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.
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 (
= 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 (
= 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.
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.
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 (
= 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.
Salmonellosis is one of the most important foodborne diseases affecting humans. To characterize the relationship between Salmonella causing human infections and their food animal reservoirs, we ...compared pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility patterns of non-typhoidal Salmonella isolated from ill humans in Pennsylvania and from food animals before retail. Human clinical isolates were received from 2005 through 2011 during routine public health operations in Pennsylvania. Isolates from cattle, chickens, swine and turkeys were recovered during the same period from federally inspected slaughter and processing facilities in the northeastern United States. We found that subtyping Salmonella isolates by PFGE revealed differences in antimicrobial susceptibility patterns and, for human Salmonella , differences in sources and invasiveness that were not evident from serotyping alone. Sixteen of the 20 most common human Salmonella PFGE patterns were identified in Salmonella recovered from food animals. The most common human Salmonella PFGE pattern, Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS), was associated with more cases of invasive salmonellosis than all other patterns. In food animals, this pattern was almost exclusively (99%) found in Salmonella recovered from chickens and was present in poultry meat in every year of the study. Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS) was associated with susceptibility to all antimicrobial agents tested in 94.7% of human and 97.2% of food animal Salmonella isolates. In contrast, multidrug resistance (resistance to three or more classes of antimicrobial agents) was observed in five PFGE patterns. Typhimurium patterns JPXX01.0003 (JPXX01.0003 ARS) and JPXX01.0018 (JPXX01.0002 ARS), considered together, were associated with resistance to five or more classes of antimicrobial agents: a mpicillin, c hloramphenicol, s treptomycin, su lfonamides and t etracycline (ACSSuT), in 92% of human and 80% of food animal Salmonella isolates. The information from our study can assist in source attribution, outbreak investigations, and tailoring of interventions to maximize their impact on prevention.
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.
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.
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.
subspecies
, a zoonotic bacterial pathogen caused a series of outbreaks with high mortality affecting swine herds in multiple locations of the USA and Canada in 2019. Further genetic analysis ...revealed that this agent clustered with ATCC 35246, a
strain associated with high mortality outbreaks in swine herds of China originally reported in 1977. Rapid and accurate diagnosis is absolutely critical for controlling and limiting further spread of this emerging disease of swine. Currently available diagnostic methods including bacteriological examination and PCR assays do not distinguish between the virulent strains and avirulent commensal strains of
, which is critical given that this pathogen is a normal inhabitant of the swine respiratory tract. Based on comparative analyses of whole genome sequences of the virulent isolates and avirulent sequences, we identified a region in the
that is highly conserved and restricted to virulent
strains. We developed and validated a novel probe-based real-time PCR targeting the conserved region of
. The assay was highly sensitive and specific to the virulent swine isolates of
subspecies
. No cross reactivity was observed with avirulent
isolates as well as other streptococcal species and a panel of porcine respiratory bacterial and viral pathogens. The PCR efficiency of the assay was 96.64 % and was able to detect as little as 20 fg of the bacterial DNA. We then validated the diagnostic sensitivity and specificity of the new PCR assay using a panel of clinical samples (
= 57) and found that the assay has 100% sensitivity and specificity as compared to bacteriological culture method. In summary, the PCR assay will be an extremely valuable tool for the rapid accurate detection of virulent swine
isolates and directly from clinical samples.
(historically called
) causes infectious coryza (IC), which is an acute respiratory disease of chickens. Recently, outbreaks of IC have been reported in Pennsylvania (PA) in broilers, layer pullets, ...and laying hens, causing significant respiratory disease and production losses. A tentative diagnosis of IC can be made based on history, clinical signs, and characteristic gross lesions. However, isolation and identification of the organism are required for a definitive diagnosis. Major challenges with the bacteriological diagnosis of
include that the organism is difficult to isolate, slow-growing, and can only be successfully isolated during the acute stage of infection and secondary bacterial infections are also common. As there were very limited whole genomes of
in the public databases, we carried out whole-genome sequencing (WGS) of PA isolates and based on the WGS data analysis; we designed a novel probe-based PCR assay targeting a highly conserved sequence in the
, the DNA repair protein gene of
. The assay includes an internal control, with a limit of detection (LOD) of 3.93 genomic copies. The PCR efficiency ranged between 90 and 97%, and diagnostic sensitivity of 98.5% compared with conventional gel-based PCR. The test was highly specific, and no cross-reactivity was observed with other species of
and a range of other common poultry respiratory viral and bacterial pathogens. Real-time PCR testing on 419 clinical samples from suspected flocks yielded 94 positives and 365 negatives in agreement with diagnostic bacterial culture-based detection. We also compared the
PCR assay with a previous
based real-time PCR assay which showed a PCR efficiency of 79%.
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