Brucella canis is a zoonotic pathogen of dogs that poses diagnostic challenges. While direct detection of B. canis by PCR or culture is ideal, serologic diagnosis is necessary for identification of carrier animals and can support a clinical diagnosis of brucellosis. Prior to 2022, B. canis seroscreening in the United States was primarily performed using a commercially available rapid slide agglutination test. However, the kit was discontinued by the manufacturer in early 2022, leaving a gap in the availability of commercial B. canis seroassays. The goal of this study was to compare the performance of three B. canis serologic tests that are currently available: VMRD Brucella ovis ELISA, Bionote Anigen Rapid C.Brucella Ab immunochromatographic lateral flow assay, and VMRD B. canis indirect fluorescent antibody (IFA) assay. A panel of 56 banked serum specimens originally submitted to the Cornell University Animal Health Diagnostic Center (the study reference laboratory) for B. canis seroscreening was distributed to 12 testing laboratories. Each sample was run on three assays developed at the reference lab: rapid slide agglutination test with 2-mercaptoethanol (2-ME RSAT), agar gel immunodiffusion test using cytoplasmic antigen (AGID II), and Canine Brucella Multiplex. Five testing labs ran the ELISA, six ran the lateral flow, and six ran the IFA. When evaluated as a screening assay, we compared the assays to the 2ME-RSAT. The ELISA had the highest sensitivity (96.8, 95%CI 83.8–99.9) but the lowest specificity (79.3, 95%CI 57.9–92.9). The sensitivity of the lateral flow was 90.6% (95%CI 75–98%) and the IFA was 87.5% (95%CI 71–96.5). Specificity for the lateral flow was 95.8% (95%CI 78.9–99.9) and IFA was 97.5% (95%CI 67.6–97.3). When compared to AGID II and Canine Brucella Multiplex, the test assays were all highly sensitive, but specificity was <90%. Interrater reliability was highest for IFA (Κ = 0.92) and lowest for the lateral flow (Κ = 0.82). Serial testing of positive samples with a more specific test, such as AGID II, will continue to be necessary when using any of the three assays tested in this study.

The detection of highly pathogenic avian influenza A virus H5N1 in dairy cows in the United States underscores the urgent need for reliable laboratory tools to support epidemiological surveillance. This study describes the development and evaluation of a fluorescent microsphere immunoassay (FMIA) for detecting IgG, IgA, and IgM antibodies against the H5 hemagglutinin protein in serum and milk samples from dairy cattle, with results compared to the NP-ELISA. H5/FMIA demonstrated 100% diagnostic sensitivity and 99.7% diagnostic specificity for anti-H5 IgG antibodies in serum. In milk samples, the assay showed comparable performance for IgG and IgA, achieving 94.7% sensitivity and 98% specificity. Paired serum and milk samples exhibited stronger correlations using H5/FMIA (r = 0.88 for IgG, r = 0.82 for IgA, and r = 0.58 for IgM) than NP-ELISA (r = 0.54). H5/FMIA IgA showed greater sensitivity in "early" infections, whereas IgG was more robust in "late" cases. These findings confirm the utility of H5/FMIA as a valuable antibody isotype-specific tool for serodiagnosis and epidemiological surveillance of H5N1 in dairy cattle.

While the vast majority of the US swine population is concentrated in 5 states (Iowa, Minnesota, North Carolina, Illinois, and Indiana), the rest is spread out across the US including pork production, hobby farms, pet pigs, and research facilities. The number of novel diseases and laboratory tests that have been established in recent years can be intimidating or overwhelming for practitioners who do not routinely work with this species. This review aims to help clinicians across the country that may not have an in-depth experience in swine medicine become more familiar with both common and novel pathogens, formulate a differential diagnosis based on the age of the animals and affected system (eg, respiratory, systemic, nervous, and enteric), select proper samples and laboratory testing, and interpret laboratory data to achieve a disease diagnosis in porcine patients.

In March 2024, highly pathogenic avian influenza A(H5N1) virus, clade 2.3.4.4b, was detected in dairy cows in the United States, and at the same time in resident cats on affected farms. To help guide sample collection and diagnosis in cats, here we report the distribution of lesions and detection of H5N1 clade 2.3.4.4b influenza A virus (IAV) infection by PCR, immunohistochemistry (IHC), and serology in samples from 4 deceased and 2 living cats from 3 separate affected dairy farms. Although gross lesions were not diagnostic, histologically, all 4 deceased cats had nonsuppurative and necrotizing encephalitis and subtle interstitial pneumonia, and some also had significant myocarditis (3 of 4), chorioretinitis (2 of 4), and sialadenitis (1 of 2). The virus was detected by IHC in the aforementioned tissues, and by PCR in each brain (Ct = 9.9-25.1), lung (17.4-32.7), oropharyngeal swab (28.3-30.5), urine (30.3-34.4), and nasal swab (33.5-34.1) collected postmortem; fecal swabs were PCR-negative. In the antemortem samples, the virus was detected by PCR in the oropharyngeal swabs (34.1-36.1), whole-blood samples (30.8-36.6), and one serum sample (31.7). Seroconversion was detected in one cat. Our results support histologic evaluation of brain, lung, eyes, and heart, and PCR testing of brain and lung for postmortem diagnosis, and show that oropharyngeal swabs, urine, serum, and whole blood are suitable samples for antemortem detection of IAV infection in clinically affected cats.

Mycoplasma hyorhinis (Mhr) and M. hyosynoviae (Mhs) are commensal organisms of the upper respiratory tract and tonsils but may also cause arthritis in pigs. In this study, 8-week-old cesarean-derived colostrum-deprived (CDCD) pigs (n = 30; 3 groups, 10 pigs per group, 2 pigs per pen) were inoculated with Mhr, Mhs, or mock-inoculated with culture medium and then pen-based oral fluids were collected at different time points over the 56 days of the experimental study. Oral fluids tested by Mhr and Mhs quantitative real-time PCRs revealed Mhr DNA between day post inoculation (DPI) 5-52 and Mhs DNA between DPI 5-15. Oral fluids were likewise tested for antibody using isotype-specific (IgG, IgA, IgM) indirect ELISAs based on a recombinant chimeric polypeptide of variable lipoproteins (A-G) for Mhr and Tween 20-extracted surface proteins for Mhs. Mhr IgA was detected at DPI 7 and, relative to the control group, significant (p < 0.05) antibody responses were detected in the Mhr group between DPI 12-15 for IgM and DPI 36-56 for both IgA and IgG. In the Mhs group, IgM was detected at DPI 10 and significant (p < 0.05) IgG and IgA responses were detected at DPI 32-56 and DPI 44-56, respectively. This study demonstrated that oral fluid could serve as an effective and convenient antemortem sample for monitoring Mhr and Mhs in swine populations.

The emergence and spread of highly pathogenic avian influenza virus A subtype H5N1 (HP H5N1-IAV), particularly clade H5N1 2.3.4.4b, pose a severe global health threat, affecting various species, including mammals. Historically, cattle have been considered less susceptible to IAV, but recent outbreaks of H5N1-IAV 2.3.4.4b in dairy farms suggest a shift in host tropism, underscoring the urgency of expanded surveillance and the need for adaptable diagnostic tools in outbreak management. This study investigated the presence of anti-nucleoprotein (NP) antibodies in serum and milk and viral RNA in milk on dairy farms affected by outbreaks in Texas, Kansas, and Michigan using a multi-species IAV ELISA and RT-qPCR. The analysis of ELISA results from a Michigan dairy farm outbreak demonstrated a positive correlation between paired serum and milk sample results, confirming the reliability of both specimen types. Our findings also revealed high diagnostic performance during the convalescent phase (up to 96%), further improving sensitivity through serial sampling. Additionally, the evaluation of diagnostic specificity using serum and milk samples from IAV-free farms showed an excellent performance (99.6%). This study underscores the efficacy of the IAV NP-blocking ELISA for detecting and monitoring H5N1-IAV 2.3.4.4b circulation in dairy farms, whose recent emergence raises significant animal welfare and zoonotic concerns, necessitating expanded surveillance efforts.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is a significant porcine respiratory disease complex pathogen, prompting many swine farms and production systems to pursue M. hyopneumoniae elimination strategies. Antibody testing is cost-effective in demonstrating sustained freedom from M. hyopneumoniae, often replacing PCR testing on deep tracheal swabs. The process typically involves testing a subpopulation of the herd using an M. hyopneumoniae screening antibody ELISA, with non-negative results further assessed through confirmatory testing, such as PCR. Recently, a commercial (Biochek) fluorescent microsphere immunoassay (FMIA) for detecting M. hyopneumoniae antibodies has been introduced as an alternative to ELISA. Its performance was compared to three commercial ELISAs (Idexx, Hipra, and Biochek) using experimental serum samples from pigs inoculated with M. hyopneumoniae, M. hyorhinis, M. hyosynoviae, M. flocculare, or mock-inoculated with Friis medium. FMIA consistently detected M. hyopneumoniae at earlier time points than the ELISAs, although two false-positive results were encountered using the manufacturer’s recommended cutoff. ROC analysis allowed for the evaluation of various cutoffs depending on testing objectives. Poisson regression of misclassification error counts detected no difference in the Biovet FMIA and Hipra ELISA but significantly fewer misclassification errors than Idexx and Biocheck ELISAs. This study showed FMIA as a suitable alternative to traditional ELISAs for screening purposes due to its superior antibody detection rate at early stages. Alternatively, adopting a more stringent cutoff to improve diagnostic specificity could position the FMIA as a viable confirmatory test option. Overall, FMIA is an optimal choice for M. hyopneumoniae antibody surveillance testing, offering versatility in testing strategies (e.g., triplex FMIA M. hyopneumoniae/PRRSV types 1 and 2) and contributing to improved diagnostic capabilities in porcine health management.

Veterinary diagnostic laboratories provide a vast catalog of tests for infectious agents and often have multiple tests and testing modalities for individual pathogens. Understanding which test to select, which sample type is best, and which animal to sample in a population are critical factors for drawing appropriate conclusions and diagnosing a disease. Each diagnostic test has its inherent diagnostic sensitivity and specificity, and, in many situations, multiple tests may be required for proper interpretation. When multiple tests are needed in a large population (eg, animal shelters, farmed animals, and breeding colonies), the cost of diagnostic testing becomes an increasing concern, and knowing when and how to use aggregate or composite samples is critical. A clinical examination is the first and argu-ably most important diagnostic procedure providing the framework for laboratory test selection. This yields not only a differential diagnosis but an expected stage of disease, which is necessary for determining the most appropriate laboratory tests and how they should be interpreted. This review will discuss the diagnostic process, strengths and limitations of commonly requested tests at a diagnostic laboratory (eg, histopathology, microbial culture, PCR, and ELISA), common sources of error in interpretation, the impact of pooling samples on different tests, and ways to help strengthen conclusions made from diagnostic test data.

Neutralizing antibodies to Porcine Epidemic Diarrhea Virus (PEDV) can be detected by 3 weeks post-infection and remain detectable through at least 24 weeks post-infection. The objective of this study was to evaluate the levels of neutralizing antibodies in sow and piglet serum and sow milk to determine the duration of neutralizing antibodies following PEDV outbreaks. Two farms were selected for the study following outbreaks of PEDV. Monthly, cohorts of sows were sampled and followed through two farrowings. Following each farrowing, samples from piglets and milk were collected. Samples were evaluated for PEDV-neutralizing antibodies by a high-throughput fluorescent neutralization assay. Although neutralizing antibodies to PEDV can be detected throughout 15 months post-outbreak, a decrease in circulating neutralizing antibody levels is noted in farms beginning at six months post-outbreak. With decreasing levels, farms may become more vulnerable to PEDV outbreaks, and practitioners can focus on this time window to implement intervention strategies.

The Mycoplasma hyorhinis (Mhr) variable lipoprotein (Vlp) family, comprising Vlps A, B, C, D, E, F, and G, are highly variable in expression, size, and cytoadhesion capabilities across Mhr strains. The ‘Vlp system’ plays a crucial role in cytoadhesion, immune evasion, and in eliciting a host immunologic response. This pilot study described the development of Vlp peptide-based ELISAs to evaluate the antigenic reactivity of individual Vlps against Mhr antisera collected throughout a longitudinal study focused on Mhr strain 38983, reproducing Mhr-associated disease under experimental conditions. Specifically, serum samples were collected at day post-inoculation 0, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, and 56 from Mhr- and mock (Friis medium)-inoculated cesarean-derived, colostrum-deprived pigs. Significant Mhr-specific IgG responses were detected at specific time points throughout the infection, with some variations for each Vlp. Overall, individual Vlp ELISAs showed consistently high accuracy rates, except for VlpD, which would likely be associated with its expression levels or the anti-Vlp humoral immune response specific to the Mhr strain used in this study. This study provides the basis and tools for a more refined understanding of these Vlp- and Mhr strain-specific variations, which is foundational in understanding the host immune response to Mhr.

Endogenous reference genes are used in gene-expression studies to "normalize" the results and, increasingly, as internal sample controls (ISC) in diagnostic quantitative polymerase chain reaction (qPCR). Three studies were conducted to evaluate the performance of a porcine-specific ISC in a commercial porcine reproductive and respiratory syndrome virus (PRRSV) reverse transcription-qPCR. Study 1 evaluated the species specificity of the ISC by testing serum from seven non-porcine domestic species (n = 34). In Study 2, the constancy of ISC detection over time (≥42 days) was assessed in oral fluid (n = 130), serum (n = 215), and feces (n = 132) collected from individual pigs of known PRRSV status. In Study 3, serum (n = 150), oral fluid (n = 150), and fecal samples (n = 75 feces, 75 fecal swabs) from commercial herds were used to establish ISC reference limits. Study 1 showed that the ISC was porcine-specific, i.e., all samples from non-porcine species were ISC negative (n = 34). In Study 2, the ISC was detected in all oral fluid, serum, and fecal samples, but differed in concentration between specimens (p < 0.05; mixed-effects regression model). The results of Study 3 were used to establish ISC reference limits for the 5th, 2.5th and 1.25th percentiles. Overall, the ISC response was consistent to the point that failure in detection is sufficient justification for re-testing and/or re-sampling.

Johne’s disease is caused by Mycobacterium avium ssp. paratu­berculosis (MAP) and can be a costly and frustrating disease in beef herds. Clinical Johne’s disease in cattle arises years after inoculation as diarrhea and progressive weight loss. Prior to reaching clinical status, subclinically infected cattle can shed bacteria into their environment, leading to continued spread of the disease within herds. The duration of subclinical infection is highly variable, and the humoral immune response and the amount of fecal shedding of MAP can vary greatly between in­dividuals and throughout the year. Currently available diagnos­tic tests (serum ELISA, fecal PCR, fecal culture) each have sig­nificant limitations. Assays may differ in their utility depending on whether they are used to confirm clinical cases of Johne’s disease or to screen healthy cattle for potential infection.

Lactogenic immunity is important for the protection of piglets against many pathogens including porcine epidemic diarrhea virus. Circulating neutralizing antibodies levels in sow sera may help determine if a detectable immune response could confer protection to piglets. Neutralizing antibodies can be detected through various diagnostic assays. This study evaluated the diagnostic characteristics of two neutralizing antibody assays for porcine epidemic diarrhea virus neutralizing antibodies in serum of challenged gilts. Four treatment groups, control, non-vaccinated, vaccinated prior to challenge, and vaccinated following challenge, were comprised of 20 gilts. Serum sample were collected from each gilt prior to and following challenge with porcine epidemic diarrhea virus. Samples were evaluated for the presence of neutralizing antibodies via a fluorescent focus neutralization assay and a high-throughput neutralization assay. Diagnostic sensitivity and specificity for the fluorescent focus neutralization and high-throughput neutralization assays for this study were optimized at a cutoff of a dilution of 80 and 80% fluorescent reduction respectively and demonstrated moderate agreement based off the kappa statistic. The focus fluorescent neutralization and high-throughput neutralization assays can be used to monitor the status of neutralizing antibodies within animals or a population of animals. The high-throughput assay has advantages over the focus fluorescent assay in that it has a higher specificity at the indicated cut-off and the nature of the results allows for more discrimination between individual results.

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae and genus Deltacoronavirus, is a major enteric pathogen in swine. Accurate PDCoV diagnosis relying on laboratory testing and antibody detection is an important approach. This study evaluated the potential of the receptor-binding subunit of the PDCoV spike protein (S1), generated using a mammalian expression system, for specific antibody detection via indirect enzyme-linked immunosorbent assay (ELISA). Serum samples were collected at day post-inoculation (DPI) −7 to 42, from pigs (n = 83) experimentally inoculated with different porcine coronaviruses (PorCoV). The diagnostic sensitivity of the PDCoV S1-based ELISA was evaluated using serum samples (n = 72) from PDCoV-inoculated animals. The diagnostic specificity and potential cross-reactivity of the assay was evaluated on PorCoV-negative samples (n = 345) and samples collected from pigs experimentally inoculated with other PorCoVs (n = 472). The overall diagnostic performance, time of detection, and detection rate over time varied across different S/P cut-offs, estimated by Receiver Operating Characteristic (ROC) curve analysis. The higher detection rate in the PDCoV group was observed after DPI 21. An S/P cut-off of 0.25 provided 100% specificity with no serological cross-reactivity against other PorCoV. These results support the use of S1 protein-based ELISA for accurate detection of PDCoV infections, transference of maternal antibodies, or active surveillance.

The diagnostic performance of a commercial Mycoplasma hyopneumoniae (MHP) serum enzyme-linked immunosorbent assay (ELISA) was evaluated for MHP antibody detection in processing fluids (n = 494) using samples from three commercial swine farms. Based on historical monitoring, one farm was considered MHP positive and two were considered MHP negative. Samples were tested at a 1:10 dilution and diagnostic sensitivities and specificities estimated for specific ELISA sample-to-positive (S:P) cutoffs. At S:P ≥ 0.40, diagnostic sensitivity and specificity were estimated as 97.6% and 100.0%, respectively. Overall, the results suggest that processing fluids can be used for MHP antibody surveillance in breeding herds.

Distinct from tests used in diagnostics, tests used in surveillance must provide for detection while avoiding false alarms, i.e., acceptable diagnostic sensitivity but high diagnostic specificity. In the case of the reproductive and respiratory syndrome virus (PRRSV), RNA detection meets these requirements during the period of viremia, but antibody detection better meets these requirements in the post-viremic stage of the infection. Using the manufacturer's recommended cut-off (S/P ≥ 0.4), the diagnostic specificity of a PRRSV oral fluid antibody ELISA (IDEXX Laboratories, Inc., Westbrook, ME, USA) evaluated in this study was previously reported as ≥ 97 %. The aim of this study was to improve its use in surveillance by identifying a cut-off that would increase diagnostic specificity yet minimally impact its diagnostic sensitivity. Three sample sets were used to achieve this goal: oral fluids (n = 596) from pigs vaccinated with a modified live PRRSV vaccine under experimental conditions, field oral fluids (n = 1574) from 94 production sites of known negative status, and field oral fluids (n = 1380) from 211 sites of unknown PRRSV status. Based on the analysis of samples of known status (experimental samples and field samples from negative sites), a cut-off of S/P ≥ 1.0 resulted in a diagnostic specificity of 99.2 (95 % CI: 98.8, 99.7) and a diagnostic sensitivity of 96.5 (95 % CI: 85.2, 99.2). Among 211 sites of unknown status, 81 sites were classified as antibody positive using the manufacturer's cut-off; 20 of which were reclassified as negative using a cut-off of S/P ≥ 1.0. Further analysis showed that these 20 sites had a small proportion of samples (18.0 %) with S/P values just exceeding the manufacturer's cut-off (x̄ = 0.5). Whereas the remainder of positive sites (n = 61) had a high proportion of samples (76.3 %) with high S/P values (x̄ = 6.6). Thus, the manufacturer's cut-off (S/P ≥ 0.4) is appropriate for diagnostic applications, but a cut-off of S/P ≥ 1.0 provided the higher specificity required for surveillance. A previously unreported finding in this study was a statistically significant association between unexpected reactors and specific production sites and animal ages or stages. While beyond the scope of this study, these data suggested that certain animal husbandry or production practices may be associated with non-specific reactions.