A Survey of Current Activities and Technologies Used to Detect Carbapenem Resistance in Bacteria Isolated from Companion Animals at Veterinary Diagnostic Laboratories-United States, 2020.

Carbapenems are antimicrobial drugs reserved for the treatment of severe multidrug-resistant Gram-negative bacterial infections. Carbapenem-resistant organisms (CROs) are an urgent public health threat and have been made reportable to public health authorities in many jurisdictions. Recent reports of CROs in companion animals and veterinary settings suggest that CROs are a One Health problem. However, standard practices of U.S. veterinary diagnostic laboratories (VDLs) to detect CROs are unknown. We assessed the capacity of VDLs to characterize carbapenem resistance in isolates from companion animals. Among 74 VDLs surveyed in 42 states, 23 laboratories (31%) from 22 states responded. Most (22/23, 96%) included ≥1 carbapenem on their primary antimicrobial susceptibility testing panel, and approximately one-third (9/23, 39%) performed phenotypic carbapenemase production testing or molecular identification of carbapenemase genes. Overall, 35% (8/23) of VDLs across eight states reported they would notify public health if a CRO was detected. Most (17/21, 81%) VDLs were not aware of CRO reporting mandates, and some expressed uncertainty about whether the scope of known mandates included CROs from veterinary sources. Although nearly all surveyed VDLs tested for carbapenem resistance, fewer had the capacity for mechanism testing or awareness of public health reporting requirements. Addressing these gaps is critical to monitoring CRO incidence and trends in veterinary medicine, preventing spread in veterinary settings, and mounting an effective One Health response. Improved collaboration and communication between public health and veterinary medicine is critical to inform infection control practices in veterinary settings and conduct a public health response when resistant isolates are detected.

Ocular disease in horses with confirmed ocular or central nervous system Borrelia infection: Case series and review of literature.

OBJECTIVE: To describe the clinical presentation, treatment, and clinical outcome of horses with ocular disease and evidence of systemic or ocular Lyme disease. ANIMALS STUDIED: Five horses met the inclusion criteria of ocular disease with evidence of B burgdorferi present in ocular or CNS tissues. PROCEDURE: The goal of this study was to describe the clinical presentation and progression of ocular disease when associated with ocular or CNS B burgdorferi infection in horses. A retrospective review of medical records was performed on horses admitted for ocular disease with evidence of B burgdorferi infection between 1998 and 2015. The diagnosis of B burgdorferi-associated uveitis was based on histopathologic lesions of lymphohistiocytic and suppurative uveitis/endophthalmitis and intralesional argyrophilic spirochetes in either ocular or CNS tissue consistent with Borrelia. Leptospiral uveitis was ruled out by PCR. RESULTS: All five horses in the current study had intraocular inflammation at the time of presentation. Medical management with anti-inflammatories was successful in controlling uveitis in the two horses in which treatment of uveitis was attempted. Systemic treatment with oral tetracyclines was unsuccessful in a single case in which treatment of Borrelia was attempted. Four horses were euthanized due to progression of neurologic disease. The surviving horse had an enucleation performed and did not show systemic signs. CONCLUSIONS: Infection with Borrelia burgdorferi should be considered in endemic areas as a differential for horses with ocular disease, in particular, uveitis. The prognosis for uveitis and neurologic disease associated with Lyme disease was poor in the current study.

Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

Detection and Characterization of Salmonella spp. in Raw Commingled Bulk Tank Milk from Dairies in Pennsylvania.

A microbiological survey was conducted to determine the presence of Salmonella spp. in raw commingled bulk tank milk (BTM) collected from the Pennsylvania dairies intended for pasteurization. The survey found 8.1% (10/123) of samples positive for Salmonella. Salmonella Cerro was the predominant serovar and genetic analysis of the Salmonella Cerro showed the existence of diverse yet closely related genotypes. Antibiotic susceptibility testing conducted on all isolates showed pan-susceptible pattern against 15 drugs covering 9 drug classes. The study helped determine the presence of Salmonella spp. in the commingled BTM, antibiotic susceptibility patterns, and serovars along with genotypic diversity among the predominant serovar, Salmonella Cerro.

CRISPR Typing and Antibiotic Resistance Correlates with Polyphyletic Distribution in Human Isolates of Salmonella Kentucky.

Although infrequently associated with reported salmonellosis in humans, Salmonella enterica, subsp. enterica serovar Kentucky (ser. Kentucky) is the most common nonclinical, nonhuman serovar reported in the United States. The goal of this study was to use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-multi-virulence-locus sequence typing (MVLST) to subtype a collection of human clinical isolates of ser. Kentucky submitted to the Pennsylvania Department of Health and to determine the extent of antibiotic resistance in these strains. This analysis highlighted the polyphyletic nature of ser. Kentucky, and separated our isolates into two groups, Group I and Group II, which were equally represented in our collection. Furthermore, antimicrobial susceptibility testing on all isolates using a National Antimicrobial Resistance Monitoring System (NARMS) panel of antibiotics demonstrated that resistance profiles could be divided into two groups. Group I isolates were resistant to cephems and penicillins, whereas Group II isolates were resistant to quinolones, gentamicin, and sulfisoxazole. Collectively, 50% of isolates were resistant to three or more classes of antibiotics and 30% were resistant to five or more classes. The correlation of antibiotic resistance with the two different lineages may reflect adaptation within two distinct reservoirs of ser. Kentucky, with differential exposure to antimicrobials.

Evaluation of Three Bacterial Identification Systems for Species Identification of Bacteria Isolated from Bovine Mastitis and Bulk Tank Milk Samples.

A study was conducted to evaluate Sensititre® Automated Reading and Incubation System 2x System (ARIS), API® (API), and Bruker MALDI-TOF MS (MALDI) bacterial species identification systems using 132 diverse bacterial isolates from bovine milk samples and bulk tank milk received at the Penn State Animal Diagnostic Laboratory. The results were compared with 16S rRNA gene sequence analysis, which served as the reference method for species identification. The ARIS, API, and MALDI identified 0%, 40%, and 33.4% of species classified as Gram-positive rod isolates belonging to genera Arthrobacter, Bacillus, Brachybacterium, Brevibacterium, and Corynebacterium, respectively. It was observed that 76.5%, 93.9%, and 96.9% of catalase-negative, Gram-positive cocci (n = 33; Aerococcus, Enterococcus, Lactococcus, Streptococcus) were correctly identified to the species level by ARIS, API, and MALDI, respectively, while 33.4%, 84.5%, and 97.7% of catalase-positive, Gram-positive cocci (n = 45; Kocuria, Staphylococcus) were correctly identified to their species by ARIS, API, and MALDI, respectively. A total of 48 isolates (Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Pantoea, Pasteurella, Providencia, Pseduomonas, Serratia) of Gram-negative bacteria were examined, of which 85.4%, 93.7%, and 95.8% of the isolates were correctly identified to the species level by ARIS, API, and MALDI, respectively. In our laboratory, the MALDI had the least costs associated with consumables and reagents compared to ARIS, API, and 16S rRNA identification methods. Identification of bacterial species was accomplished in <2 h using MALDI and 24 h for ARIS, API, and 16S rRNA identification systems.

Statewide surveillance of Ixodes scapularis (Acari: Ixodidae) for the presence of the human pathogen Borrelia miyamotoi (Spirochaetales: Spirochaetaceae), a relapsing fever spirochete in Pennsylvania, USA, 2019-2020.

Borrelia miyamotoi disease is an emerging tick-borne human illness in the United States caused by Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) bacterium. With Pennsylvania reporting thousands of tick-borne disease cases annually, determining the minimum infection rate (MIR) of B. miyamotoi in Ixodes scapularis (Say, Acari: Ixodidae) adults within Pennsylvania is of utmost importance. Active surveillance was performed from October 2019 to April 2020 to collect a minimum of 50 I. scapularis ticks from every county within Pennsylvania and then screened for B. miyamotoi via qPCR. Ticks were collected from all 67 counties with the majority of those being adult I. scapularis. Additional ticks collected were Dermacentor albipictus (Packard, Acari: Ixodidae), Haemaphysalis longicornis (Neumann, Acari: Ixodidae), and immature I. scapularis. Adult I. scapularis were pooled and tested for B. miyamotoi. MIR for positive B. miyamotoi pools and density of infected adult I. scapularis varied by county, with positive pools from 38 Pennsylvania counties. This is the first statewide evaluation of B. miyamotoi in Pennsylvania in questing adult I. scapularis. These prevalence and distribution data will aid health care practitioners within the state of Pennsylvania and the northeast United States to understand potential risk and bring awareness to the lesser known human Borrelia illness, Borrelia miyamotoi disease.

Molecular Characterization of Non-H5 and Non-H7 Avian Influenza Viruses from Non-Mallard Migratory Waterbirds of the North American Flyways, 2006-2011.

The surveillance of migratory waterbirds (MWs) for avian influenza virus (AIV) is indispensable for the early detection of a potential AIV incursion into poultry. Surveying AIV infections and virus subtypes in understudied MW species could elucidate their role in AIV ecology. Oropharyngeal-cloacal (OPC) swabs were collected from non-mallard MWs between 2006 and 2011. OPC swabs (n = 1158) that molecularly tested positive for AIV (Cts ≤ 32) but tested negative for H5 and H7 subtypes were selected for virus isolation (VI). The selected samples evenly represented birds from all four North American flyways (Pacific, Central, Mississippi, and Atlantic). Eighty-seven low pathogenic AIV isolates, representing 31 sites in 17 states, were recovered from the samples. All isolates belonged to the North American lineage. The samples representing birds from the Central Flyway had the highest VI positive rate (57.5%) compared to those from the other flyways (10.3-17.2%), suggesting that future surveillance can focus on the Central Flyway. Of the isolates, 43.7%, 12.6%, and 10.3% were obtained from blue-winged teal, American wigeon, and American black duck species, respectively. Hatch-year MWs represented the majority of the isolates (70.1%). The most common H and N combinations were H3N8 (23.0%), H4N6 (18.4%), and H4N8 (18.4%). The HA gene between non-mallard and mallard MW isolates during the same time period shared 85.5-99.5% H3 identity and 89.3-99.7% H4 identity. Comparisons between MW (mallard and non-mallard) and poultry H3 and H4 isolates also revealed high similarity (79.0-99.0% and 88.7-98.4%), emphasizing the need for continued AIV surveillance in MWs.

Inter-laboratory comparison of real-time quaking-induced conversion (RT-QuIC) for the detection of chronic wasting disease prions in white-tailed deer retropharyngeal lymph nodes.

The rapid geographic spread of chronic wasting disease (CWD) in white-tailed deer (WTD; Odocoileus virginianus) increases the need for the development and validation of new detection tests. Real-time quaking-induced conversion (RT-QuIC) has emerged as a sensitive tool for CWD prion detection, but federal approval in the United States has been challenged by practical constraints on validation and uncertainty surrounding RT-QuIC robustness between laboratories. To evaluate the effect of inter-laboratory variation on CWD prion detection using RT-QuIC, we conducted a multi-institution comparison on a shared anonymized sample set. We hypothesized that RT-QuIC can accurately and reliably detect the prions that cause CWD in postmortem samples from medial retropharyngeal lymph node (RPLN) tissue despite variation in laboratory protocols. Laboratories from 6 U.S. states (Michigan, Minnesota, Missouri, New York, Pennsylvania, Wisconsin) were enlisted to compare the use of RT-QuIC in determining CWD prion status (positive or negative) among 50 anonymized RPLNs of known prion status. Our sample set included animals of 3 codon 96 WTD genotypes known to affect CWD progression and detection (G96G, G96S, S96S). All 6 laboratories successfully identified the true disease status consistently for all 3 tested codon 96 genotypes. Our results indicate that RT-QuIC is a suitable test for the detection of CWD prions in RPLN tissues in several genotypes of WTD.

Improved molecular detection of Neorickettsia risticii with a duplex real-time PCR assay in the diagnosis of Potomac horse fever.

Neorickettsia risticii, an obligate intracellular bacterium, is the causative agent of Potomac horse fever (PHF). Diagnosis of PHF is based on demonstration of serum antibodies, isolation of N. risticii, and/or detection of nucleic acid by a PCR assay. An existing real-time PCR assay targeting the N. risticii 16S rRNA has been validated using blood samples from horses with colitis, and snails; to our knowledge, the performance of the assay for other sample types has not been reported. We describe here a modification of the 16S rRNA gene assay by the addition of a set of primers and probe targeting the N. risticii p51 gene to form a duplex assay. We validated the new assay using diagnostic specimens from 56 horses with suspected PHF. The assay consistently detected down to 5 copies of synthetic targets, and did not show any cross-reaction with common equine enteric pathogens. Although we did not establish the diagnostic sensitivity and specificity of the duplex assay, results for both gene targets were in complete agreement, with the exception of 4 fecal samples that tested positive for the 16S rRNA gene only. Further analysis indicated that testing of fecal samples using our 16S rRNA gene assay alone can produce a false-positive result.

SARS-CoV-2 Infection Dynamics in the Pittsburgh Zoo Wild Felids with Two Viral Variants (Delta and Alpha) during the 2021-2022 Pandemic in the United States.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported in multiple animal species besides humans. The goal of this study was to report clinical signs, infection progression, virus detection and antibody response in a group of wild felids housed in adjacent but neighboring areas at the Pittsburgh Zoo. Initially, five African lions (Panthera leo krugeri) housed together exhibited respiratory clinical signs with viral shedding in their feces in March of 2021 coinciding with infection of an animal keeper. During the second infection wave in December 2021, four Amur tigers (Panthera tigris altaica) and a Canadian lynx (Lynx canadensis) showed clinical signs and tested positive for viral RNA in feces. In infected animals, viral shedding in feces was variable lasting up to 5 weeks and clinical signs were observed for up to 4 weeks. Despite mounting an antibody response to initial exposure, lions exhibited respiratory clinical signs during the second infection wave, but none shed the virus in their feces. The lions were positive for alpha variant (B.1.1.7 lineage) during the first wave and the tiger and lynx were positive for delta variant (AY.25.1. lineage) during the second wave. The viruses recovered from felids were closely related to variants circulating in human populations at the time of the infection. Cheetahs (Acinonyx jubatus) in the park did not show either the clinical signs or the antibody response.

SARS-CoV-2 Prevalence and Variant Surveillance among Cats in Pittsburgh, Pennsylvania, USA.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects many mammals, and SARS-CoV-2 circulation in nonhuman animals may increase the risk of novel variant emergence. Cats are highly susceptible to SARS-CoV-2 infection, and there were cases of virus transmission between cats and humans. The objective of this study was to assess the prevalence of SARS-CoV-2 variant infection of cats in an urban setting. We investigated the prevalence of SARS-CoV-2 variant infections in domestic and community cats in the city of Pittsburgh (n = 272). While no cats tested positive for SARS-CoV-2 viral RNA, 35 cats (12.86%) tested SARS-CoV-2-antibody-positive. Further, we compared a cat-specific experimental lateral flow assay (eLFA) and species-agnostic surrogate virus neutralization assay (sVNT) for SARS-CoV-2 antibody detection in cats (n = 71). The eLFA demonstrated 100% specificity compared to sVNT. The eLFA also showed 100% sensitivity for sera with >90% inhibition and 63.63% sensitivity for sera with 40-89% inhibition in sVNT. Using a variant-specific pseudovirus neutralization assay (pVNT) and antigen cartography, we found the presence of antibodies to pre-Omicron and Omicron SARS-CoV-2 variants. Hence, this approach proves valuable in identifying cat exposure to different SARS-CoV-2 variants. Our results highlight the continued exposure of cats to SARS-CoV-2 and warrant coordinated surveillance efforts.

Prevalence of Salmonella cerro in laboratory-based submissions of cattle and comparison with human infections in Pennsylvania, 2005-2010.

The aim of this study was to identify Salmonella serotypes infecting cattle in Pennsylvania, to compare infection rates for the predominant serotype, Salmonella enterica serotype Cerro, with the infection rates for the same serotype in humans, and to study the clonal diversity and antimicrobial resistance for this serotype in cattle from 2005 to 2010. Clonal diversity among the selected isolates was studied using pulsed-field gel electrophoresis (PFGE) and repetitive (rep)-polymerase chain reaction (PCR). Salmonella Cerro showed the single largest increase as a cause of cattle infections over the study period. The proportional distribution of Salmonella Cerro serotype among laboratory-submitted Salmonella positive cases in cattle was 36.1% in the year 2010 compared to 14.3% in 2005. A simultaneous decrease in serotype Newport infections was also observed in cattle (25% in 2005, to 10.1% in 2010). Studies of clonal diversity for cattle and human isolates revealed a predominant PFGE type but showed some variability. All tested isolates (n = 60) were susceptible to sulfamethoxazole-trimethoprim, but 2% of cattle isolates (n = 1/50) and 20% of human isolates (n = 2/10) showed resistance to tetracycline and sulfisoxazole. One human isolate showed additional resistance to ampicillin and gentamicin. This study suggests an increase in Salmonella Cerro infections in the cattle population and a decrease in Salmonella Newport infections. The increase in Cerro infections appears to be restricted to the cattle population, but occasional human infections occur.

Prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infection in horses from Pennsylvania (2017-2019) using antibody and organism-based detection.

OBJECTIVE: To determine the prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infections in Pennsylvania horses. ANIMALS: 271 horses. PROCEDURES: A survey was conducted with PCR and serology to evaluate anaplasmosis and Lyme disease infections in horses from Pennsylvania that were suspected for tick-borne infection. RESULTS: A phagocytophilum was detected in 19/271 (7.0%) Pennsylvania horses tested by the duplex PCR. B burgdorferi was not detected in any horse blood tested by PCR. Overall, 120/271 (44.3%) horses tested positive for presence of A phagocytophilum antibodies by at least the IDEXX SNAP 4Dx Plus lateral flow immunosorbent (SNAP) or indirect fluorescent antibody (IFA) assay, with 69 (25.5%) testing positive by both SNAP and IFA; 43 (15.9%) tested positive by IFA only, and 8 (3.0%) tested positive by SNAP only. Similarly, 209/271 (77.1%) horses tested positive for the presence of B burgdorferi antibodies by at least 1 test, with 139 (51.3%) testing positive by both SNAP and IFA; 45 (16.6%) tested positive by SNAP only, and 25 (9.2%) tested positive by IFA. CLINICAL RELEVANCE: Both A phagocytophilum and B burgdorferi are important tick-borne infections. The study provides prevalence data for both A phagocytophilum and B burgdorferi and compares test performance. For serologic detection, IFA detected antibodies to A phagocytophilum in a higher proportion (41.3%) of horses compared to SNAP (28.4%), while SNAP detected antibodies to B burgdorferi in a higher proportion (67.9%) of horses compared to IFA (60.5%). Both diseases showed a high seroprevalence in all areas surveyed.

Detection of chronic wasting disease in feces and recto-anal mucosal associated lymphoid tissues with RT-QuIC in a naturally infected farmed white-tailed deer herd.

Chronic wasting disease (CWD) is an infectious prion disease affecting the cervids, including white-tailed deer (WTD) (Odocoileus virginianus). CWD is typically diagnosed postmortem in farmed cervids by immunohistochemistry (IHC). Amplification-based detection methods are newer generation tests currently being evaluated to improve the detection of prion disease. In addition to improving sensitivity, antemortem detection by amplification assays is a focus for improving disease control and management. In this study, we evaluate the use of real-time quaking-induced conversion (RT-QuIC) to detect CWD in fecal and recto-anal mucosal-associated lymphoid tissue (RAMALT) samples from naturally infected farmed WTD herds at postmortem. We successfully detected the presence of CWD prions in WTD RAMALT with a specificity of 100% and a sensitivity of 85.7% (n = 71) and in feces with a specificity of 100% and a sensitivity of 60% (n = 69), utilizing RT-QuIC on samples collected postmortem. Seeding activity detected in RAMALT (15.3 ± 4.2%, n = 18) was much stronger than in feces (44.4 ± 4.2%, n = 15), as measured by cycle threshold (Ct) and rise in relative fluorescence in samples collected from the same WTD. Prion detection in the RAMALT (94.7%) and feces (70.5%) was highest when both obex and retropharyngeal lymph nodes (RPLNs) were positive for CWD via IHC. In the study group, we were also able to test prion protein gene variants and associated disease susceptibility. A majority of tested WTD were the CWD genotype (96 GG) and also harbored the highest percentage of positive animals (43.7%). The second highest population of WTD was the genotype 96 GS and had a CWD positivity rate of 37.5%. Each of these groups showed no difference in RAMALT or fecal detection of CWD.

Transmission of SARS-CoV-2 from humans to a 16-year-old domestic cat with comorbidities in Pennsylvania, USA.

BACKGROUND AND OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), besides causing human infection, has been shown to naturally infect several susceptible animal species including large cats (tigers, lions, pumas, spotted leopards), dogs, cats, ferrets, gorillas and minks. Cats and minks are continuing to be the most reported species with SARS-CoV-2 infections among animals but it needs to be investigated further. METHODS AND RESULTS: We report the detection of SARS-CoV-2 from a domestic cat that exhibited respiratory disease after being exposed to SARS-CoV-2 virus from humans in the same household. SARS-CoV-2 RNA was detected in two oropharyngeal swabs collected at two time points, 11 days apart; the first, when the cat was reported to be sick and the second, before euthanasia due to poor prognosis. The viral nucleic acid detected at two time points showed no genomic variation and resembled the clade GH circulating in humans in the United States. Clinical and pathological findings noted in this 16-year-old cat were consistent with respiratory and cardiac insufficiency. CONCLUSIONS: SARS-CoV-2 viral infection was likely an incidental clinical finding, as the virus was not detected in fixed lungs, heart, or kidney tissues. Only fresh lung tissue collected at necropsy showed the presence of viral nucleic acid, albeit at a very low level. Further research is needed to clarify the clinical course of SARS-CoV-2 in companion animals of advanced age and underlying cardiac disease.

Development and Validation of Indirect Enzyme-Linked Immunosorbent Assays for Detecting Antibodies to SARS-CoV-2 in Cattle, Swine, and Chicken.

Multiple domestic and wild animal species are susceptible to SARS-CoV-2 infection. Cattle and swine are susceptible to experimental SARS-CoV-2 infection. The unchecked transmission of SARS-CoV-2 in animal hosts could lead to virus adaptation and the emergence of novel variants. In addition, the spillover and subsequent adaptation of SARS-CoV-2 in livestock could significantly impact food security as well as animal and public health. Therefore, it is essential to monitor livestock species for SARS-CoV-2 spillover. We developed and optimized species-specific indirect ELISAs (iELISAs) to detect anti-SARS-CoV-2 antibodies in cattle, swine, and chickens using the spike protein receptor-binding domain (RBD) antigen. Serum samples collected prior to the COVID-19 pandemic were used to determine the cut-off threshold. RBD hyperimmunized sera from cattle (n = 3), swine (n = 6), and chicken (n = 3) were used as the positive controls. The iELISAs were evaluated compared to a live virus neutralization test using cattle (n = 150), swine (n = 150), and chicken (n = 150) serum samples collected during the COVID-19 pandemic. The iELISAs for cattle, swine, and chicken were found to have 100% sensitivity and specificity. These tools facilitate the surveillance that is necessary to quickly identify spillovers into the three most important agricultural species worldwide.

Detection by real-time quaking-induced conversion (RT-QuIC), ELISA, and IHC of chronic wasting disease prion in lymph nodes from Pennsylvania white-tailed deer with specific PRNP genotypes.

Chronic wasting disease (CWD) is a prion-mediated, transmissible disease of cervids, including deer (Odocoileus spp.), which is characterized by spongiform encephalopathy and death of the prion-infected animals. Official surveillance in the United States using immunohistochemistry (IHC) and ELISA entails the laborious collection of lymphoid and/or brainstem tissue after death. New, highly sensitive prion detection methods, such as real-time quaking-induced conversion (RT-QuIC), have shown promise in detecting abnormal prions from both antemortem and postmortem specimens. We compared RT-QuIC with ELISA and IHC for CWD detection utilizing deer retropharyngeal lymph node (RLN) tissues in a diagnostic laboratory setting. The RLNs were collected postmortem from hunter-harvested animals. RT-QuIC showed 100% sensitivity and specificity for 50 deer RLN (35 positive by both IHC and ELISA, 15 negative) included in our study. All deer were also genotyped for PRNP polymorphism. Most deer were homozygous at codons 95, 96, 116, and 226 (QQ/GG/AA/QQ genotype, with frequency 0.86), which are the codons implicated in disease susceptibility. Heterozygosity was noticed in Pennsylvania deer, albeit at a very low frequency, for codons 95GS (0.06) and 96QH (0.08), but deer with these genotypes were still found to be CWD prion-infected.

Evaluation of a high-throughput nucleic acid extraction method for the detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples by PCR.

Johne's disease (paratuberculosis) is an economically important disease of cattle worldwide. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious gram-positive bacterium. PCR is increasingly used in diagnostic laboratories for the detection of MAP in fecal samples given the rapid test turnaround time and sensitivity and specificity comparable to fecal culture. However, efficient extraction of DNA for sensitive detection of MAP by PCR is affected by the complex lipid-rich cell wall of MAP and the presence of PCR inhibitors in feces. We evaluated a high-throughput nucleic acid extraction method (MagMAX core nucleic acid purification kit with mechanical lysis module) in conjunction with an hspX gene PCR for the detection of MAP from bovine fecal samples, which resulted in correct identification of all negative (13 of 13) and positive (35 of 35) proficiency test samples obtained from the National Veterinary Services Laboratories. In addition, all 6 negative and 50 of 51 positive diagnostic specimens tested were categorized correctly.

Detection of Anaplasma Phagocytophilum in Horses With Suspected Tick-Borne Disease in Northeastern United States by Metagenomic Sequencing.

Metagenomic sequencing of clinical diagnostic specimens has a potential for unbiased detection of infectious agents, diagnosis of polymicrobial infections and discovery of emerging pathogens. Herein, next generation sequencing (NGS)-based metagenomic approach was used to investigate the cause of illness in a subset of horses recruited for a tick-borne disease surveillance study during 2017-2019. Blood samples collected from 10 horses with suspected tick-borne infection and five apparently healthy horses were subjected to metagenomic analysis. Total genomic DNA extracted from the blood samples were enriched for microbial DNA and subjected to shotgun next generation sequencing using Nextera DNA Flex library preparation kit and V2 chemistry sequencing kit on the Illumina MiSeq sequencing platform. Overall, 0.4-0.6 million reads per sample were analyzed using Kraken metagenomic sequence classification program. The taxonomic classification of the reads indicated that bacterial genomes were overrepresented (0.5 to 1%) among the total microbial reads. Most of the bacterial reads (~91%) belonged to phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Tenericutes in both groups. Importantly, 10-42.5% of Alphaproteobacterial reads in 5 of 10 animals with suspected tick-borne infection were identified as Anaplasma phagocytophilum. Of the 5 animals positive for A. phagocytophilum sequence reads, four animals tested A. phagocytophilum positive by PCR. Two animals with suspected tick-borne infection and A. phagocytophilum positive by PCR were found negative for any tick-borne microbial reads by metagenomic analysis. The present study demonstrates the usefulness of the NGS-based metagenomic analysis approach for the detection of blood-borne microbes.