryovial tubes and filtered by Millipore filter paper of 0.22µm pore size, labeled, and stored at -80°C until needed for further tests. About 0.5ml of filtered tissue suspensions, which were suspected to contain FMDV, were inoculated into confluent cultured baby hamster kidney (BHK-21) monolayer cells grown in 25cm 2 tissue culture flasks and incubated at 37°C for 1hour for adsorption of virus. Then, 8ml of DMEM maintenance media (2% fetal calf serum) was added to the infected cell and incubated at 37°C with 5% CO 2 in a humidified incubator for 24–48 hours, and monitored for cytopathic effect (CPE) while normal non-infected cells were served as controls. Cytopathic effect was observed after 48 hours in the positive cases, although in some samples it was observed within 24 hours through an inverted microscope. CPE was characterized by a fast destruction of the monolayer cells, cell rounding, and infected cells were disrupted and detached from the flask, which was mostly seen within 48 hours of inoculation. If no CPE was observed after 48 hours, the sample was considered as ‘no virus detected’ (NVD) and the culture was frozen at -80°C, then thawed and centrifuged at 3000 rpm for 10 min to collect supernatant for the second passage; this was repeated for the third passage and if no CPE was observed at 48 hours, then the sample was considered negative for FMDV as described by Souleyet al. ( 2018 ). Once CPE was completed in the cultures, the fluids were tested for FMDV using ELISA and rRT-PCR testing. Finally, isolated samples were labeled according to a system specified by OIE standards (OIE, 2012 ). The first three letters signify the sample’s country of origin, ETH for Ethiopia, followed by a number representing the number of the sample in the batch of samples, and finally the last two digits of the year in which the sampling was carried out. Serotyping of FMD virus Foot and mouth disease virus serotypes were identified using FMDV antigen detection and serotyping sandwich ELISA (IZSLER, Brescia, Italy), which was performed with selected combinations of anti-FMDV monoclonal antibodies (MAbs), used as coated and conjugated antibodies. The test was applied for the detection and typing of the FMD virus. The kit was designed for the detection and typing of FMD viruses of types O, A, SAT1, and SAT2. A pan-FMD test was used to detect any isolates of serotypes O, A, C, and Asia1, and in addition, some of the SAT serotypes were included in the kit to complement the specific typing and to detect FMD viruses that might have escaped binding to the selected type-specific MAb.The microplates were supplied pre-coated with catchedMAbs to detect 10 samples at a time with one positive and one negative control for each serotype. The controls were already incorporated into the ELISA microplate trapped by the respective captured MAbs. The cell-culture isolates were subjected to the test assay and serotyped. The test was carried out as per the manufacturer’s recommendation. Briefly, the first samples were diluted in ½ in diluent buffer, and 50µl per well of each sample was distributed to 8 wells of a column (from A-H rows). Then, 50µl of diluent buffer per well was added to all wells of 11 and 12 columns (positive and negative control, respectively), and plates were incubated at room temperature for 1 hour. After incubation, all the fluids in the wells were discarded, and the plates were tapped hard to remove all the residual fluid. Then, 200µl washing solutions per well were added in all wells and incubated for 3 min at room temperature; subsequently, wells were emptied and washed repeatedly twice (three washing cycles in total). Then all residual fluids were removed by tapping on a clean absorbent paper, and 50µl per well of conjugates (conjugate A) was added into rows from A to F, and the same volume of conjugate B was added into rows G and H. Then, the plates were covered and incubated at room temperature for 1hour. After incubation, the last washing was performed for 5 minutes, and 50µl per well of substrate(chromogen) was added to all wells, and plates were covered and left at room temperature for 20minutes in the dark; the time was calibrated when the first well was filled. The reaction was stopped by adding 50µl per well of stop solution (sulfuric acid), and the wells’ contents were properly mixed before reading. Immediately after stopping, read the optical density (OD) of each well at 450 nm wavelength using a microplate reader. Criteria for test validity: The positive inactivated controls were expected to give OD values of ≥ 1.0 unit, while the negative control for serotypes O, A, C, Asia 1, and Pan-FMDV was expected to give OD values < 0.1 unit, and the negative control for serotypes SAT1 and SAT2 were expected to give OD values ≤ 0.2unit. Results for the samples examined were interpreted, after subtraction of the OD value of each negative control from the OD value measured with the corresponding captured Mab. Molecular identification RNA extraction An epithelial tissue suspension was prepared by grinding samples using a sterile tissue-grinding device (mortar and pestle) and silica sand to disrupt viral cells. For every 29 samples, the procedure was repeated by cleaning materials for the next activity and transferring the suspension of the ground part into the centrifuge tube. The suspension was centrifuged at 3000 revolutions per minute (rpm) for 10 minutes, and the supernatant was taken for molecular work. Total viral RNA was extracted from digested tissue supernatants and oro-pharyngeal fluid suspensions using QIAamp® Viral RNA Mini Kit (QIAGEN, Cat. No.52906, Hilden, Germany)in accordance with the manufacturer’s instructions. Briefly, lysine solution buffer and carrier RNA were prepared and mixed, then 560µl AVL buffer containing carrier RNA was dispensed into sterilized centrifuge tubes. The samples prepared were vortexed to mix properly, then 140µl of the sample suspension was transferred into microcentrifuge tubes containing AVL buffer and carrier RNA, and then the tubes were vortexed for 1 second and incubated at room temperature for 10 min for lysing purposes. Then, 560µl absolute alcohol (ethanol) was added to centrifuge tubes containing AVL buffer, carrier RNA, and samples. The tubes were mixed by vortexing for 1 second. A 630µl of the solution was added into QIAamp mini spin columns in 2ml collection tubes, then centrifuged at 8000 rpm for 1 minute, and the filtrate fluid was discarded, then the column filter was transferred to new collection tubes. This procedure was repeated twice. Then, 500µl of wash Buffer AW-1 was added to the QIAamp mini spin column tubes and centrifuged again at 8000 rpm for 1 minute. The filtrate was discarded, and the columns were placed into fresh 2ml collection tubes. Then 500µl of wash buffer AW-2 was added to the columns and centrifuged at 14000 rpm for 3 minutes, and the filtrate was discarded. Then, the columns were centrifuged at 14000 rpm for 1 minute to dry. A 60µl of AVE buffer was added into the column and incubated at room temperature for 1 minute, then centrifuged at 8000 rpm for 1 minute to elute RNA from the QIAamp mini spin column, and then the QIAamp mini spin column was removed. Finally, extracted RNA was kept at + 4°C for immediate use or stored at -80°C for further molecular characterization of FMDV. Detection of viral RNA by rRT- PCR The presence of viral RNA was screened by using an Applied Biosystems 7500 fast one-step real-time PCR thermocycler machine. The extracted RNA was converted to cDNA using a reverse transcriptase enzyme, and the cDNA was amplified using FMDV-specific forward and reverse primers. Detection of the 3D (pol) region of FMDV was performed using the Qiamp viral RNA mini kit as per instructions and the selected catalog number of 52906. The nucleotide sequences of forward primer (FMD-3DF)of 5’-ACTGGGTTTTACAAACCTGTGA-3’, reverse primer (FMD-3DR) of 5’-GCGAGTCCTGCCACGGA-3’, and TaqMan probe 5’-6-FAM-TCCTTTGCACGCCGTGGGAC-TAMRA-3’ were used in this assay. The probe labeled with 6- (FAM) at the 5’ end and the quencher tetra-methyl-rhodamine (TAMRA) at the 3’end in a real-time RT-PCR reaction that detected the 3Dpol gene sequence in all the FMDV serotypes. In brief, a master mix was prepared by mixing 12.5µl of 2x - reaction, 2µl of forward primer, 2µl of reverse primer, 1µl of RNAse-free water, 1.5µl of Taqman probe, 0.5µl Rox and 0.5µl of superscript ®III RT to make a total of 20µl per sample for each reaction of PCR per well per plate including the positive and negative control master mix, then thoroughly mixed by pulse vortexing. 5µl of RNA sample template (extracted RNA)was added to each reaction. Then, the plate was sealed with adhesive sealant and loaded into the thermal cycler machine for processing according to QIAmgen one-step RT-PCR kit protocols. The amplification reaction was set by creating a plate sheet for the PCR machine to run using the 7500 Fast System SDS Software. Accordingly, the amplification was carried out with a final reaction volume of 25µl containing a 20µl master mix and 5µl RNA template submitted to a thermal profile of one cycle reverse transcription, and FMDV was detected through cycle threshold (Ct) values based on baseline and graphs. The one-step RT-PCR amplification started with reverse transcription (RT) cDNA synthesis at 50°C for 30 min; followed by activation or denaturation of reverse transcriptase at 95°C for 10 min; then annealing and extension were conducted at 95°C for 15seconds and 60°C for 1min, respectively. In such a way, the clinical samples detected at a cycle threshold (Ct) value < 35 were declared as positive, whereas Ct values 35 indicated that the samples were negative. Data Management and Analysis Data generated from field and laboratory work were coded, entered, and stored in a Microsoft Excel-2019 spreadsheet and analyzed using STATA version 14.0 for Windows (Stata Corp., College Station, TX, USA). Descriptive statistics were used to summarize data, and the prevalence was calculated for all associated risk factors. Both univariable and multivariable logistic regression were used to assess the association between factors and sero-positivity of FMDV. For multivariable logistic regression analysis, all putative risk factors having a critical p-value < 0.25 in univariable analysis were selected. After selecting the final model of logistic regression, the beta (β) coefficients of each independent variable were observed to estimate the odds ratio (OR), which is used for assessing the strength of association. Statistically significant association between variables was considered to exist if the computed p-value at 95% confidence interval and 5% degree of precision is < 0.05. In cell culture results, CPE development and molecular detection results were recorded and tabulated. RESULTS Sero-prevalence of FMD In this study, a total of 654 cattle sera were tested using the 3ABC non-structural competitive ELISA test, and 178 sera were found to be positive. Thus, as shown in Table 1 , the overall sero-prevalence of FMD was 27.2% (95% CI: 24.0–30.0) in the study areas. Higher sero-prevalence of FMD was observed in Moretinajiru district (39.3%) than in cattle found in Shashemene (20.0%) district, with a statistically significant difference (p < 0.05). Table 1 FMD prevalence of cattle in the selected districts from March 2022 to February 2023 District Number of Examined Number of Positive Prevalence (95% CI) p-value MoretinaJiru 389 153 39.3% (34.0–44.0) 0.001 Shashemene 265 53 20.0% (15.0–25.0) Total 654 178 27.2% (24.0–30.0) Factors Associated with FMD Several factors, including district, age, breed, sex, herd size, body condition, and production system, were considered for the risk factor analysis. Sero-prevalence of antibodies against FMDV was compared between different age groups of cattle. From the results, it is evident that cattle older than 4 years had a greater sero-prevalence of 37.6% when compared to cattle between 2–4 years and below 2 years of age, whose sero-prevalence was estimated to be 26.9% and 21.9%, respectively. Additionally, cattle within a large herd size (37.1%) had a larger prevalence than the medium (25.0%) and the small cattle herd size, with a significant difference (p < 0.05). The prevalence was also found to be higher in large herd size (37.1%) as compared with medium herd size (25.0%) and small animal herd size (19.1%). Sero-prevalence was found to be significantly different in the production system. The highest sero-prevalence was observed in the extensive production system (41.3%), as compared to the intensive production system (19.0%) and the semi-intensive production system (27.2%) (Table 2 ). Univariable logistic regression was run to identify the possible individual risk factors for sero-positivity to FMDV antibody. Univariable logistic regression analysis shows that districts, age, herd size, and production system have statistically significant associations with FMD infection (P < 0.05). However, age, sex, and breed were not significantly associated with FMDV sero-positivity (P 0.05) as shown in Table 2 . The variables with a p-value of less than 0.25 in univariable analysis were taken to the multivariable analysis to control confounders and to see their independent effect on FMDV sero-positivity. Accordingly, districts, age, herd size, and production system were subjected to multivariable analysis. Table 2 Univariable logistic regression analysis of factors for FMDV sero-positivity Factors Categories No. Tested No. Positive Prevalence (%) OR (95%CI) P-value District Shashemene 265 53 20 Ref Moretinajiru 389 153 39.3 2.75 (1.9–3.9) 0.001* Sex Male 134 33 24.6 Ref Female 520 145 27.9 1.183(0.7–1.8) 0.450 Age < 2 years 178 39 21.9 Ref 2–4 years 375 101 26.9 1.636(1-2.6) 0.037* 4 years 101 38 37.6 2.15(1.26–3.6) 0.005* Breed Local 181 42 23.2 Ref Cross 349 95 27.2 1.321 (0.85-2) 0.218 Exotic 124 41 33.1 1.635(1-2.7) 0.58 Herd size Small 136 26 19.1 Ref Medium 332 83 25 1.769(1.2–2.6) 0.004* Large 186 69 37.1 2.495(1.4–4.2) 0.001* Bo.Condition Good 183 39 21.3 Ref Medium 295 85 28.5 1.142 (0.8–1.7) 0.523 Poor 176 55 31.2 1.678 (1-2.7) 0.33 Pro.System Intensive 216 41 19 Ref Semi-intensive 312 85 27.2 1.877(1.2–2.9) 0.004* Extensive 126 52 41.3 2.999(1.8–4.9) 0.001* Keys: *-Significant; CI-Confidence Interval; OR-Odd Ratio; Ref-Reference; No. tested-Number of tested; No. Positive number of positive, Pro. system-Production System, Bo.condition-Body condition In the current study, multivariable logistic regression analysis revealed that study districts, age, herd size, and production systems remained as independent predictors of FMDV sero-positivity with significant differences (P < 0.05). Foot and mouth disease sero-positivity was significantly higher in Moretinajiru than in the Shashemene district. Thus, cattle found in Moretinajiru were 2.18 times more likely to develop the disease than those cattle found in the Shashemene district. Concerning herd size, cattle from large herd sizes were 2.2 times more likely to develop the disease than animals from small herd sizes. Similarly, cattle from medium herd size were 1.6 times more likely to be at higher risk than small animal herd for FMD infection. The odds of being positive for FMDV were also 4 times as high as those reared in an extensive production system and 2.1 times higher in animals that were reared in a semi-intensive production system, as compared with intensive production systems, with a significant difference (P < 0.05). Hence, the age groups of cattle were found statistically significant, which means cattle older than 4years were more than 2 times, and cattle with age group from 2–4 years were 1.56 times more likely at higher risk for FMD infection as compared with cattle below two years of age. For a more in-depth understanding, see Table 3 . Table 3 Multivariable logistic regression analysis of different risk factors Factors Categories No. Tested No. Positive Prevalence (%) OR (95% CI) P-value District Shashemene 265 53 20 Ref Moretinajiru 389 153 39.3 2.18 (1.5–3.1) 0.001* Age < 2 years 178 39 21.9 Ref 2–4 years 375 101 26.9 1.566 (0.96–2.5) 0.071 4 years 101 38 37.6 2.147(1.2–3.8) 0.009* Herd size Small 136 26 19.1 Ref Medium 332 83 25 1.59 (1.06–2.4) 0.026* Large 186 69 37.1 2.21 (1.3–3.8) 0.005* Production system Intensive 216 41 19 Ref Semi-intensive 312 85 27.2 2.099 (1.3–3.3) 0.001* Extensive 126 52 41.3 4.043 (1.8–5.08) 0.001* Keys: *-Significant; OR-Odds Ratio; CI-Confidence Interval; No. Tested-Number of tested; No. Positive-Number of positive; Ref-Reference Clinical Examination Among 157 animals observed in three districts during outbreak investigation based on reports, 66 (42.0%) of cattle were found to be clinically infected with FMD. Using this report-wise, information was collected from such disease-affected areas. Cattle were carefully examined for the presence of typical clinical signs of FMD. The observed clinical signs in sick cattle were vesicles and erosions in gums, dorsum of the tongue, and in the inter-digital spaces and coronary band, erosions, profuse salivation, and lameness that were suggestive of FMD cases, see Table 4 . Table 4 Summary of FMD outbreaks that occurred in selected study areas. Sites of outbreaks District Kebele No. of examined animals No. of infected animals No. of deaths occurred Shashemene Ale luilu 27 9 - Faji sole 15 5 2 Denisa 13 7 - Adamitullu and Jidokombolcha Wolenbula 30 10 - Naka 19 11 1 Moretinajiru Yimedeb 21 7 - Jihurzuria 20 6 1 Mangudo 12 5 - Total 157 66 (42.0%) 4 (6.0%) FMD Virus Isolation Out of 66 samples collected from different outbreaks, 25 (37.9%) representative samples were subjected to virus isolation using Baby Hamster Kidney (BHK-21) cell culture, while the remaining 41 (62.1%) samples were not inoculated on the cells because two or more samples were collected from the same outbreak in the study areas. The current study result revealed that out of 25 clinical samples inoculated on BHK-21 cell culture, CPE was observed on 19 (76.0%) samples (Table 5 ), while the virus did not grow (CPE was not observed) on the rest of 6 (24.0%) samples even at the third passage. The current results indicated that the virus isolated from clinical samples on BHK-21 cells showed CPE, which was characterized by the fast destruction of the monolayer cells. Infected cells were round, formed singly, and the destruction of the cells was mostly seen within 48 hours of inoculation. As time progressed, there was sloughing of cells or monolayer detachment from the wall of the cell culture flask, and some cells were severely damaged within 72 hours post-inoculation. Finally, cell death, which indicated the presence of the virus, but negative samples did not show CPE, which might not induce the morphological change of cells. Moreover, the morphology of uninfected control BHK-21 cell lines was compared with that of FMD-infected BHK-21 cells, as shown in Fig. 3 . Samples that showed typical CPE (positive cases) were used for serotype identification of the virus involved in the outbreak cases using antigen detection sandwich ELISA. Table 5 FMDV isolated from cattle in different outbreaks in the selected areas from March 2022 to February 2023 Site of Outbreak Date of collection Type of sample Name of isolated virus District Kebele With CPE Without CPE Shashemene Ale Luilu 20/09/2022 21/09/2022 21/09/2022 Tissue Tissue OP ETH-03-22 ETH-05-22 - - - ETH-06-22 Mudeta 11/10/2022 11/10/2022 Tissue OP ETH-08-22 ETH-09-22 - - Faji Sole 19/10/2022 19/10/2022 24/10/2022 Tissue Tissue OP ETH-10-22 ETH-11-22 ETH-13-22 - - - Denisa 26/01/2023 26/01/2023 Tissue OP - - ETH-40-23 ETH-41-23 Adamitullu and Jidokombolcha Naka 27/10/2022 27/10/2022 Tissue Tissue ETH-15-22 ETH-16-22 - - Welenbula 28/10/2022 28/10/2022 OP Tissue - - ETH-18-22 ETH-20-22 DestaAbjata 17/01/2023 17/01/2023 Tissue OP ETH-37-23 ETH-38-23 - - Moretinajiru Yimedeb 26/12/2022 27/12/2022 Tissue Tissue ETH-22-22 ETH-23-22 - - Jihurzuria 11/01/2023 11/01/2023 12/01/2023 12/01/2023 Tissue - ETH-27-23 ETH-28-23 ETH-30-23 ETH-25-23 - - - Mangudo 13/01/2023 13/01/2023 14/01/2023 Tissue ETH-32-23 ETH-33-23 ETH-35-23 - - - Total 19 6 ETH-Ethiopia; OP-Oro-pharyngeal fluid C and D show rounding and cell detachment from the flask and lysis of cells (indicated by arrows) against respective controls (A and B). Serotyping of FMDV Isolates Foot and mouth disease virus serotyping was executed using an antigen detection sandwich ELISA. The antigen detection ELISA test was employed for the identification and serotyping of FMDV. A total of 19 positive samples’ suspensions that exhibited FMDV cytopathic effect (CPE) on BHK-21 cell were needed to be tested for the detection of serotype identification using sandwich ELISA on a microplate containing 96 wells. Out of 19 samples subjected to serotyping by sandwich ELISA, 14 (73.7%) samples were found positive, and three serotypes (O, SAT2, and A) were identified. The outbreaks were predominantly caused by FMDV serotype O (50%), followed by SAT2 (42.86%)and serotype A (7.14%). The results demonstrated that serotype O was found in samples taken from MoretinaJiru district, whereas serotype SAT2 and A were found in samples taken from Shashemene, AdamiTullu, and Jidokombolcha districts. Samples collected from the Shashemene district were serotyped into “SAT2”, whereas samples collected from the Adami Tullu and Jidokombolcha districts were serotyped into “SAT2” and “A”, and samples collected from outbreaks in the Moretinajiru district were serotyped into “O”. Furthermore, bovine epithelial tissues collected during the outbreak at Shashemene district were serotyped into “SAT2”, whereas oro-pharyngeal fluid samples collected from clinical cases in the same district became more negative. All the results of FMD virus serotypes were summarized in Table 6 as indicated below. Table 6 Serotypes of FMDV identified in the study areas Site of outbreak Date of collection Positive CPE Type of sample Virus name Serotype No serotype District Kebele Shashemene Ale Luilu 20/09/2022 21/09/2022 2 Tissue ETH-03-22 ETH-05-22 SAT2 - - NVD Mudeta 11/10/2022 11/10/2022 2 Tissue OP ETH-08-22 ETH-09-22 SAT2 SAT2 - - Faji Sole 19/10/2022 19/10/2022 24/10/2022 3 Tissue Tissue OP ETH-10-22 ETH-11-22ETH-13-22 SAT2 SAT2 - - - NVD Adamitullu and Jidokombolcha Naka 27/10/2022 27/10/2022 2 Tissue Tissue ETH-15-22 ETH-16-22 SAT2 A - - Destaabjata 17/01/2023 17/01/2023 2 Tissue OP ETH-37-23 ETH-38-23 - - NVD Moretinajiru Yimedeb 26/12/2022 27/12/2022 2 Tissue Tissue ETH-22-22 ETH-23-22 O - - NVD JihurZuria 11/01/2023 12/01/2023 12/01/2023 3 Tissue ETH-27-23 ETH-28-23 ETH-30-23 O O O - - - Mangudo 13/01/2023 13/01/2023 14/01/2023 3 Tissue ETH-32-23 ETH-33-23 ETH-35-23 O O O - - - Total 19 Keys OP- Oro-pharyngeal fluid; NVD-No virus detected. Molecular Detection FMDV Gene Detection by Real-Time RT-PCR The presence of FMD viral genetic material was detected by using the real-time reverse-transcription polymerase chain reaction (rRT-PCR) method. The rRT-PCR has been widely adopted by FMD reference laboratories as a principal tool for FMDV detection, offering high sensitivity and rapid sample throughput (Shaw et al., 2007 ; Reid et al ., 2009). The extracted RNA virus from clinical samples was tested by the rRT-PCR method targeting the 3D regions of the FMD virus genome to determine the presence of viral RNA in clinical samples (Callahan et al., 2002 ). The rRT-PCR could determine the serotype of the FMD virus. However, serotypes were unable to be identified due to a lack of primers and probes. For this limitation, virus isolation (VI) and antigen detection ELISA (Ag-ELISA) were implemented for the identification of FMD virus serotypes. Out of the total 29 suspected clinical samples tested with RT- PCR, 17(58.6%) were found positive for FMDV nucleic acid with Ct values ranging from 18-34.1 with a cutoff value of 35. Ct values < 35 were considered positive, and a positive control was detected at a 20.75 Ct value, while samples with Ct values 35 were considered negative results(Fig. 4 ). The remaining samples that did not show Ct values were considered NVD. The lowest Ct value was 18, recorded in epithelial tissue samples collected from Shashemene district, while the highest Ct value, 43, was obtained from oro-pharyngeal fluid samples collected from Moretinajiru district (Fig. 4 ). The oro-pharyngeal fluid samples had Ct values higher than epithelial tissue suspensions, indicating that there were higher levels of viral RNA concentration in the epithelial tissue samples than in the oral swab samples. In most of the epithelial samples tested, the Ct values were lower than the values of oropharyngeal fluid samples. In the above figure, the real-time RT-PCR graph was sketched with Ct values between 18 and 34.1, which were considered positive with respect to the positive control, whereas sample35 were considered negative as compared to the negative control. DISCUSSION Foot and Mouth Disease (FMD) is an important highly contagious transboundary viral disease that affects cloven-hoofed domesticated and wild animals in Asia and Africa (Zeedan et al .,2020). The result presented here suggests that FMD is the main bottleneck for livestock production in the study areas. The current study revealed that an overall sero-prevalence of FMD was 27.2%, which agreed with the findings of various researchers who reported 24.22% in Adama and Asella (Mishamo et al ., 2018), 26.8% in Gamo Zone (Mesfin et al ., 2022), 28.2% in East Shewa Zone (Yasmin et al ., 2023), 26% in Eritrea (Dekker et al., 2015 ), and 22.5% in Kenya (Chepkwony et al., 2021 ).On the other hand, the finding of this study was higher than the reports of Esayase et al . (2010) in South Omo Zone(8.18%), Gezahegn (2014) in Central Ethiopia (14.5%), Dinaol et al . (2016) in Eastern Showa Zone (10.88%), and Kebede et al . (2018) in West and South West Shoa Zones (15.0%).On the contrary, the prevalence finding was much lower than the previous results reported by Asamenew et al . (2016) with 42.7% in Borena Zone, Lencho et al . (2020) with 40.4% in West Shoa Zone, Beksisa et al . (2020) with 49.2% in Welmera District, and Kalkidan et al . (2023) with 85.1% in Addis Ababa. Variation in the prevalence can be due to differences in sample size, geographical location, and management system. In the present study, a statistically significant association was found between study districts and FMD sero-prevalence. FMD sero-prevalence obtained in the study areas was39.3% and 20.0% in Moretinajiru and Shashemene districts, respectively. Thus, cattle found in Moretinajiru district were 2.18 times more likely to develop the disease than those found in Shashemene district. Variation in the prevalence can be due to differences in animal immune status, sample size, management system, sampling procedure used, geographical location, interaction of cattle with other animals, and the existence of recent outbreak cases. FMD prevalence variation occurred based on differences in individual animals’ breed, immune status, interaction of cattle with other animals like small ruminants, and production system (Mishamo et al ., 2018). In this study, prevalence varied between age groups in a statistically significant manner. This finding was in line with the results of Asamenew et al . (2016) and Wungak et al. ( 2016 ), who reported that FMDV seropositivity in animals increased as animals aged. In contradiction to this finding, Esayas et al . (2009) in the BenchiMaji zone and Dinaol et al . (2016) in the Eastern Showa zone reported no statistically significant difference in the sero-prevalence of FMD in different age groups. The risk of FMD infection in 4 years increased by 2.15 times, making them more likely to have a chance of contracting FMD than < 2 years old. The odds of being seropositive for FMDV antibodies were 1.566 times higher in 2–4 years as compared with < 2 years. The possible justification for the age association with FMD sero-prevalence could be due to adult cattle have acquired through exposure over time and could get access to mix with other herds at communal pasture land and market places. Relatively low sero-prevalence in animal groups below 2 years old might be indicative of the existence of passive maternal immunity and low frequency of exposure (Abdulaji et al ., 2011), and young animals were often managed separately at around the homestead, so that young animals have a low frequency of exposure to the virus (Teshager and Wossene, 2021). In the current study, herds with a large number of animals (37.1%) had a significantly higher sero-prevalence than herds with medium and small numbers of animals (25.0%, 19.1%, respectively. This finding was in line with previous studies conducted by Esay et al . (2009), Tolosa et al. ( 2015 ), and Mishamo et al . (2018), who reported that higher sero-prevalence of FMD was observed in larger herd sizes. In this finding, those animals from medium herd size and large herd size were 1.59and 2.213 times more likely to develop the disease as compared to those animals from small herd size, while keeping the other factors constant. This significant difference might be because the probability of getting the disease increases as herd size increases due to the crowding of animals, which facilitates the frequency of direct contact and hence enhances the chances of transmission easily because of the contagious nature of the disease(Mohammed, 2022). The findings of this study also revealed that relatively higher sero-prevalence of FMDV was observed in extensive management systems (41.3%) than semi-intensive (27.2%) and intensive (19.0%) management systems, with a statistically significant difference. This finding was agreed with in the report of Fanos et al . (2022), who stated that seropositivity increased in animals raised under an extensive management system (31.7%), followed by semi-intensive (25.0%) and intensively (23.0%) managed animals. The current study showed that extensively and semi-intensively managed cattle were 4 and 2 times more likely to have a chance of contracting FMD, respectively, than intensively managed cattle. When animals feed outside of the farm, they will have more exposure to the virus because of contact with infected cattle at communal grazing and watering points (Belege et al ., 2019). This could be attributed to the crowding of animals, which can facilitate the frequency of direct contact and hence enhance the chances of transmission. There is also an undeniable fact that the spread of the disease from one herd to another herd and from one area to another is almost frequently due to the movement of an infected animal from an infected herd to a non-infected susceptible herd (Constable et al., 2017 ). In this study of the total number of 157 cattle examined, 66 (42%) animals manifested clinical signs that were suggestive of FMD. The current findings were related to the previous clinical findings of Haileleul (2011), who reported that 36.9%of animals manifested clinical signs during the outbreak investigation of FMD. This was justified by previous reports of Kitching et al . (2005) and McLaws et al. ( 2006 ), who described that variations in clinical manifestation and its severity were associated with the virus strains, infection dose of the virus, species affected, susceptibility of the host, farming system, and previous exposure of the animal. In this study, the BHK-21 cell line was used to isolate FMD virus from clinical samples. Out of 25 field samples subjected to cell culture,19(76%) field samples showed FMDV-induced CPE after 24–48 hours of incubation in a humidified incubator at 37°C and 5% CO 2 . The CPE was characterized by rounding of cells, infected cells detaching from flask surfaces, and fast destruction of the cell monolayer, which agreed with previous reports of Yeneneh (2014), Beksisa (2017), and Mishamo et al . (2018). However, the other 6 (24%) samples did not show CPE even after the third passage. This could be due to the death of the virus during transportation or the presence of a very small quantity of live virus that could not overcome the challenge of the cell culture environment (Sentayehu et al ., 2014). In addition, samples had been taken at the late stage of recovery during sample collection (Metages, 2018) and in the laboratory; the cold chain system might not be properly maintained, which leads to the loss of FMDV, and the samples might have been taken from animals that had other vesicular diseases with similar clinical signs like vesicular diseases. In the current study, from 19 samples serotyped by antigen-capturing sandwich ELISA, three serotypes (O, SAT2, and A) of FMD viruses were isolated. The most dominant serotype was O (50%), followed by SAT2 (42.86%)and A (7.14%), respectively. These results were consistent with previous reports of Jemberu et al. ( 2016 ), who indicated that serotype O (70%) was followed by serotype SAT2 (20.8%) during outbreaks in Ethiopia. In support of these findings, studies conducted by Noureldin and Elfadil ( 2014 ) in Khartoum state of Sudan indicated that serotype O (82.6%) and SAT2 (40%) were the main circulating FMDVs in the cattle and the most prevalent serotype in the world (Kitching et al., 2007 ). Serotype O was isolated from the samples collected from the Moretina Jiru district, which was the most prevalent serotype in this finding. Previously, this serotype was reported from samples collected from Shewarobit and South Wollo districts (Metages, 2018) of Amhara Regional State, which was in proximity to this study area. Serotypes SAT2 and A were isolated from samples collected from Adami Tulu Jidokombolcha and Shashemene districts of Oromia Regional State. According to the report of Mishamo et al . (2018), serotype A was identified indifferent districts of the Arsi zone in Oromia regional state that were neighbors to Adami Tulu Jidokombolcha and Shashemene districts. Thus, because of the presence of unrestricted animal movement, a high rate of contact between animals at market areas, communal grazing places, and at watering points, which was enhanced by windborne transmission of disease serotypes from one site to another, could easily happen. In the present study, detection of the FMDV genome was conducted from 29 fields, and isolated samples were tested by rRT-PCR targeting the 3D region. Of the 17 (58.6%) samples were positive for FMD viral RNA. The remaining samples were detected as negative, which indicated the absence of a viral genome. From the result, the lowest (18) and the highest (43) Ct values were recorded in epithelial and oropharyngeal samples collected from Moretinajiru and Adami Tulu Jidokombolcha areas, respectively. The Ct values from epithelial samples were lower than those from oropharyngeal fluid samples. This might indicate that lower concentration of FMDV in oro-pharyngeal fluid samples than in freshly collected epithelial samples due to sampling time, since they were collected from cattle in the later stages of outbreaks. The possible reason for the absence of detectable viral genome in some samples indicated that sampling was done at a late phase of infection, with low virus load in the tissues, and low quantity/quality of tissues that could have hampered the amplification by RT-PCR. Moreover, it might be due to the degradation of the RNA genome that the bacterial RNAses or other degradative enzymes might have degraded the viral genome during sample storage or transportation. These reasons could be the possible causes of bringing in either weak or no detectable signals by RT-PCR. CONCLUSION AND RECOMMENDATIONS The present study was designed to estimate the sero-prevalence, isolation, and molecular detection of FMDV circulating in selected districts of Oromia and Amhara regional states, Ethiopia. Thus, the result of 3ABC-ELISA showed an overall sero-prevalence of 27.2%. The detection of antibodies in the cattle sera suggested that FMDV was circulating in the study areas. In this study, district, age, herd size, and production system were the predictors of FMDV sero-positivity. The research has also shown that typical clinical signs of FMD were identified, and the case was confirmed by cultural isolation, and FMDV was detected with real-time PCR, which is the first report in the study areas. In the current study, serotypes O, SAT2, and A were identified, and serotype O was the dominant one. The findings of this research provide insights for the identification of topotypes circulated in the study areas and vaccine-matching studies of field isolates to evaluate vaccine protection potential, which have paramount importance for effective vaccine development. However, this study is limited by the absence of genetic sequencing of FMDV isolates, which was important for vaccine matching. Finally, the presence of huge susceptible animals, age variability, breed susceptibility, lack of molecular information for vaccination programs, free movement, communal grazing places, and high contact of animals at common points were identified as major risk factors that could contribute to the occurrence of FMD. Therefore, in line with the above conclusion, the following recommendations are forwarded: Animals should be vaccinated with appropriate strains of vaccines targeting circulating serotypes. Continuous surveillance, serotyping, and molecular characterization of FMDV needs to be conducted to check the introduction and circulation of new serotypes of the virus in different parts of the country. It is necessary to train field veterinarians on disease outbreak reporting, and the reporting system should also be strengthened by digitalizing it for easy access to disease outbreak areas. Declarations Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . Funding This research did not receive specific funding from any funding agency in the public, commercial, or not-for-profit sectors. Acknowledgments The authors would like to thank the Animal Health Institute, Ethiopia, for their logistical support and for granting full access to their laboratories. Ethical Approval and Consent to Participate This study was conducted as an observational sero-surveillance and outbreak investigation of Foot-and-Mouth Disease (FMD) in cattle, as part of routine veterinary public health and disease control activities in Ethiopia. The study did not involve any experimental interventions such as deliberate infection, treatment, vaccination, or euthanasia of animals. Blood, epithelial, and oro-pharyngeal samples were collected from naturally infected or suspected animals using standard, minimally invasive diagnostic procedures. All sampled animals were handled appropriately and returned to their owners immediately after sample collection without further intervention. No animals were anesthetized, euthanized, or sacrificed for the purpose of this study. As the work constituted routine disease surveillance and outbreak investigation and did not involve experimental manipulation of animals, formal ethical approval from an Institutional Review Board (IRB) or Institutional Animal Care and Use Committee (IACUC) was not required under applicable national veterinary guidelines. All procedures were conducted in accordance with internationally accepted animal welfare principles, and all efforts were made to minimize stress and discomfort during sample collection. All animals included in the study were privately owned, and informed verbal consent was obtained from the animal owners prior to sampling. Clinical trial number: Not applicable. Consent to Publication The supplementary images and figures included in this study (PCR results, cell culture images, and study area maps) do not contain any human participants, personal data, or identifiable individual information. Therefore, specific written consent for publication was not required. Data Availability Statement The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. References Abdulaji Mohamoud E, Tessema, Degefu H. Sero-prevalence of bovine foot and mouth disease (FMD) in Awbere and Babille districts of Jijiga zone, Somalia regional state, Eastern Ethiopia. Afr J Microbiol Res. 2011;5(21):3559–63. Adamitullu and Jidokombolcha Livestock and Fisheries Resource Office (AJLFO). 2022. 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Lencho Megersa B, Ahmed G, Mulatu MS, and Gelma Boneya. 2020. Sero-prevalenceassociated risk factors of footmouth disease in cattle in West Shewa Zone, Ethiopia. Veterinary Medicine International , Volume 2020, Article ID 6821809, 6 pages. Martha Kidemu M, Gebreyesus M, Semere A, Worku AA. Traditional Ecological Knowledge for Climate Change Assessment and Rainfall Prediction: A Case of Adami Tulu Jido, Kombolcha District, Oromia Region, Ethiopia. Int J Nat Resource Ecol Manage. 2020;5(2):43–8. McLaws M, Ribble C, Martin W, Stephen C. Factors associated with the clinical diagnosis of foot and mouth disease during the 2001 epidemic in the UK. Prev Vet Med. 2006;77:65–81. Mesfin Sahle, Venter EH, Dwarka RM, Vosloo W. Molecular epidemiology of serotype O foot-and-mouth disease virus isolated from cattle in Ethiopia between 1979 and 2001. Onderstepoort J Vet Res. 2004;71:129–38. Mesfin Shurbe B, Simeon W, Seyoum A, Muluneh E, Tora, and Edget Abayneh. Sero-prevalence and associated risk factors for foot and mouth disease virus sero-positivity in cattle in selected districts of Gamo zone, Southern Ethiopia. Front Veterinary Sci. 2022;9:931643. MetagesYirgalem. 2018. Molecular characterization of foot and mouth disease viruses in cattle from outbreaks that occurred in different parts of Ethiopia. MVSc Thesis, Addis Ababa University, Bishoftu, Ethiopia. Mishamo Sulayeman, FufaDawo, BedasoMammo D, Gizaw, and DerejeShegu. Isolation, molecular characterization, and sero-prevalence study of foot-and-mouth disease virus circulating in Central Ethiopia. BMC Vet Res. 2018;14(1):110. Mohammed AliyeTunfuri. 2022. Epidemiology and economic impact of foot and mouth disease of cattle in selected districts of Arsi and Bale Zone, Oromia Regional State, Ethiopia. MVSc Thesis, Addis Ababa University, Bishoftu, Ethiopia. Molla Wassie E, Aman Z, Gebreegizabher, Temesgen W. Spatial and temporal distribution of foot and mouth disease outbreaks in the Amhara region of Ethiopia in the period 1999 to 2016. BMC Vet Res. 2020;16:185. MoretinaJiruWoreda Livestock and Fisheries Resource Office (MWLFRO). 2022. Annual report on livestock population of the district, Shewa, Ethiopia. Noureldin M, Elfadil A. 2014. Prevalence and risk factors of foot and mouth disease of cattle in Khartoum State, Sudan. Master's Thesis, Sudan University of Science and Technology. OIE. 2004. Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds, and bees): 5th edition, volume I. Office international des Epizooties (OIE), Paris, France. Pp. 111–128. OIE. 2012. Foot and mouth disease, manual of diagnostic tests and vaccines for terrestrial animals, version adopted by the World Assembly of Delegates of the OIE in May 2012, OIE Terrestrial , pp. 1–29. OIE. 2016. Foot and mouth disease vaccination and post-vaccination monitoring guidelines. OIE. Foot and Mouth Disease Foot and Mouth Disease. March; 2021. pp. 1–10. Reid S, Ebert K, Bachanek-Bankowska K, Batten C, Sanders A, Wright C, Shaw. A.. Sentayhu Menda S, Jenberie H, Negussie G, Ayelet, Amsalu K. Molecular epidemiology of foot and mouth disease virus outbreaks in Ethiopia in 2011/2012. Acad J Anim Dis. 2014;3:8–16. Shashemene District Livestock and Fisheries Resource Office (SDLFRO). 2022. Annual report of livestock population, Shashemene, Ethiopia. Shashemene Woreda Agricultural Development Office (SWADO). Shashemene Woreda Agricultural Plan for the Year 2015/2016. Ethiopia: Shashemene; 2015. Shaw A, Reid S, Ebert K, Hutchings G, Ferris N, King D. Implementation of a one-step real-time RT-PCR protocol for the diagnosis of foot and mouth disease. J Virol Methods. 2007;143:81–5. Shiferaw Jenbere K, Moses, Etana M. Participatory appraisal of foot and mouth disease in the Afar pastoral area, Northeast Ethiopia: implications for understanding disease ecology and control strategy. Trop Anim Health Prod. 2010;42(2):193–201. Souley K, Elliot F, King D, Hyera J, Knowles N, Ludi A, Mioulet V, Matlho G, De Clercq K, Thys E. Outbreak investigations and molecular characterization of foot and mouth disease viruses circulating in South-west Niger. Trans-bound Emerg Dis. 2018;65:146–57. Teshager Dubie and Wossene Negash. Sero-prevalence of bovine foot and mouth disease (FMD) and its associated risk factors in selected districts of Afar Region, Ethiopia. Veterinary Med Sci. 2021;7(5):1678–87. Thrusfield M. Veterinary epidemiology. 4th ed. Oxford, UK.: Blackwell Science; 2018. pp. 270–95. Tolosa T, Beyene B, Rufael T, Hailu B, Teklue T. Foot and mouth disease in selected districts of western Ethiopia: sero-prevalence and associated risk factors, Department of Microbiology and Veterinary Public Health, Jimma University, Jimma, Ethiopia. Rev Sci Tech Off Int Epiz. 2015;34(3):939–52. Vosloo W, Bastos A, Sangare S, Hargreaves O, S., and, Thomson R. Review of the status and control of foot and mouth disease in Sub-Saharan Africa. Rev Sci Tech Off Int Epizt. 2002;21:437–49. Wungak YS, Olugasa BO, Ishola OO, Lazarus DD, Ularamu GH. Foot and mouth disease (FMD) prevalence and exposure factors associated with sero-positivity of cattle in North-Central, Nigeria. Afr J Biotechnol. 2016;15(24):1224–32. Yasmin Jibril A, Alemayehu, AyelechMuluneh and HaileleulNegussie. Foot and mouth disease in Adama and Boset districts, East Shewa zone, Ethiopia: Sero-prevalence and virus serotyping. Ethiop Veterinary J. 2023;27(1):143–56. Yeneneh T, Khan F, and Esayas Gelaye. Molecular characterization of foot-and-mouth disease viruses collected from Northern and Central Ethiopia during the 2018 outbreak. Veterinary World. 2020;13(3):542–8. Yeneneh Tesfaye. 2014. Isolation, molecular characterization, and vaccine matching of foot and mouth disease virus circulating in Ethiopia. MSc Thesis, Addis Ababa University, Bishofu, Ethiopia. Zeedan GSG, Mahmoud AH, Abdalhamed AM, Khafagi MH. Diagnosis of Foot and Mouth Disease in Cattle and Buffaloes in Different Governorates of Egypt. World Vet J. 2020;10(1):43–52. Additional Declarations No competing interests reported. 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for sero-prevalence determination\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9365906/v1/7ca0875d8a7e202f1bfbbe0a.png"},{"id":108796409,"identity":"409eb4af-3d0e-419c-8efd-a5b23c1efdfd","added_by":"auto","created_at":"2026-05-08 13:28:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":457530,"visible":true,"origin":"","legend":"\u003cp\u003ePicture showing FMDV isolation on the BHK-21 cell line from March 2022 to February 2023\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9365906/v1/b5ac5e84d34b03a74389bcb2.png"},{"id":108796384,"identity":"2ff43d09-5c7f-49da-a0d1-7d6d03b22b7a","added_by":"auto","created_at":"2026-05-08 13:27:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":520701,"visible":true,"origin":"","legend":"\u003cp\u003eReal-time RT-PCR result showing amplification curve\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9365906/v1/ef54d038f2bce7443958036b.png"},{"id":108796504,"identity":"ddce9ceb-e58f-49b9-a547-3946adce12b8","added_by":"auto","created_at":"2026-05-08 13:28:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1750203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9365906/v1/fcf18ed5-5071-4bd1-a75d-a41f90f57b64.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sero-prevalence and an Outbreak Investigation of Foot and Mouth Disease with Isolation and Identification of Circulating Virus from Cattle in Selected Districts of Oromia and Amhara Regional States, Ethiopia","fulltext":[{"header":"INTRODUCTION","content":"\n\u003ch3\u003eBackground\u003c/h3\u003e\n\u003cp\u003eEthiopia is one of the countries that possesses a huge number of livestock populations in the African. According to the Central Statistical Agency (2021), the country has approximately 70\u0026nbsp;million head of cattle, 42.9\u0026nbsp;million head of sheep, 52.5\u0026nbsp;million head of goats, 2.15\u0026nbsp;million head of horses, 10.80\u0026nbsp;million head of donkeys, 0.38\u0026nbsp;million head of mules, 8.1\u0026nbsp;million head of camels, and 56.87\u0026nbsp;million head of poultry at the country level. Even though the country is endowed with huge livestock populations, the contribution of livestock to the national economy is minimal compared to its potential. The main constraint is the spread of many infectious diseases, including foot and mouth disease (FMD), which drastically reduces the production and productivity of livestock (Belege \u003cem\u003eet al\u003c/em\u003e., 2019).\u003c/p\u003e \u003cp\u003eFoot and mouth disease is an important contagious viral disease caused by foot and mouth disease virus (FMDV), which is a positive-sense, single-stranded, small non-enveloped RNA virus belonging to genus \u003cem\u003eAphthovirus\u003c/em\u003e of family \u003cem\u003ePicornaviridae\u003c/em\u003e (Knowles and Samuel, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; OIE,2012). According to immunological classification, there are seven distinct serotypes, namely, O, A, C, Southern African Territories (SAT)1, SAT2, SAT3, and Asia 1, as well as over 60 subtypes (OIE, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These serotypes do not provide any cross-immunity among each other after immunization or infection, so there is no universal vaccine that can confer protection against all serotypes (Knight-Jones et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe disease spreads directly through contact with an infected host or mechanically by indirect contact with contaminated fomites such as clothing, shoes, vehicles, and veterinary instruments. In addition to contact, viruses can be transmitted orally or through the respiratory tract to a new susceptible animal. Aerosol transmission is the most common method of spreading within a herd. The reasons for the rapidity of disease spread are due to the presence of susceptible populations, the production of high titers in respiratory secretions, the large volumes of droplets, aerosols of virus shed by infected animals, and the rapid replication cycle with very high virus yields (Mesfin \u003cem\u003eet al\u003c/em\u003e., 2004; Belegio \u003cem\u003eet al\u003c/em\u003e., 2019).\u003c/p\u003e \u003cp\u003eFoot and mouth disease is widely distributed in most developing countries, particularly in large areas of Africa, Asia, and South America. It has shown an extraordinary ability to cross international boundaries and cause epidemics in previously free areas (Knowles et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). FMDV serotypes are not distributed evenly around the world. Serotype O is the most common serotype in most parts of the world, including Ethiopia (Endris \u003cem\u003eet al.\u003c/em\u003e, 2020). In most of the sub-Saharan African countries, serotypes O, A, SAT1, and SAT2 are still in circulation. According to Vosloo et al. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), serotype SAT3 has been recorded only in Uganda. The introduction of FMD in disease-free countries is mainly due to the import of live animals, animal products, and feed of animal origin (Tolosa et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Knight-Jones et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFoot and mouth disease is the major endemic disease in Ethiopia with abundant socio-economic importance because of reduced production, deaths in newborn animals, the huge cost of veterinary services, and restricted animal and meat movement locally and between countries (Knight-Jones and Rushton, 2013). According to the report of Jemberu et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the total annual costs of FMD under the official control program were estimated at 1354 (864\u0026ndash;2042) million ETB. The major cost (94%) was due to production losses, the rest were export losses, and control costs (Gezahegn \u003cem\u003eet al\u003c/em\u003e., 2014).\u003c/p\u003e \u003cp\u003eStudies undertaken in Ethiopia revealed that the disease is still endemic and occurs in different parts of the country, mainly due to a lack of effective vaccines, the absence of animal movement control, and a lack of systematic disease surveillance and reliable epidemiological data (Shiferaw \u003cem\u003eet al\u003c/em\u003e., 2010). The disease was first recorded in Ethiopia in 1957; ever since, it has remained endemic in the country, causing several outbreaks each year (Ayelet \u003cem\u003eet al\u003c/em\u003e., 2012). According to the report of Gelagay \u003cem\u003eet al\u003c/em\u003e. (2009), 4 of the 7 serotypes, namely A, O, SAT1, and SAT2, were circulated in all regions of the country.\u003c/p\u003e \u003cp\u003eGenerally, studies undertaken on FMD revealed the existence of a disease with sero-prevalence varying from 5.66% to 85.1% (Kalkidan \u003cem\u003eet al\u003c/em\u003e., 2023; Betelihem \u003cem\u003eet al\u003c/em\u003e., 2023), which indicated the status of FMD in different parts of Ethiopia. Several sero-prevalence studies (Gezahegn \u003cem\u003eet al\u003c/em\u003e., 2014; Yasmin \u003cem\u003eet al\u003c/em\u003e., 2023) and a few molecular detection studies (Sentayehu \u003cem\u003eet al\u003c/em\u003e., 2014; Tesfaye \u003cem\u003eet al\u003c/em\u003e., 2020) have been done on FMD in different parts of the country.\u003c/p\u003e \u003cp\u003eThe study areas were known for their abundance of cattle, and there was high cattle movement towards central Ethiopia for trading purposes and across different borders in the surrounding districts in search of food and water. This leads to an increase in the distribution of the disease in the area. Moreover, the absence of updated epidemiological studies and the detection of circulating serotypes in the current study areas aggravate the problems. However, the constraints of disease might be solved with appropriate monitoring, surveillance, and regular vaccination strategies. Hence, by considering the above problems, the current study was planned to conduct an epidemiological study and serotyping of circulating FMDV isolates from suspected cattle, which are useful for vaccine strain selection, tracing the source of outbreaks, the implementation of good control programs, and the eradication of the disease in the country.\u003c/p\u003e\n\u003ch3\u003eGeneral Objective\u003c/h3\u003e\n\u003cp\u003eTo estimate the sero-prevalence, isolation, and molecular detection of foot and mouth disease virus from cattle circulating in selected districts of Oromia and Amhara regional states from March 2022 to February 2023.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSpecific Objectives\u003c/h2\u003e \u003cp\u003eTo estimate the sero-prevalence and determine risk factors associated with FMD in the study areas.\u003c/p\u003e \u003cp\u003eTo identify FMD serotypes responsible for FMD outbreaks in the selected areas\u003c/p\u003e \u003cp\u003eTo isolate and molecularly detect FMDV from lesions of diseased cattle in the study areas from March 2022 to February 2023\u003c/p\u003e \u003c/div\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStudy Areas\u003c/h2\u003e \u003cp\u003eThe study was carried out from March 2022 to February 2023 in the selected districts. The selected districts were Shashemene district from West Arsi zone, Adami Tullu and Jido Kombolcha district from East Shewa zone of Oromia, and Moretinajiru district from North Shewa Zone of Amhara regional state (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdamiTullu and JidoKombolchadistrict is located in the Great Rift Valley between 38\u0026deg;25\u0026prime;E and 38\u0026deg; 55\u0026prime;E and 7\u0026deg;35\u0026prime;N and 8\u0026deg;05\u0026prime;N. It is located about 160 km from Addis Ababa, Southwest of Lake Ziway at an altitude of 1500\u0026ndash;2300 meters above sea level. The annual rainfall varies from 600 to 800mm, and it is characterized by bimodal rainfall (Martha \u003cem\u003eet al\u003c/em\u003e., 2020). The area is characterized by arid and semiarid climatic conditions, and it has a rapidly growing population. This district has a livestock population of 345356 cattle, 78976 sheep, 157598 goats, 19645 horses, 49687 donkeys, and 51764 poultry (AJLFRO, 2022). Other agricultural production systems in this area comprise small-scale irrigated vegetable and fruit production as well as livestock farming systems. In some areas, agro-pastoral livelihoods are also present. Feed availability and quality, especially during the dry season, are important constraints in livestock production, and it determines the physical performance of the livestock sector (Holden and Bekele, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). This district has substandard veterinary practices, and unqualified animal health experts provide services, so that animals do not receive the essential preventive care and timely treatment they need. There were FMD vaccination services in the area where the disease outbreak occurred, and unfortunately, they gave the vaccine even to diseased animals.\u003c/p\u003e \u003cp\u003eShashemene district is located at 250 km south of Addis Ababa. It is situated at 7\u003csup\u003eo\u003c/sup\u003e05\u0026prime; to 7\u003csup\u003eo\u003c/sup\u003e19\u0026prime;N and 38\u003csup\u003eo\u003c/sup\u003e23\u0026prime; to 38\u003csup\u003eo\u003c/sup\u003e41\u0026prime;E. Its climate is characterized by an annual temperature ranging from 12\u0026deg;C to 27\u0026deg;C. It has an altitude range from 1,685 to 2,722 meters above sea level. The district has an estimated 267234 cattle, 87345 sheep, 111156 goats, 43245 equine, and 56264 poultry (SDLFRO, 2022). The agro-climatic conditions of the district are favorable for agriculture with two rainy seasons. It has an annual rainfall ranging from 700 mm to 950 mm (SWADO, 2015). Animals in this district did not receive timely, appropriate, and high-quality treatment when they fell ill or were injured. This leads to unnecessary animal suffering and economic losses for owners. Veterinary services in this district were limited, especially since the problems were serious outside Sheshemene town due to the limited number of licensed veterinarians, especially in rural and remote areas, and uneven distribution of services (number of clinics). Vaccination service was given in the study areas, particularly for FMD, but insufficient animal coverage was given regardless of the serotypes circulating in the area.\u003c/p\u003e \u003cp\u003eThe third study area was the Moretina Jiru district. The coordinates for the study area are 39\u0026deg; 19\u0026rsquo; 24\u0026rsquo;\u0026rsquo; E and 10\u0026deg; 6\u0026rsquo; 2\u0026rsquo;\u0026rsquo;N with an altitude range from 1,500 to 2694 m above sea level. The area is located at 195 km North East of Addis Ababa and receives an annual rainfall of 850 mm, while the temperature varies from 5.2\u0026deg;C in November to 28.8\u0026deg;C (Atinkugn, 2020). The livestock populations in the district are composed of 172536 goats, 51043 sheep, 124959 cattle, and 64868 equine (MWLFRO, 2022). The livestock farming system in this area is mainly a mixed farming system for a source of meat, milk, manure, hide, and skin, in addition to crop production. Cattle fattening is an effective system for poverty alleviation and has become an important business for smallholder farmers in this study area. Mainly, oxen used for draught power are put into a feedlot period to improve body condition under traditional feedlot conditions, as a source of income. The animal management system, particularly health care services in this district, focused on preventive measures like vaccination and early disease reporting. However, it was challenged with a shortage of licensed veterinarians, particularly in rural areas, uneven distribution of services, lack of infrastructure, and high costs of services. Many livestock owners, especially smallholder farmers, lack awareness about the importance of regular animal vaccination. This contributes to poor uptake of vaccination services. These systemic issues result in many animals not receiving necessary care.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy population\u003c/h3\u003e\n\u003cp\u003eThe study populations were cattle with various sexes, age groups, and breeds with different management and production systems selected from two districts of the West Arsi and North Shewa Zone of ORS and ARS, respectively. In this study, the age of cattle was categorized as \u0026lt;\u0026thinsp;2 years, 2\u0026ndash;4 years, and 4 years old according to Bereket \u003cem\u003eet al\u003c/em\u003e. (2010). The herd size of the study cattle was categorized as large (30 animals), medium (15\u0026ndash;30 animals), and small (\u0026lt;\u0026thinsp;15 animals) according to Amare et al. (2019). The body condition of cattle was also categorized as poor, medium, and good (Ibrahim, 2015).\u003c/p\u003e\n\u003ch3\u003eInclusion and Exclusion Criteria\u003c/h3\u003e\n\u003cp\u003eFor the sero-prevalence study, due to the presence of maternal antibodies in cattle below six months, only those older than six months were included. For isolation and molecular detection of FMDV, only those that manifested clinical signs of FMD irrespective of age group, sex, and breed were considered, while cattle without any suspected FMD clinical signs or not experienced the disease were excluded from the study on purposive sampling.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStudy Variables\u003c/h2\u003e \u003cp\u003eIn this study, the variables were considered as independent and dependent variables, in which the independent variable is hypothesized to affect the dependent variable (outcome). The outcome variable was the sero-positivity of cattle tested by the 3ABC non-structural competitive ELISA test. Whereas the explanatory variables were animal-related factors such as sex, age, breed, and body condition, and mixed factors including study area, herd size, and production systems, which were assessed to determine the influence on FMDV sero-positivity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Design and Duration\u003c/h3\u003e\n\u003cp\u003eA cross-sectional study was conducted from March 2022 to February 2023 in the selected districts of Oromia and Amhara regional states to estimate sero-prevalence, isolate, and molecularly detect FMDV from cattle. For the isolation and molecular detection of the virus, field tissue and oropharyngeal lesions were collected from active cases of disease outbreak based on reports.\u003c/p\u003e\n\u003ch3\u003eSampling Technique\u003c/h3\u003e\n\u003cp\u003eA multistage sampling method was used to select the sampling unit. The study districts (Moretinajiru and Shashemene) were selected based on livestock population, agro-ecologies, and route of trade, accessibility, and relative security. A total of 14 kebeles were selected; 9 kebeles were selected in Shashemene district from 36 kebeles, and 5 kebeles were selected in MoretinaJiru district from 17 kebeles.\u003c/p\u003e \u003cp\u003eAccording to the livestock proportion to households in Shashemene and Moretinajiru, the average cattle size per household was 5 for Shashemene and 4 for Moretinajiru district. Then, household selection for each respective kebele was carried out by a simple random method. Hence, 53 and 97 households were selected from each of the selected kebeles of Shashemene and Moretinajiru districts, respectively. So, a total of 150 households were selected, and in each household, 5 animals in Shashemene and 4 animals in Moretinajiru were sampled. Then, if the randomly selected household owned fewer than 5 and 4 cattle for Shashemene and Moretinajiru, respectively, the household was rejected, and the next randomized household was sampled. Finally, sampling of individual animals was carried out by using a simple random sampling method from Shashemene and Moretinajiru districts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDuring sampling, risk factors related to cattle, such as age, sex, breed, body condition, herd size, and production system were also recorded. Following a report of the disease outbreak, an investigation was carried out purposively in and around specific outbreak areas. A thorough physical examination was conducted on clinically sick animals to record clinical signs and disease conditions. Those animals that had obvious clinical signs and symptoms suggestive of FMD were sampled.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSample Size Determination\u003c/h2\u003e \u003cp\u003eThe sample size for this study was estimated based on the formula described by Thrusfield(2018) using 95% confidence interval and 5% desired absolute precision with previous sero prevalence findings of 44% (Molla \u003cem\u003eet al\u003c/em\u003e., 2020) and 22.2% (Getnet \u003cem\u003eet al\u003c/em\u003e., 2016) in Moretinajiru and Shashemene districts, respectively, using the formula:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eN= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\left(1.96\\right)2\\:\\text{x}\\:\\text{P}\\text{e}\\text{x}\\text{p}\\:(1-\\text{P}\\text{e}\\text{x}\\text{p})}{\\text{d}2}\\)\u003c/span\u003e\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eWhere N\u0026thinsp;=\u0026thinsp;required sample size, Pexp\u0026thinsp;=\u0026thinsp;expected prevalence, and d\u003csup\u003e2\u003c/sup\u003e=desired absolute precision. Thus, substituting the respective values in the formula, a total of 654 (389 from Moretinajiru and 265 from Shashemene) cattle were considered for the sero-prevalence part of this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStudy Methods\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eClinical examination\u003c/h2\u003e \u003cp\u003eSoon after arrival at the specific outbreak site, based on outbreak reports received, the animals were examined from a distance for evidence of salivation and lameness in each animal owner\u0026rsquo;s homestead, since there was no crush in the area. In each outbreak, animals that have manifested signs of disease, such as vesicles on the tongue or ruptured vesicles in the oral cavity, on the feet and teat, as well as excessive salivation, lameness, anorexia, and a temperature rise, were considered clinically sick animals.\u003c/p\u003e \u003cp\u003eAnimals with salivation and lameness were restrained for thorough examination and specimen collection. The oral cavities of salivating animals were examined for evidence of any intact and ruptured vesicles, erosions, and ulcers on the tongue, dental pad, hard palate, gum, and mucosa of the mouth cavity. The hooves of lamed animals were thoroughly washed with clean water and carefully examined for the presence of lesions, and the coronary bands and inter-digital spaces of the hooves were examined. Animals were also examined their teat and external genitalia for the presence of any vesicular lesion (OIE, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and transportation\u003c/h2\u003e \u003cp\u003eBlood samples were collected from the jugular vein using plain vacutainer tubes and venoject needles for serological analysis, and tissue and oro-pharyngeal (OP) samples were aseptically collected from clinically suspected cases for molecular detection. Briefly, the jugular vein area was disinfected using 70% alcohol, and approximately 3\u0026ndash;5 mL of blood was collected using a venoject needle and vacutainer tubes. Sampling was conducted as part of routine veterinary surveillance and outbreak investigation activities using standard minimally invasive procedures. All animals were handled in accordance with accepted animal welfare practices, and informed verbal consent was obtained from animal owners prior to sample collection.\u003c/p\u003e \u003cp\u003eIn such a way, the collected blood sample was labeled with date of collection, specific identification number, and species of the animals and transported to DebreBerhan Agricultural Research Center and Hawassa University Veterinary Medicine Microbiology Laboratory with a cool ice box for temporary storage. In the laboratory, the blood sample collected with plain vacutainer tubes was allowed to stand in a slant position for 24 hours at room temperature to collect serum. After 24 hours, samples from which serum was not clearly separated were centrifuged at 5000 rpm for three minutes to remove the remaining red blood cells and collect clear serum. Then, the serum was harvested using a sterile micropipette into cryogenic vials and stored at -20\u0026deg;C in aliquots. Finally, the samples were transported to AHI, being chilled in a cool icebox for serological analysis.\u003c/p\u003e \u003cp\u003eFor epithelial tissue sample collection, epithelial tissues that were freshly ruptured or unruptured vesicles were collected from FMD-suspected cattle, usually from the tongue, buccal mucosa, or feet, by using sterile forceps and scissors. The samples were collected and stored in cryovials containing virus transport medium composed of equal amounts of glycerol and 0.04-M phosphate-buffered saline (PBS) solution (pH 7.2\u0026ndash;7.6) with some antibiotics and antifungal (OIE, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOro-pharyngeal samples were collected from previously FMD-suspected, infected, and asymptomatic cattle that were found near the affected herd. Samples were collected in advance or convalescent cases by a probang cup and poured into a 20 ml bottle. The fluid was added to a 5 ml tube containing about 2ml of transport medium (OIE, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Both tissue and OP fluid samples were properly labeled and immediately transported at 4\u0026deg;C using an ice pack box from the collection site to Animal Health Institute (AHI), Sebeta, Ethiopia, for sample processing and other laboratory investigations. All the samples were coded by laboratory code at the reception room and transferred to the laboratory upon arrival. Tissue samples were immediately stored at -80\u0026ordm;C until processed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSerological diagnostic tests\u003c/h2\u003e \u003cp\u003eSerum samples were screened for antibodies against conserved non-structural protein (NSP) of the FMDV using FMD non-structural protein ELISA (3ABC-ELISA) to identify FMDV sero-positive or sero-negative animals. A commercially available test kit (ID Screen \u0026reg; FMD NSP Competition, ID.VetInnovative Diagnostics, Louis Pasteur, Grables, France) was used. The test plates of the kit contain FMDV NSP captured by the coated 3ABC-specific monoclonal antibodies (MAbs). The assay was performed according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003cp\u003eIn brief, all reagents were allowed to come to room temperature(21\u003csup\u003eo\u003c/sup\u003eC\u0026plusmn;5\u003csup\u003eo\u003c/sup\u003eC) and were homogenized by vortex before use. The test was carried out in 3ABC antigen-coated 96 wells of micro plates. Then, the procedure commences as follows: 50\u0026micro;l of dilution buffer 18 was added to each microplate well. A 30\u0026micro;l of positive control (to A1 and B1 wells)and 30\u0026micro;lof negative control (to C1 and D1 wells) were added to respective plate wells. Subsequently, 30\u0026micro;l of each sample was added to the remaining wells using a multichannel pipette, sealed with adhesive plastic plate sealants to prevent cross-contamination and evaporation of the samples during incubation. Then, it was incubated at 37\u0026deg;C for 2 hours. The sera were discarded from the plates after incubation and washed 5 times by adding 300\u0026micro;l of wash solution immediately to avoid drying between washes. Then, 100\u0026micro;l of the conjugate 1X was added to each well after washing and incubated for 30minutes at 21\u0026deg;C. After incubation, the contents were discarded from the plates, and each well was washed 5 times by adding 300\u0026micro;l of wash solution. Then, 100\u0026micro;l of the substrate solution (Tetramethylbenzidine)was added to each plate well and again incubated for 15 minutes at 21\u0026deg;C in a dark place.\u003c/p\u003e \u003cp\u003eFinally, after a final incubation, the substrate reaction was stopped by adding 100\u0026micro;l stop solution, and the color reactions were quantified by measuring the optical density (OD) of each well at 450 nm wavelength using an ELISA reader. The test was validated if the mean value of the negative control OD (OD\u003csub\u003eNC\u003c/sub\u003e) was greater than 0.7 and the mean value of the positive control OD (OD\u003csub\u003ePC\u003c/sub\u003e) was less than 30% of the negative control OD.\u003c/p\u003e \u003cp\u003eThe interpretation for each sample was based on the competition percentage (S/N%):\u003c/p\u003e \u003cp\u003eS/N%= \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eOD sample\u003c/span\u003e x100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOD\u003csub\u003eNC\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eSamples presenting S/N% less than or equal to 50% were considered positive, and those greater than 50% were considered negative.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eVirus isolation\u003c/h2\u003e \u003cp\u003eVirus isolation (VI) was conducted in a laminar air flow class II biosafety cabinet. The collected samples were processed and cultured on a BHK-21 cell monolayer with three subsequent passages as follows: The tissue epithelium samples were first taken from the transport media and blotted dry on absorbent paper to reduce the glycerol content that is toxic to cell culture. About 1gram of epithelial tissue was ground with sterile sand by a sterile pestle and mortar with a small volume of tissue culture medium and antibiotics (penicillin, streptomycin, and Amphotericin B solution). Dulbecco's Modified Eagle Medium (DMEM) was added until a final volume of nine times that of the added epithelial sample was reached, giving a 10% suspension (OIE, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The suspension was clarified on a bench centrifuge at 3000 rpm for 10 minutes. The supernatant of the suspension was collected in sterile cryovial tubes and filtered by Millipore filter paper of 0.22\u0026micro;m pore size, labeled, and stored at -80\u0026deg;C until needed for further tests.\u003c/p\u003e \u003cp\u003eAbout 0.5ml of filtered tissue suspensions, which were suspected to contain FMDV, were inoculated into confluent cultured baby hamster kidney (BHK-21) monolayer cells grown in 25cm\u003csup\u003e2\u003c/sup\u003e tissue culture flasks and incubated at 37\u0026deg;C for 1hour for adsorption of virus. Then, 8ml of DMEM maintenance media (2% fetal calf serum) was added to the infected cell and incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified incubator for 24\u0026ndash;48 hours, and monitored for cytopathic effect (CPE) while normal non-infected cells were served as controls.\u003c/p\u003e \u003cp\u003eCytopathic effect was observed after 48 hours in the positive cases, although in some samples it was observed within 24 hours through an inverted microscope. CPE was characterized by a fast destruction of the monolayer cells, cell rounding, and infected cells were disrupted and detached from the flask, which was mostly seen within 48 hours of inoculation. If no CPE was observed after 48 hours, the sample was considered as \u0026lsquo;no virus detected\u0026rsquo; (NVD) and the culture was frozen at -80\u0026deg;C, then thawed and centrifuged at 3000 rpm for 10 min to collect supernatant for the second passage; this was repeated for the third passage and if no CPE was observed at 48 hours, then the sample was considered negative for FMDV as described by Souleyet al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOnce CPE was completed in the cultures, the fluids were tested for FMDV using ELISA and rRT-PCR testing. Finally, isolated samples were labeled according to a system specified by OIE standards (OIE, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The first three letters signify the sample\u0026rsquo;s country of origin, ETH for Ethiopia, followed by a number representing the number of the sample in the batch of samples, and finally the last two digits of the year in which the sampling was carried out.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSerotyping of FMD virus\u003c/h2\u003e \u003cp\u003eFoot and mouth disease virus serotypes were identified using FMDV antigen detection and serotyping sandwich ELISA (IZSLER, Brescia, Italy), which was performed with selected combinations of anti-FMDV monoclonal antibodies (MAbs), used as coated and conjugated antibodies. The test was applied for the detection and typing of the FMD virus. The kit was designed for the detection and typing of FMD viruses of types O, A, SAT1, and SAT2. A pan-FMD test was used to detect any isolates of serotypes O, A, C, and Asia1, and in addition, some of the SAT serotypes were included in the kit to complement the specific typing and to detect FMD viruses that might have escaped binding to the selected type-specific MAb.The microplates were supplied pre-coated with catchedMAbs to detect 10 samples at a time with one positive and one negative control for each serotype. The controls were already incorporated into the ELISA microplate trapped by the respective captured MAbs. The cell-culture isolates were subjected to the test assay and serotyped.\u003c/p\u003e \u003cp\u003eThe test was carried out as per the manufacturer\u0026rsquo;s recommendation. Briefly, the first samples were diluted in \u0026frac12; in diluent buffer, and 50\u0026micro;l per well of each sample was distributed to 8 wells of a column (from A-H rows). Then, 50\u0026micro;l of diluent buffer per well was added to all wells of 11 and 12 columns (positive and negative control, respectively), and plates were incubated at room temperature for 1 hour. After incubation, all the fluids in the wells were discarded, and the plates were tapped hard to remove all the residual fluid. Then, 200\u0026micro;l washing solutions per well were added in all wells and incubated for 3 min at room temperature; subsequently, wells were emptied and washed repeatedly twice (three washing cycles in total). Then all residual fluids were removed by tapping on a clean absorbent paper, and 50\u0026micro;l per well of conjugates (conjugate A) was added into rows from A to F, and the same volume of conjugate B was added into rows G and H. Then, the plates were covered and incubated at room temperature for 1hour. After incubation, the last washing was performed for 5 minutes, and 50\u0026micro;l per well of substrate(chromogen) was added to all wells, and plates were covered and left at room temperature for 20minutes in the dark; the time was calibrated when the first well was filled. The reaction was stopped by adding 50\u0026micro;l per well of stop solution (sulfuric acid), and the wells\u0026rsquo; contents were properly mixed before reading. Immediately after stopping, read the optical density (OD) of each well at 450 nm wavelength using a microplate reader.\u003c/p\u003e \u003cp\u003eCriteria for test validity: The positive inactivated controls were expected to give OD values of \u0026ge;\u0026thinsp;1.0 unit, while the negative control for serotypes O, A, C, Asia 1, and Pan-FMDV was expected to give OD values\u0026thinsp;\u0026lt;\u0026thinsp;0.1 unit, and the negative control for serotypes SAT1 and SAT2 were expected to give OD values\u0026thinsp;\u0026le;\u0026thinsp;0.2unit. Results for the samples examined were interpreted, after subtraction of the OD value of each negative control from the OD value measured with the corresponding captured Mab.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMolecular identification\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eRNA extraction\u003c/strong\u003e \u003cp\u003eAn epithelial tissue suspension was prepared by grinding samples using a sterile tissue-grinding device (mortar and pestle) and silica sand to disrupt viral cells. For every 29 samples, the procedure was repeated by cleaning materials for the next activity and transferring the suspension of the ground part into the centrifuge tube. The suspension was centrifuged at 3000 revolutions per minute (rpm) for 10 minutes, and the supernatant was taken for molecular work. Total viral RNA was extracted from digested tissue supernatants and oro-pharyngeal fluid suspensions using QIAamp\u0026reg; Viral RNA Mini Kit (QIAGEN, Cat. No.52906, Hilden, Germany)in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eBriefly, lysine solution buffer and carrier RNA were prepared and mixed, then 560\u0026micro;l AVL buffer containing carrier RNA was dispensed into sterilized centrifuge tubes. The samples prepared were vortexed to mix properly, then 140\u0026micro;l of the sample suspension was transferred into microcentrifuge tubes containing AVL buffer and carrier RNA, and then the tubes were vortexed for 1 second and incubated at room temperature for 10 min for lysing purposes. Then, 560\u0026micro;l absolute alcohol (ethanol) was added to centrifuge tubes containing AVL buffer, carrier RNA, and samples. The tubes were mixed by vortexing for 1 second. A 630\u0026micro;l of the solution was added into QIAamp mini spin columns in 2ml collection tubes, then centrifuged at 8000 rpm for 1 minute, and the filtrate fluid was discarded, then the column filter was transferred to new collection tubes. This procedure was repeated twice. Then, 500\u0026micro;l of wash Buffer AW-1 was added to the QIAamp mini spin column tubes and centrifuged again at 8000 rpm for 1 minute. The filtrate was discarded, and the columns were placed into fresh 2ml collection tubes. Then 500\u0026micro;l of wash buffer AW-2 was added to the columns and centrifuged at 14000 rpm for 3 minutes, and the filtrate was discarded. Then, the columns were centrifuged at 14000 rpm for 1 minute to dry. A 60\u0026micro;l of AVE buffer was added into the column and incubated at room temperature for 1 minute, then centrifuged at 8000 rpm for 1 minute to elute RNA from the QIAamp mini spin column, and then the QIAamp mini spin column was removed. Finally, extracted RNA was kept at +\u0026thinsp;4\u0026deg;C for immediate use or stored at -80\u0026deg;C for further molecular characterization of FMDV.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDetection of viral RNA by rRT- PCR\u003c/strong\u003e \u003cp\u003eThe presence of viral RNA was screened by using an Applied Biosystems 7500 fast one-step real-time PCR thermocycler machine. The extracted RNA was converted to cDNA using a reverse transcriptase enzyme, and the cDNA was amplified using FMDV-specific forward and reverse primers. Detection of the 3D (pol) region of FMDV was performed using the Qiamp viral RNA mini kit as per instructions and the selected catalog number of 52906.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe nucleotide sequences of forward primer (FMD-3DF)of 5\u0026rsquo;-ACTGGGTTTTACAAACCTGTGA-3\u0026rsquo;, reverse primer (FMD-3DR) of 5\u0026rsquo;-GCGAGTCCTGCCACGGA-3\u0026rsquo;, and TaqMan probe 5\u0026rsquo;-6-FAM-TCCTTTGCACGCCGTGGGAC-TAMRA-3\u0026rsquo; were used in this assay. The probe labeled with 6- (FAM) at the 5\u0026rsquo; end and the quencher tetra-methyl-rhodamine (TAMRA) at the 3\u0026rsquo;end in a real-time RT-PCR reaction that detected the 3Dpol gene sequence in all the FMDV serotypes.\u003c/p\u003e \u003cp\u003eIn brief, a master mix was prepared by mixing 12.5\u0026micro;l of 2x - reaction, 2\u0026micro;l of forward primer, 2\u0026micro;l of reverse primer, 1\u0026micro;l of RNAse-free water, 1.5\u0026micro;l of Taqman probe, 0.5\u0026micro;l Rox and 0.5\u0026micro;l of superscript \u0026reg;III RT to make a total of 20\u0026micro;l per sample for each reaction of PCR per well per plate including the positive and negative control master mix, then thoroughly mixed by pulse vortexing. 5\u0026micro;l of RNA sample template (extracted RNA)was added to each reaction. Then, the plate was sealed with adhesive sealant and loaded into the thermal cycler machine for processing according to QIAmgen one-step RT-PCR kit protocols.\u003c/p\u003e \u003cp\u003eThe amplification reaction was set by creating a plate sheet for the PCR machine to run using the 7500 Fast System SDS Software. Accordingly, the amplification was carried out with a final reaction volume of 25\u0026micro;l containing a 20\u0026micro;l master mix and 5\u0026micro;l RNA template submitted to a thermal profile of one cycle reverse transcription, and FMDV was detected through cycle threshold (Ct) values based on baseline and graphs. The one-step RT-PCR amplification started with reverse transcription (RT) cDNA synthesis at 50\u0026deg;C for 30 min; followed by activation or denaturation of reverse transcriptase at 95\u0026deg;C for 10 min; then annealing and extension were conducted at 95\u0026deg;C for 15seconds and 60\u0026deg;C for 1min, respectively. In such a way, the clinical samples detected at a cycle threshold (Ct) value\u0026thinsp;\u0026lt;\u0026thinsp;35 were declared as positive, whereas Ct values 35 indicated that the samples were negative.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eData Management and Analysis\u003c/h2\u003e \u003cp\u003eData generated from field and laboratory work were coded, entered, and stored in a Microsoft Excel-2019 spreadsheet and analyzed using STATA version 14.0 for Windows (Stata Corp., College Station, TX, USA). Descriptive statistics were used to summarize data, and the prevalence was calculated for all associated risk factors. Both univariable and multivariable logistic regression were used to assess the association between factors and sero-positivity of FMDV. For multivariable logistic regression analysis, all putative risk factors having a critical p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.25 in univariable analysis were selected. After selecting the final model of logistic regression, the beta (β) coefficients of each independent variable were observed to estimate the odds ratio (OR), which is used for assessing the strength of association. Statistically significant association between variables was considered to exist if the computed p-value at 95% confidence interval and 5% degree of precision is \u0026lt;\u0026thinsp;0.05. In cell culture results, CPE development and molecular detection results were recorded and tabulated.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eSero-prevalence of FMD\u003c/h2\u003e \u003cp\u003eIn this study, a total of 654 cattle sera were tested using the 3ABC non-structural competitive ELISA test, and 178 sera were found to be positive. Thus, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the overall sero-prevalence of FMD was 27.2% (95% CI: 24.0\u0026ndash;30.0) in the study areas. Higher sero-prevalence of FMD was observed in Moretinajiru district (39.3%) than in cattle found in Shashemene (20.0%) district, with a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFMD prevalence of cattle in the selected districts from March 2022 to February 2023\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistrict\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of Examined\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of Positive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrevalence (95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoretinaJiru\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39.3% (34.0\u0026ndash;44.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShashemene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.0% (15.0\u0026ndash;25.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e654\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.2% (24.0\u0026ndash;30.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eFactors Associated with FMD\u003c/h2\u003e \u003cp\u003eSeveral factors, including district, age, breed, sex, herd size, body condition, and production system, were considered for the risk factor analysis. Sero-prevalence of antibodies against FMDV was compared between different age groups of cattle. From the results, it is evident that cattle older than 4 years had a greater sero-prevalence of 37.6% when compared to cattle between 2\u0026ndash;4 years and below 2 years of age, whose sero-prevalence was estimated to be 26.9% and 21.9%, respectively.\u003c/p\u003e \u003cp\u003eAdditionally, cattle within a large herd size (37.1%) had a larger prevalence than the medium (25.0%) and the small cattle herd size, with a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The prevalence was also found to be higher in large herd size (37.1%) as compared with medium herd size (25.0%) and small animal herd size (19.1%). Sero-prevalence was found to be significantly different in the production system. The highest sero-prevalence was observed in the extensive production system (41.3%), as compared to the intensive production system (19.0%) and the semi-intensive production system (27.2%) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnivariable logistic regression was run to identify the possible individual risk factors for sero-positivity to FMDV antibody. Univariable logistic regression analysis shows that districts, age, herd size, and production system have statistically significant associations with FMD infection (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, age, sex, and breed were not significantly associated with FMDV sero-positivity (P 0.05) as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The variables with a p-value of less than 0.25 in univariable analysis were taken to the multivariable analysis to control confounders and to see their independent effect on FMDV sero-positivity. Accordingly, districts, age, herd size, and production system were subjected to multivariable analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUnivariable logistic regression analysis of factors for FMDV sero-positivity\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCategories\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. Tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. Positive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePrevalence (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOR (95%CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDistrict\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShashemene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMoretinajiru\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.75 (1.9\u0026ndash;3.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e145\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.183(0.7\u0026ndash;1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.450\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u0026ndash;4 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.636(1-2.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.037*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.15(1.26\u0026ndash;3.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.005*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eBreed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCross\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e349\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.321 (0.85-2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.218\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExotic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.635(1-2.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHerd size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.769(1.2\u0026ndash;2.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.004*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.495(1.4\u0026ndash;4.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eBo.Condition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e295\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.142 (0.8\u0026ndash;1.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.523\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePoor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.678 (1-2.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePro.System\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntensive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSemi-intensive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.877(1.2\u0026ndash;2.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.004*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExtensive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.999(1.8\u0026ndash;4.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eKeys: *-Significant; CI-Confidence Interval; OR-Odd Ratio; Ref-Reference; No. tested-Number of tested; No. Positive number of positive, Pro. system-Production System, Bo.condition-Body condition\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the current study, multivariable logistic regression analysis revealed that study districts, age, herd size, and production systems remained as independent predictors of FMDV sero-positivity with significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eFoot and mouth disease sero-positivity was significantly higher in Moretinajiru than in the Shashemene district. Thus, cattle found in Moretinajiru were 2.18 times more likely to develop the disease than those cattle found in the Shashemene district. Concerning herd size, cattle from large herd sizes were 2.2 times more likely to develop the disease than animals from small herd sizes. Similarly, cattle from medium herd size were 1.6 times more likely to be at higher risk than small animal herd for FMD infection. The odds of being positive for FMDV were also 4 times as high as those reared in an extensive production system and 2.1 times higher in animals that were reared in a semi-intensive production system, as compared with intensive production systems, with a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eHence, the age groups of cattle were found statistically significant, which means cattle older than 4years were more than 2 times, and cattle with age group from 2\u0026ndash;4 years were 1.56 times more likely at higher risk for FMD infection as compared with cattle below two years of age. For a more in-depth understanding, see Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMultivariable logistic regression analysis of different risk factors\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFactors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCategories\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. Tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. Positive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePrevalence (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOR (95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDistrict\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShashemene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMoretinajiru\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.18 (1.5\u0026ndash;3.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u0026ndash;4 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.566 (0.96\u0026ndash;2.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.071\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.147(1.2\u0026ndash;3.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.009*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHerd size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.59 (1.06\u0026ndash;2.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.026*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.21 (1.3\u0026ndash;3.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.005*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eProduction system\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntensive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSemi-intensive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.099 (1.3\u0026ndash;3.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExtensive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.043 (1.8\u0026ndash;5.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eKeys: *-Significant; OR-Odds Ratio; CI-Confidence Interval; No. Tested-Number of tested; No. Positive-Number of positive; Ref-Reference\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eClinical Examination\u003c/h2\u003e \u003cp\u003eAmong 157 animals observed in three districts during outbreak investigation based on reports, 66 (42.0%) of cattle were found to be clinically infected with FMD. Using this report-wise, information was collected from such disease-affected areas. Cattle were carefully examined for the presence of typical clinical signs of FMD. The observed clinical signs in sick cattle were vesicles and erosions in gums, dorsum of the tongue, and in the inter-digital spaces and coronary band, erosions, profuse salivation, and lameness that were suggestive of FMD cases, see Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of FMD outbreaks that occurred in selected study areas.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSites of outbreaks\u003c/p\u003e \u003cp\u003eDistrict Kebele\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of examined animals\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. of infected animals\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo. of deaths occurred\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eShashemene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAle luilu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFaji sole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDenisa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAdamitullu and Jidokombolcha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWolenbula\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaka\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMoretinajiru\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYimedeb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJihurzuria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMangudo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66 (42.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (6.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eFMD Virus Isolation\u003c/h2\u003e \u003cp\u003eOut of 66 samples collected from different outbreaks, 25 (37.9%) representative samples were subjected to virus isolation using Baby Hamster Kidney (BHK-21) cell culture, while the remaining 41 (62.1%) samples were not inoculated on the cells because two or more samples were collected from the same outbreak in the study areas.\u003c/p\u003e \u003cp\u003eThe current study result revealed that out of 25 clinical samples inoculated on BHK-21 cell culture, CPE was observed on 19 (76.0%) samples (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), while the virus did not grow (CPE was not observed) on the rest of 6 (24.0%) samples even at the third passage. The current results indicated that the virus isolated from clinical samples on BHK-21 cells showed CPE, which was characterized by the fast destruction of the monolayer cells. Infected cells were round, formed singly, and the destruction of the cells was mostly seen within 48 hours of inoculation.\u003c/p\u003e \u003cp\u003eAs time progressed, there was sloughing of cells or monolayer detachment from the wall of the cell culture flask, and some cells were severely damaged within 72 hours post-inoculation. Finally, cell death, which indicated the presence of the virus, but negative samples did not show CPE, which might not induce the morphological change of cells. Moreover, the morphology of uninfected control BHK-21 cell lines was compared with that of FMD-infected BHK-21 cells, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Samples that showed typical CPE (positive cases) were used for serotype identification of the virus involved in the outbreak cases using antigen detection sandwich ELISA.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFMDV isolated from cattle in different outbreaks in the selected areas from March 2022 to February 2023\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSite of Outbreak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDate of collection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eType of sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eName of isolated virus\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistrict\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKebele\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWith CPE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWithout CPE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eShashemene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAle Luilu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20/09/2022\u003c/p\u003e \u003cp\u003e21/09/2022\u003c/p\u003e \u003cp\u003e21/09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETH-03-22\u003c/p\u003e \u003cp\u003eETH-05-22\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003eETH-06-22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMudeta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11/10/2022\u003c/p\u003e \u003cp\u003e11/10/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETH-08-22\u003c/p\u003e \u003cp\u003eETH-09-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFaji Sole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19/10/2022\u003c/p\u003e \u003cp\u003e19/10/2022\u003c/p\u003e \u003cp\u003e24/10/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETH-10-22\u003c/p\u003e \u003cp\u003eETH-11-22\u003c/p\u003e \u003cp\u003eETH-13-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDenisa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26/01/2023\u003c/p\u003e \u003cp\u003e26/01/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-40-23\u003c/p\u003e \u003cp\u003eETH-41-23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAdamitullu and Jidokombolcha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaka\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27/10/2022\u003c/p\u003e \u003cp\u003e27/10/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETH-15-22\u003c/p\u003e \u003cp\u003eETH-16-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWelenbula\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28/10/2022\u003c/p\u003e \u003cp\u003e28/10/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOP\u003c/p\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-18-22\u003c/p\u003e \u003cp\u003eETH-20-22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDestaAbjata\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17/01/2023\u003c/p\u003e \u003cp\u003e17/01/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETH-37-23\u003c/p\u003e \u003cp\u003eETH-38-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMoretinajiru\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYimedeb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26/12/2022\u003c/p\u003e \u003cp\u003e27/12/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETH-22-22\u003c/p\u003e \u003cp\u003eETH-23-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJihurzuria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11/01/2023\u003c/p\u003e \u003cp\u003e11/01/2023\u003c/p\u003e \u003cp\u003e12/01/2023\u003c/p\u003e \u003cp\u003e12/01/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003eETH-27-23\u003c/p\u003e \u003cp\u003eETH-28-23 ETH-30-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-25-23\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMangudo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13/01/2023\u003c/p\u003e \u003cp\u003e13/01/2023\u003c/p\u003e \u003cp\u003e14/01/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETH-32-23\u003c/p\u003e \u003cp\u003eETH-33-23\u003c/p\u003e \u003cp\u003eETH-35-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eETH-Ethiopia; OP-Oro-pharyngeal fluid\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eC and D show rounding and cell detachment from the flask and lysis of cells (indicated by arrows) against respective controls (A and B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSerotyping of FMDV Isolates\u003c/h2\u003e \u003cp\u003eFoot and mouth disease virus serotyping was executed using an antigen detection sandwich ELISA. The antigen detection ELISA test was employed for the identification and serotyping of FMDV. A total of 19 positive samples\u0026rsquo; suspensions that exhibited FMDV cytopathic effect (CPE) on BHK-21 cell were needed to be tested for the detection of serotype identification using sandwich ELISA on a microplate containing 96 wells.\u003c/p\u003e \u003cp\u003eOut of 19 samples subjected to serotyping by sandwich ELISA, 14 (73.7%) samples were found positive, and three serotypes (O, SAT2, and A) were identified. The outbreaks were predominantly caused by FMDV serotype O (50%), followed by SAT2 (42.86%)and serotype A (7.14%). The results demonstrated that serotype O was found in samples taken from MoretinaJiru district, whereas serotype SAT2 and A were found in samples taken from Shashemene, AdamiTullu, and Jidokombolcha districts.\u003c/p\u003e \u003cp\u003eSamples collected from the Shashemene district were serotyped into \u0026ldquo;SAT2\u0026rdquo;, whereas samples collected from the Adami Tullu and Jidokombolcha districts were serotyped into \u0026ldquo;SAT2\u0026rdquo; and \u0026ldquo;A\u0026rdquo;, and samples collected from outbreaks in the Moretinajiru district were serotyped into \u0026ldquo;O\u0026rdquo;. Furthermore, bovine epithelial tissues collected during the outbreak at Shashemene district were serotyped into \u0026ldquo;SAT2\u0026rdquo;, whereas oro-pharyngeal fluid samples collected from clinical cases in the same district became more negative. All the results of FMD virus serotypes were summarized in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e as indicated below.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerotypes of FMDV identified in the study areas\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSite of outbreak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDate\u0026nbsp;of collection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive CPE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eType\u0026nbsp;of sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVirus name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSerotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo serotype\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistrict\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKebele\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eShashemene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAle Luilu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20/09/2022\u003c/p\u003e \u003cp\u003e21/09/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-03-22 ETH-05-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSAT2\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003eNVD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMudeta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11/10/2022\u003c/p\u003e \u003cp\u003e11/10/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-08-22 ETH-09-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSAT2\u003c/p\u003e \u003cp\u003eSAT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFaji Sole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19/10/2022\u003c/p\u003e \u003cp\u003e19/10/2022\u003c/p\u003e \u003cp\u003e24/10/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-10-22\u003c/p\u003e \u003cp\u003eETH-11-22ETH-13-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSAT2\u003c/p\u003e \u003cp\u003eSAT2\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003eNVD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAdamitullu and Jidokombolcha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaka\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27/10/2022\u003c/p\u003e \u003cp\u003e27/10/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-15-22\u003c/p\u003e \u003cp\u003eETH-16-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSAT2\u003c/p\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDestaabjata\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17/01/2023\u003c/p\u003e \u003cp\u003e17/01/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eOP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-37-23 ETH-38-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNVD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMoretinajiru\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYimedeb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26/12/2022\u003c/p\u003e \u003cp\u003e27/12/2022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-22-22 ETH-23-22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eO\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003eNVD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJihurZuria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11/01/2023\u003c/p\u003e \u003cp\u003e12/01/2023\u003c/p\u003e \u003cp\u003e12/01/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-27-23 ETH-28-23 ETH-30-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eO\u003c/p\u003e \u003cp\u003eO\u003c/p\u003e \u003cp\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMangudo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13/01/2023\u003c/p\u003e \u003cp\u003e13/01/2023\u003c/p\u003e \u003cp\u003e14/01/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eETH-32-23 ETH-33-23\u003c/p\u003e \u003cp\u003eETH-35-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eO\u003c/p\u003e \u003cp\u003eO\u003c/p\u003e \u003cp\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eKeys\u003c/strong\u003e \u003cp\u003eOP- Oro-pharyngeal fluid; NVD-No virus detected.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Detection\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eFMDV Gene Detection by Real-Time RT-PCR\u003c/strong\u003e \u003cp\u003eThe presence of FMD viral genetic material was detected by using the real-time reverse-transcription polymerase chain reaction (rRT-PCR) method. The rRT-PCR has been widely adopted by FMD reference laboratories as a principal tool for FMDV detection, offering high sensitivity and rapid sample throughput (Shaw et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Reid \u003cem\u003eet al\u003c/em\u003e., 2009). The extracted RNA virus from clinical samples was tested by the rRT-PCR method targeting the 3D regions of the FMD virus genome to determine the presence of viral RNA in clinical samples (Callahan et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The rRT-PCR could determine the serotype of the FMD virus. However, serotypes were unable to be identified due to a lack of primers and probes. For this limitation, virus isolation (VI) and antigen detection ELISA (Ag-ELISA) were implemented for the identification of FMD virus serotypes.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eOut of the total 29 suspected clinical samples tested with RT- PCR, 17(58.6%) were found positive for FMDV nucleic acid with Ct values ranging from 18-34.1 with a cutoff value of 35. Ct values\u0026thinsp;\u0026lt;\u0026thinsp;35 were considered positive, and a positive control was detected at a 20.75 Ct value, while samples with Ct values 35 were considered negative results(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The remaining samples that did not show Ct values were considered NVD. The lowest Ct value was 18, recorded in epithelial tissue samples collected from Shashemene district, while the highest Ct value, 43, was obtained from oro-pharyngeal fluid samples collected from Moretinajiru district (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe oro-pharyngeal fluid samples had Ct values higher than epithelial tissue suspensions, indicating that there were higher levels of viral RNA concentration in the epithelial tissue samples than in the oral swab samples. In most of the epithelial samples tested, the Ct values were lower than the values of oropharyngeal fluid samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the above figure, the real-time RT-PCR graph was sketched with Ct values between 18 and 34.1, which were considered positive with respect to the positive control, whereas sample35 were considered negative as compared to the negative control.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eFoot and Mouth Disease (FMD) is an important highly contagious transboundary viral disease that affects cloven-hoofed domesticated and wild animals in Asia and Africa (Zeedan \u003cem\u003eet al\u003c/em\u003e.,2020). The result presented here suggests that FMD is the main bottleneck for livestock production in the study areas. The current study revealed that an overall sero-prevalence of FMD was 27.2%, which agreed with the findings of various researchers who reported 24.22% in Adama and Asella (Mishamo \u003cem\u003eet al\u003c/em\u003e., 2018), 26.8% in Gamo Zone (Mesfin \u003cem\u003eet al\u003c/em\u003e., 2022), 28.2% in East Shewa Zone (Yasmin \u003cem\u003eet al\u003c/em\u003e., 2023), 26% in Eritrea (Dekker et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and 22.5% in Kenya (Chepkwony et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).On the other hand, the finding of this study was higher than the reports of Esayase \u003cem\u003eet al\u003c/em\u003e. (2010) in South Omo Zone(8.18%), Gezahegn (2014) in Central Ethiopia (14.5%), Dinaol \u003cem\u003eet al\u003c/em\u003e. (2016) in Eastern Showa Zone (10.88%), and Kebede \u003cem\u003eet al\u003c/em\u003e. (2018) in West and South West Shoa Zones (15.0%).On the contrary, the prevalence finding was much lower than the previous results reported by Asamenew \u003cem\u003eet al\u003c/em\u003e. (2016) with 42.7% in Borena Zone, Lencho \u003cem\u003eet al\u003c/em\u003e. (2020) with 40.4% in West Shoa Zone, Beksisa \u003cem\u003eet al\u003c/em\u003e. (2020) with 49.2% in Welmera District, and Kalkidan \u003cem\u003eet al\u003c/em\u003e. (2023) with 85.1% in Addis Ababa. Variation in the prevalence can be due to differences in sample size, geographical location, and management system.\u003c/p\u003e \u003cp\u003eIn the present study, a statistically significant association was found between study districts and FMD sero-prevalence. FMD sero-prevalence obtained in the study areas was39.3% and 20.0% in Moretinajiru and Shashemene districts, respectively. Thus, cattle found in Moretinajiru district were 2.18 times more likely to develop the disease than those found in Shashemene district. Variation in the prevalence can be due to differences in animal immune status, sample size, management system, sampling procedure used, geographical location, interaction of cattle with other animals, and the existence of recent outbreak cases. FMD prevalence variation occurred based on differences in individual animals\u0026rsquo; breed, immune status, interaction of cattle with other animals like small ruminants, and production system (Mishamo \u003cem\u003eet al\u003c/em\u003e., 2018).\u003c/p\u003e \u003cp\u003eIn this study, prevalence varied between age groups in a statistically significant manner. This finding was in line with the results of Asamenew \u003cem\u003eet al\u003c/em\u003e. (2016) and Wungak et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who reported that FMDV seropositivity in animals increased as animals aged. In contradiction to this finding, Esayas\u003cem\u003eet al\u003c/em\u003e. (2009) in the BenchiMaji zone and Dinaol \u003cem\u003eet al\u003c/em\u003e. (2016) in the Eastern Showa zone reported no statistically significant difference in the sero-prevalence of FMD in different age groups. The risk of FMD infection in 4 years increased by 2.15 times, making them more likely to have a chance of contracting FMD than \u0026lt;\u0026thinsp;2 years old. The odds of being seropositive for FMDV antibodies were 1.566 times higher in 2\u0026ndash;4 years as compared with \u0026lt;\u0026thinsp;2 years. The possible justification for the age association with FMD sero-prevalence could be due to adult cattle have acquired through exposure over time and could get access to mix with other herds at communal pasture land and market places. Relatively low sero-prevalence in animal groups below 2 years old might be indicative of the existence of passive maternal immunity and low frequency of exposure (Abdulaji \u003cem\u003eet al\u003c/em\u003e., 2011), and young animals were often managed separately at around the homestead, so that young animals have a low frequency of exposure to the virus (Teshager and Wossene, 2021).\u003c/p\u003e \u003cp\u003eIn the current study, herds with a large number of animals (37.1%) had a significantly higher sero-prevalence than herds with medium and small numbers of animals (25.0%, 19.1%, respectively. This finding was in line with previous studies conducted by Esay \u003cem\u003eet al\u003c/em\u003e. (2009), Tolosa et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and Mishamo \u003cem\u003eet al\u003c/em\u003e. (2018), who reported that higher sero-prevalence of FMD was observed in larger herd sizes. In this finding, those animals from medium herd size and large herd size were 1.59and 2.213 times more likely to develop the disease as compared to those animals from small herd size, while keeping the other factors constant. This significant difference might be because the probability of getting the disease increases as herd size increases due to the crowding of animals, which facilitates the frequency of direct contact and hence enhances the chances of transmission easily because of the contagious nature of the disease(Mohammed, 2022).\u003c/p\u003e \u003cp\u003eThe findings of this study also revealed that relatively higher sero-prevalence of FMDV was observed in extensive management systems (41.3%) than semi-intensive (27.2%) and intensive (19.0%) management systems, with a statistically significant difference. This finding was agreed with in the report of Fanos \u003cem\u003eet al\u003c/em\u003e. (2022), who stated that seropositivity increased in animals raised under an extensive management system (31.7%), followed by semi-intensive (25.0%) and intensively (23.0%) managed animals.\u003c/p\u003e \u003cp\u003eThe current study showed that extensively and semi-intensively managed cattle were 4 and 2 times more likely to have a chance of contracting FMD, respectively, than intensively managed cattle. When animals feed outside of the farm, they will have more exposure to the virus because of contact with infected cattle at communal grazing and watering points (Belege \u003cem\u003eet al\u003c/em\u003e., 2019). This could be attributed to the crowding of animals, which can facilitate the frequency of direct contact and hence enhance the chances of transmission. There is also an undeniable fact that the spread of the disease from one herd to another herd and from one area to another is almost frequently due to the movement of an infected animal from an infected herd to a non-infected susceptible herd (Constable et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study of the total number of 157 cattle examined, 66 (42%) animals manifested clinical signs that were suggestive of FMD. The current findings were related to the previous clinical findings of Haileleul (2011), who reported that 36.9%of animals manifested clinical signs during the outbreak investigation of FMD. This was justified by previous reports of Kitching \u003cem\u003eet al\u003c/em\u003e. (2005) and McLaws et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), who described that variations in clinical manifestation and its severity were associated with the virus strains, infection dose of the virus, species affected, susceptibility of the host, farming system, and previous exposure of the animal.\u003c/p\u003e \u003cp\u003eIn this study, the BHK-21 cell line was used to isolate FMD virus from clinical samples. Out of 25 field samples subjected to cell culture,19(76%) field samples showed FMDV-induced CPE after 24\u0026ndash;48 hours of incubation in a humidified incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The CPE was characterized by rounding of cells, infected cells detaching from flask surfaces, and fast destruction of the cell monolayer, which agreed with previous reports of Yeneneh (2014), Beksisa (2017), and Mishamo \u003cem\u003eet al\u003c/em\u003e. (2018). However, the other 6 (24%) samples did not show CPE even after the third passage. This could be due to the death of the virus during transportation or the presence of a very small quantity of live virus that could not overcome the challenge of the cell culture environment (Sentayehu \u003cem\u003eet al\u003c/em\u003e., 2014). In addition, samples had been taken at the late stage of recovery during sample collection (Metages, 2018) and in the laboratory; the cold chain system might not be properly maintained, which leads to the loss of FMDV, and the samples might have been taken from animals that had other vesicular diseases with similar clinical signs like vesicular diseases.\u003c/p\u003e \u003cp\u003eIn the current study, from 19 samples serotyped by antigen-capturing sandwich ELISA, three serotypes (O, SAT2, and A) of FMD viruses were isolated. The most dominant serotype was O (50%), followed by SAT2 (42.86%)and A (7.14%), respectively. These results were consistent with previous reports of Jemberu et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who indicated that serotype O (70%) was followed by serotype SAT2 (20.8%) during outbreaks in Ethiopia. In support of these findings, studies conducted by Noureldin and Elfadil (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) in Khartoum state of Sudan indicated that serotype O (82.6%) and SAT2 (40%) were the main circulating FMDVs in the cattle and the most prevalent serotype in the world (Kitching et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Serotype O was isolated from the samples collected from the Moretina Jiru district, which was the most prevalent serotype in this finding. Previously, this serotype was reported from samples collected from Shewarobit and South Wollo districts (Metages, 2018) of Amhara Regional State, which was in proximity to this study area.\u003c/p\u003e \u003cp\u003eSerotypes SAT2 and A were isolated from samples collected from Adami Tulu Jidokombolcha and Shashemene districts of Oromia Regional State. According to the report of Mishamo \u003cem\u003eet al\u003c/em\u003e. (2018), serotype A was identified indifferent districts of the Arsi zone in Oromia regional state that were neighbors to Adami Tulu Jidokombolcha and Shashemene districts. Thus, because of the presence of unrestricted animal movement, a high rate of contact between animals at market areas, communal grazing places, and at watering points, which was enhanced by windborne transmission of disease serotypes from one site to another, could easily happen.\u003c/p\u003e \u003cp\u003eIn the present study, detection of the FMDV genome was conducted from 29 fields, and isolated samples were tested by rRT-PCR targeting the 3D region. Of the 17 (58.6%) samples were positive for FMD viral RNA. The remaining samples were detected as negative, which indicated the absence of a viral genome. From the result, the lowest (18) and the highest (43) Ct values were recorded in epithelial and oropharyngeal samples collected from Moretinajiru and Adami Tulu Jidokombolcha areas, respectively.\u003c/p\u003e \u003cp\u003eThe Ct values from epithelial samples were lower than those from oropharyngeal fluid samples. This might indicate that lower concentration of FMDV in oro-pharyngeal fluid samples than in freshly collected epithelial samples due to sampling time, since they were collected from cattle in the later stages of outbreaks. The possible reason for the absence of detectable viral genome in some samples indicated that sampling was done at a late phase of infection, with low virus load in the tissues, and low quantity/quality of tissues that could have hampered the amplification by RT-PCR. Moreover, it might be due to the degradation of the RNA genome that the bacterial RNAses or other degradative enzymes might have degraded the viral genome during sample storage or transportation. These reasons could be the possible causes of bringing in either weak or no detectable signals by RT-PCR.\u003c/p\u003e"},{"header":"CONCLUSION AND RECOMMENDATIONS","content":"\u003cp\u003eThe present study was designed to estimate the sero-prevalence, isolation, and molecular detection of FMDV circulating in selected districts of Oromia and Amhara regional states, Ethiopia. Thus, the result of 3ABC-ELISA showed an overall sero-prevalence of 27.2%. The detection of antibodies in the cattle sera suggested that FMDV was circulating in the study areas. In this study, district, age, herd size, and production system were the predictors of FMDV sero-positivity. The research has also shown that typical clinical signs of FMD were identified, and the case was confirmed by cultural isolation, and FMDV was detected with real-time PCR, which is the first report in the study areas. In the current study, serotypes O, SAT2, and A were identified, and serotype O was the dominant one. The findings of this research provide insights for the identification of topotypes circulated in the study areas and vaccine-matching studies of field isolates to evaluate vaccine protection potential, which have paramount importance for effective vaccine development. However, this study is limited by the absence of genetic sequencing of FMDV isolates, which was important for vaccine matching. Finally, the presence of huge susceptible animals, age variability, breed susceptibility, lack of molecular information for vaccination programs, free movement, communal grazing places, and high contact of animals at common points were identified as major risk factors that could contribute to the occurrence of FMD.\u003c/p\u003e \u003cp\u003eTherefore, in line with the above conclusion, the following recommendations are forwarded:\u003c/p\u003e \u003cp\u003eAnimals should be vaccinated with appropriate strains of vaccines targeting circulating serotypes.\u003c/p\u003e \u003cp\u003eContinuous surveillance, serotyping, and molecular characterization of FMDV needs to be conducted to check the introduction and circulation of new serotypes of the virus in different parts of the country.\u003c/p\u003e \u003cp\u003eIt is necessary to train field veterinarians on disease outbreak reporting, and the reporting system should also be strengthened by digitalizing it for easy access to disease outbreak areas.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive specific funding from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Animal Health Institute, Ethiopia, for their logistical support and for granting full access to their laboratories.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted as an observational sero-surveillance and outbreak investigation of Foot-and-Mouth Disease (FMD) in cattle, as part of routine veterinary public health and disease control activities in Ethiopia. The study did not involve any experimental interventions such as deliberate infection, treatment, vaccination, or euthanasia of animals.\u003c/p\u003e\n\u003cp\u003eBlood, epithelial, and oro-pharyngeal samples were collected from naturally infected or suspected animals using standard, minimally invasive diagnostic procedures. All sampled animals were handled appropriately and returned to their owners immediately after sample collection without further intervention. No animals were anesthetized, euthanized, or sacrificed for the purpose of this study.\u003c/p\u003e\n\u003cp\u003eAs the work constituted routine disease surveillance and outbreak investigation and did not involve experimental manipulation of animals, formal ethical approval from an Institutional Review Board (IRB) or Institutional Animal Care and Use Committee (IACUC) was not required under applicable national veterinary guidelines.\u003c/p\u003e\n\u003cp\u003eAll procedures were conducted in accordance with internationally accepted animal welfare principles, and all efforts were made to minimize stress and discomfort during sample collection. All animals included in the study were privately owned, and informed verbal consent was obtained from the animal owners prior to sampling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supplementary images and figures included in this study (PCR results, cell culture images, and study area maps) do not contain any human participants, personal data, or identifiable individual information. Therefore, specific written consent for publication was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdulaji Mohamoud E, Tessema, Degefu H. Sero-prevalence of bovine foot and mouth disease (FMD) in Awbere and Babille districts of Jijiga zone, Somalia regional state, Eastern Ethiopia. Afr J Microbiol Res. 2011;5(21):3559\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdamitullu and Jidokombolcha Livestock and Fisheries Resource Office (AJLFO). 2022. The annual report on livestock population.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmare BerheKidane, KefenaEffaDelesa YY, Mummed, Tadesse M. 2019. 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Foot and mouth disease vaccination and post-vaccination monitoring guidelines.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOIE. Foot and Mouth Disease Foot and Mouth Disease. March; 2021. pp. 1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReid S, Ebert K, Bachanek-Bankowska K, Batten C, Sanders A, Wright C, Shaw. A..\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSentayhu Menda S, Jenberie H, Negussie G, Ayelet, Amsalu K. Molecular epidemiology of foot and mouth disease virus outbreaks in Ethiopia in 2011/2012. Acad J Anim Dis. 2014;3:8\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShashemene District Livestock and Fisheries Resource Office (SDLFRO). 2022. Annual report of livestock population, Shashemene, Ethiopia.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShashemene Woreda Agricultural Development Office (SWADO). Shashemene Woreda Agricultural Plan for the Year 2015/2016. Ethiopia: Shashemene; 2015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaw A, Reid S, Ebert K, Hutchings G, Ferris N, King D. Implementation of a one-step real-time RT-PCR protocol for the diagnosis of foot and mouth disease. J Virol Methods. 2007;143:81\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiferaw Jenbere K, Moses, Etana M. Participatory appraisal of foot and mouth disease in the Afar pastoral area, Northeast Ethiopia: implications for understanding disease ecology and control strategy. Trop Anim Health Prod. 2010;42(2):193\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSouley K, Elliot F, King D, Hyera J, Knowles N, Ludi A, Mioulet V, Matlho G, De Clercq K, Thys E. Outbreak investigations and molecular characterization of foot and mouth disease viruses circulating in South-west Niger. Trans-bound Emerg Dis. 2018;65:146\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeshager Dubie and Wossene Negash. Sero-prevalence of bovine foot and mouth disease (FMD) and its associated risk factors in selected districts of Afar Region, Ethiopia. Veterinary Med Sci. 2021;7(5):1678\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThrusfield M. Veterinary epidemiology. 4th ed. Oxford, UK.: Blackwell Science; 2018. pp. 270\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTolosa T, Beyene B, Rufael T, Hailu B, Teklue T. Foot and mouth disease in selected districts of western Ethiopia: sero-prevalence and associated risk factors, Department of Microbiology and Veterinary Public Health, Jimma University, Jimma, Ethiopia. Rev Sci Tech Off Int Epiz. 2015;34(3):939\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVosloo W, Bastos A, Sangare S, Hargreaves O, S., and, Thomson R. Review of the status and control of foot and mouth disease in Sub-Saharan Africa. Rev Sci Tech Off Int Epizt. 2002;21:437\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWungak YS, Olugasa BO, Ishola OO, Lazarus DD, Ularamu GH. Foot and mouth disease (FMD) prevalence and exposure factors associated with sero-positivity of cattle in North-Central, Nigeria. Afr J Biotechnol. 2016;15(24):1224\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYasmin Jibril A, Alemayehu, AyelechMuluneh and HaileleulNegussie. Foot and mouth disease in Adama and Boset districts, East Shewa zone, Ethiopia: Sero-prevalence and virus serotyping. Ethiop Veterinary J. 2023;27(1):143\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeneneh T, Khan F, and Esayas Gelaye. Molecular characterization of foot-and-mouth disease viruses collected from Northern and Central Ethiopia during the 2018 outbreak. Veterinary World. 2020;13(3):542\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeneneh Tesfaye. 2014. Isolation, molecular characterization, and vaccine matching of foot and mouth disease virus circulating in Ethiopia. MSc Thesis, Addis Ababa University, Bishofu, Ethiopia.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeedan GSG, Mahmoud AH, Abdalhamed AM, Khafagi MH. Diagnosis of Foot and Mouth Disease in Cattle and Buffaloes in Different Governorates of Egypt. World Vet J. 2020;10(1):43\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ethiopia, Cattle, FMD Virus, Molecular Detection, Sero-prevalence","lastPublishedDoi":"10.21203/rs.3.rs-9365906/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9365906/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFoot and Mouth Disease (FMD)is a highly contagious transboundary viral disease of cloven-hoofed animals. It is one of the most economically important animal diseases in developing countries, including Ethiopia. A cross-sectional study was conducted from March 2022 to February 2023to estimate sero-prevalence, isolate, and molecular detection of foot and mouth disease virus (FMDV) circulating in selected districts of Oromia and Amhara regional states, Ethiopia. Multistage sampling was conducted to select the required sample size of cattle for the sero-prevalence study, and purposive sampling was implemented for collecting field epithelial tissues and oro-pharyngeal fluid samples for FMDV isolation and molecular detection. A total of 654 cattle sera samples were collected and tested for antibodies against FMDV using 3ABC-ELISA. The result showed an overall sero-prevalence of FMDVof 27.2%(CI\u0026thinsp;=\u0026thinsp;24.0%-30.0%). The multivariable logistic regression analysis revealed that FMDV sero-positivity was significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) affected by factors such as districts, production system, herd size, and age. Cattle that were kept in large herd sizes were 2.2 times more likely to be infected with FMDV than those in small herds (OR\u0026thinsp;=\u0026thinsp;2.2, 95% CI\u0026thinsp;=\u0026thinsp;1.3\u0026ndash;3.8). The odds of being positive for FMDV were also 4 times higher in cattle in the extensive production systems than those in intensive production systems. From 25 epithelial tissue and oro-pharyngeal samples, which were inoculated into monolayer BHK-21 cell lines, 19 (76%) samples exhibited foot and mouth disease virus cytopathic effect, and the viruses were isolated. In this study, three serotypes: serotype O (50%), SAT2 (42.86%), and serotype A (7.14%) were identified using antigen detection ELISA. Out of 29 samples tested by rRT-PCR, 17(58.6%) were positive for FMDV genome with Ct values ranging from 18-34.1. The current study findings revealed that FMD was prevalent in the study districts. Thus, the control strategy should be designed in such a way that early reporting of the disease outbreaks, regular sero-surveillance, and effective targeted vaccination. Further study on sequencing and characterizing the circulating virus to a lineage level is also recommended.\u003c/p\u003e","manuscriptTitle":"Sero-prevalence and an Outbreak Investigation of Foot and Mouth Disease with Isolation and Identification of Circulating Virus from Cattle in Selected Districts of Oromia and Amhara Regional States, Ethiopia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-08 13:27:00","doi":"10.21203/rs.3.rs-9365906/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-18T03:52:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T07:01:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328756004177802514178491609604416120632","date":"2026-05-06T00:57:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332490696285351315475014500268155748982","date":"2026-05-04T04:21:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-30T07:45:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-24T12:17:40+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-20T16:44:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-18T09:33:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2026-04-18T09:27:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"166f5f5a-2f8b-4d97-83f9-c6378c03f68a","owner":[],"postedDate":"May 8th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T03:52:19+00:00","index":60,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T07:01:35+00:00","index":59,"fulltext":""},{"type":"reviewerAgreed","content":"328756004177802514178491609604416120632","date":"2026-05-06T00:57:40+00:00","index":57,"fulltext":""},{"type":"reviewerAgreed","content":"332490696285351315475014500268155748982","date":"2026-05-04T04:21:59+00:00","index":55,"fulltext":""},{"type":"reviewersInvited","content":"17","date":"2026-04-30T07:45:32+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T13:27:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-08 13:27:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9365906","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9365906","identity":"rs-9365906","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}