Oral fluid as a material for serological diagnostics of caseous lymphadenitis in goats

Oral fluid as a material for serological diagnostics of caseous lymphadenitis in goats | 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 Article Oral fluid as a material for serological diagnostics of caseous lymphadenitis in goats Kinga Biernacka, Marcin Mickiewicz, Zofia Nowek, Agata Moroz-Fik, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6065536/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Corynebacterium pseudotuberculosis causes caseous lymphadenitis (CLA) in goats and sheep. This study assessed oral fluid (OF) as a minimally invasive sample for CLA serodiagnosis by detecting antibodies to PLD, CP40, and PknG using Western blot (WB). Ninety-six goats from a CLA-affected herd in Poland were tested. Paired serum and OF samples were analyzed via ELISA (serum only) and WB (serum and OF). The rPLD-ELISA had 70–80% diagnostic sensitivity, depending on the cut-off. WB on serum showed high sensitivity (98%) and good agreement with ELISA. WB on OF had slightly lower sensitivity (90%), with differences in antigen detection. OF shows promise for CLA diagnosis, with PLD antibodies being the most relevant. Fewer OF samples had CP40 and PknG antibodies, likely due to antibody class differences or detection limitations. Further investigation into immunoglobulin classes present in different sample types and optimized secondary antibodies is essential for developing an effective OF-based diagnostic test for CLA. Biological sciences/Microbiology/Pathogens Health sciences/Diseases/Infectious diseases/Bacterial infection Health sciences/Medical research Corynebacterium pseudotuberculosis CLA diagnostics goats immunoassays Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Corynebacterium pseudotuberculosis ( C. pseudotuberculosis ) is a Gram-positive facultatively anaerobic coccobacillus. It causes several diseases, including caseous lymphadenitis (CLA) in sheep and goats, and ulcerative lymphangitis in horses and cattle 1 , 2 , 3 , 4 . C. pseudotuberculosis also affects camels 5 , 6 , 7 , alpacas 8 , 9 , 10 , and wild ruminants 11 , 12 , 13 , 14 . It has some zoonotic potential, although few cases in people have been reported 15 . CLA occurs worldwide and is economically significant in small ruminants 10 , 16 . The disease follows a chronic course with a long incubation period and presumably a lifelong carrier state following recovery, although this issue has not been ultimately settled. Several months after the infection, clinical signs, including mainly abscesses in the superficial lymph nodes, become apparent – such form of the disease is called the external form of CLA. Some animals develop abscesses in the internal lymph nodes or internal organs, referred to as the internal form of CLA 16 , 17 , 18 , 19 . Over the last decades, CLA has become a widespread disease in goat herds in Poland 20 . Its seroprevalence was first determined in 1996, and it was shown to have increased from 13% to over 60% in 2002 21 . A recent investigation has shown that the herd-level seroprevalence increased to approximately 70% 20 . Isolation of C. pseudotuberculosis from abscesses and its identification using microbiological or molecular methods is regarded as the gold standard of CLA diagnostics 16 , 22 , 23 . However, due to a long incubation period (2–6 months), unknown, but presumably long duration of carrier state, and internal form of the disease, serological tests play an important role, especially, in the evaluation of the status of herds as well as of individual animals before introduction into a herd 19 . Routine serological diagnostics is mainly based on immunoenzymatic assays (ELISA) however, western-blot (WB) is considered the serological reference standard 24 . Several antigens of C. pseudotuberculosis with potential diagnostic applicability have been described 25 , 26 , 27 . Phospholipase D (PLD), an approximately 30 kDa protein, is the main exotoxin and the major virulence factor favouring the infection process 28 . PLD is characterized by both very high immunogenicity and antigenicity which makes it a suitable component of vaccines 29 and antigen of serological tests 30 . A recombinant form of PLD (rPLD) has been used for developing commercial ELISAs (rPLD-ELISA) and they are commonly used for serological diagnostics of CLA 31 , 32 . Other antigenic enzymes are a CP40, an approximately 40 kDa protein, secreted endo-b-N-acetylglucosaminidase 33 , 34 and a serine/threonine protein kinase G (PknG) 35 , 36 , an approximately 70 kDa protein homologous to an important virulence factor of Mycobacterium tuberculosis 30 , 37 , 38 . In some preliminary studies based on in-house ELISAs, CP40 has shown high diagnostic specificity (dSp) of 97% but very low diagnostic sensitivity (dSe) of only 43% 30 while PknG turned out to be both highly sensitive and specific (94% and 97%, respectively) 39 . Oral fluid (OF) is a mixture of saliva and serum transudate that enters the mouth by crossing the buccal and gingival mucosa from the capillaries 40 , 41 . Therefore, OF contains antibodies from the systemic immune system (from the passage of serum antibodies) as well as locally produced antibodies from the secretory immune system in the salivary glands 42 , 43 , 44 . OF also contains feed particles, cell detritus, tracheal-nasal secretions, gastrointestinal reflux, and other particles from the environment currently being chewed by an animal. In ruminants, contrary to other species, the composition of OF is also vastly affected by rumination, which involves the regurgitation of material from the reticulo-rumen to the buccal cavity, where solid material is re-masticated and re-insalivated before being swallowed. Serological tests based on OF are becoming increasingly popular in non-ruminating animal species, especially in pigs. Such tests are used for diagnosing classical swine fever 45 , 46 , 47 , porcine reproductive and respiratory syndrome 48 , 49 , 50 , porcine circovirus type 2 51 (Prickett et al., 2011) and Toxoplasma gondii infections 52 , 53 . Although, theoretically, rumination can hinder diagnostic application of OF, it has so far been shown to be a suitable material for serological diagnostics of foot-and-mouth disease 54 and Schmallenberg virus infection 55 in ruminants. Additionally, an in-house OF-based ELISA for M. bovis infection in goats has been developed 56 . OF offers several advantages over traditional diagnostic specimens such as blood or tissue. The OF collection procedure is minimally invasive and welfare-friendly, with much less discomfort or stress for the animals than blood collection 43 . Moreover, it can be performed by non-veterinarian personnel with minimal training and does not require the consent of the Ethics Committee when carried out within the frame of scientific studies. Contrary to another type of non-invasive sample – milk – OF can also be collected from males and non-lactating females. Our study aimed to evaluate the potential usefulness of OF as a material for serological identification of C. pseudotuberculosis -infected goats based on the detection of antibodies to PLD, CP40, and PknG using WB. Additionally, the dSe of rPLD-ELISA and WB performed using serum samples was estimated. 2. Results 2.1. Antigenic structure of the reference strain Multiple bands were visible in the gel, with the most prominent bands located at heights corresponding to approximately 30 kDa (two bands), 35 kDa (one band), 40 kDa (one band), 70 kDa (multiple bands) (Fig. 1 ). Based on previous findings, the locations of the bands corresponded to: LexA protein (28 kDa), PLD (30 kDa or 31.4 kDa) TrxR (35 kDa), CP40 (40 kDa), and PknG (70 kDa) 30 , 57 . The band that corresponds to CP40 was the most visible. 2.2. Diagnostic sensitivity of rPLD-ELISA In the rPLD-ELISA, 67 serum samples (69.8%) were positive, 10 (10.4%) – inconclusive, and 19 (19.8%) – negative. S/P% of the aforementioned samples ranged from 50.1–157.5% (median of 88.9%), from 40.1–49.0% (median of 46.0%), and from 0.6–39.9% (median of 34.2%), respectively. The dSe of rPLD-ELISA was 69.8% (CI 95%: 60.0%, 78.1%) if only serum samples with S/P% >50% were considered positive and 80.2% (CI 95%: 71.1%, 87.0%) if all serum samples with S/P% >40% were considered positive. 2.3. Agreement between rPLD-ELISA and WB on serum samples In WB, antibodies to any of the three antigens were detected in the serum of 94 / 96 goats (dSe = 97.9%; CI 95%: 92.7%, 99.4%). The agreement between rPLD-ELISA and WB was good: AC 1 = 61.4% (CI 95%: 49.1%, 73.8%; P o = 71.9%) if only serum samples with S/P% >50% in ELISA were considered positive and AC 1 = 75.4% (CI 95%: 65.5%, 85.3%; P o = 80.2%) if all serum samples with S/P% >40% in ELISA were considered positive. 2.4. Comparison of diagnostic sensitivity of WB on serum and OF samples Antibodies to any of the three antigens were detected in the OF samples of 86 / 96 goats (dSe = 89.6%; CI 95%: 81.9%, 94.2%). The proportion of positive OF samples was significantly lower than that of positive sera (by 8.3%; CI 95%: 2.3%, 15.8%; P = 0.003). Antibodies to PLD were present in a similar proportion of serum and OF samples ( P = 0.302). Antibodies to CP40 and PknG were detected in a significantly lower proportion of OF than serum samples ( P < 0.001 in both cases) (Table 1 ). Table 1 Results of the western blot on paired serum and oral fluid (OF) samples of 96 goats affected by caseous lymphadenitis Antigen Serum Oral fluid McNemar’s test P -value Difference between proportions in serum and OF (CI 95%) Number of positive results Proportion (CI 95%) [%] Number of positive results Proportion (CI 95%) [%] PLD 89 92.7 (85.7, 96.4) a 84 87.5 (79.4, 92.7) a 0.302 5.2 (-3.2, 13.9) CP40 82 85.4 (77.0, 91.1) ab 10 10.4 (5.8, 18.1) c < 0.001 75.0 (63.6, 82.4) PknG 73 76.0 (66.6, 83.5) b 31 32.3 (23.8, 42.2) b < 0.001 43.8 (31.2, 54.2) P -value d < 0.001 < 0.001 a,b,c proportions denoted by the same letter do not differ significantly d Cochrane Q test followed by McNemar’s test with Bonferroni correction for multiple comparisons – p-values reported in the text In the serum samples, antibodies to PLD were detected significantly more often than antibodies to PknG ( P BC = 0.004), whereas the differences between the proportion of samples with antibodies to PLD and CP40 or between the proportion of samples with antibodies to CP40 and PknG were not significant ( P BC = 0.438 and P BC = 0.372, respectively). In the OF samples, antibodies to PLD were detected significantly more often than antibodies to PknG ( P BC < 0.001) or CP40 ( P BC < 0.001), and antibodies to PknG were detected significantly more often than antibodies to CP40 ( P BC < 0.001). The latter were detected in only 10% of OF samples (Table 1 ). Most serum samples were positive for antibodies to all three antigens (63.5%), followed by sera positive for antibodies to PLD and CP40 (16.7%). Only 2 / 94 seropositive goats (2.1%) did not have antibodies to PLD (instead, they had antibodies to CP40) (Fig. 2 ). Over half of OF samples were only positive for antibodies to PLD (56.3%), followed by sera positive for antibodies to PLD and PknG (20.8%), and sera positive for antibodies to all three antigens (9.4%). Only 2 / 86 goats with positive results of OF (2.3%) did not have antibodies to PLD (instead they had antibodies to PknG) (Fig. 2 ). Combination of PLD with any of the two other antigens did not significantly increase the dSe of WB either in serum (Fig. 3 a) or OF (Fig. 3 b). Combination of CP40 and PknG (without PLD) performed significantly worse than any other combination in OF ( P < 0.001) but not in serum. The difference between dSe of a combination of PLD and CP40 compared to dSe of PLD alone (98% vs. 93%, respectively) was closest to statistical significance ( P = 0.074). The agreement between WB performed using serum and OF was high (P o = 88.5%; AC 1 = 86.6%, CI 95%: 79.2%, 94.1%). The high agreement resulted only from similar results with respect to antibodies to PLD since the agreement between serum and OF concerning PknG was poor, and it was even significantly negative in terms of CP40 (Table 2 ). Table 2 Number of serum and oral fluid (OF) samples with positive result of the western-blot (WB) for antibodies to certain antigens of Corynebacterium pseudotuberculosis : phospholipase D (PLD), CP40, and protein kinase G (PknG) Result of WB on biological material Number of samples in which C. pseudotuberculosis antigens were detected Serum Oral fluid PLD CP40 PknG Positive Positive 79 9 27 Positive Negative 10 73 46 Negative Positive 5 1 4 Negative Negative 2 13 19 Observed agreement between serum and OF [%] 84.4 22.9 47.9 Chance-corrected agreement between serum and OF (Gwet’s AC 1 & CI 95%) [%] 81.0 (72.1, 89.8) -53.9 (-70.7, -37.1) -3.5 (-23.3, 16.4) In terms of samples classified as negative, distribution of samples without any band and with weak bands was balanced, except for PLD in serum samples where samples with weak bands significantly predominated ( P = 0.047) and CP40 in OF samples where samples without any band significantly predominated ( P = 0.001) (Fig. 4 ). 3. Discussion Our study shows for the first time that the OF of goats contains antibodies to C. pseudotuberculosis and can serve as a material for serological diagnostics of CLA in this species. Antibodies to PLD appear to be the only ones of potential clinical usefulness. Surprisingly, antibodies to CP40 and PknG are detectable in OF of a low proportion of CLA-affected goats. The main difficulty encountered when investigating the diagnostic accuracy of tests for CLA is the determination of the true health status of an animal. This problem results from an ambiguous definition of an animal diseased with CLA. If a CLA-diseased animal is defined as the one with abscess(es) confirmed bacteriologically to be of C. pseudotuberculosis origin, serological tests turn out to be perfectly sensitive but rather unspecific like in the studies of Ellis et al. and Costa et al. 32 , 58 . This is because abscesses usually develop after several months of infection, which is long enough for an animal to mount a humoral immune response, detectable in serological tests 17 , 19 . However, in many animals infected with C. pseudotuberculosis , apparent abscesses develop much later, or even never, which results in positive results of serology in many, by definition, “healthy” animals. In terms of evaluating the diagnostic accuracy of the serological tests, classifying only animals with abscesses as CLA-diseased animals does not seem reasonable because bacteriology, not serology, is the diagnostic test of choice in such animals. Another approach to determining true health status is to define a CLA-diseased animal as the one with antibodies to C. pseudotuberculosis . In this situation, the most acknowledged serological test is chosen as a gold standard – it is usually WB as it detects antibodies to the broadest array of pathogen’s antigens. Animals positive in this test are considered as CLA-diseased. This approach has been commonly applied to evaluate the accuracy of serological tests for CLA 24 , 30 , 59 . Obviously, no serological gold standard is able to overcome the problem of delayed seroconversion. Furthermore, the assumption is that persistence of antibodies parallels the lifelong infection. This is highly likely to be true as C. pseudotuberculosis can remain viable inside phagocytes and establish long-lasting intracellular infection 19 , however, we do not know any study directly evidencing the lifelong nature of C. pseudotuberculosis infection. In our study, we simply assumed that all adult goats born and raised or kept in the herd affected by clinical form of CLA for several years must have become infected with C. pseudotuberculosis and their infection is lifelong. It is a strict and conservative approach and it could have obviously underestimated the dSe of ELISA, especially when we take note of the fact that our estimate of dSe of ELISA is quite low (70%-80%) compared to many previous studies 24 , 27 , 30 . However, if we consider WB performed with serum as the gold standard, an increase in the estimated dSe of ELISA is negligible (from to 69.8–71.3% when only serum samples with S/P% >50% are considered positive and from 80.2–80.9% when all serum samples with S/P% >40% are considered positive). Therefore, we suspect that there may be some variability in the diagnostic performance of rPLD-ELISA caused by unknown animal- or pathogen-related factors. Our study does not allow for drawing any conclusions regarding dSp as only diseased animals have been enrolled. Virtually 90% of OF samples contained antibodies to PLD, approximately 30% to PknG, and only 10% to CP40. It means that in an eight times larger number of samples, antibodies to CP40 were detectable in serum than in OF, and a two times larger number to PknG. It can be explained by the fact that in various mammalian species, antibody levels in serum are significantly higher for many pathogens than in OF. Thus, the concentration of antibodies is reflected in their detectability in the sample 44 , 60 , 61 , 62 . CP40 has a high antigenic potential, stimulating the production of specific antibodies detectable in the serum of goats and sheep 30 . Also, in our study, 85% of goats had antibodies to CP40 in serum but only 10% in OF. Further, 28 goats had a weak band in WB, indicating that some low concentrations of the antibodies could be present in OF. The existence of some physical or physiological barrier for antibodies to CP40 and PknG between systemic circulation and OF is one of the explanations, yet highly unlikely. More probable is that antibodies to PLD are not only of IgG class but also of IgA class and are produced locally at higher concentrations while antibodies to CP40 and PknG are mostly of systemic origin and reach OF in low amounts. If that is correct, WB shows true differences in antibody concentrations between serum and OF. However, another explanation may be a technical shortcoming of our WB protocol. Maybe antibodies to PLD are mostly of IgG class and systemic origin, and they are efficiently detected by rabbit anti-goat IgG secondary antibodies, which we used in our study. At the same time, antibodies to CP40 and PknG are mostly local IgA secretory antibodies. In this situation, using anti-goat IgA secondary antibodies together with anti-goat IgG secondary antibodies could increase the detectability of anti-CP40 and anti-PknG antibodies. Both scenarios are feasible as, according to the datasheet, the rabbit anti-goat IgG (H/L) antibody recognizes the heavy and light chains of goat IgG but may react with the light chains of other goat immunoglobulins. Determination of the class of antibodies in OF could clarify this problem and this aspect warrants further investigation. Showing that specific antibodies are detectable in WB is the first step on the long pathway to develop ELISA for CLA with OF as diagnostic material. An indirect ELISA based on the whole-bacterium antigen used in our study was developed over 20 years ago by Kaba et al. and has performed very well on goat sera ever since 24 . Results of our study indicate that using more targeted antigen e.g. rPLD may be worth considering as antibodies to PLD are most abundant or at least most effectively detected in OF. Developing an efficient serological diagnostic test based on non-invasively collected material would be of value. Stress-reducing procedures become increasingly popular in modern veterinary medicine and animal disease control programs based on such procedures have been shown to play a useful role 63 . The method of OF collection employed in our study differs from those described previously. For pigs, a rope hung in the pen is recommended because pigs are eager to interact with the rope and chew it. In ruminants, more active OF collection using cotton swabs 54 , 56 or commercial plastic shafts 55 (Copan Flocked Swabs) has been performed. We used a sponge held in metal forceps. This was an extremely inexpensive and efficient method as by squeezing the sponge in a syringe, we obtained between 1 and 3 ml of OF without centrifugation. This amount was enough to perform several rounds of testing. Last but not least, we encountered some problems regarding the identification of PknG owing to inconsistent information regarding its molecular mass which can be found in the literature. According to Barral et al. 30 , PknG weighs 69 kDa, although, in fact, the corresponding electrophoresis band lay somewhere between 55 and 70 kDa (Fig. 1 in Barral et al. 30 ). On the other hand, in the studies of Santana-Jorge et al. 36 and Silva et al. 64 PknG was claimed to weigh 83 kDa, while the band on the electrophoresis gel presented in Fig. 1 of Silva et al. 64 was located slightly above 70 kDa. We decided to stick to the molecular mass indicated on the pictures of electrophoresis gels and mentioned by Barral et al. 30 . Concluding, OF appears to be a promising material for non-invasive and non-stressing serological testing of goats for CLA. However, classes of anti- C. pseudotuberculosis immunoglobulins predominating in OF should be determined first, to allow for the most optimal selection of secondary antibodies to be used in the OF-targeted serological assay. 4. Material and Methods 4.1. Animals and sample collection The study enrolled 96 adult (> 1-year-old) Saanen goats, 92 females and 4 males, from the dairy herd affected by CLA. The goats had been under strict regular observation since August 2022, and serum and OF samples were collected in November 2022. The herd was located in north-eastern Poland. The herd had been bought by the current owner and moved from south-eastern Poland in 2019; however, only one year earlier, the goats had been purchased from Austria by the previous owner. At the moment of the study, the goats were kept in two spacious and high concrete-wooden barns and were irregularly grazed on a small backyard pasture during summer months. They produced milk mainly for on-farm cheese production. In 2018, a few months after moving from Austria, CLA had been diagnosed first in a male goat with a superficial abscess. In the following years, the disease gradually spread over the entire herd. It was repeatedly confirmed by positive results of bacteriological culture of swabs collected from abscesses of the superficial lymph nodes. The herd was tested for caprine arthritis-encephalitis (CAE) using an indirect ELISA (ID Screen MVV-CAEV Indirect Screening test, IDvet Innovative Diagnostics, Grabels, France) twice in 2018 and in 2022 and all goats tested negative. Of 96 goats enrolled in the study, 26 (27.1%) had had at least one abscess confirmed by bacteriological culture caused by C. pseudotuberculosis between August and November 2022. According to the owner’s account, the superficial abscesses had been regularly observed in the goats from the very moment of their purchase. Given that all goats enrolled in this study had been either born and raised in this herd or kept in the herd for at least 3 years (since the purchase of the herd by the current owner in 2019), we classified all of them as infected with C. pseudotuberculosis and affected by CLA (either clinically or subclinically). Therefore, the proportion of goats whose serum or OF samples tested positive in the test (rPLD-ELISA with serum, WB with serum, or WB with OF) was considered an estimate of dSe of the test. Paired samples of blood and OF were collected from each goat at the same moment. The blood was collected from the jugular vein into 10-ml dry tubes (BD Vacutainer, BP-Plymouth, UK) and transported to the laboratory. After 24 h storage in the refrigerator (4–8°C), blood was centrifuged at 3000 rpm for 10 minutes, serum was harvested to 1.5-ml Eppendorf tubes and stored at -20°C until testing using rPLD-ELISA and WB. The OF was collected with a 5×1×1 cm fragment of polypropylene sponge held in stainless steel forceps. It was inserted into the oral cavity and kept there for approximately 30 sec. so that an animal could thoroughly chew it up. Then, the sponge was placed in a 20-ml clogged syringe to prevent fluid leakage, and in such a form, the material was transported to the laboratory. There, OF was squeezed out of sponges into 1.5-ml Eppendorf tubes. OF samples were stored at -20°C until testing with WB. OF samples were not centrifuged. Forceps were washed in the mild detergent (soap) between animals. We confirmed that all experiments in this study were performed in accordance with the relevant guidelines and regulations. All the procedures of the study followed the ARRIVE guidelines. Blood collection was performed as part of routine herd diagnostics for infectious diseases and was approved by the Local Ethics Committee in Warsaw (Approval No. WAW2/048/2021). OF was collected non-invasively and this procedure did not require the Ethics Committee approval according to Polish legal regulations (the Act on the Protection of Animals Used for Scientific or Educational Purposes of 15 January 2015). 4.2. Commercial rPLD-ELISA Serum samples were tested using a commercial indirect ELISA based on rPLD (ID Screen CLA, IDvet Innovative Diagnostics, Grabels, France) according to the manufacturer’s manual. Briefly, serum samples were diluted 1:20 in the dilution buffer, and reading was performed at 450 nm using the Epoch Microplate Spectrophotometer (BioTek, USA). The optical density (OD) of samples was corrected by the OD of the manufacturer’s positive and negative control serum, and the sample-to-positive control ratio (S/P%) was interpreted as follows: S/P% ≤40% – negative result, S/P% >40–50% – inconclusive result, and S/P% >50% – positive result. 4.3. Western blot The positive control serum was obtained from an adult female goat from this herd several months before the study. The goat had multiple abscesses in the superficial lymph nodes, from which C. pseudotuberculosis was isolated in a bacteriological culture. The goat was also positive in the commercial rPLD-ELISA. The negative control serum and OF were obtained from an apparently healthy adult goat from another herd with no history of CLA and negative rPLD-ELISA result. The whole-bacterium antigen for WB was prepared from the C. pseudotuberculosis isolate obtained from an abscess of a goat from the same herd according to the method described by Kaba et al. 24 with minor modifications. Briefly, the C. pseudotuberculosis isolate was cultivated on Columbia blood agar (Graso Biotech, Poland) for 24 h at 37°C in the aerobic atmosphere, then 5 ml of Brain Heart Infusion (BHI) broth (Graso Biotech, Poland) was inoculated with a few colonies, and incubated in the same conditions with agitation. This culture was transferred into a 50 ml BHI broth, and incubation was continued under the same conditions for 24 h. Next, 2 liters of BHI broth were inoculated with the 50 ml culture and incubated in the same conditions for 16 h. Bacterial cells were harvested by centrifugation (7000 rpm, 10 min) and then washed three times in PBS (phosphate buffered saline; 150 mM NaCl, 1.5 mM KH 2 PO 4 , 9 mM Na 2 HPO 4 × 12H 2 O, 2.5 mM KCl, pH 7.2) with 0.2% Tween-80 (Merck, Darmstadt, Germany). The cell pellet was resuspended in 12 ml of the antigen preparation buffer (0.5 M Tris pH 6.8; 5.2% SDS; 8.7% 2-mercaptoethanol), boiled (100°C) for 10 min, and then centrifuged (at 10,000 rpm for 15 min). The collected supernatant represented the ELISA solid phase antigen and was stored in 500 ml aliquots at -80°C. The antigen was electrophoresed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to standard procedures. Briefly, antigens were heated at 98°C for 5 min in sample buffer containing 0.0625 M Tris (pH 6.8), 2.0% (wt/vol) sodium dodecyl sulphate, 25.0% Glycerol, 5.0% (vol/vol) b-mercaptoethanol, and 0.00125% bromophenol blue. Antigens were separated on polyacrylamide gels (stacking gel: 4%, running gel: 4%). The protein standard Precision Plus Protein Standards Dual Color (Bio Rad Laboratories, USA) was used. Electrophoresis was performed under a voltage of 60 V for stacking gel and 120 V for running gel. SDS gels were stained with Coomassie blue (2.5% brilliant blue in 50% methanol, 10% acetic acid) prior to analysis. Subsequently, the electrophoresed antigen was transferred from the gel to the supported nitrocellulose membrane (Bio-Rad, USA) in the TransBlot Turbo Transfer system (Bio-Rad, USA). Membranes were washed in TBST (a mixture of tris-buffered saline and Polysorbate 20) for 10 min. and incubated in the Everyblot Blocking Buffer (Bio-Rad, USA) for 20 min. Then, the membranes were washed 3 times for 10 min. An estimation of the suitable dilutions of the tested samples was performed. Finally, serum samples were diluted 1:100 in the blocking buffer. OF samples were centrifuged at 2600 rpm for 5 min. to separate the liquid from the solid particles and then diluted 1:5 in the blocking buffer. One-hour incubation of serum and OF samples on membranes was performed in the Mini Protean Multi II (Bio-Rad, USA). Then, the membranes were washed 3 times for 10 min. in TBST. In the following 1-hour incubation process, secondary antibodies (Rabbit anti-goat IgG H/L conjugated to alkaline phosphatase, Bio-Rad, USA) were used at a dilution of 1:4500 for membranes incubated with sera and 1:4000 for membranes incubated with OF. After the incubation, membranes were washed twice for 5 min. in TBST, once for 5 min. in TBS, and twice for 5 min. in alkaline phosphatase color development buffer (Bio-Rad, USA). The results were visualized using Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad, USA). The Precision Plus Protein™ Standards Dual Color (Bio-Rad, USA) ladder was used to estimate protein sizes. The results of WB were visually reviewed by a single investigator (KB) and classified as i) any band absent, ii) weak band present, and iii) strong band present, separately at the level of each of 3 antigens: PLD – approximately 30 kDa, CP40 – approximately 40 kDa, PknG – approximately 70 kDa. When a band at the level of a relevant antigen was absent or weak the result was classified as negative; only a strong band was considered a positive result. The serum or OF sample was considered positive in WB if a strong band at the level of at least 1 of the aforementioned antigens was present. 4.4. Statistical analysis Categorical variables were presented as counts (n) and proportions (%), and compared between paired groups using the McNemar’s test (2 groups). In the case of comparing > 2 groups, the Cochrane Q test was carried out as an omnibus test. If significant, post-hoc analyses were performed using the McNemar’s test with Bonferroni correction (p BC ). The 95% confidence intervals (CI 95%) for proportions were calculated using the Wilson score method 65 and for the difference between proportions using the Newcombe's method 57 . Proportions of negative results without bands and with weak bands were compared using the log-likelihood ratio goodness-of-fit test 66 . Change-corrected agreement of results of rPLD-ELISA and WB and of WB between serum and OF was assessed using the Gwet’s AC 1 coefficient of agreement 67 . The chance-corrected agreement was categorized as follows: AC 1 = 0.81-1.0 – very good agreement, 0.61–0.80 – good (substantial), 0.41–0.60 – moderate, 0.21–0.40 – fair, ≤ 0.20 – poor agreement 68 . Observed agreement (P o ) was also presented. All significance tests were two-sided and a significance level (α) was set at 0.05. Statistical analysis was carried out in TIBCO Statistica 13.3 (TIBCO Software Inc., Palo Alto, CA). Declarations Ethic statement This study did not involve experimental procedures on animals. Blood collection was performed as part of routine herd diagnostics for infectious diseases and was approved by the Local Ethics Committee in Warsaw (Approval No. WAW2/048/2021). Oral fluid was collected non-invasively and this procedure did not require the Ethics Committee approval according to Polish legal regulations (the Act on the Protection of Animals Used for Scientific or Educational Purposes of 15 January 2015) Declaration of Competing Interest None of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Funding sources The publication was (co)financed by Science development fund of the Warsaw University of Life Sciences – SGGW. Contributions Kinga Biernacka: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing – original draft; Marcin Mickiewicz: Conceptualization, Methodology, Investigation; Zofia Nowek: investigation; Agata Moroz-Fik: Methodology, Investigation; Magdalena Rzewuska: Methodology, Investigation; Resources; Writing – review & editing; Ewelina Kwiecień: Methodology, Investigation; Małgorzata Murawska: Methodology, Investigation; Tomasz Nalbert: Investigation; Olga Szaluś-Jordanow: Investigation; Lucjan Witkowski: Investigation; Iwona Markowska-Daniel: Conceptualization, Writing – review & editing; Emilia Bagnicka: Conceptualization, Writing – review & editing; Michał Czopowicz: Conceptualization, Methodology, Software, Data curation, Formal analysis, Visualization; Supervision, Resources, Validation, Writing – original draft; Jarosław Kaba: Conceptualization, Methodology, Formal analysis, Supervision, Resources; Writing – review & editing. Data Availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. References Yeruham, I., Elad, D., Van-Ham, M., Shpigel, N. Y. & Perl, S. Corynebacterium pseudotuberculosis infection in Israeli cattle: clinical and epidemiological studies. Vet. Rec. 140 , 423–427 (1997). https://doi.org/10.1136/vr.140.16.423. Connor, K. M., Fontaine, M. C., Rudge, K., Baird, G. J. & Donachie, W. Molecular genotyping of multinational ovine and caprine Corynebacterium pseudotuberculosis isolates using pulsed-field gel electrophoresis. Vet. Res. 38 , 613–623 (2007). https://doi.org/10.1051/vetres:2007013. Boysen, C., Davis, E. G., Beard, L. A., Lubbers, B. V. & Raghavan, R. K. Bayesian geostatistical analysis and ecoclimatic determinants of Corynebacterium pseudotuberculosis infection among horses. PLoS One 10 , e0140666 (2015). https://doi.org/10.1371/journal.pone.0140666. Mathewos, M. & Fesseha, H. Cytopathological and bacteriological studies on caseous lymphadenitis in cattle slaughtered at Bishoftu municipal abattoir, Ethiopia. Vet. Med. Sci. 8 , 1211–1218 (2022). https://doi.org/10.1002/vms3.744. Radwan, A. I., El-Magawry, S., Hawari, A., Al-Bekairi, S. I. & Rebleza, R. M. Corynebacterium pseudotuberculosis infection in camels ( Camelus dromedarius ) in Saudi Arabia. Trop. Anim. Health Prod. 21 , 229–230 (1989). https://doi.org/10.1007/BF02261094. Tejedor, M. T., Martin, J. L., Corbera, J. A., Shulz, U. & Gutierrez, C. Pseudotuberculosis in dromedary camels in the Canary Islands. Trop. Anim. Health Prod. 36 , 459–462 (2004). https://doi.org/10.1023/B:TROP.0000035012.63821.12. Terab, A. M. A. et al. Pathology, bacteriology and molecular studies on caseous lymphadenitis in Camelus dromedarius in the Emirate of Abu Dhabi, UAE, 2015–2020. PLoS One 16 , e0252893 (2021). https://doi.org/10.1371/journal.pone.0252893. Anderson, D. E., Rings, D. M. & Kowalski, J. Infection with Corynebacterium pseudotuberculosis in five alpacas. J. Am. Vet. Med. Assoc. 225 , 1743–1747 (2004). https://doi.org/10.2460/javma.2004.225.1743. Sprake, P. & Gold, J. R. Corynebacterium pseudotuberculosis liver abscess in a mature alpaca ( Lama pacos ). Can. Vet. J. 53 , 387–390 (2012). Sting, R. et al. Corynebacterium pseudotuberculosis infections in alpacas ( Vicugna pacos ). Animals 12 , 1612 (2022). https://doi.org/10.3390/ani12131612. Stauber, E., Armstrong, P., Chamberlain, K. & Gorgen, B. Caseous lymphadenitis in a white-tailed deer. J. Wildl. Dis. 9 , 56–57 (1973). https://doi.org/10.7589/0090-3558-9.1.56. Morales, N. et al. Corynebacterium pseudotuberculosis infection in Patagonian huemul ( Hippocamelus bisulcus ). J. Wildl. Dis. 53 , 621 (2017). https://doi.org/10.7589/2016-09-213. Varela-Castro, L. et al. Endemic caseous lymphadenitis in a wild Caprinae population. Vet. Rec. 180 , 405 (2017). https://doi.org/10.1136/vr.103925. Di Donato, A. et al. First report of caseous lymphadenitis by Corynebacterium pseudotuberculosis and pulmonary verminosis in a roe deer ( Capreolus capreolus Linnaeus, 1758) in Italy. Animals 14 , 566 (2024). https://doi.org/10.3390/ani14040566. Bastos, LB. Corynebacterium pseudotuberculosis : immunological responses in animal models and zoonotic potential. J. Clin. Cell. Immunol. S4 , 005 (2012). https://doi.org/10.4172/2155-9899.S4-005. Smith, M. C. & Sherman, D. M. Goat Medicine . 3rd edn. (Wiley Blackwell, 2023). Ashfaq, M. K. & Campbell, S. G. Experimentally induced caseous lymphadenitis in goats. Am. J. Vet. Res. 41 , 1789–1792 (1980). Williamson, L. H. Caseous lymphadenitis in small ruminants. Vet. Clin. North Am. Food Anim. Pract. 17 , 359–371 (2001). https://doi.org/10.1016/s0749-0720(15)30033-5. Baird, G. J. & Fontaine, M. C. Corynebacterium pseudotuberculosis and its role in ovine caseous lymphadenitis. J. Comp. Pathol. 137 , 179–210 (2007). https://doi.org/10.1016/j.jcpa.2007.07.002. Kaba, J. et al. Herd-level true seroprevalence of caseous lymphadenitis and paratuberculosis in the goat population of Poland. Prev. Vet. Med. 230 , 106278 (2024). https://doi.org/10.1016/j.prevetmed.2024.106278. Kaba, J. et al. Evaluation of the risk factors influencing the spread of caseous lymphadenitis in goat herds. Pol. J. Vet. Sci. 14 , (2011). https://doi.org/10.2478/v10181-011-0035-6. Dorella, F. A., Pacheco, L. G. C., Oliveira, S. C., Miyoshi, A. & Azevedo, V. Corynebacterium pseudotuberculosis : microbiology, biochemical properties, pathogenesis and molecular studies of virulence. Vet. Res. 37 , 201–218 (2006). https://doi.org/10.1051/vetres:2005056. Almeida, S. et al. Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi. BMC Vet. Res. 13 , 290 (2017). https://doi.org/10.1186/s12917-017-1210-5. Kaba, J. Development of an ELISA for the diagnosis of Corynebacterium pseudotuberculosis infections in goats. Vet. Microbiol. 78 , 155–163 (2001). https://doi.org/10.1016/S0378-1135(00)00284-4. Dorella, F. A. et al. Antigens of Corynebacterium pseudotuberculosis and prospects for vaccine development. Expert Rev. Vaccines 8 , 205–213 (2009). https://doi.org/10.1586/14760584.8.2.205. Galvão, C. E. et al. Identification of new Corynebacterium pseudotuberculosis antigens by immunoscreening of gene expression library. BMC Microbiol. 17 , 202 (2017). https://doi.org/10.1186/s12866-017-1110-7. Silva, M. T. D. O. et al. The combination of Corynebacterium pseudotuberculosis recombinant proteins rPLD, rCP01850 and rCP09720 for improved detection of caseous lymphadenitis in sheep by ELISA. J. Med. Microbiol. 68 , 1759–1765 (2019). https://doi.org/10.1099/jmm.0.001096. Pépin, M., Boisramé, A. & Marly, J. Corynebacterium pseudotuberculosis : biochemical properties, production of toxin and virulence of ovine and caprine strains. Ann. Rech. Vet. 20 , 111–115 (1989). De Pinho, BR. et al. A novel approach for an immunogen against Corynebacterium pseudotuberculosis infection: An Escherichia coli bacterin expressing phospholipase D. Microb. Pathog. 151 , 104746 (2021). https://doi.org/10.1016/j.micpath.2021.104746. Barral, T. D. et al. A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep. Microb. Biotechnol. 12 , 1313–1323 (2019). https://doi.org/10.1111/1751-7915.13454. Sting, R. et al. Clinical and serological investigations on caseous lymphadenitis in goat breeding herds in Baden-Wuerttemberg. Berl. Munch. Tierarztl. Wochenschr. 130 , 136–143 (2017). https://doi.org/10.2376/0005-9366-16024. Costa, L. et al. Utility assessment of an enzyme‐linked immunosorbent assay for detection of subclinical cases of caseous lymphadenitis in small ruminant flocks. Vet. Med. Sci. 6 , 796–803 (2020). https://doi.org/10.1002/vms3.297. Silva, J. W. et al. Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis. BMC Vet. Res. 10 , 965 (2014). https://doi.org/10.1186/s12917-014-0304-6. Shadnezhad, A., Naegeli, A. & Collin, M. CP40 from Corynebacterium pseudotuberculosis is an endo-β-N-acetylglucosaminidase. BMC Microbiol. 16 , 261 (2016). https://doi.org/10.1186/s12866-016-0884-3. Trost, E. et al. The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence. BMC Genomics 11 , 728 (2010). https://doi.org/10.1186/1471-2164-11-728. Santana-Jorge, K. T. O. et al. Putative virulence factors of Corynebacterium pseudotuberculosis FRC41: vaccine potential and protein expression. Microb. Cell Fact. 15 , 83 (2016). https://doi.org/10.1186/s12934-016-0479-6. Walburger, A. et al. Protein Kinase G from pathogenic Mycobacteria promotes survival within macrophages. Science 304 , 1800–1804 (2004). https://doi.org/10.1126/science.1099384. Warner, D. F. & Mizrahi, V. The survival kit of Mycobacterium tuberculosis . Nat. Med. 13 , 282–284 (2007). https://doi.org/10.1038/nm0307-282. Farias, A. P. F. D. et al. rSodC is a potential antigen to diagnose Corynebacterium pseudotuberculosis by enzyme-linked immunoassay. AMB Expr. 10 , 186 (2020). https://doi.org/10.1186/s13568-020-01125-0. Atkinson, J. et al. Guidelines for saliva nomenclature and collection. Ann. N. Y. Acad. Sci. 694 , XI–XII (1993). Chiappin, S., Antonelli, G., Gatti, R. & De Palo, E. F. Saliva specimen: A new laboratory tool for diagnostic and basic investigation. Clin. Chim. Acta 383 , 30–40 (2007). https://doi.org/10.1016/j.cca.2007.04.011 Mestecky, J. Saliva as a manifestation of the common mucosal immune system. Ann. N. Y. Acad. Sci. 694 , 184–194 (1993). https://doi.org/10.1111/j.1749-6632.1993.tb18352.x. Henao-Diaz, A., Giménez-Lirola, L. & Baum, D. H. Guidelines for oral fluid-based surveillance of viral pathogens in swine. Porc. Health Manag. 6 , 28 (2020). https://doi.org/10.1186/s40813-020-00168-w. Page, L. J., Lagunas-Acosta, J., Castellana, E. T. & Messmer, B. T. Accurate prediction of serum antibody levels from noninvasive saliva/nasal samples. Biotechniques. 74 , 131–136 (2023). https://doi.org/10.2144/btn-2022-0106. Mur, L. et al. Potential use of oral fluid samples for serological diagnosis of African swine fever. Vet. Microbiol. 165 , 135–139 (2013). https://doi.org/10.1016/j.vetmic.2012.12.034. Giménez-Lirola, L. G. et al. Detection of African swine fever virus antibodies in serum and oral fluid specimens using a recombinant protein 30 (p30) dual matrix indirect ELISA. PLoS One 11 , e0161230 (2016). https://doi.org/10.1371/journal.pone.0161230. Panyasing, Y., Thanawongnuwech, R., Ji, J., Giménez-Lirola, L. & Zimmerman, J. Detection of classical swine fever virus (CSFV) E2 and Erns antibody (IgG, IgA) in oral fluid specimens from inoculated (ALD strain) or vaccinated (LOM strain) pigs. Vet. Microbiol. 224 , 70–77 (2018). https://doi.org/10.1016/j.vetmic.2018.08.024. Kittawornrat, A. et al. Kinetics of the porcine reproductive and respiratory syndrome virus (PRRSV) humoral immune response in swine serum and oral fluids collected from individual boars. BMC Vet. Res. 9 , 61 (2013). https://doi.org/10.1186/1746-6148-9-61. Rotolo, M. L. et al. Detection of porcine reproductive and respiratory syndrome virus (PRRSV)-specific IgM-IgA in oral fluid samples reveals PRRSV infection in the presence of maternal antibody. Vet. Microbiol. 214 , 13–20 (2018). https://doi.org/10.1016/j.vetmic.2017.11.011. Henao-Diaz, A., Giménez-Lirola, L., Magtoto, R., Ji, J. & Zimmerman, J. Evaluation of three commercial porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody ELISAs using samples of known status. Res. Vet. Sci. 125 , 113–118 (2019). https://doi.org/10.1016/j.rvsc.2019.05.019. Prickett, J. R. et al. Prolonged detection of PCV2 and anti-PCV2 antibody in oral fluids following experimental inoculation: prolonged detection of PCV2 infection by oral fluid. Transbound. Emerg. Dis. 58 , 121–127 (2011). https://doi.org/10.1111/j.1865-1682.2010.01189.x. Campero, L. M. et al. Detection of antibodies to Toxoplasma gondii in oral fluid from pigs. Int. J. Parasitol. 50 , 349–355 (2020). https://doi.org/10.1016/j.ijpara.2019.11.002. Kauter, J. et al. Detection of Toxoplasma gondii -specific antibodies in pigs using an oral fluid-based commercial ELISA: advantages and limitations. Int. J. Parasitol. 53 , 523–530 (2023). https://doi.org/10.1016/j.ijpara.2022.11.003. Archetti, I. L., Amadori, M., Donn, A., Salt, J. & Lodetti, E. Detection of foot-and-mouth disease virus-infected cattle by assessment of antibody response in oropharyngeal fluids. J. Clin. Microbiol. 33 , 79–84 (1995). https://doi.org/10.1128/jcm.33.1.79-84.1995. Lazutka, J., Spakova, A., Sereika, V., Lelesius, R., Sasnauskas, K. & Petraityte-Burneikiene, R. Saliva as an alternative specimen for detection of Schmallenberg virus-specific antibodies in bovines. BMC Vet. Res. 11 , 237 (2015). https://doi.org/10.1186/s12917-015-0552-0. Ortega, J. et al. Evaluation of P22 ELISA for the detection of Mycobacterium bovis -specific antibody in the oral fluid of goats. Front. Vet. Sci. 8 , 674636 (2021). https://doi.org/10.3389/fvets.2021.674636. Hodgson, A. L., Bird, P. & Nisbet, I. T. Cloning, nucleotide sequence, and expression in Escherichia coli of the phospholipase D gene from Corynebacterium pseudotuberculosis . J. Bacteriol. 172 , 1256–1261 (1990). https://doi.org/10.1128/jb.172.3.1256-1261.1990. Ellis, J. A., Hawk, D. A., Holler, L. D., Mills, K. W. & Pratt, D. L. Differential antibody responses to Corynebacterium pseudotuberculosis in sheep with naturally acquired caseous lymphadenitis. J. Am. Vet. Med. Assoc. 196 , 1609–1613 (1990). ter Laak, E. A., Bosch, J., Bijl, G. C. & Schreuder, B. E. Double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot analysis used for control of caseous lymphadenitis in goats and sheep. Am. J. Vet. Res. 53 , 1125–1132 (1992). Hunt, A. J. et al. The testing of saliva samples for HIV-1 antibodies: reliability in a non-clinic setting. Sex. Transm. Infect. 69 , 29–30 (1993). https://doi.org/10.1136/sti.69.1.29. Langenhorst, R. et al. Development of a fluorescent microsphere immunoassay for detection of antibodies against porcine reproductive and respiratory syndrome virus using oral fluid samples as an alternative to serum-based assays. Clin. Vaccine Immunol. 19 , 180–189 (2011). https://doi.org/10.1128/CVI.05372-11. Nokes, D. J. et al. Has oral fluid the potential to replace serum for the evaluation of population immunity levels? A study of measles, rubella and hepatitis B in rural Ethiopia. Bull. World Health Organ. 79 , 588–595 (2001). Nagel‐Alne, G. E., Valle, P. S., Krontveit, R. & Sølverød, L. S. Caprine arthritis encephalitis and caseous lymphadenitis in goats: use of bulk tank milk ELISAs for herd‐level surveillance. Vet. Rec. 176 , 173–173 (2015). https://doi.org/10.1136/vr.102605. Silva, M. T. D. O. et al. NanH and PknG putative virulence factors as a recombinant subunit immunogen against Corynebacterium pseudotuberculosis infection in mice. Vaccine. 38 , 8099–8106 (2020). https://doi.org/10.1016/j.vaccine.2020.11.010. Altman, D. G., Machin, D., Bryant, T. N. & Gardner, M. J. Statistics with Confidence . 2nd edn. (BMJ Books, 2000). Zar, J. H. Biostatistical Analysis . 5th edn. (Pearson Prentice Hall, 2010). Gwet, K. L. Computing inter‐rater reliability and its variance in the presence of high agreement. Br. J. Math. Stat. Psychol. 61 , 29–48 (2008). https://doi.org/10.1348/000711006X126600. Altman, D. G. (1990). Practical statistics for medical research (1st ed.). Chapman and Hall/CRC. https://doi.org/10.1201/9780429258589 Additional Declarations No competing interests reported. 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Sciences-SGGW","correspondingAuthor":false,"prefix":"","firstName":"Jarosław","middleName":"","lastName":"Kaba","suffix":""}],"badges":[],"createdAt":"2025-02-19 15:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6065536/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6065536/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-06957-z","type":"published","date":"2025-07-02T15:58:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78668287,"identity":"07e5fcb8-f2ae-44e7-a07d-deace97cece2","added_by":"auto","created_at":"2025-03-17 11:52:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":828858,"visible":true,"origin":"","legend":"\u003cp\u003eCoomassie blue stained SDS PAGE and immunoblots showing the protein distribution of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e strain that was used as a whole-bacterium antigen in this study.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6065536/v1/785be7d7b856a3dd67f06775.png"},{"id":78668290,"identity":"723c06fc-1a30-4888-8566-4a018eece5f3","added_by":"auto","created_at":"2025-03-17 11:52:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":197552,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of various combinations of antibodies to phospholipase D (PLD), CP40, and protein kinase G (PknG) in serum and oral fluid (OF) of 96 goats affected by caseous lymphadenitis. The number of samples is shown above the bars.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6065536/v1/4a92696f9971e84c6d35e24c.png"},{"id":78669260,"identity":"f42fec01-baea-439b-97e4-8b2408395bc6","added_by":"auto","created_at":"2025-03-17 12:08:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":285444,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic sensitivity of various combinations of three antigens of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e(phospholipase D (PLD), CP40, and protein kinase G (PknG)) compared to PLD alone in serum (a) and oral fluid (b). Asterisk (*) indicates a combination significantly different from the others.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6065536/v1/c1ad131bfa0791a74d1c1af4.png"},{"id":78668294,"identity":"2923400b-90d8-4a45-88b7-2e4068d45b13","added_by":"auto","created_at":"2025-03-17 11:52:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":190624,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the western-blot on serum and oral fluid samples classified as negative. Numbers of samples without any band (blue) and with weak bands (grey) are indicated on bars. Asterisk (*) indicates situations in which one of the aforementioned classes significantly predominated.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6065536/v1/ec511d1f4d3aed543366f3fd.png"},{"id":86180582,"identity":"62f6dca0-daf2-4916-acfa-ea1dd371c11d","added_by":"auto","created_at":"2025-07-07 16:22:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2694521,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6065536/v1/3710b551-9221-4bb5-93c1-24e6f37ef928.pdf"},{"id":78668965,"identity":"141a642c-c6af-4202-99b7-77d3bc8f5465","added_by":"auto","created_at":"2025-03-17 12:00:33","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":80483,"visible":true,"origin":"","legend":"","description":"","filename":"wholeblotusedforFig.1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-6065536/v1/527aa676edc048e6349828af.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oral fluid as a material for serological diagnostics of caseous lymphadenitis in goats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e (\u003cem\u003eC. pseudotuberculosis\u003c/em\u003e) is a Gram-positive facultatively anaerobic coccobacillus. It causes several diseases, including caseous lymphadenitis (CLA) in sheep and goats, and ulcerative lymphangitis in horses and cattle\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e also affects camels\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, alpacas\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and wild ruminants\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. It has some zoonotic potential, although few cases in people have been reported\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. CLA occurs worldwide and is economically significant in small ruminants\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The disease follows a chronic course with a long incubation period and presumably a lifelong carrier state following recovery, although this issue has not been ultimately settled. Several months after the infection, clinical signs, including mainly abscesses in the superficial lymph nodes, become apparent \u0026ndash; such form of the disease is called the external form of CLA. Some animals develop abscesses in the internal lymph nodes or internal organs, referred to as the internal form of CLA\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Over the last decades, CLA has become a widespread disease in goat herds in Poland\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Its seroprevalence was first determined in 1996, and it was shown to have increased from 13% to over 60% in 2002\u003csup\u003e21\u003c/sup\u003e. A recent investigation has shown that the herd-level seroprevalence increased to approximately 70% \u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIsolation of \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e from abscesses and its identification using microbiological or molecular methods is regarded as the gold standard of CLA diagnostics\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, due to a long incubation period (2\u0026ndash;6 months), unknown, but presumably long duration of carrier state, and internal form of the disease, serological tests play an important role, especially, in the evaluation of the status of herds as well as of individual animals before introduction into a herd\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Routine serological diagnostics is mainly based on immunoenzymatic assays (ELISA) however, western-blot (WB) is considered the serological reference standard\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Several antigens of \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e with potential diagnostic applicability have been described\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Phospholipase D (PLD), an approximately 30 kDa protein, is the main exotoxin and the major virulence factor favouring the infection process \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. PLD is characterized by both very high immunogenicity and antigenicity which makes it a suitable component of vaccines \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and antigen of serological tests \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. A recombinant form of PLD (rPLD) has been used for developing commercial ELISAs (rPLD-ELISA) and they are commonly used for serological diagnostics of CLA\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Other antigenic enzymes are a CP40, an approximately 40 kDa protein, secreted endo-b-N-acetylglucosaminidase\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and a serine/threonine protein kinase G (PknG)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, an approximately 70 kDa protein homologous to an important virulence factor of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In some preliminary studies based on in-house ELISAs, CP40 has shown high diagnostic specificity (dSp) of 97% but very low diagnostic sensitivity (dSe) of only 43% \u003csup\u003e30\u003c/sup\u003e while PknG turned out to be both highly sensitive and specific (94% and 97%, respectively) \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOral fluid (OF) is a mixture of saliva and serum transudate that enters the mouth by crossing the buccal and gingival mucosa from the capillaries\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Therefore, OF contains antibodies from the systemic immune system (from the passage of serum antibodies) as well as locally produced antibodies from the secretory immune system in the salivary glands \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. OF also contains feed particles, cell detritus, tracheal-nasal secretions, gastrointestinal reflux, and other particles from the environment currently being chewed by an animal. In ruminants, contrary to other species, the composition of OF is also vastly affected by rumination, which involves the regurgitation of material from the reticulo-rumen to the buccal cavity, where solid material is re-masticated and re-insalivated before being swallowed. Serological tests based on OF are becoming increasingly popular in non-ruminating animal species, especially in pigs. Such tests are used for diagnosing classical swine fever\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, porcine reproductive and respiratory syndrome\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, porcine circovirus type 2 51 (Prickett et al., 2011) and \u003cem\u003eToxoplasma gondii\u003c/em\u003e infections\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Although, theoretically, rumination can hinder diagnostic application of OF, it has so far been shown to be a suitable material for serological diagnostics of foot-and-mouth disease\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e and Schmallenberg virus infection\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e in ruminants. Additionally, an in-house OF-based ELISA for \u003cem\u003eM. bovis\u003c/em\u003e infection in goats has been developed\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. OF offers several advantages over traditional diagnostic specimens such as blood or tissue. The OF collection procedure is minimally invasive and welfare-friendly, with much less discomfort or stress for the animals than blood collection\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Moreover, it can be performed by non-veterinarian personnel with minimal training and does not require the consent of the Ethics Committee when carried out within the frame of scientific studies. Contrary to another type of non-invasive sample \u0026ndash; milk \u0026ndash; OF can also be collected from males and non-lactating females.\u003c/p\u003e \u003cp\u003eOur study aimed to evaluate the potential usefulness of OF as a material for serological identification of \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e-infected goats based on the detection of antibodies to PLD, CP40, and PknG using WB. Additionally, the dSe of rPLD-ELISA and WB performed using serum samples was estimated.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Antigenic structure of the reference strain\u003c/h2\u003e\n \u003cp\u003eMultiple bands were visible in the gel, with the most prominent bands located at heights corresponding to approximately 30 kDa (two bands), 35 kDa (one band), 40 kDa (one band), 70 kDa (multiple bands) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Based on previous findings, the locations of the bands corresponded to: LexA protein (28 kDa), PLD (30 kDa or 31.4 kDa) TrxR (35 kDa), CP40 (40 kDa), and PknG (70 kDa)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The band that corresponds to CP40 was the most visible.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Diagnostic sensitivity of rPLD-ELISA\u003c/h2\u003e\n \u003cp\u003eIn the rPLD-ELISA, 67 serum samples (69.8%) were positive, 10 (10.4%) \u0026ndash; inconclusive, and 19 (19.8%) \u0026ndash; negative. S/P% of the aforementioned samples ranged from 50.1\u0026ndash;157.5% (median of 88.9%), from 40.1\u0026ndash;49.0% (median of 46.0%), and from 0.6\u0026ndash;39.9% (median of 34.2%), respectively. The dSe of rPLD-ELISA was 69.8% (CI 95%: 60.0%, 78.1%) if only serum samples with S/P% \u0026gt;50% were considered positive and 80.2% (CI 95%: 71.1%, 87.0%) if all serum samples with S/P% \u0026gt;40% were considered positive.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Agreement between rPLD-ELISA and WB on serum samples\u003c/h2\u003e\n \u003cp\u003eIn WB, antibodies to any of the three antigens were detected in the serum of 94 / 96 goats (dSe\u0026thinsp;=\u0026thinsp;97.9%; CI 95%: 92.7%, 99.4%). The agreement between rPLD-ELISA and WB was good: AC\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;61.4% (CI 95%: 49.1%, 73.8%; P\u003csub\u003eo\u003c/sub\u003e = 71.9%) if only serum samples with S/P% \u0026gt;50% in ELISA were considered positive and AC\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;75.4% (CI 95%: 65.5%, 85.3%; P\u003csub\u003eo\u003c/sub\u003e = 80.2%) if all serum samples with S/P% \u0026gt;40% in ELISA were considered positive.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Comparison of diagnostic sensitivity of WB on serum and OF samples\u003c/h2\u003e\n \u003cp\u003eAntibodies to any of the three antigens were detected in the OF samples of 86 / 96 goats (dSe\u0026thinsp;=\u0026thinsp;89.6%; CI 95%: 81.9%, 94.2%). The proportion of positive OF samples was significantly lower than that of positive sera (by 8.3%; CI 95%: 2.3%, 15.8%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). Antibodies to PLD were present in a similar proportion of serum and OF samples (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.302). Antibodies to CP40 and PknG were detected in a significantly lower proportion of OF than serum samples (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 in both cases) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResults of the western blot on paired serum and oral fluid (OF) samples of 96 goats affected by caseous lymphadenitis\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAntigen\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSerum\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eOral fluid\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMcNemar\u0026rsquo;s test\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eDifference between proportions in serum and OF (CI 95%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of positive results\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProportion (CI 95%) [%]\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNumber of positive results\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProportion (CI 95%) [%]\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePLD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92.7 (85.7, 96.4)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e87.5 (79.4, 92.7)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.302\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.2 (-3.2, 13.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCP40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85.4 (77.0, 91.1)\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.4 (5.8, 18.1)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75.0 (63.6, 82.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePknG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.0 (66.6, 83.5)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.3 (23.8, 42.2)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.8 (31.2, 54.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ea,b,c\u003c/sup\u003e proportions denoted by the same letter do not differ significantly\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ed\u003c/sup\u003e Cochrane Q test followed by McNemar\u0026rsquo;s test with Bonferroni correction for multiple comparisons \u0026ndash; p-values reported in the text\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eIn the serum samples, antibodies to PLD were detected significantly more often than antibodies to PknG (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eBC\u003c/em\u003e\u003c/sub\u003e = 0.004), whereas the differences between the proportion of samples with antibodies to PLD and CP40 or between the proportion of samples with antibodies to CP40 and PknG were not significant (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eBC\u003c/em\u003e\u003c/sub\u003e = 0.438 and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eBC\u003c/em\u003e\u003c/sub\u003e = 0.372, respectively). In the OF samples, antibodies to PLD were detected significantly more often than antibodies to PknG (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eBC\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.001) or CP40 (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eBC\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.001), and antibodies to PknG were detected significantly more often than antibodies to CP40 (\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eBC\u003c/em\u003e\u003c/sub\u003e \u0026lt; 0.001). The latter were detected in only 10% of OF samples (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eMost serum samples were positive for antibodies to all three antigens (63.5%), followed by sera positive for antibodies to PLD and CP40 (16.7%). Only 2 / 94 seropositive goats (2.1%) did not have antibodies to PLD (instead, they had antibodies to CP40) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Over half of OF samples were only positive for antibodies to PLD (56.3%), followed by sera positive for antibodies to PLD and PknG (20.8%), and sera positive for antibodies to all three antigens (9.4%). Only 2 / 86 goats with positive results of OF (2.3%) did not have antibodies to PLD (instead they had antibodies to PknG) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eCombination of PLD with any of the two other antigens did not significantly increase the dSe of WB either in serum (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) or OF (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). Combination of CP40 and PknG (without PLD) performed significantly worse than any other combination in OF (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not in serum. The difference between dSe of a combination of PLD and CP40 compared to dSe of PLD alone (98% vs. 93%, respectively) was closest to statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.074).\u003c/p\u003e\n \u003cp\u003eThe agreement between WB performed using serum and OF was high (P\u003csub\u003eo\u003c/sub\u003e = 88.5%; AC\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;86.6%, CI 95%: 79.2%, 94.1%). The high agreement resulted only from similar results with respect to antibodies to PLD since the agreement between serum and OF concerning PknG was poor, and it was even significantly negative in terms of CP40 (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNumber of serum and oral fluid (OF) samples with positive result of the western-blot (WB) for antibodies to certain antigens of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e: phospholipase D (PLD), CP40, and protein kinase G (PknG)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eResult of WB on biological material\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eNumber of samples in which \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e antigens were detected\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOral fluid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePLD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCP40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePknG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eObserved agreement between serum and OF [%]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eChance-corrected agreement between serum and OF (Gwet\u0026rsquo;s AC\u003csub\u003e1\u003c/sub\u003e \u0026amp; CI 95%) [%]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81.0\u003c/p\u003e\n \u003cp\u003e(72.1, 89.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-53.9\u003c/p\u003e\n \u003cp\u003e(-70.7, -37.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.5\u003c/p\u003e\n \u003cp\u003e(-23.3, 16.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eIn terms of samples classified as negative, distribution of samples without any band and with weak bands was balanced, except for PLD in serum samples where samples with weak bands significantly predominated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.047) and CP40 in OF samples where samples without any band significantly predominated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eOur study shows for the first time that the OF of goats contains antibodies to \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e and can serve as a material for serological diagnostics of CLA in this species. Antibodies to PLD appear to be the only ones of potential clinical usefulness. Surprisingly, antibodies to CP40 and PknG are detectable in OF of a low proportion of CLA-affected goats.\u003c/p\u003e \u003cp\u003eThe main difficulty encountered when investigating the diagnostic accuracy of tests for CLA is the determination of the true health status of an animal. This problem results from an ambiguous definition of an animal diseased with CLA. If a CLA-diseased animal is defined as the one with abscess(es) confirmed bacteriologically to be of \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e origin, serological tests turn out to be perfectly sensitive but rather unspecific like in the studies of Ellis et al. and Costa et al. \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. This is because abscesses usually develop after several months of infection, which is long enough for an animal to mount a humoral immune response, detectable in serological tests\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, in many animals infected with \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e, apparent abscesses develop much later, or even never, which results in positive results of serology in many, by definition, \u0026ldquo;healthy\u0026rdquo; animals. In terms of evaluating the diagnostic accuracy of the serological tests, classifying only animals with abscesses as CLA-diseased animals does not seem reasonable because bacteriology, not serology, is the diagnostic test of choice in such animals. Another approach to determining true health status is to define a CLA-diseased animal as the one with antibodies to \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e. In this situation, the most acknowledged serological test is chosen as a gold standard \u0026ndash; it is usually WB as it detects antibodies to the broadest array of pathogen\u0026rsquo;s antigens. Animals positive in this test are considered as CLA-diseased. This approach has been commonly applied to evaluate the accuracy of serological tests for CLA\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Obviously, no serological gold standard is able to overcome the problem of delayed seroconversion. Furthermore, the assumption is that persistence of antibodies parallels the lifelong infection. This is highly likely to be true as \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e can remain viable inside phagocytes and establish long-lasting intracellular infection\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, however, we do not know any study directly evidencing the lifelong nature of \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e infection. In our study, we simply assumed that all adult goats born and raised or kept in the herd affected by clinical form of CLA for several years must have become infected with \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e and their infection is lifelong. It is a strict and conservative approach and it could have obviously underestimated the dSe of ELISA, especially when we take note of the fact that our estimate of dSe of ELISA is quite low (70%-80%) compared to many previous studies\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, if we consider WB performed with serum as the gold standard, an increase in the estimated dSe of ELISA is negligible (from to 69.8\u0026ndash;71.3% when only serum samples with S/P% \u0026gt;50% are considered positive and from 80.2\u0026ndash;80.9% when all serum samples with S/P% \u0026gt;40% are considered positive). Therefore, we suspect that there may be some variability in the diagnostic performance of rPLD-ELISA caused by unknown animal- or pathogen-related factors. Our study does not allow for drawing any conclusions regarding dSp as only diseased animals have been enrolled.\u003c/p\u003e \u003cp\u003eVirtually 90% of OF samples contained antibodies to PLD, approximately 30% to PknG, and only 10% to CP40. It means that in an eight times larger number of samples, antibodies to CP40 were detectable in serum than in OF, and a two times larger number to PknG. It can be explained by the fact that in various mammalian species, antibody levels in serum are significantly higher for many pathogens than in OF. Thus, the concentration of antibodies is reflected in their detectability in the sample\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCP40 has a high antigenic potential, stimulating the production of specific antibodies detectable in the serum of goats and sheep \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Also, in our study, 85% of goats had antibodies to CP40 in serum but only 10% in OF. Further, 28 goats had a weak band in WB, indicating that some low concentrations of the antibodies could be present in OF. The existence of some physical or physiological barrier for antibodies to CP40 and PknG between systemic circulation and OF is one of the explanations, yet highly unlikely. More probable is that antibodies to PLD are not only of IgG class but also of IgA class and are produced locally at higher concentrations while antibodies to CP40 and PknG are mostly of systemic origin and reach OF in low amounts. If that is correct, WB shows true differences in antibody concentrations between serum and OF. However, another explanation may be a technical shortcoming of our WB protocol. Maybe antibodies to PLD are mostly of IgG class and systemic origin, and they are efficiently detected by rabbit anti-goat IgG secondary antibodies, which we used in our study. At the same time, antibodies to CP40 and PknG are mostly local IgA secretory antibodies. In this situation, using anti-goat IgA secondary antibodies together with anti-goat IgG secondary antibodies could increase the detectability of anti-CP40 and anti-PknG antibodies. Both scenarios are feasible as, according to the datasheet, the rabbit anti-goat IgG (H/L) antibody recognizes the heavy and light chains of goat IgG but may react with the light chains of other goat immunoglobulins. Determination of the class of antibodies in OF could clarify this problem and this aspect warrants further investigation.\u003c/p\u003e \u003cp\u003eShowing that specific antibodies are detectable in WB is the first step on the long pathway to develop ELISA for CLA with OF as diagnostic material. An indirect ELISA based on the whole-bacterium antigen used in our study was developed over 20 years ago by Kaba et al. and has performed very well on goat sera ever since \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Results of our study indicate that using more targeted antigen e.g. rPLD may be worth considering as antibodies to PLD are most abundant or at least most effectively detected in OF. Developing an efficient serological diagnostic test based on non-invasively collected material would be of value. Stress-reducing procedures become increasingly popular in modern veterinary medicine and animal disease control programs based on such procedures have been shown to play a useful role\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The method of OF collection employed in our study differs from those described previously. For pigs, a rope hung in the pen is recommended because pigs are eager to interact with the rope and chew it. In ruminants, more active OF collection using cotton swabs\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e or commercial plastic shafts\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e (Copan Flocked Swabs) has been performed. We used a sponge held in metal forceps. This was an extremely inexpensive and efficient method as by squeezing the sponge in a syringe, we obtained between 1 and 3 ml of OF without centrifugation. This amount was enough to perform several rounds of testing.\u003c/p\u003e \u003cp\u003eLast but not least, we encountered some problems regarding the identification of PknG owing to inconsistent information regarding its molecular mass which can be found in the literature. According to Barral et al. \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, PknG weighs 69 kDa, although, in fact, the corresponding electrophoresis band lay somewhere between 55 and 70 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e in Barral et al.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e). On the other hand, in the studies of Santana-Jorge et al. \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and Silva et al. \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e PknG was claimed to weigh 83 kDa, while the band on the electrophoresis gel presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e of Silva et al. \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e was located slightly above 70 kDa. We decided to stick to the molecular mass indicated on the pictures of electrophoresis gels and mentioned by Barral et al. \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConcluding, OF appears to be a promising material for non-invasive and non-stressing serological testing of goats for CLA. However, classes of anti-\u003cem\u003eC. pseudotuberculosis\u003c/em\u003e immunoglobulins predominating in OF should be determined first, to allow for the most optimal selection of secondary antibodies to be used in the OF-targeted serological assay.\u003c/p\u003e"},{"header":"4. Material and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Animals and sample collection\u003c/h2\u003e \u003cp\u003eThe study enrolled 96 adult (\u0026gt;\u0026thinsp;1-year-old) Saanen goats, 92 females and 4 males, from the dairy herd affected by CLA. The goats had been under strict regular observation since August 2022, and serum and OF samples were collected in November 2022. The herd was located in north-eastern Poland. The herd had been bought by the current owner and moved from south-eastern Poland in 2019; however, only one year earlier, the goats had been purchased from Austria by the previous owner. At the moment of the study, the goats were kept in two spacious and high concrete-wooden barns and were irregularly grazed on a small backyard pasture during summer months. They produced milk mainly for on-farm cheese production. In 2018, a few months after moving from Austria, CLA had been diagnosed first in a male goat with a superficial abscess. In the following years, the disease gradually spread over the entire herd. It was repeatedly confirmed by positive results of bacteriological culture of swabs collected from abscesses of the superficial lymph nodes. The herd was tested for caprine arthritis-encephalitis (CAE) using an indirect ELISA (ID Screen MVV-CAEV Indirect Screening test, IDvet Innovative Diagnostics, Grabels, France) twice in 2018 and in 2022 and all goats tested negative. Of 96 goats enrolled in the study, 26 (27.1%) had had at least one abscess confirmed by bacteriological culture caused by \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e between August and November 2022. According to the owner\u0026rsquo;s account, the superficial abscesses had been regularly observed in the goats from the very moment of their purchase. Given that all goats enrolled in this study had been either born and raised in this herd or kept in the herd for at least 3 years (since the purchase of the herd by the current owner in 2019), we classified all of them as infected with \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e and affected by CLA (either clinically or subclinically). Therefore, the proportion of goats whose serum or OF samples tested positive in the test (rPLD-ELISA with serum, WB with serum, or WB with OF) was considered an estimate of dSe of the test.\u003c/p\u003e \u003cp\u003ePaired samples of blood and OF were collected from each goat at the same moment. The blood was collected from the jugular vein into 10-ml dry tubes (BD Vacutainer, BP-Plymouth, UK) and transported to the laboratory. After 24 h storage in the refrigerator (4\u0026ndash;8\u0026deg;C), blood was centrifuged at 3000 rpm for 10 minutes, serum was harvested to 1.5-ml Eppendorf tubes and stored at -20\u0026deg;C until testing using rPLD-ELISA and WB. The OF was collected with a 5\u0026times;1\u0026times;1 cm fragment of polypropylene sponge held in stainless steel forceps. It was inserted into the oral cavity and kept there for approximately 30 sec. so that an animal could thoroughly chew it up. Then, the sponge was placed in a 20-ml clogged syringe to prevent fluid leakage, and in such a form, the material was transported to the laboratory. There, OF was squeezed out of sponges into 1.5-ml Eppendorf tubes. OF samples were stored at -20\u0026deg;C until testing with WB. OF samples were not centrifuged. Forceps were washed in the mild detergent (soap) between animals.\u003c/p\u003e \u003cp\u003e We confirmed that all experiments in this study were performed in accordance with the relevant guidelines and regulations. All the procedures of the study followed the ARRIVE guidelines. Blood collection was performed as part of routine herd diagnostics for infectious diseases and was approved by the Local Ethics Committee in Warsaw (Approval No. WAW2/048/2021). OF was collected non-invasively and this procedure did not require the Ethics Committee approval according to Polish legal regulations (the Act on the Protection of Animals Used for Scientific or Educational Purposes of 15 January 2015).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Commercial rPLD-ELISA\u003c/h2\u003e \u003cp\u003eSerum samples were tested using a commercial indirect ELISA based on rPLD (ID Screen CLA, IDvet Innovative Diagnostics, Grabels, France) according to the manufacturer\u0026rsquo;s manual. Briefly, serum samples were diluted 1:20 in the dilution buffer, and reading was performed at 450 nm using the Epoch Microplate Spectrophotometer (BioTek, USA). The optical density (OD) of samples was corrected by the OD of the manufacturer\u0026rsquo;s positive and negative control serum, and the sample-to-positive control ratio (S/P%) was interpreted as follows: S/P% \u0026le;40% \u0026ndash; negative result, S/P% \u0026gt;40\u0026ndash;50% \u0026ndash; inconclusive result, and S/P% \u0026gt;50% \u0026ndash; positive result.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Western blot\u003c/h2\u003e \u003cp\u003eThe positive control serum was obtained from an adult female goat from this herd several months before the study. The goat had multiple abscesses in the superficial lymph nodes, from which \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e was isolated in a bacteriological culture. The goat was also positive in the commercial rPLD-ELISA. The negative control serum and OF were obtained from an apparently healthy adult goat from another herd with no history of CLA and negative rPLD-ELISA result.\u003c/p\u003e \u003cp\u003eThe whole-bacterium antigen for WB was prepared from the \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e isolate obtained from an abscess of a goat from the same herd according to the method described by Kaba et al. \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e with minor modifications. Briefly, the \u003cem\u003eC. pseudotuberculosis\u003c/em\u003e isolate was cultivated on Columbia blood agar (Graso Biotech, Poland) for 24 h at 37\u0026deg;C in the aerobic atmosphere, then 5 ml of Brain Heart Infusion (BHI) broth (Graso Biotech, Poland) was inoculated with a few colonies, and incubated in the same conditions with agitation. This culture was transferred into a 50 ml BHI broth, and incubation was continued under the same conditions for 24 h. Next, 2 liters of BHI broth were inoculated with the 50 ml culture and incubated in the same conditions for 16 h. Bacterial cells were harvested by centrifugation (7000 rpm, 10 min) and then washed three times in PBS (phosphate buffered saline; 150 mM NaCl, 1.5 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 9 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e \u0026times; 12H\u003csub\u003e2\u003c/sub\u003eO, 2.5 mM KCl, pH 7.2) with 0.2% Tween-80 (Merck, Darmstadt, Germany). The cell pellet was resuspended in 12 ml of the antigen preparation buffer (0.5 M Tris pH 6.8; 5.2% SDS; 8.7% 2-mercaptoethanol), boiled (100\u0026deg;C) for 10 min, and then centrifuged (at 10,000 rpm for 15 min). The collected supernatant represented the ELISA solid phase antigen and was stored in 500 ml aliquots at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe antigen was electrophoresed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to standard procedures. Briefly, antigens were heated at 98\u0026deg;C for 5 min in sample buffer containing 0.0625 M Tris (pH 6.8), 2.0% (wt/vol) sodium dodecyl sulphate, 25.0% Glycerol, 5.0% (vol/vol) b-mercaptoethanol, and 0.00125% bromophenol blue. Antigens were separated on polyacrylamide gels (stacking gel: 4%, running gel: 4%). The protein standard Precision Plus Protein Standards Dual Color (Bio Rad Laboratories, USA) was used. Electrophoresis was performed under a voltage of 60 V for stacking gel and 120 V for running gel. SDS gels were stained with Coomassie blue (2.5% brilliant blue in 50% methanol, 10% acetic acid) prior to analysis.\u003c/p\u003e \u003cp\u003eSubsequently, the electrophoresed antigen was transferred from the gel to the supported nitrocellulose membrane (Bio-Rad, USA) in the TransBlot Turbo Transfer system (Bio-Rad, USA). Membranes were washed in TBST (a mixture of tris-buffered saline and Polysorbate 20) for 10 min. and incubated in the Everyblot Blocking Buffer (Bio-Rad, USA) for 20 min. Then, the membranes were washed 3 times for 10 min. An estimation of the suitable dilutions of the tested samples was performed. Finally, serum samples were diluted 1:100 in the blocking buffer. OF samples were centrifuged at 2600 rpm for 5 min. to separate the liquid from the solid particles and then diluted 1:5 in the blocking buffer. One-hour incubation of serum and OF samples on membranes was performed in the Mini Protean Multi II (Bio-Rad, USA). Then, the membranes were washed 3 times for 10 min. in TBST. In the following 1-hour incubation process, secondary antibodies (Rabbit anti-goat IgG H/L conjugated to alkaline phosphatase, Bio-Rad, USA) were used at a dilution of 1:4500 for membranes incubated with sera and 1:4000 for membranes incubated with OF. After the incubation, membranes were washed twice for 5 min. in TBST, once for 5 min. in TBS, and twice for 5 min. in alkaline phosphatase color development buffer (Bio-Rad, USA). The results were visualized using Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad, USA). The Precision Plus Protein\u0026trade; Standards Dual Color (Bio-Rad, USA) ladder was used to estimate protein sizes.\u003c/p\u003e \u003cp\u003eThe results of WB were visually reviewed by a single investigator (KB) and classified as i) any band absent, ii) weak band present, and iii) strong band present, separately at the level of each of 3 antigens: PLD \u0026ndash; approximately 30 kDa, CP40 \u0026ndash; approximately 40 kDa, PknG \u0026ndash; approximately 70 kDa. When a band at the level of a relevant antigen was absent or weak the result was classified as negative; only a strong band was considered a positive result. The serum or OF sample was considered positive in WB if a strong band at the level of at least 1 of the aforementioned antigens was present.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Statistical analysis\u003c/h2\u003e \u003cp\u003eCategorical variables were presented as counts (n) and proportions (%), and compared between paired groups using the McNemar\u0026rsquo;s test (2 groups). In the case of comparing\u0026thinsp;\u0026gt;\u0026thinsp;2 groups, the Cochrane Q test was carried out as an omnibus test. If significant, post-hoc analyses were performed using the McNemar\u0026rsquo;s test with Bonferroni correction (p\u003csub\u003eBC\u003c/sub\u003e). The 95% confidence intervals (CI 95%) for proportions were calculated using the Wilson score method\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e and for the difference between proportions using the Newcombe's method \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Proportions of negative results without bands and with weak bands were compared using the log-likelihood ratio goodness-of-fit test \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Change-corrected agreement of results of rPLD-ELISA and WB and of WB between serum and OF was assessed using the Gwet\u0026rsquo;s AC\u003csub\u003e1\u003c/sub\u003e coefficient of agreement \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. The chance-corrected agreement was categorized as follows: AC\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.81-1.0 \u0026ndash; very good agreement, 0.61\u0026ndash;0.80 \u0026ndash; good (substantial), 0.41\u0026ndash;0.60 \u0026ndash; moderate, 0.21\u0026ndash;0.40 \u0026ndash; fair, \u0026le;\u0026thinsp;0.20 \u0026ndash; poor agreement \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Observed agreement (P\u003csub\u003eo\u003c/sub\u003e) was also presented. All significance tests were two-sided and a significance level (α) was set at 0.05. Statistical analysis was carried out in TIBCO Statistica 13.3 (TIBCO Software Inc., Palo Alto, CA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthic statement\u003c/h2\u003e\n\u003cp\u003eThis study did not involve experimental procedures on animals. Blood collection was performed as part of routine herd diagnostics for infectious diseases and was approved by the Local Ethics Committee in Warsaw (Approval No. WAW2/048/2021). Oral fluid was collected non-invasively and this procedure did not require the Ethics Committee approval according to Polish legal regulations (the Act on the Protection of Animals Used for Scientific or Educational Purposes of 15 January 2015)\u003c/p\u003e\n\u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eNone of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.\u003c/p\u003e\u003ch2\u003eFunding sources\u003c/h2\u003e \u003cp\u003eThe publication was (co)financed by Science development fund of the Warsaw University of Life Sciences \u0026ndash; SGGW.\u003c/p\u003e\n\u003ch2\u003eContributions \u003c/h2\u003e\n\u003cp\u003eKinga Biernacka: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing \u0026ndash; original draft; Marcin Mickiewicz: Conceptualization, Methodology, Investigation; Zofia Nowek: investigation; Agata Moroz-Fik: Methodology, Investigation; Magdalena Rzewuska: Methodology, Investigation; Resources; Writing \u0026ndash; review \u0026amp; editing; Ewelina Kwiecień: Methodology, Investigation; Małgorzata Murawska: Methodology, Investigation; Tomasz Nalbert: Investigation; Olga Szaluś-Jordanow: Investigation; Lucjan Witkowski: Investigation; Iwona Markowska-Daniel: Conceptualization, Writing \u0026ndash; review \u0026amp; editing; Emilia Bagnicka: Conceptualization, Writing \u0026ndash; review \u0026amp; editing; Michał Czopowicz: Conceptualization, Methodology, Software, Data curation, Formal analysis, Visualization; Supervision, Resources, Validation, Writing \u0026ndash; original draft; Jarosław Kaba: Conceptualization, Methodology, Formal analysis, Supervision, Resources; Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYeruham, I., Elad, D., Van-Ham, M., Shpigel, N. Y. \u0026amp; Perl, S. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e infection in Israeli cattle: clinical and epidemiological studies. \u003cem\u003eVet. Rec.\u003c/em\u003e\u003cstrong\u003e140\u003c/strong\u003e, 423\u0026ndash;427 (1997). https://doi.org/10.1136/vr.140.16.423.\u003c/li\u003e\n\u003cli\u003eConnor, K. M., Fontaine, M. C., Rudge, K., Baird, G. J. \u0026amp; Donachie, W. Molecular genotyping of multinational ovine and caprine \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e isolates using pulsed-field gel electrophoresis. \u003cem\u003eVet. Res.\u003c/em\u003e\u003cstrong\u003e38\u003c/strong\u003e, 613\u0026ndash;623 (2007). https://doi.org/10.1051/vetres:2007013.\u003c/li\u003e\n\u003cli\u003eBoysen, C., Davis, E. G., Beard, L. A., Lubbers, B. V. \u0026amp; Raghavan, R. K. Bayesian geostatistical analysis and ecoclimatic determinants of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e infection among horses. \u003cem\u003ePLoS One\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, e0140666 (2015). https://doi.org/10.1371/journal.pone.0140666.\u003c/li\u003e\n\u003cli\u003eMathewos, M. \u0026amp; Fesseha, H. Cytopathological and bacteriological studies on caseous lymphadenitis in cattle slaughtered at Bishoftu municipal abattoir, Ethiopia. \u003cem\u003eVet. Med. Sci.\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 1211\u0026ndash;1218 (2022). https://doi.org/10.1002/vms3.744.\u003c/li\u003e\n\u003cli\u003eRadwan, A. I., El-Magawry, S., Hawari, A., Al-Bekairi, S. I. \u0026amp; Rebleza, R. M. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e infection in camels (\u003cem\u003eCamelus dromedarius\u003c/em\u003e) in Saudi Arabia. \u003cem\u003eTrop. Anim. Health Prod.\u003c/em\u003e\u003cstrong\u003e21\u003c/strong\u003e, 229\u0026ndash;230 (1989). https://doi.org/10.1007/BF02261094.\u003c/li\u003e\n\u003cli\u003eTejedor, M. T., Martin, J. L., Corbera, J. A., Shulz, U. \u0026amp; Gutierrez, C. Pseudotuberculosis in dromedary camels in the Canary Islands. \u003cem\u003eTrop. Anim. Health Prod.\u003c/em\u003e\u003cstrong\u003e36\u003c/strong\u003e, 459\u0026ndash;462 (2004). https://doi.org/10.1023/B:TROP.0000035012.63821.12.\u003c/li\u003e\n\u003cli\u003eTerab, A. M. A. et al. Pathology, bacteriology and molecular studies on caseous lymphadenitis in \u003cem\u003eCamelus dromedarius\u003c/em\u003e in the Emirate of Abu Dhabi, UAE, 2015\u0026ndash;2020. \u003cem\u003ePLoS One\u003c/em\u003e\u003cstrong\u003e16\u003c/strong\u003e, e0252893 (2021). https://doi.org/10.1371/journal.pone.0252893.\u003c/li\u003e\n\u003cli\u003eAnderson, D. E., Rings, D. M. \u0026amp; Kowalski, J. Infection with \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e in five alpacas. \u003cem\u003eJ. Am. Vet. Med. Assoc.\u003c/em\u003e\u003cstrong\u003e225\u003c/strong\u003e, 1743\u0026ndash;1747 (2004). https://doi.org/10.2460/javma.2004.225.1743.\u003c/li\u003e\n\u003cli\u003eSprake, P. \u0026amp; Gold, J. R. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e liver abscess in a mature alpaca (\u003cem\u003eLama pacos\u003c/em\u003e). \u003cem\u003eCan. Vet. J.\u003c/em\u003e\u003cstrong\u003e53\u003c/strong\u003e, 387\u0026ndash;390 (2012).\u003c/li\u003e\n\u003cli\u003eSting, R. et al. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e infections in alpacas (\u003cem\u003eVicugna pacos\u003c/em\u003e). \u003cem\u003eAnimals\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 1612 (2022). https://doi.org/10.3390/ani12131612.\u003c/li\u003e\n\u003cli\u003eStauber, E., Armstrong, P., Chamberlain, K. \u0026amp; Gorgen, B. Caseous lymphadenitis in a white-tailed deer. \u003cem\u003eJ. Wildl. Dis.\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 56\u0026ndash;57 (1973). https://doi.org/10.7589/0090-3558-9.1.56.\u003c/li\u003e\n\u003cli\u003eMorales, N. et al. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e infection in Patagonian huemul (\u003cem\u003eHippocamelus bisulcus\u003c/em\u003e). \u003cem\u003eJ. Wildl. Dis.\u003c/em\u003e\u003cstrong\u003e53\u003c/strong\u003e, 621 (2017). https://doi.org/10.7589/2016-09-213.\u003c/li\u003e\n\u003cli\u003eVarela-Castro, L. et al. Endemic caseous lymphadenitis in a wild Caprinae population. \u003cem\u003eVet. Rec.\u003c/em\u003e\u003cstrong\u003e180\u003c/strong\u003e, 405 (2017). https://doi.org/10.1136/vr.103925.\u003c/li\u003e\n\u003cli\u003eDi Donato, A. et al. First report of caseous lymphadenitis by \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e and pulmonary verminosis in a roe deer (\u003cem\u003eCapreolus capreolus\u003c/em\u003e Linnaeus, 1758) in Italy. \u003cem\u003eAnimals\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 566 (2024). https://doi.org/10.3390/ani14040566.\u003c/li\u003e\n\u003cli\u003eBastos, LB. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e: immunological responses in animal models and zoonotic potential. \u003cem\u003eJ. Clin. Cell. Immunol.\u003c/em\u003e\u003cstrong\u003eS4\u003c/strong\u003e, 005 (2012). https://doi.org/10.4172/2155-9899.S4-005.\u003c/li\u003e\n\u003cli\u003eSmith, M. C. \u0026amp; Sherman, D. M. \u003cem\u003eGoat Medicine\u003c/em\u003e. 3rd edn. (Wiley Blackwell, 2023).\u003c/li\u003e\n\u003cli\u003eAshfaq, M. K. \u0026amp; Campbell, S. G. Experimentally induced caseous lymphadenitis in goats. \u003cem\u003eAm. J. Vet. Res.\u003c/em\u003e\u003cstrong\u003e41\u003c/strong\u003e, 1789\u0026ndash;1792 (1980).\u003c/li\u003e\n\u003cli\u003eWilliamson, L. H. Caseous lymphadenitis in small ruminants. \u003cem\u003eVet. Clin. North Am. Food Anim. Pract.\u003c/em\u003e\u003cstrong\u003e17\u003c/strong\u003e, 359\u0026ndash;371 (2001). https://doi.org/10.1016/s0749-0720(15)30033-5.\u003c/li\u003e\n\u003cli\u003eBaird, G. J. \u0026amp; Fontaine, M. C. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e and its role in ovine caseous lymphadenitis. \u003cem\u003eJ. Comp. Pathol.\u003c/em\u003e\u003cstrong\u003e137\u003c/strong\u003e, 179\u0026ndash;210 (2007). https://doi.org/10.1016/j.jcpa.2007.07.002.\u003c/li\u003e\n\u003cli\u003eKaba, J. et al. Herd-level true seroprevalence of caseous lymphadenitis and paratuberculosis in the goat population of Poland. \u003cem\u003ePrev. Vet. Med.\u003c/em\u003e\u003cstrong\u003e230\u003c/strong\u003e, 106278 (2024). https://doi.org/10.1016/j.prevetmed.2024.106278.\u003c/li\u003e\n\u003cli\u003eKaba, J. et al. Evaluation of the risk factors influencing the spread of caseous lymphadenitis in goat herds. \u003cem\u003ePol. J. Vet. Sci.\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, (2011). https://doi.org/10.2478/v10181-011-0035-6.\u003c/li\u003e\n\u003cli\u003eDorella, F. A., Pacheco, L. G. C., Oliveira, S. C., Miyoshi, A. \u0026amp; Azevedo, V. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e: microbiology, biochemical properties, pathogenesis and molecular studies of virulence. \u003cem\u003eVet. Res.\u003c/em\u003e\u003cstrong\u003e37\u003c/strong\u003e, 201\u0026ndash;218 (2006). https://doi.org/10.1051/vetres:2005056.\u003c/li\u003e\n\u003cli\u003eAlmeida, S. et al. Quadruplex PCR assay for identification of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e differentiating biovar Ovis and Equi. \u003cem\u003eBMC Vet. Res.\u003c/em\u003e\u003cstrong\u003e13\u003c/strong\u003e, 290 (2017). https://doi.org/10.1186/s12917-017-1210-5.\u003c/li\u003e\n\u003cli\u003eKaba, J. Development of an ELISA for the diagnosis of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e infections in goats. \u003cem\u003eVet. Microbiol.\u003c/em\u003e\u003cstrong\u003e78\u003c/strong\u003e, 155\u0026ndash;163 (2001). https://doi.org/10.1016/S0378-1135(00)00284-4.\u003c/li\u003e\n\u003cli\u003eDorella, F. A. et al. Antigens of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e and prospects for vaccine development. \u003cem\u003eExpert Rev. Vaccines\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 205\u0026ndash;213 (2009). https://doi.org/10.1586/14760584.8.2.205.\u003c/li\u003e\n\u003cli\u003eGalv\u0026atilde;o, C. E. et al. Identification of new \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e antigens by immunoscreening of gene expression library. \u003cem\u003eBMC Microbiol.\u003c/em\u003e\u003cstrong\u003e17\u003c/strong\u003e, 202 (2017). https://doi.org/10.1186/s12866-017-1110-7.\u003c/li\u003e\n\u003cli\u003eSilva, M. T. D. O. et al. The combination of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e recombinant proteins rPLD, rCP01850 and rCP09720 for improved detection of caseous lymphadenitis in sheep by ELISA. \u003cem\u003eJ. Med. Microbiol.\u003c/em\u003e\u003cstrong\u003e68\u003c/strong\u003e, 1759\u0026ndash;1765 (2019). https://doi.org/10.1099/jmm.0.001096.\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;pin, M., Boisram\u0026eacute;, A. \u0026amp; Marly, J. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e: biochemical properties, production of toxin and virulence of ovine and caprine strains. \u003cem\u003eAnn. Rech. Vet.\u003c/em\u003e\u003cstrong\u003e20\u003c/strong\u003e, 111\u0026ndash;115 (1989).\u003c/li\u003e\n\u003cli\u003eDe Pinho, BR. et al. A novel approach for an immunogen against \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e infection: An \u003cem\u003eEscherichia coli\u003c/em\u003e bacterin expressing phospholipase D. \u003cem\u003eMicrob. Pathog.\u003c/em\u003e\u003cstrong\u003e151\u003c/strong\u003e, 104746 (2021). https://doi.org/10.1016/j.micpath.2021.104746.\u003c/li\u003e\n\u003cli\u003eBarral, T. D. et al. A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep. \u003cem\u003eMicrob. Biotechnol.\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 1313\u0026ndash;1323 (2019). https://doi.org/10.1111/1751-7915.13454.\u003c/li\u003e\n\u003cli\u003eSting, R. et al. Clinical and serological investigations on caseous lymphadenitis in goat breeding herds in Baden-Wuerttemberg. \u003cem\u003eBerl. Munch. Tierarztl. Wochenschr.\u003c/em\u003e\u003cstrong\u003e130\u003c/strong\u003e, 136\u0026ndash;143 (2017). https://doi.org/10.2376/0005-9366-16024.\u003c/li\u003e\n\u003cli\u003eCosta, L. et al. Utility assessment of an enzyme‐linked immunosorbent assay for detection of subclinical cases of caseous lymphadenitis in small ruminant flocks. \u003cem\u003eVet. Med. Sci.\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 796\u0026ndash;803 (2020). https://doi.org/10.1002/vms3.297.\u003c/li\u003e\n\u003cli\u003eSilva, J. W. et al. \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis. \u003cem\u003eBMC Vet. Res.\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 965 (2014). https://doi.org/10.1186/s12917-014-0304-6.\u003c/li\u003e\n\u003cli\u003eShadnezhad, A., Naegeli, A. \u0026amp; Collin, M. CP40 from \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e is an endo-\u0026beta;-N-acetylglucosaminidase. \u003cem\u003eBMC Microbiol.\u003c/em\u003e\u003cstrong\u003e16\u003c/strong\u003e, 261 (2016). https://doi.org/10.1186/s12866-016-0884-3.\u003c/li\u003e\n\u003cli\u003eTrost, E. et al. The complete genome sequence of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence. \u003cem\u003eBMC Genomics\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 728 (2010). https://doi.org/10.1186/1471-2164-11-728.\u003c/li\u003e\n\u003cli\u003eSantana-Jorge, K. T. O. et al. Putative virulence factors of \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e FRC41: vaccine potential and protein expression. \u003cem\u003eMicrob. Cell Fact.\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 83 (2016). https://doi.org/10.1186/s12934-016-0479-6.\u003c/li\u003e\n\u003cli\u003eWalburger, A. et al. Protein Kinase G from pathogenic \u003cem\u003eMycobacteria\u003c/em\u003e promotes survival within macrophages. \u003cem\u003eScience\u003c/em\u003e\u003cstrong\u003e304\u003c/strong\u003e, 1800\u0026ndash;1804 (2004). https://doi.org/10.1126/science.1099384.\u003c/li\u003e\n\u003cli\u003eWarner, D. F. \u0026amp; Mizrahi, V. The survival kit of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e. \u003cem\u003eNat. Med.\u003c/em\u003e\u003cstrong\u003e13\u003c/strong\u003e, 282\u0026ndash;284 (2007). https://doi.org/10.1038/nm0307-282.\u003c/li\u003e\n\u003cli\u003eFarias, A. P. F. D. et al. rSodC is a potential antigen to diagnose \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e by enzyme-linked immunoassay. \u003cem\u003eAMB Expr.\u003c/em\u003e\u003cstrong\u003e10\u003c/strong\u003e, 186 (2020). https://doi.org/10.1186/s13568-020-01125-0.\u003c/li\u003e\n\u003cli\u003eAtkinson, J. et al. Guidelines for saliva nomenclature and collection. \u003cem\u003eAnn. N. Y. Acad. Sci.\u003c/em\u003e\u003cstrong\u003e694\u003c/strong\u003e, XI\u0026ndash;XII (1993).\u003c/li\u003e\n\u003cli\u003eChiappin, S., Antonelli, G., Gatti, R. \u0026amp; De Palo, E. F. Saliva specimen: A new laboratory tool for diagnostic and basic investigation. \u003cem\u003eClin. Chim. Acta\u003c/em\u003e\u003cstrong\u003e383\u003c/strong\u003e, 30\u0026ndash;40 (2007). https://doi.org/10.1016/j.cca.2007.04.011\u003c/li\u003e\n\u003cli\u003eMestecky, J. Saliva as a manifestation of the common mucosal immune system. \u003cem\u003eAnn. N. Y. Acad. Sci.\u003c/em\u003e\u003cstrong\u003e694\u003c/strong\u003e, 184\u0026ndash;194 (1993). https://doi.org/10.1111/j.1749-6632.1993.tb18352.x.\u003c/li\u003e\n\u003cli\u003eHenao-Diaz, A., Gim\u0026eacute;nez-Lirola, L. \u0026amp; Baum, D. H. Guidelines for oral fluid-based surveillance of viral pathogens in swine. \u003cem\u003ePorc. Health Manag.\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 28 (2020). https://doi.org/10.1186/s40813-020-00168-w.\u003c/li\u003e\n\u003cli\u003ePage, L. J., Lagunas-Acosta, J., Castellana, E. T. \u0026amp; Messmer, B. T. Accurate prediction of serum antibody levels from noninvasive saliva/nasal samples. \u003cem\u003eBiotechniques.\u003c/em\u003e\u003cstrong\u003e74\u003c/strong\u003e, 131\u0026ndash;136 (2023). https://doi.org/10.2144/btn-2022-0106. \u003c/li\u003e\n\u003cli\u003eMur, L. et al. Potential use of oral fluid samples for serological diagnosis of African swine fever. \u003cem\u003eVet. Microbiol.\u003c/em\u003e\u003cstrong\u003e165\u003c/strong\u003e, 135\u0026ndash;139 (2013). https://doi.org/10.1016/j.vetmic.2012.12.034.\u003c/li\u003e\n\u003cli\u003eGim\u0026eacute;nez-Lirola, L. G. et al. Detection of African swine fever virus antibodies in serum and oral fluid specimens using a recombinant protein 30 (p30) dual matrix indirect ELISA. \u003cem\u003ePLoS One\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, e0161230 (2016). https://doi.org/10.1371/journal.pone.0161230.\u003c/li\u003e\n\u003cli\u003ePanyasing, Y., Thanawongnuwech, R., Ji, J., Gim\u0026eacute;nez-Lirola, L. \u0026amp; Zimmerman, J. Detection of classical swine fever virus (CSFV) E2 and Erns antibody (IgG, IgA) in oral fluid specimens from inoculated (ALD strain) or vaccinated (LOM strain) pigs. \u003cem\u003eVet. Microbiol.\u003c/em\u003e\u003cstrong\u003e224\u003c/strong\u003e, 70\u0026ndash;77 (2018). https://doi.org/10.1016/j.vetmic.2018.08.024.\u003c/li\u003e\n\u003cli\u003eKittawornrat, A. et al. Kinetics of the porcine reproductive and respiratory syndrome virus (PRRSV) humoral immune response in swine serum and oral fluids collected from individual boars. \u003cem\u003eBMC Vet. Res.\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 61 (2013). https://doi.org/10.1186/1746-6148-9-61.\u003c/li\u003e\n\u003cli\u003eRotolo, M. L. et al. Detection of porcine reproductive and respiratory syndrome virus (PRRSV)-specific IgM-IgA in oral fluid samples reveals PRRSV infection in the presence of maternal antibody. \u003cem\u003eVet. Microbiol.\u003c/em\u003e\u003cstrong\u003e214\u003c/strong\u003e, 13\u0026ndash;20 (2018). https://doi.org/10.1016/j.vetmic.2017.11.011.\u003c/li\u003e\n\u003cli\u003eHenao-Diaz, A., Gim\u0026eacute;nez-Lirola, L., Magtoto, R., Ji, J. \u0026amp; Zimmerman, J. Evaluation of three commercial porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody ELISAs using samples of known status. \u003cem\u003eRes. Vet. Sci.\u003c/em\u003e\u003cstrong\u003e125\u003c/strong\u003e, 113\u0026ndash;118 (2019). https://doi.org/10.1016/j.rvsc.2019.05.019.\u003c/li\u003e\n\u003cli\u003ePrickett, J. R. et al. Prolonged detection of PCV2 and anti-PCV2 antibody in oral fluids following experimental inoculation: prolonged detection of PCV2 infection by oral fluid. \u003cem\u003eTransbound. Emerg. Dis.\u003c/em\u003e\u003cstrong\u003e58\u003c/strong\u003e, 121\u0026ndash;127 (2011). https://doi.org/10.1111/j.1865-1682.2010.01189.x.\u003c/li\u003e\n\u003cli\u003eCampero, L. M. et al. Detection of antibodies to \u003cem\u003eToxoplasma gondii\u003c/em\u003e in oral fluid from pigs. \u003cem\u003eInt. J. Parasitol.\u003c/em\u003e\u003cstrong\u003e50\u003c/strong\u003e, 349\u0026ndash;355 (2020). https://doi.org/10.1016/j.ijpara.2019.11.002.\u003c/li\u003e\n\u003cli\u003eKauter, J. et al. Detection of \u003cem\u003eToxoplasma gondii\u003c/em\u003e-specific antibodies in pigs using an oral fluid-based commercial ELISA: advantages and limitations. \u003cem\u003eInt. J. Parasitol.\u003c/em\u003e\u003cstrong\u003e53\u003c/strong\u003e, 523\u0026ndash;530 (2023). https://doi.org/10.1016/j.ijpara.2022.11.003.\u003c/li\u003e\n\u003cli\u003eArchetti, I. L., Amadori, M., Donn, A., Salt, J. \u0026amp; Lodetti, E. Detection of foot-and-mouth disease virus-infected cattle by assessment of antibody response in oropharyngeal fluids. \u003cem\u003eJ. Clin. Microbiol.\u003c/em\u003e\u003cstrong\u003e33\u003c/strong\u003e, 79\u0026ndash;84 (1995). https://doi.org/10.1128/jcm.33.1.79-84.1995.\u003c/li\u003e\n\u003cli\u003eLazutka, J., Spakova, A., Sereika, V., Lelesius, R., Sasnauskas, K. \u0026amp; Petraityte-Burneikiene, R. Saliva as an alternative specimen for detection of Schmallenberg virus-specific antibodies in bovines. \u003cem\u003eBMC Vet. Res.\u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, 237 (2015). https://doi.org/10.1186/s12917-015-0552-0.\u003c/li\u003e\n\u003cli\u003eOrtega, J. et al. Evaluation of P22 ELISA for the detection of \u003cem\u003eMycobacterium bovis\u003c/em\u003e-specific antibody in the oral fluid of goats. \u003cem\u003eFront. Vet. Sci.\u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 674636 (2021). https://doi.org/10.3389/fvets.2021.674636.\u003c/li\u003e\n\u003cli\u003eHodgson, A. L., Bird, P. \u0026amp; Nisbet, I. T. Cloning, nucleotide sequence, and expression in \u003cem\u003eEscherichia coli\u003c/em\u003e of the phospholipase D gene from \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e. \u003cem\u003eJ. Bacteriol.\u003c/em\u003e\u003cstrong\u003e172\u003c/strong\u003e, 1256\u0026ndash;1261 (1990). https://doi.org/10.1128/jb.172.3.1256-1261.1990.\u003c/li\u003e\n\u003cli\u003eEllis, J. A., Hawk, D. A., Holler, L. D., Mills, K. W. \u0026amp; Pratt, D. L. Differential antibody responses to \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e in sheep with naturally acquired caseous lymphadenitis. \u003cem\u003eJ. Am. Vet. Med. Assoc.\u003c/em\u003e\u003cstrong\u003e196\u003c/strong\u003e, 1609\u0026ndash;1613 (1990).\u003c/li\u003e\n\u003cli\u003eter Laak, E. A., Bosch, J., Bijl, G. C. \u0026amp; Schreuder, B. E. Double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot analysis used for control of caseous lymphadenitis in goats and sheep. \u003cem\u003eAm. J. Vet. Res.\u003c/em\u003e\u003cstrong\u003e53\u003c/strong\u003e, 1125\u0026ndash;1132 (1992).\u003c/li\u003e\n\u003cli\u003eHunt, A. J. et al. The testing of saliva samples for HIV-1 antibodies: reliability in a non-clinic setting. \u003cem\u003eSex. Transm. Infect.\u003c/em\u003e\u003cstrong\u003e69\u003c/strong\u003e, 29\u0026ndash;30 (1993). https://doi.org/10.1136/sti.69.1.29.\u003c/li\u003e\n\u003cli\u003eLangenhorst, R. et al. Development of a fluorescent microsphere immunoassay for detection of antibodies against porcine reproductive and respiratory syndrome virus using oral fluid samples as an alternative to serum-based assays. \u003cem\u003eClin. Vaccine Immunol.\u003c/em\u003e\u003cstrong\u003e19\u003c/strong\u003e, 180\u0026ndash;189 (2011). https://doi.org/10.1128/CVI.05372-11.\u003c/li\u003e\n\u003cli\u003eNokes, D. J. et al. Has oral fluid the potential to replace serum for the evaluation of population immunity levels? A study of measles, rubella and hepatitis B in rural Ethiopia. \u003cem\u003eBull. World Health Organ.\u003c/em\u003e\u003cstrong\u003e79\u003c/strong\u003e, 588\u0026ndash;595 (2001).\u003c/li\u003e\n\u003cli\u003eNagel‐Alne, G. E., Valle, P. S., Krontveit, R. \u0026amp; S\u0026oslash;lver\u0026oslash;d, L. S. Caprine arthritis encephalitis and caseous lymphadenitis in goats: use of bulk tank milk ELISAs for herd‐level surveillance. \u003cem\u003eVet. Rec.\u003c/em\u003e\u003cstrong\u003e176\u003c/strong\u003e, 173\u0026ndash;173 (2015). https://doi.org/10.1136/vr.102605.\u003c/li\u003e\n\u003cli\u003eSilva, M. T. D. O. et al. NanH and PknG putative virulence factors as a recombinant subunit immunogen against \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e infection in mice. \u003cem\u003eVaccine.\u003c/em\u003e\u003cstrong\u003e38\u003c/strong\u003e, 8099\u0026ndash;8106 (2020). https://doi.org/10.1016/j.vaccine.2020.11.010.\u003c/li\u003e\n\u003cli\u003eAltman, D. G., Machin, D., Bryant, T. N. \u0026amp; Gardner, M. J. \u003cem\u003eStatistics with Confidence\u003c/em\u003e. 2nd edn. (BMJ Books, 2000).\u003c/li\u003e\n\u003cli\u003eZar, J. H. \u003cem\u003eBiostatistical Analysis\u003c/em\u003e. 5th edn. (Pearson Prentice Hall, 2010).\u003c/li\u003e\n\u003cli\u003eGwet, K. L. Computing inter‐rater reliability and its variance in the presence of high agreement. \u003cem\u003eBr. J. Math. Stat. Psychol.\u003c/em\u003e\u003cstrong\u003e61\u003c/strong\u003e, 29\u0026ndash;48 (2008). https://doi.org/10.1348/000711006X126600.\u003c/li\u003e\n\u003cli\u003eAltman, D. G. (1990). \u003cem\u003ePractical statistics for medical research\u003c/em\u003e (1st ed.). Chapman and Hall/CRC. https://doi.org/10.1201/9780429258589\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Corynebacterium pseudotuberculosis, CLA, diagnostics, goats, immunoassays","lastPublishedDoi":"10.21203/rs.3.rs-6065536/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6065536/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eCorynebacterium pseudotuberculosis\u003c/em\u003e causes caseous lymphadenitis (CLA) in goats and sheep. This study assessed oral fluid (OF) as a minimally invasive sample for CLA serodiagnosis by detecting antibodies to PLD, CP40, and PknG using Western blot (WB). Ninety-six goats from a CLA-affected herd in Poland were tested. Paired serum and OF samples were analyzed via ELISA (serum only) and WB (serum and OF). The rPLD-ELISA had 70\u0026ndash;80% diagnostic sensitivity, depending on the cut-off. WB on serum showed high sensitivity (98%) and good agreement with ELISA. WB on OF had slightly lower sensitivity (90%), with differences in antigen detection. OF shows promise for CLA diagnosis, with PLD antibodies being the most relevant. Fewer OF samples had CP40 and PknG antibodies, likely due to antibody class differences or detection limitations. Further investigation into immunoglobulin classes present in different sample types and optimized secondary antibodies is essential for developing an effective OF-based diagnostic test for CLA.\u003c/p\u003e","manuscriptTitle":"Oral fluid as a material for serological diagnostics of caseous lymphadenitis in goats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 11:52:28","doi":"10.21203/rs.3.rs-6065536/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-02T08:07:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-01T15:28:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-21T19:30:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91131086024027627447949374162947955905","date":"2025-04-17T15:06:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238540393173124715237054324954957134495","date":"2025-03-28T05:49:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-28T05:36:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-14T18:01:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-13T03:16:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-13T03:13:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-19T15:32:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c124afb3-705f-474c-8cca-8f0b7b5e097c","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45704093,"name":"Biological sciences/Microbiology/Pathogens"},{"id":45704094,"name":"Health sciences/Diseases/Infectious diseases/Bacterial infection"},{"id":45704095,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-07-07T16:18:09+00:00","versionOfRecord":{"articleIdentity":"rs-6065536","link":"https://doi.org/10.1038/s41598-025-06957-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-02 15:58:45","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2025-03-17 11:52:28","video":"","vorDoi":"10.1038/s41598-025-06957-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-06957-z","workflowStages":[]},"version":"v1","identity":"rs-6065536","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6065536","identity":"rs-6065536","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}