Experimental co-infection of calves with SARS-CoV-2 Delta and Omicron variants of concern.

Since emerging in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly crossed the species barrier with natural infections reported in various domestic and wild animal species. The emergence and global spread of SARS-CoV-2 variants of concern (VOCs) has expanded the range of susceptible host species. Previous experimental infection studies in cattle using Wuhan-like SARS-CoV-2 isolates suggested that cattle were not likely amplifying hosts for SARS-CoV-2. However, SARS-CoV-2 sero- and RNA-positive cattle have since been identified in Europe, India, and Africa. Here, we investigated the susceptibility and transmission of the Delta and Omicron SARS-CoV-2 VOCs in cattle. Eight Holstein calves were co-infected orally and intranasally with a mixed inoculum of SARS-CoV-2 VOCs Delta and Omicron BA.2. Twenty-four hours post-challenge, two sentinel calves were introduced to evaluate virus transmission. The co-infection resulted in a high proportion of calves shedding SARS-CoV-2 RNA at 1- and 2-days post-challenge (DPC). Extensive tissue distribution of SARS-CoV-2 RNA was observed at 3 and 7 DPC and infectious virus was recovered from two calves at 3 DPC. Next-generation sequencing revealed that only the SARS-CoV-2 Delta variant was detected in clinical samples and tissues. Similar to previous experimental infection studies in cattle, we observed only limited seroconversion and no clear evidence of transmission to sentinel calves. Together, our findings suggest that cattle are more permissive to infection with SARS-CoV-2 Delta than Omicron BA.2 and Wuhan-like isolates but, in the absence of horizontal transmission, are not likely to be reservoir hosts for currently circulating SARS-CoV-2 variants.

Validation of a real-time PCR panel for detection and quantification of nine pathogens commonly associated with canine infectious respiratory disease.

Canine infectious respiratory disease (CIRD) is a complicated respiratory syndrome in dogs [1], [2], [3]. A panel PCR was developed [4] to detect nine pathogens commonly associated with CIRD: Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica; canine adenovirus type 2, canine herpesvirus 1, canine parainfluenza virus, canine distemper virus, canine influenza virus and canine respiratory coronavirus [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. To evaluate diagnostic performance of the assay, 740 nasal swab and lung tissue samples were collected and tested with the new assay, and compared to an older version of the assay detecting the same pathogens except that it does not differentiate the two Mycoplasma species. Results indicated that the new assay had the same level of specificity, but with higher diagnostic sensitivity and had identified additional samples with potential co-infections. To confirm the new assay is detecting the correct pathogens, samples with discrepant results between the two assays were sequence-confirmed. Spiking a high concertation target to samples carrying lower concentrations of other targets was carried out and the results demonstrated that there was no apparent interference among targets in the same PCR reaction. Another spike-in experiment was used to determine detection sensitivity between nasal swab and lung tissue samples, and similar results were obtained.•A nine-pathogen CIRD PCR panel assay had identified 139 positives from 740 clinical samples with 60 co-infections;•High-concentration target does not have apparent effect on detecting low-concentration targets;•Detection sensitivity were similar between nasal swab and lung tissue samples.

Isolation of porcine reproductive and respiratory syndrome virus from feed ingredients and complete feed, with subsequent RT-qPCR analysis.

We used virus isolation (VI) to determine tissue culture infectivity and reverse-transcription quantitative PCR (RT-qPCR) to determine the stability of porcine reproductive and respiratory syndrome virus 2 (PRRSV) strain P129 in solvent-extracted soybean meal (SBM), dried distillers grains with solubles (DDGS), complete swine feed (FEED), or medium (DMEM) at 4°C, 23°C, or 37°C for up to 3 d. Samples of each treatment were taken at regular intervals and processed. Supernatant was titrated and used to inoculate confluent MARC-145 cells to determine infectivity. RNA was extracted from each supernatant sample and tested by RT-qPCR to determine any change in detectable virus RNA across matrix type, temperature, and time. An interaction (p = 0.028) was observed for matrix × temperature × hour for live virus detected by VI. At 4°C, the concentration of infectious virus was greatest in DMEM, intermediate in SBM, and lowest in DDGS and FEED. DMEM also had the greatest concentration of infectious PRRSV at 23°C over time; a higher infectious virus concentration was maintained in SBM for longer than in DDGS or FEED. At 37°C, a greater concentration of infectious virus was sustained in DMEM than in the feedstuffs, with concentrations decreasing until 48 h post-inoculation. Only matrix type influenced the quantity of viral RNA detected by RT-qPCR (p = 0.032). More viral RNA was detected in the virus control than in DDGS; SBM and FEED were intermediate. By VI, we found that infectious virus could be harbored in SBM, DDGS, and FEED for a short time.

2023 International African Swine Fever Workshop: Critical Issues That Need to Be Addressed for ASF Control.

The 2023 International African Swine Fever Workshop (IASFW) took place in Beijing, China, on 18-20 September 2023. It was jointly organized by the U.S.-China Center for Animal Health (USCCAH) at Kansas State University (KSU) and the Chinese Veterinary Drug Association (CVDA) and sponsored by the United States Department of Agriculture Foreign Agricultural Service (USDA-FAS), Harbin Veterinary Research Institute, and Zoetis Inc. The objective of this workshop was to provide a platform for ASF researchers around the world to unite and share their knowledge and expertise on ASF control and prevention. A total of 24 outstanding ASF research scientists and experts from 10 countries attended this meeting. The workshop included presentations on current ASF research, opportunities for scientific collaboration, and discussions of lessons and experiences learned from China/Asia, Africa, and Europe. This article summarizes the meeting highlights and presents some critical issues that need to be addressed for ASF control and prevention in the future.

Development of an Indirect ELISA for the Detection of SARS-CoV-2 Antibodies in Cats.

Companion animals are susceptible to a variety of coronaviruses, and recent studies show that felines are highly susceptible to SARS-CoV-2 infection. RT-PCR diagnostic is currently the method of choice to detect the presence of SARS-CoV-2-specific viral nucleic acids in animal samples during an active infection; however, serological assays are critical to determine whether animals were exposed to the virus and to determine the seroprevalence of SARS-CoV-2-specific antibodies in a defined population. In this study, we utilized recombinant nucleocapsid (N) protein and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 expressed in E. coli (N) and mammalian cells (N, RBD) to develop indirect ELISA (iELISA) tests using well-characterized SARS-CoV-2-positive and -negative cat serum panels from previous experimental cat challenge studies. The optimal conditions for the iELISA tests were established based on checkerboard dilutions of antigens and antibodies. The diagnostic sensitivity for the detection of feline antibodies specific for the N or RBD proteins of the iELISA tests was between 93.3 and 97.8%, respectively, and the diagnostic specificity 95.5%. The iELISAs developed here can be used for high-throughput screening of cat sera for both antigens. The presence of SARS-CoV-2-specific antibodies in a BSL-2 biocontainment environment, unlike virus neutralization tests with live virus which have to be performed in BSL-3 laboratories.

Development of a three-panel multiplex real-time PCR assay for simultaneous detection of nine canine respiratory pathogens.

Infectious respiratory disease is one of the most common diseases in dogs worldwide. Several bacterial and viral pathogens can serve as causative agents of canine infectious respiratory disease (CIRD), including Mycoplasma cynos, Mycoplasma canis, Bordetella bronchiseptica, canine adenovirus type 2 (CAdV-2), canine herpesvirus 1 (CHV-1), canine parainfluenza virus (CPIV), canine distemper virus (CDV), canine influenza virus (CIA) and canine respiratory coronavirus (CRCoV). Since these organisms cause similar clinical symptoms, disease diagnosis based on symptoms alone can be difficult. Therefore, a quick and accurate test is necessary to rapidly identify the presence and relative concentrations of causative CIRD agents. In this study, a multiplex real-time PCR panel assay was developed and composed of three subpanels for detection of the aforementioned pathogens. Correlation coefficients (R2) were >0.993 for all singleplex and multiplex real-time PCR assays with the exception of one that was 0.988; PCR amplification efficiencies (E) were between 92.1% and 107.8% for plasmid DNA, and 90.6-103.9% for RNA templates. In comparing singular and multiplex PCR assays, the three multiplex reactions generated similar R2 and E values to those by corresponding singular reactions, suggesting that multiplexing did not interfere with the detection sensitivities. The limit of detection (LOD) of the multiplex real-time PCR for DNA templates was 5, 2, 3, 1, 1, 1, 4, 24 and 10 copies per microliter for M. cynos, M. canis, B. brochiseptica, CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively; and 3, 2, 6, 17, 4 and 8 copies per microliter for CAdV-2, CHV-1, CPIV, CDV, CIA and CRCoV, respectively, when RNA templates were used for the four RNA viruses. No cross-detection was observed among the nine pathogens. For the 740 clinical samples tested, the newly designed PCR assay showed higher diagnostic sensitivity compared to an older panel assay; pathogen identities from selected samples positive by the new assay but undetected by the older assay were confirmed by Sanger sequencing. Our data showed that the new assay has higher diagnostic sensitivity while maintaining the assay's specificity, as compared to the older version of the panel assay.

Susceptibility of sheep to experimental co-infection with the ancestral lineage of SARS-CoV-2 and its alpha variant.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 by experimental and/or natural infections. Sheep are a commonly farmed domestic ruminant that have not been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cells and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived kidney cells support SARS-CoV-2 replication. Furthermore, the experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs at 1 and 3-days post challenge (DPC); viral RNA was also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled naïve sheep was not highly efficient; however, viral RNA was detected in respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used a challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern, to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection and that the alpha variant outcompeted the lineage A strain.

Infection and transmission of ancestral SARS-CoV-2 and its alpha variant in pregnant white-tailed deer.

ABSTRACTSARS-CoV-2 was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks have demonstrated the significant role of intermediate hosts in viral maintenance and transmission. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (WTD) are amongst the most abundant and geographically widespread wild ruminant species in the US. Recently, WTD fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult WTD. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the alpha variant of concern (VOC) B.1.1.7 through co-infection of WTD. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult WTD are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in WTD, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from the genome of virus present in tissues of principal infected deer, fetuses and contact animals.

Susceptibility of sheep to experimental co-infection with the ancestral lineage of SARS-CoV-2 and its alpha variant.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 (COVID-19) in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 infection by experimental and/or natural infections. Domestic and large cats, mink, ferrets, hamsters, deer mice, white-tailed deer, and non-human primates have been shown to be highly susceptible, whereas other species such as mice, dogs, pigs, and cattle appear to be refractory to infection or have very limited susceptibility. Sheep (Ovis aries) are a commonly farmed domestic ruminant that have not previously been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cell cultures and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived cell cultures support SARS-CoV-2 replication. Furthermore, experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs primarily at 1-day post challenge (DPC), and also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was also observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled naive sheep was not highly efficient; hovewer, viral RNA was detected in some of the respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern (VOC), to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection, and that the alpha VOC outcompeted the ancestral lineage A strain.

Infection and transmission of ancestral SARS-CoV-2 and its alpha variant in pregnant white-tailed deer.

SARS-CoV-2, a novel Betacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult white-tailed deer are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.

Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories.

The exquisite sensitivity of in vitro amplification assays such as real-time polymerase chain reaction (rtPCR) requires the establishment of thorough and robust laboratory practices. To this end, an American Association of Veterinary Laboratory Diagnosticians (AAVLD) committee of subject matter experts was convened to develop a set of best practices for performance of nucleic acid amplification assays. Consensus advice for the performance of preanalytical, analytical, and postanalytical steps is presented here, along with a review of supporting literature.

Suggested guidelines for validation of real-time PCR assays in veterinary diagnostic laboratories.

This consensus document presents the suggested guidelines developed by the Laboratory Technology Committee (LTC) of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) for development, validation, and modification (methods comparability) of real-time PCR (rtPCR) assays. These suggested guidelines are presented with reference to the World Organisation for Animal Health (OIE) guidelines for validation of nucleic acid detection assays used in veterinary diagnostic laboratories. Additionally, our proposed practices are compared to the guidelines from the Foods Program Regulatory Subdivision of the U.S. Food and Drug Administration (FDA) and from the American Society for Veterinary Clinical Pathology (ASVCP). The LTC suggestions are closely aligned with those from the OIE and comply with version 2021-01 of the AAVLD Requirements for an Accredited Veterinary Medical Diagnostic Laboratory, although some LTC recommendations are more stringent and extend beyond the AAVLD requirements. LTC suggested guidelines are substantially different than the guidelines recently published by the U.S. FDA for validation and modification of regulated tests used for detection of pathogens in pet food and animal-derived products, such as dairy. Veterinary diagnostic laboratories that perform assays from the FDA Bacteriological Analytical Method (BAM) manual must be aware of the different standard.

Development of a differential multiplex real-time PCR assay for porcine circovirus type 2 (PCV2) genotypes PCV2a, PCV2b and PCV2d.

A multiplex quantitative real-time polymerase chain reaction (mqPCR) assay was developed and validated for detection and differentiation of porcine circovirus type 2 (PCV2) genotypes, PCV2a, PCV2b and PCV2d. Single nucleotide polymorphism in primers or probes was deployed for different genotype detections, while conserved sequence in the 3' end of a primer and in the middle of a probe was used for the targeted genotype. In silico analysis of 2601 PCV2 ORF2 sequences showed that the predicted strain coverage of the assay was 93.4 % (409/438) for PCV2a, 95.1 % (1161/1221) for PCV2b and 93.6 % (882/942) for PCV2d strains. The PCR amplification efficiencies were 94.5 %, 100.2 %, and 99.2 % for PCV2a, PCV2b and PCV2d, respectively, with correlation coefficients >0.995 for all genotypes. The limits of detection (LOD) were 1.58 × 10-4 TCID50/mL for PCV2a, 5.62 × 10-4 TCID50/mL for PCV2b, and 3.16 × 10-3 TCID50/mL for PCV2d. Sanger sequencing of 74 randomly selected PCV2 positive clinical samples confirmed the genotypes of strains identified by the mqPCR. Validation with clinical samples co-positive for target and non-target pathogens demonstrated that the mqPCR assay specifically detected targeted viruses without cross reacting to each other or to other common porcine viruses.

The NC229 multi-station research consortium on emerging viral diseases of swine: Solving stakeholder problems through innovative science and research.

The NC229 research consortium was created in 1999 in response to the emergence of porcine reproductive and respiratory syndrome virus (PRRSV), a viral agent responsible for devastating economic losses to the swine industry. The project follows the traditional "consortium" approach for Multistate Agricultural Research driven through the US State Agricultural Experiment Stations (SAES), wherein stakeholder-driven needs to combat swine infectious diseases are identified and scientific solutions pursued by combining funds from federal, state, commodity groups, and the animal health industry. The NC229 consortium was the main driving force in successfully competing for a USDA multi-station Coordinated Agricultural Project (PRRS CAP-I) in 2004-2008, immediately followed by a renewal for 2010-2014 (PRRS CAP-II)-, resulting in an overall record achievement of almost $10 million dollars. The CAP funding was not only useful for quality research, extension, and education in PRRS and related diseases, but also instrumental in enabling the group to leverage swine industry funding of more than $34 million dollars, distributed between creative research and extension on PRRS during the last 20 years. The North American/International PRRS Symposium, now recognized by the community as a highly effective platform for the exchange of basic research findings and fundamental translational technology, is directly derived from the NC229 consortium. Other significant offshoots from NC229 include the PHGC (PRRS Host Genomic Consortium), a platform for discoveries on the role of host genetics during PRRSV infection, since 2007. Since 2009, the NC229 consortium has expanded its collective research interests beyond PRRSV to include nine other emerging viral diseases of swine. In the current project (2019-2024), African Swine Fever Virus (ASFV) retains a central focus, with the goal of harnessing the group's expertise in promoting preparedness for the global control of ASFV.

Serologic investigation of exposure to influenza A virus H3N2 infection in dogs and cats in the United States.

We investigated, in a cross-sectional study, the prevalence of antibodies against canine influenza A virus (CIV) H3N2 in serum samples collected from dogs and cats using a commercial ELISA and a hemagglutination inhibition (HI) test. Samples were obtained from 519 cats and dogs from 13 states within the United States. Data were analyzed for potential risk factors with positive sera (vs. negative sera) by logistic regression. Odds ratios and their 95% confidence intervals (CIs) were calculated by exponentiation of the regression coefficients. Ten dogs (2.21%; 95% CI: 1.05-3.98%) and 6 cats (8.96%; 95% CI: 3.36-18.48%) tested seropositive for CIV H3N2 by HI. One feline sample (1.49%; 95% CI: 0.04-8.04%) and 16 canine samples (3.53%; 95% CI: 2.01-5.61%) tested seropositive by ELISA for influenza A virus. There were no apparent associations between seropositivity and putative risk factors. All positive animals were from Indiana or Illinois; however, CIV H3N2 seroprevalence was not common in Illinois and Indiana.

Immunogenicity of PtpA secreted during Mycobacterium avium ssp. paratuberculosis infection in cattle.

AIMS: Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease. To survive within host macrophages, the pathogen secretes a battery of proteins to interfere with the immunological response of the host. One of these proteins is tyrosine phosphate A (PtpA), which has been identified as a secreted protein critical for survival of its close relative M. tuberculosis within infected macrophages. METHODS AND RESULTS: In this study, the immune response to recombinant PtpA used as an antigen was investigated in a cohort of ∼1000 cows infected with MAP compared to negative control animals using ELISA. The sera from MAP-infected cows had significantly higher levels of antibodies against PtpA when compared to uninfected cows. CONCLUSIONS: The data presented here indicate that the antibodies produced against PtpA are sensitive enough to detect infected animals before the appearance of the disease symptoms. SIGNIFICANCE AND IMPACT OF STUDY: The use of PtpA as an antigen can be developed as an early diagnostic test. Moreover, PtpA is a candidate antigen for detection of humoral immune responses in cows infected with MAP.

Genomic Sequence of a Swine Pasivirus Type 1 Strain Identified in U.S. Swine.

We report for the first time in the United States the identification of a swine pasivirus (SPaV) strain with a genomic sequence identity of less than 80% to other SPaVs reported in Europe and China, using a next-generation sequencing (NGS) technique in sow tissues collected from an animal study conducted in 2001, suggesting virus circulation in domestic swine.

A cross sectional study evaluating the prevalence of Coxiella burnetii, potential risk factors for infection, and agreement between diagnostic methods in goats in Indiana.

Coxiella burnetii is the etiologic agent of the zoonotic disease Q fever and is considered to be endemic in domestic ruminants. Small ruminants in particular are important reservoirs for human infection. Serologic and molecular methods are both available for diagnosis of infection with C. burnetii, but there has been little research evaluating the prevalence of this organism in small ruminants outside of the context of clinical disease outbreaks. The objectives of this study were to estimate seroprevalence of C. burnetii and the prevalence of shedding of C. burnetii DNA in milk by goats in Indiana, USA, to evaluate potential risk factors for association with C. burnetii exposure and shedding, and to assess the level of agreement between the enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR) tests used to estimate prevalence. A total of 649 does over 1 year of age and not pregnant at the time of sampling were included in the study. Serum samples were collected from 608 does representing 89 farms. Milk samples were collected from 387 does representing 85 farms. Both milk and serum samples were collected from 356 does representing 80 farms. The estimated individual seroprevalence and shedding prevalence in milk adjusted for clustering were 3.1% (n=23/608, 95% CI: 1.2-7.0%) and 2.5% (n=9/387, 9.5% CI: 1.0-5.6%) respectively. Estimated adjusted herd level C. burnetii seroprevalence and herd level shedding prevalence were 11.5% (n=10/89, 95% CI: 6.4-20.1%) and 7.0% (n=6/85, 95% CI: 3.3-14.6%) respectively. Based on a generalized estimating equation model (GEE), meat breeds of goat had 7.0 times increased odds of shedding C. burnetii DNA in milk samples as compared to dairy breeds. Agreement between tests as determined by Cohen's kappa was poor at both the individual (kappa=0.04, 95% CI: -0.1 to 0.2) and herd (kappa=0.2, 95% CI: -0.1 to 0.5) levels. This indicates that serologic screening alone is unlikely to prevent the introduction of does shedding C. burnetii into herds.

Identification of several clades of novel single-stranded circular DNA viruses with conserved stem-loop structures in pig feces.

Metagenomic analysis of fecal samples collected from diarrheal swine detected sequences encoding a replication initiator protein (Rep). The genomes of ten novel single-stranded DNA viruses were determined, and they exhibited a similar genome organization. The two putative open reading frames (ORFs) encoding Rep and the capsid protein are bidirectionally transcribed and separated by two intergenic regions. Stem-loop structure(s) typical of genomes that undergo the rolling-circle DNA replication mechanism were observed. Phylogenetically, these ten genomes are in a monophyletic clade with the previously described porcine stool-associated virus (PoSCV) but are divergent enough to be further classified into to six distinct virus clades.