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Zika virus (ZIKV) remains a public health concern, with epidemics in endemic regions and sporadic outbreaks in new areas posing significant threats. Several mosquito-borne flaviviruses that can cause human illness, including West Nile, Usutu, and St. Louis encephalitis, have associations with birds. However, the susceptibility of chickens to ZIKV and their role in viral epidemiology is not currently known. We investigated the susceptibility of chickens to experimental ZIKV infection using chickens ranging from 1-day-old chicks to 6-week-old birds. ZIKV caused no clinical signs in chickens of all age groups tested. Viral RNA was detected in the blood and tissues during the first 5 days post-inoculation in 1-day and 4-day-old chicks inoculated with a high viral dose, but ZIKV was undetectable in 6-week-old birds at all timepoints. Minimal antibody responses were observed in 6-week-old birds, and while present in younger chicks, they waned by 28 days post-infection. Innate immune responses varied significantly between age groups. Robust type I interferon and inflammasome responses were measured in older chickens, while limited innate immune activation was observed in younger chicks. Signal transducer and activator of transcription 2 (STAT2) is a major driver of host restriction to ZIKV, and chicken STAT2 is distinct from human STAT2, potentially contributing to the observed resistance to ZIKV infection. The rapid clearance of the virus in older chickens coincided with an effective innate immune response, highlighting age-dependent susceptibility. Our study indicates that chickens are not susceptible to productive ZIKV infection and are unlikely to play a role in the ZIKV epidemiology. [ABSTRACT FROM AUTHOR]

The antimicrobial-resistant gram-negative enteric bacterial isolates were analyzed to find its resistance to ampicillin, centiofur, chloramphenicol, florfenicol, spectinomycin and tetracycline. The strains of Escherichia coli isolated from cattle were found to exhibit multidrug resistance.

Healthy calves from a dairy herd in central Pennsylvania were analyzed each month over five-month period for fecal shedding of ceftiofur-resistant gram-negative bacteria. Ceftiofur resistant Escherichia coli isolates were characterized by antimicrobial resistance, serotype, pulse-field gel electrophoresis subtypes, beta-lactamase genes and virulence genes and the high prevalence of multidrug-resistant nonpathogenic Escherichia coli in calves could be significant source of resistant genes to other bacteria that share the same environment.

Listeria monocylogenes is an intracellular food borne pathogen that contaminates a variety of food, causing listeriosis, which can be fatal. A multi-virulence locus sequence-typing (MVLST) scheme was developed to subtype Listeria monocytogenes and results were compared with other schemes developed in the past.

Many animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and could act as reservoirs; however, transmission in free-living animals has not been documented. White-tailed deer, the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 is circulating in deer, 283 retro-pharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021 were assayed for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 (33.2%) deer samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, following the November 2020 peak of human cases in Iowa, and coinciding with the onset of winter and the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples collected over a 7-wk period. Whole genome sequencing of all 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%) and B.1.311 (n = 19; 20%) accounting for ~75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple human-to-deer transmission events followed by subsequent deer-to-deer spread. These discoveries have important implications for the long-term persistence of the SARS-CoV-2 pandemic. Our findings highlight an urgent need for a robust and proactive "One Health" approach to obtain enhanced understanding of the ecology, molecular evolution, and dissemination of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Avian influenza viruses (AIVs) cause major economic losses to the global poultry industry. Many host factors have been identified that act as regulators of the inflammatory response and virus replication in influenza A virus (IAV) infected cells including nucleotide-binding oligomerization domain (NOD) like receptor (NLR) family proteins. Evidence is emerging that NLRC5, the largest NLR member, is a regulator of host immune responses against invading pathogens including viruses; however, its role in the avian immune system and AIV pathogenesis has not been fully explored. In this study, we found that NLRC5 is activated by a range of low and highly pathogenic AIVs in primary chicken lung cells and a chicken macrophage cell line. Further, siRNA mediated NLRC5 knockdown in chicken macrophages resulted in a significant reduction in AIV replication which was associated with the upregulation of genes associated with activated NFκB signaling pathway. The knockdown of NLRC5 enhanced the expression of genes known to be associated with viral defense and decreased innate cytokine gene expression following AIV infection. Overall, our investigation strongly suggests that NLRC5 is a pro-viral factor during IAV infection in chicken and may contribute to pathogenesis through innate cytokine regulation. Further studies are warranted to investigate the IAV protein(s) that may regulate activation of NLRC5. [ABSTRACT FROM AUTHOR]

Uterine examinations provide an inexpensive and reliable postmortem alternative to monitor pregnancy rates in free-ranging elk (Cervus canadensis). However, this technique may be insensitive during early pregnancies (i.e., <20 d postconception), relies on proper collection of tissues, and may not be comparable to antemortem approaches used throughout the rest of the year. To circumvent some of these issues, the sensitivity and specificity of a commercially available serum pregnancy-specific protein B (PSPB) enzyme-linked immunosorbent assay (ELISA) was determined relative to uterine examination. From 2013 to 2017, paired serum samples and uteri were collected from 245 harvested free-ranging cow elk in Pennsylvania, US in November. Uteri were examined to determine whether the cow was pregnant, and, if so, gestation age was estimated based on embryo crown-rump (CR) length. The serum PSPB ELISA testing was then performed. Since harvested elk could not be retested, samples with optical densities close to the threshold for pregnancy determination (i.e., high-recheck samples) were considered as both not pregnant and pregnant, and analyses were performed separately under each scenario. Overall, the PSPB ELISA had a sensitivity of 95% (high-recheck considered pregnant) and 93% (high-recheck considered not pregnant), and a specificity of 91% (high-recheck considered pregnant) and 93% (high-recheck considered not pregnant) relative to uterine examinations. Based on CR length, gestation age was <14 to 55 d. Our results indicated the PSPB ELISA was an accurate serum-based pregnancy test for elk. [ABSTRACT FROM AUTHOR]

Salmonella enterica resistance to extended-spectrum cephalosporins (ESC) conferred by cefotaximases (bla CTX-M) is a growing concern in the United States. Among food-producing animals, poultry are a major reservoir of ESC-resistant Salmonella. A retrospective study was carried out to further characterize 38 ceftiofur-resistant clinical Salmonella enterica isolates obtained from poultry during 2007–2018. Of the isolates tested, 31 displayed resistance to ceftriaxone and harbored bla CMY-2, whereas 7 isolates demonstrated resistance or reduced susceptibility to cefepime in addition to ceftriaxone resistance. These 7 isolates displayed extended-spectrum β-lactamase activity, harbored bla CTX-M-1, and were recovered only from recent poultry diagnostic submissions made in 2011–2018 as opposed to the 31 isolates that were recovered in 2007–2018. Further characterization of the bla CTX-M-1 gene determined that it was located on conjugative IncN/ST1 and IncI1/ST87 plasmids in the isolates from commercial turkeys and broilers, respectively. These plasmids have been responsible for extensive spread of bla CTX-M-1 in livestock, poultry, and humans in Europe. Potential transfer of IncN and IncI1 plasmids and/or nontyphoidal Salmonella carrying these plasmids through the food chain, or by other means to humans, may result in treatment failures. Our study demonstrates the importance of further characterization of ceftiofur-resistant S. enterica isolates detected by veterinary diagnostic laboratories to identify the sources of bla CTX-M-1 and to mitigate the spread of ESC-resistant Salmonella in the poultry production pyramid. [ABSTRACT FROM AUTHOR]

Tularemia is an endemic zoonotic disease in many parts of the world including Asia. A cross-sectional study was conducted to determine genome-based prevalence of Francisella tularensis (Ft) in soil, assess an association between its occurrence in soil and likely predictors i.e., macro and micro-nutrients and several categorical variables, and determine seroconversion in small and large ruminants. The study included a total of 2,280 soil samples representing 456 villages in eight districts of the Punjab Province of Pakistan followed by an analysis of serum antibodies in 707 ruminants. The genome of Ft was detected in 3.25% (n = 74, 95% CI: 2.60–4.06) of soil samples. Soluble salts (OR: 1.276, 95% CI: 1.043–1.562, p = 0.015), Ni (OR: 2.910, 95%CI: 0.795–10.644, p = 0.106), Mn (OR:0.733, 95% CI:0.565–0.951, p = 0.019), Zn (OR: 4.922, 95% CI:0.929–26.064, p = 0.061) and nutrients clustered together as PC-1 (OR: 4.76, 95% CI: 2.37–9.54, p = 0.000) and PC-3 (OR: 0.357, 95% CI: 0.640, p = 0.001) were found to have a positive association for the presence of Ft in soil. The odds of occurrence of Ft DNA in soil were higher at locations close to a water source, including canals, streams or drains, [χ2 = 6.7, OR = 1.19, 95% CI:1.05–3.09, p = 0.004] as well as places where animals were present [χ2 = 4.09, OR = 2.06, 95% CI: 1.05–4.05, p = 0.02]. The seroconversion was detected in 6.22% (n = 44, 95% CI: 4.67–8.25) of domestic animals. An occurrence of Ft over a wide geographical region indicates its expansion to enzootic range, and demonstrates the need for further investigation among potential disease reservoirs and at-risk populations, such as farmers and veterinarians. [ABSTRACT FROM AUTHOR]

Salmonella Enteritidis (SE) is one of the most common causes of bacterial food-borne illnesses in the world. Despite the SE’s ability to colonize and infect a wide-range of host, the most common source of infection continues to be the consumption of contaminated shell eggs and egg-based products. To date, the role of the source of SE infection has not been studied as it relates to SE pathogenesis and resulting disease. Using a streptomycin-treated mouse model of human colitis, this study examined the virulence of SE grown in egg yolk and Luria Bertani (LB) broth, and mouse feces collected from mice experimentally infected with SEE1 (SEE1 passed through mice). Primary observations revealed that the mice infected with SE grown in egg yolk displayed greater illness and disease markers than those infected with SE passed through mice or grown in LB broth. Furthermore, the SE grown in egg yolk achieved higher rates of colonization in the mouse intestines and extra-intestinal organs of infected mice than the SE from LB broth or mouse feces. Our results here indicate that the source of SE infection may contribute to the overall pathogenesis of SE in a second host. These results also suggest that reservoir-pathogen dynamics may be critical for SE’s ability to establish colonization and priming for virulence potential. [ABSTRACT FROM AUTHOR]

A multidisciplinary, collaborative project was conducted to determine the prevalence and distribution of soil-borne zoonotic pathogens in Lahore district of Pakistan and ascertain its Public Health Significance. Using a grid-based sampling strategy, soil samples (n = 145) were collected from villages (n = 29, 5 samples/village) and examined for Bacillus anthracis, Burkholderia mallei/pseudomallei, Coxiella burnetii, Francisella tularensis, and Yersinia pestis using real time PCR assays. Chemical analysis of soil samples was also performed on these samples. The relationship between soil composition and absence or presence of the pathogen, and seven risk factors was evaluated. DNA of B. anthracis (CapB), B. mallei/pseudomallei (chromosomal gene), C. burnetii (IS1111, transposase gene), and F. tularensis (lipoprotein/outer membrane protein) was detected in 9.6, 1.4, 4.8, and 13.1% of soil samples, respectively. None of the samples were positive for protective antigen plasmid (PA) of B. anthracis and Y. pestis (plasminogen activating factor, pPla gene). The prevalence of B. anthracis (CapB) was found to be associated with organic matter, magnesium (Mg), copper (Cu), chromium (Cr), manganese (Mn), cobalt (Co), cadmium (Cd), sodium (Na), ferrous (Fe), calcium (Ca), and potassium (K). Phosphorous (P) was found to be associated with prevalence of F. tularensis while it were Mg, Co, Na, Fe, Ca, and K for C. burnetii. The odds of detecting DNA of F. tularensis were 2.7, 4.1, and 2.7 higher when soil sample sites were >1 km from animal markets, >500m from vehicular traffic roads and animal density of <1000 animals, respectively. While the odds of detecting DNA of C. burnetii was 32, 11.8, and 5.9 higher when soil sample sites were >500m from vehicular traffic roads, presence of ground cover and animal density of <1000 animals, respectively. In conclusion, the distribution pattern of the soil-borne pathogens in and around the areas of Lahore district puts both human and animal populations at a high risk of exposure. Further studies are needed to explore the genetic nature and molecular diversity of prevailing pathogens together with their seroconversion in animals and humans. [ABSTRACT FROM AUTHOR]

A total of 23 clinical isolates of Fusobacterium spp. were recovered at necropsy over a 2-year period from the respiratory tract of white-tailed deer (Odocoileus virginianus). Isolates were identified as Fusobacterium varium (18/23), Fusobacterium necrophorum subsp. funduliforme (3/23), and Fusobacterium necrophorum subsp. necrophorum (2/23). Using polymerase chain reaction–based detection of virulence genes, all F. necrophorum isolates were positive for the promoter region of the leukotoxin operon and the hemagglutinin-related protein gene, while all F. varium isolates were negative. The presence of the leukotoxin gene in F. necrophorum isolates and the absence of this gene in F. varium isolates were confirmed by Southern hybridization using 2 separate probes. Toxicity to bovine polymorphonuclear leukocytes was observed with all F. necrophorum isolates, but was not observed in any F. varium isolates. Susceptibility to antimicrobials was markedly different for F. varium as compared to F. necrophorum. In summary, no evidence of leukotoxin production was detected in any of the 23 F. varium isolates used in the current study. The data suggests that F. varium, the most common species isolated, may be a significant pathogen in deer with a different virulence mechanism than F. necrophorum. [ABSTRACT FROM PUBLISHER]

The article discusses a study which tested Mycoplasma bovis isolates submitted to the Pennsylvania Animal Diagnostic Laboratory from December 2007 to December 2008 for antimicrobial susceptibility to ceftiofur, enrofloxacin, erythromycin, florfenicol, spectinomycin, tetracycline, and oxytetracycline. Broth microdilution method and flow cytometry were used to determine the isolates' minimum inhibitory concentration (MIC) ranges. The most effective antimicrobials against M. bovis are also cited.

The article present a study which examined the antigenic variants of Escherichia (E.) coli strains from pigs. Findings showed that the F18 fimbriae carried by E. coli to colonize the small intestine of pigs consists of the F18ab and F18ac antigens. The study concludes that both F18ab- and F18ac-positive strains may carry genes for Shiga toxins and enterotoxins, and are involved in post-weaning diarrhea or edema in pigs.

Objective-To determine current management practices used by white-tailed deer farms in Pennsylvania and identify animal health problems that exist in these herds. Design-Cross-sectional study. Study Population-Owners and managers of 233 farms in Pennsylvania that raised white- tailed deer. Procedures-A self-administered questionnaire was mailed to participants. Results-Herds ranged in size from 1 to 350 deer. Land holdings ranged from 0.07 to 607 hectares (0.17 to 1,500 acres). Stocking density ranged from 0.1 to 118.6 deer/hectare (0.04 to 48 deer/acre). Most (84%) respondents raised deer for breeding or hunting stock; 13% raised deer exclusively as pets or for hobby purposes, and purpose varied by herd size. Multiple associations were identified between management or disease factors and herd size. The use of vaccines, use of veterinary and diagnostic services, use of pasture, and use of artificial insemination increased as herd size increased. The most common conditions in herds of all sizes were respiratory tract disease, diarrhea, parasitism, and sudden death. The prevalence of respiratory tract disease increased as herd size increased. Conclusions and Clinical Relevance-Results suggested that many aspects of herd management for white-tailed deer farms in Pennsylvania were associated with herd size, but that regardless of herd size, many preventive medicine practices were improperly used or underused in many herds. [ABSTRACT FROM AUTHOR]

Clostridium perfringens is a Gram-positive sporulating anaerobic bacterium that is responsible for a wide spectrum of diseases in animals, birds and humans. The virulence of C. perfringens is associated with the production of several enterotoxins and exotoxins. β2-toxin is a 28 kDa exotoxin produced by C. perfringens. It is implicated in necrotic enteritis in animals and humans, a disease characterized by a sudden acute onset with lethal hemorrhagic mucosal ulceration. The recombinant expression, purification and crystallization of β2-toxin using the batch-under-oil technique are reported here. Native X-ray diffraction data were obtained to 2.9 Å resolution on a synchrotron beamline at the F2 station at Cornell High Energy Synchrotron Source (CHESS) using an ADSC Quantum-210 CCD detector. The crystals belong to space group R3, with a dimer in the asymmetric unit; the unit-cell parameters are a =  b = 103.71, c = 193.48 Å, α = β = 90, γ = 120° using the hexagonal axis setting. A self-rotation function shows that the two molecules are related by a noncrystallographic twofold axis with polar angles ω = 90.0, ϕ = 210.3°. [ABSTRACT FROM AUTHOR]

A multi-virulence-locus sequence typing (MVLST) scheme was developed for subtyping Listeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST). A set of 28 strains (eight different serotypes and three known genetic lineages) of L. monocytogenes was selected from a strain collection (n > 1,000 strains) to represent the genetic diversity of this species. Internal fragments (ca. 418 to 469 bp) of three virulence genes (prfA, inlB, and inlC) and three virulenceassociated genes (dal, lisR, and clpP) were sequenced and analyzed. Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types. Comparison of MVLST with automated EcoRI-RT and PFGE with ApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970). Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype ½a and 4b strains than MLST. Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology of L. monocytogenes. [ABSTRACT FROM AUTHOR]

Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)—CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)—was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs. [ABSTRACT FROM AUTHOR]

• A probe-based real-time PCR assay for Neospora caninum detection is presented. • Assay demonstrates analytical sensitivity of 3 genomic copies of N. caninum DNA. • Assay results agree with established conventional PCR assay in bovine clinical samples. Neospora caninum is an apicomplexan protozoan parasite that is a leading cause of abortion in cattle. Detection of parasite-specific DNA by PCR is a highly sensitive method for identifying the presence of N. caninum in a variety of tissues. We developed and validated a probe-based real-time PCR assay targeting the conserved Nc5 gene of N. caninum. Using N. caninum strain Nc-1 genomic DNA and a synthetic gene fragment as amplification standards, we determined the PCR amplification efficiency and the limit of detection to be 95.60% and 3 copies, respectively. Five pathogens frequently associated with bovine abortions, namely bovine viral diarrhea virus types I and II, bovine alphaherpesvirus-1, Chlamydia, and Leptospira , were tested to ensure analytical exclusivity. A total of 103 clinical samples from aborted fetuses were tested concurrently with a standard conventional PCR and the new probe-based real-time PCR assay. All tested samples showed 100% agreement between these two assays. In conclusion, the probe-based real-time PCR assay facilitates accurate and rapid detection of N. caninum from abortions in cattle. [ABSTRACT FROM AUTHOR]

Bovine herpesvirus-1 (BoHV-1) is a major pathogen affecting cattle worldwide causing primarily respiratory illness referred to as infectious bovine rhinotracheitis (IBR), along with reproductive disorders including abortion and infertility in cattle. While modified live vaccines (MLVs) effectively induce immune response against BoHV-1, they are implicated in disease outbreaks in cattle. Current diagnostic methods cannot distinguish between MLVs and field strains of BoHV-1. We performed whole genome sequencing of 18 BoHV-1 isolates from Pennsylvania and Minnesota along with five BoHV-1 vaccine strains using the Illumina Miseq platform. Based on nucleotide polymorphisms (SNPs) the sequences were clustered into three groups with two different vaccine groups and one distinct cluster of field isolates. Using this information, we developed a novel SNP-based PCR assay that can allow differentiation of vaccine and clinical strains and help accurately determine the incidence of BoHV-1 and the association of MLVs with clinical disease in cattle. [ABSTRACT FROM AUTHOR]

Glanders is an infectious and contagious bacterial disease of equines. A little is known about its seroprevalence and risk factors in working equines in countries where the disease is endemic. Also, there are no reports on prevalence of the disease in areas where there is a prior evidence of Burkholderia (B.) mallei detection in soil. A cross-sectional study was conducted in selected districts (n = 09) of Punjab province of Pakistan during 2014–2015. A total of 1008 serum samples were screened for detection of antibodies to B. mallei with complement fixation test followed by western blot. The overall seroprevalence was found to be 3.17% (95% CI: 2.25–4.44). The seropositivity was significantly higher from the sampling sites where B. mallei was detected in soil [OR: 10.66 (95% CI: 4.42–31.66), p = 0.00]. Other risk factors significantly associated with animal seropositivity were: age group [OR: 1.78 (95% CI: 4.58–15.56), p = 0.00], location in urban area [OR: 2.99 (95% CI: 1.46–6.51), p = 0.00],body condition [OR: 3.47 (95% CI: 1.64–7.99), p = 0.00], presence of farcy lesion[OR: 7.71 (95% CI: 3.47–19.50), p = 0.00], proximity to water bodies [OR: 7.71 (95% CI: 3.47–19.50), p = 0.00]; domestic animal population [OR: 3.20 (95% CI: 1.24–10.87), p = 0.03] and number of households in sampling area [OR: 4.18 (95%CI: 1.82–11.30), p = 0.00]. The study provides an estimate of prevalence of glanders and a potential link between animal seropositivity and presence of B. mallei in soil. The risk factors identified in this study can be used in surveillance and disease awareness. The high prevalence of disease in draught horses and contact of infected animals with their care-takers in developing countries signify need to initiate progressive control of the disease using one health approach. [ABSTRACT FROM AUTHOR]

Influenza A viruses (IAVs) continue to threaten animal and human health with constant emergence of novel variants. While aquatic birds are a major reservoir of most IAVs, the role of other terrestrial birds in the evolution of IAVs is becoming increasingly evident. Since 2006, several reports of IAV isolations from emus have surfaced and avian influenza infection of emus can lead to the selection of mammalian like PB2-E627K and PB2-D701N mutants. However, the potential of emus to be co-infected with avian and mammalian IAVs is not yet understood. As a first step, we investigated sialic acid (SA) receptor distribution across major organs and body systems of emu and found a widespread co-expression of both SAα2,3Gal and SAα2,6Gal receptors in various tissues that are compatible with avian and human IAV binding. Our results suggest that emus could allow genetic recombination and hence play an important role in the evolution of IAVs. [ABSTRACT FROM AUTHOR]

Coxiella burnetii causes query (Q) fever, an important zoonotic disease with worldwide significance. The role of environment in the ecology of C. burnetti , and its influence on seroconversion in animals has not been elucidated in Pakistan. We carried out a cross-sectional study in Punjab province to (1) determine the prevalence and distribution of C. burnetii in soil using an ISIIII gene-based real time-polymerase chain reaction (RT-PCR) assay, (2) analyze association between the occurrence of C. burnetii in soil and its predictors i.e. soil characteristics (macro- and micro-nutrients) and several likely risk factors including the seroconversion in small ruminants at places where its genome had or had not been detected, and (3) predict homology and genetic diversity of the identified strains using sequences originated from different hosts worldwide. A total of 2425 soil samples from nine districts of Punjab province were processed. C. burnetii DNA was detected in 47 samples (1.94%, 95% CI: ±0.55) originating from 35 villages of studied districts (7.22%, 95% CI: ±2.30). The highest prevalence was found in Attock (7.11%, 95% CI: ±3.36), followed by Lahore (4.83%, 95% CI: ±3.49), Sahiwal (4.70%, 95% CI: ±2.6), Dera Ghazi Khan (2.33%, 95% CI: ±2.02), Faisalabad (1.35%, 95% CI: ±1.18) and Sheikhupura (0.68%, 95% CI: ±0.94). The odds of detecting bacterial DNA in soil was increased with a unit increase in organic matter [2.511 (95% CI: 1.453–4.340), p = 0.001] and sodium [1.013 (95% CI: 1.005–1.022), p = 0.001], whereas, calcium [0.984 (95% CI: 0.975–0.994), p = 0.002] and potassium [0.994 (95% CI: 0.990–0.999), p = 0.011] had protective effect where a unit increase in each analyte decreased odds for its occurrence by 1.0% approximately. Likewise, for categorical variables (risk factors), the odds of detecting C. burnetii were higher at locations >500 m away from a main road [1.95 (95% CI: 1.06–3.78), p = 0.04]. The enzyme-linked immunosorbent assay (ELISA) revealed an increased prevalence of antibodies in sheep (17.9%, 95% CI: ±5.54) compared with goats (16.4%, 95% CI: ±4.34). When determining the association between soil DNA and C. burnetii antibodies in small ruminants, the odds of detecting these antibodies were significant in sheep at the livestock barns [2.81 (95% CI: 1.20–7.37), p = 0.02]. The IS1111 gene-based sequence analysis revealed a clustering of the DNA into two distinct groups with much genetic divergence (0.76–68.70%): the first group that contained sequences from Lahore district clustered with human and buffalo origin isolates, whereas the second group that contained the sequences from the remaining study districts clustered with goat-, rodent- and human-origin isolates. This study provides the first evidence of the presence of C . burnetii in the environment in Punjab province, Pakistan. Future studies are needed to ascertain the bacteria’s molecular epidemiology over a wide geographical area, type the isolates, and evaluates the potential risks to human populations, particularly farmers and veterinarians. [ABSTRACT FROM AUTHOR]

Abstract: Mycoplasma bovis is an etiologic agent of pneumonia, arthritis, and otitis in young calves, such as those found in the special-fed veal industry. We conducted a blinded, controlled trial of two commercially available M. bovis bacterin vaccines for the prevention of respiratory disease in calves associated with M. bovis infection. Calves were randomly assigned to a subcutaneous treatment of vaccine A (n =50), adjuvant A (n =50), vaccine B (n =50), or 0.9% sterile saline solution (n =50) beginning at 27 days of age. Upper-respiratory tract colonization was not impacted by vaccination status. Vaccine A significantly reduced the presence of lung lesions (p =0.0325), however there was no significant reduction of M. bovis in lung lesions. Vaccine B did not significantly reduce total lung lesions or M. bovis-specific lung lesions. The relative risk was determined to be 0.56, 1.0, and 1.36 for vaccine A, adjuvant A, and vaccine B, respectively. There was no association between the total specific antibody isotype (IgM, IgG1, IgG2, IgA) concentrations or M. bovis antibodies and the M. bovis-associated morbidity in the veal calves. Under the field conditions of this study, observed vaccine efficacy for vaccine A and vaccine B was 44% and less than 1%, respectively. [Copyright &y& Elsevier]

Abstract: Extraintestinal pathogenic Escherichia coli (ExPEC) are known to cause important diseases of humans and animals, and they have been shown to carry a variety of plasmids associated with increased virulence and decreased antimicrobial susceptibility. Here, the completed DNA sequence of a human uropathogenic E. coli (UPEC; O6:H31 isolate) plasmid, pEC14_114, was determined. The plasmid was 114,222bp in length and was highly similar to plasmid sequences or draft contiguous sequences from three other human cystitis-associated UPEC isolates. pEC14_114 contained 141 coding regions, including a number of genes associated with mobile genetic elements, F-type transfer, plasmid maintenance and stability, colicin immunity, and plasmid replication. This plasmid also possessed a “genetic load” region containing genes with predicted similarity to iron acquisition systems and virulence factors. The prevalence of pEC14-associated genes was determined for a collection of 1456 E. coli isolates, including those from food products, humans, dogs, cats, pigs, chickens, and turkeys. pEC14_114-associated genes were found significantly more often (16–35%) among human UPEC and neonatal meningitis-associated isolates than among food- and animal-source isolates (0–8%). Overall, this plasmid represents a novel IncFIB/FIIA plasmid type associated with human ExPEC belonging to the B2 phylogenetic group. The overall role of this plasmid, if any, in human ExPEC infections remains to be determined. [Copyright &y& Elsevier]

We report a complete genome sequence of bovine coronavirus (BCoV) isolated from a goat in the state of Pennsylvania in 2022. BCoV often causes calf scours and winter dysentery in cattle., Competing Interests: The authors declare no conflict of interest.

Staphylococcus pseudintermedius is a pathogen of veterinary importance, as it is the major causative agent of superficial pyoderma in dogs. We present the complete genome sequences of six strains of S. pseudintermedius derived from dogs affected with epidermal collarettes and superficial bacterial folliculitis, which are two variants of superficial pyoderma.

Avibacterium paragallinarum (historically called Hemophilus paragallinarum ) 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 A. paragallinarum 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 A. paragallinarum 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 recN , the DNA repair protein gene of A. paragallinarum . 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 Avibacterium 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 recN PCR assay with a previous HPG-2 based real-time PCR assay which showed a PCR efficiency of 79%., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kuchipudi, Yon, Surendran Nair, Byukusenge, Barry, Nissly, Williams, Pierre, Mathews, Walner-Pendleton, Dunn, Barnhart, Loughrey, Davison, Kelly, Tewari and Jayarao.)

Draft genome sequences of two outbreak isolates of Streptococcus equi subsp. zooepidemicus from a Pennsylvania swine herd affected with high mortality and morbidity are reported here. The genome analysis revealed that the isolates are closely related to a virulent strain originally identified in China., (Copyright © 2020 Surendran Nair et al.)

Avibacterium paragallinarum , the causative agent of infectious coryza, causes significant economic losses to the poultry industry due to increased culling rates in growing chickens and decreased egg production in layers. We present the complete genome sequences of seven strains of Avibacterium paragallinarum isolated from poultry farms in Pennsylvania during 2019., (Copyright © 2020 Byukusenge et al.)

Nontyphoidal Salmonella enterica strains are major foodborne pathogens with global public health importance. Foodborne salmonellosis can be life-threatening, especially in sub-Saharan Africa. We report the complete genome sequences of 20 nontyphoidal Salmonella enterica strains isolated in Rwanda. The reported 20 bacterial chromosomes and 8 plasmids each belong to 1 of 9 nontyphoidal Salmonella serotypes., (Copyright © 2019 Byukusenge et al.)

Avian avulavirus 1 infects multiple avian hosts, and rare reports of human infection have been noted throughout the last century. Here, we report the complete genome sequences of three isolates of avulavirus 1 collected from poultry farmers in Pakistan exhibiting mild respiratory signs., (Copyright © 2018 Shabbir et al.)

Infectious pancreatic necrosis (IPN) is an acute contagious systemic disease affecting several fish species and a critical disease in the salmonid fish farming industry. Here, we report the complete genome sequence of IPN virus (IPNV) RNA segments A and B, isolated from a farmed brook trout in Pennsylvania., (Copyright © 2018 Palchak et al.)

Influenza A viruses (IAVs) continue to threaten animal and human health globally. Bats are asymptomatic reservoirs for many zoonotic viruses. Recent reports of two novel IAVs in fruit bats and serological evidence of avian influenza virus (AIV) H9 infection in frugivorous bats raise questions about the role of bats in IAV epidemiology. IAVs bind to sialic acid (SA) receptors on host cells, and it is widely believed that hosts expressing both SA α2,3-Gal and SA α2,6-Gal receptors could facilitate genetic reassortment of avian and human IAVs. We found abundant co-expression of both avian (SA α2,3-Gal) and human (SA α2,6-Gal) type SA receptors in little brown bats (LBBs) that were compatible with avian and human IAV binding. This first ever study of IAV receptors in a bat species suggest that LBBs, a widely-distributed bat species in North America, could potentially be co-infected with avian and human IAVs, facilitating the emergence of zoonotic strains.

The Pennsylvania Egg Quality Assurance Program (EQAP) provided the framework for Salmonella Enteritidis (SE) control programs, including the Food and Drug Administration (FDA) mandated Final Egg Rule, for commercial layer facilities throughout the United States. Although flocks with ≥3000 birds must comply with the FDA Final Egg Rule, smaller flocks are exempted from the rule. As a result, eggs produced by small layer flocks may pose a greater public health risk than those from larger flocks. It is also unknown if the EQAPs developed with large flocks in mind are suitable for small- and medium-sized flocks. Therefore, a study was performed to evaluate the effectiveness of best management practices included in EQAPs in reducing SE contamination of small- and medium-sized flocks by longitudinal monitoring of their environment and eggs. A total of 59 medium-sized (3000 to 50,000 birds) and small-sized (<3000 birds) flocks from two major layer production states of the United States were enrolled and monitored for SE by culturing different types of environmental samples and shell eggs for two consecutive flock cycles. Isolated SE was characterized by phage typing, pulsed-field gel electrophoresis (PFGE), and clustered regularly interspaced short palindromic repeats-multi-virulence-locus sequence typing (CRISPR-MVLST). Fifty-four Salmonella isolates belonging to 17 serovars, 22 of which were SE, were isolated from multiple sample types. Typing revealed that SE isolates belonged to three phage types (PTs), three PFGE fingerprint patterns, and three CRISPR-MVLST SE Sequence Types (ESTs). The PT8 and JEGX01.0004 PFGE pattern, the most predominant SE types associated with foodborne illness in the United States, were represented by a majority (91%) of SE. Of the three ESTs observed, 85% SE were typed as EST4. The proportion of SE-positive hen house environment during flock cycle 2 was significantly less than the flock cycle 1, demonstrating that current EQAP practices were effective in reducing SE contamination of medium and small layer flocks.

This report presents the complete genome sequences of two Salmonella enterica serovar Enteritidis strains bearing the pulsed-field gel electrophoresis profile JEGX01.0004, which were isolated from the internal contents of eggs., (Copyright © 2015 Moreau et al.)

A study was conducted to determine the prevalence of Clostridium difficile and characterize C. difficile isolates from human stool and retail grocery meat samples. Human stool samples (n=317) were obtained from a clinical laboratory and meat samples (n=303) were collected from 8 retail grocery stores from October 2011 through September 2012 from Centre County of Pennsylvania and were examined for C. difficile. C. difficile was isolated from 16.7% of stool samples (n=317) and 6.9%, 11.5%, 14.5%, and 7.8% of beef (n=72), pork (n=78), turkey (n=76), and chicken (n=77) samples, respectively. Six different toxin gene profiles were detected in all human and meat isolates of C. difficile based on the presence or absence of toxin genes tcdA, tcdB, and cdtA and cdtB. Interestingly, 75.6% of the human C. difficile isolates lacked any deletion in the tcdC gene (139-bp), whereas a 39-bp deletion was observed in 61.3% of the C. difficile strains isolated from meat samples. C. difficile from meat samples were more susceptible to clindamycin, moxifloxacin, vancomycin, and metronidazole than C. difficile isolates from human samples. Twenty-five different ribotypes were identified in human and meat C. difficile isolates. In conclusion, significant genotypic and phenotypic differences were observed between human and meat isolates of C. difficile; however, a few C. difficile isolates from meat-in particular ribotypes 078, PA01, PA05, PA16, and PA22 with unique profiles (toxin gene, tcdC gene size and antimicrobial resistance profiles)-were similar to human C. difficile isolates.

There is a growing concern of a public health risk associated with non-O157 Shiga toxin-producing Escherichia coli (STEC) since E. coli serogroups O26, O45, O103, O111, O121, and O145 are frequently implicated in outbreaks of human illness worldwide. Recently, the Food Safety and Inspection Service of the U.S. Department of Agriculture declared these six STEC O groups to be adulterants in beef. We describe here a rapid, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for the detection of these top six non-O157 STEC O groups. The assays were tested against 174 reference E. coli O groups, with 60 clinical isolates belonging to the target O groups and 10 non-E coli strains belonging to the family Enterobacteriaceae. Assays for serogroups O103, O111, and O121 exhibited 100% specificity, while assays for serogroups O26 and O45 had 98.2% specificity, and O145 had 99.1% specificity. ELISA conducted using artificially inoculated ground beef samples displayed 100% accuracy. The sensitivity of the assay was 5×10(5) colony-forming unit (CFU)/mL, with limits of detection in the range of 1-10 CFU/25 g of ground beef sample following enrichment. The findings of the study suggest that the assay described is simple and rapid, and can be employed to detect target STEC O groups in beef and other food samples. In addition, the assay provides a conceptual framework that can be adapted for the development of similar tests for the rapid detection of other serogroups of E. coli.

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.

Rapid, sensitive, and highly specific flow-cytometric assays were developed for the detection of the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) O groups in ground beef. The analytical sensitivity of the assays was 2 × 10(3) target cells in a bacterial mixture of 10(5) CFU/ml, and the limit of detection in ground beef was 1 to 10 CFU following 8 h of enrichment. The assays may be utilized for rapid detection of STEC O groups in meat.

A study was conducted in two parts to determine the prevalence of toxigenic Clostridium difficile in veal calves and retail meat. The first part of the study focused on the veal production continuum (farm to abattoir). Fifty calves from 4 veal herds (n=200) were followed for 18-22 weeks from the time of arrival on the veal farm to the time of slaughter. Fecal samples were collected from calves every 4-6 weeks. Half of the calves included in the study (n=100) were followed to the abattoir where carcass swabs were collected post slaughter. Fecal samples and carcass swabs were screened for genes encoding C. difficile toxins TcdA, TcdB, and CDT by using real-time polymerase chain reaction (PCR). Carcass swabs were also screened for toxigenic C. difficile by using traditional culture methods. In the second part of the study, ground veal products (n=50 samples) purchased from local grocery stores were examined for toxigenic C. difficile by using real-time PCR and traditional culture methods. Fecal samples from 56 of 200 (28%) calves tested positive for C. difficile toxin genes at least once over the course of the study. Calf age (p=0.011) influenced prevalence of C. difficile toxin genes in calf feces. Toxin genes of C. difficile were detected in one carcass swab by multiplex real-time PCR only. Toxigenic C. difficile was detected by PCR and culture in four (8%) and three (6%) ground veal samples, respectively. The findings of the study reveal that toxigenic C. difficile was most prevalent in veal calves (12%) just before slaughter, although viable toxigenic C. difficile was not recovered from veal carcasses. On the contrary, viable toxigenic C. difficle was recovered from 6% retail meat, thus suggesting that contamination occurs either during or after veal fabrication.

Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)-was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs.