The Susceptibility of Chickens to Zika Virus: A Comprehensive Study on Age-Dependent Infection Dynamics and Host Responses.

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.

Complete Genome Sequence of a Bovine Coronavirus Isolated from a Goat in Pennsylvania, USA.

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.

Transmission history of SARS-CoV-2 in humans and white-tailed deer.

The emergence of a novel pathogen in a susceptible population can cause rapid spread of infection. High prevalence of SARS-CoV-2 infection in white-tailed deer (Odocoileus virginianus) has been reported in multiple locations, likely resulting from several human-to-deer spillover events followed by deer-to-deer transmission. Knowledge of the risk and direction of SARS-CoV-2 transmission between humans and potential reservoir hosts is essential for effective disease control and prioritisation of interventions. Using genomic data, we reconstruct the transmission history of SARS-CoV-2 in humans and deer, estimate the case finding rate and attempt to infer relative rates of transmission between species. We found no evidence of direct or indirect transmission from deer to human. However, with an estimated case finding rate of only 4.2%, spillback to humans cannot be ruled out. The extensive transmission of SARS-CoV-2 within deer populations and the large number of unsampled cases highlights the need for active surveillance at the human-animal interface.

Multiple spillovers from humans and onward transmission of SARS-CoV-2 in white-tailed deer.

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 retropharyngeal 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.

Complete Genome Sequences of Six Staphylococcus pseudintermedius Strains from Dogs with Superficial Pyoderma in Georgia, USA.

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.

A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry.

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%.

Draft Genome Sequences of Two Virulent Streptococcus equi subsp. zooepidemicus Swine Isolates from Pennsylvania.

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.

Complete Genome Sequences of Seven Avibacterium paragallinarum Isolates from Poultry Farms in Pennsylvania, USA.

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.

NLRC5 Serves as a Pro-viral Factor During Influenza Virus Infection in Chicken Macrophages.

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.

Detection of CTX-M-1 extended-spectrum beta-lactamase among ceftiofur-resistant Salmonella enterica clinical isolates of poultry.

Salmonella enterica resistance to extended-spectrum cephalosporins (ESC) conferred by cefotaximases (blaCTX-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 blaCMY-2, whereas 7 isolates demonstrated resistance or reduced susceptibility to cefepime in addition to ceftriaxone resistance. These 7 isolates displayed extended-spectrum ß-lactamase activity, harbored blaCTX-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 blaCTX-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 blaCTX-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 blaCTX-M-1 and to mitigate the spread of ESC-resistant Salmonella in the poultry production pyramid.

Assessment of a Commercially Available Serum Pregnancy-specific Protein B Test in Free-ranging Elk (Cervus canadensis) in Pennsylvania, USA.

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.

A probe-based real-time PCR assay for the detection of Neospora caninum in clinical samples from cattle.

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.

Cross Sectional Study and Risk Factors Analysis of Francisella tularensis in Soil Samples in Punjab Province of Pakistan.

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.

Complete Genome Sequences of 20 Nontyphoidal Salmonella Isolates from Rwanda.

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.

Whole-genome sequence analysis reveals unique SNP profiles to distinguish vaccine and wild-type strains of bovine herpesvirus-1 (BoHV-1).

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.

Complete Genome Sequences of Three Related Avian Avulavirus 1 Isolates from Poultry Farmers in Pakistan.

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.

Whole-Genome Sequence of Infectious Pancreatic Necrosis Virus Isolated from Farmed Brook Trout (Salvelinus fontinalis) in Pennsylvania.

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.

A Retrospective Study of Salmonella Enteritidis Isolated from Commercial Layer Flocks.

Consumption of shell eggs has been associated with Salmonella Enteritidis (SE) infections in humans in the United States. Because of this, the Pennsylvania Egg Quality Assurance Program (PEQAP) was developed and implemented in 1994. The PEQAP involves periodic flock testing and management practices to minimize SE contamination of shell eggs. Subsequently, the U.S. Food and Drug Administration (FDA) introduced a mandatory federal program in 2010 and 2012 for shell egg producers modeled closely after PEQAP to reduce the incidence and prevalence of SE during production, storage, and transport nationwide. In this study, a retrospective epidemiologic analysis was conducted by characterizing SE isolated from commercial layer environment samples and shell eggs submitted to the Animal Diagnostic Laboratory at The Pennsylvania State University using phage typing and pulsed-field gel electrophoresis (PFGE). The objective of this study was to determine the relatedness of SE isolates from hen house environments and shell eggs and to optimize the existing protocols of egg quality assurance programs by identifying the best layer-house environmental sampling time points in order to minimize SE contamination of shell eggs. A total of 94 SE isolates from 65 hen flocks on 35 premises in Pennsylvania recovered during 2007 to 2015 were used in this study. The SE phage type 8 and PFGE fingerprint type JEGX01.0004 most commonly associated with human SE infection was also the predominant type present in layer-house environments and shell eggs. This reconfirms hen house environmental monitoring is an effective method to identify SE-infected flocks. Further, the PEQAP program allowed SE detection of infected flocks earlier than the FDA program as it included an additional environmental test at 29-31 wk of age, enabling the earlier prevention of SE-contaminated shell eggs going to the market. Therefore, it is recommended to refine the sampling time points of the current FDA Egg Rule by adding hen house environmental testing at 29-31 wk of age.

Seroprevalence and risk factors of glanders in working equines - Findings of a cross-sectional study in Punjab province of Pakistan.

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.

An immuno-chromatographic lateral flow assay (LFA) for rapid on-the-farm detection of classical swine fever virus (CSFV).

Classical swine fever (CSF) is a highly contagious and potentially fatal disease of domestic pigs. Classical swine fever is routinely diagnosed by clinical signs, serology, detection of CSF virus (CSFV) nucleic acid by PCR and virus isolation. Most of the current CSF diagnostic methods are expensive and have an extended turnaround time. In the majority of the CSF endemic countries, lack of easy access to diagnostic facilities is a major problem for swine producers trying to obtain early diagnosis and often results in the entire herd being infected. The acute form of CSF can show non-specific signs of illness, leaving CSF often undiagnosed. Hence there is an urgent need for a rapid and reliable pen side diagnostic assay for the better detection and control of this economically important disease of swine. We developed an immuno-chromatographic lateral flow assay (LFA) for on the farm detection of CSFV. A CSFV isolate [CSFV/AP/TRP2/2009 (TS2)] of genotype 1.1 was used for the production of monoclonal antibodies (mAbs) for the LFA's development. The virus detection level of the LFA device was 36.8 TCID50/ml of CSFV. The sensitivity and specificity of LFA in comparison with PCR were 80.36% and 87.10%, respectively. The positive and negative predictive values of the LFA device were 91.84% and 87.10%, respectively. In conclusion, the CSFV-LFA is a reliable and convenient resource for preliminary on the farm detection of classic swine fever.