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Clinical reports of Zika Virus (ZIKV) RNA detection in breast milk have been described, but evidence conflicts as to whether this RNA represents infectious virus. We infected post-parturient AG129 murine dams deficient in type I and II interferon receptors with ZIKV. ZIKV RNA was detected in pup stomach milk clots (SMC) as early as 1 day post maternal infection (dpi) and persisted as late as 7 dpi. In mammary tissues, ZIKV replication was demonstrated by immunohistochemistry in multiple cell types including cells morphologically consistent with myoepithelial cells. No mastitis was seen histopathologically. In the SMC and tissues of the nursing pups, no infectious virus was detected via focus forming assay. However, serial passages of fresh milk supernatant yielded infectious virus, and immunohistochemistry showed ZIKV replication protein associated with degraded cells in SMC. These results suggest that breast milk may contain infectious ZIKV. However, breast milk transmission (BMT) does not occur in this mouse strain that is highly sensitive to ZIKV infection. These results suggest a low risk for breast milk transmission of ZIKV, and provide a platform for investigating ZIKV entry into milk and mechanisms which may prevent or permit BMT.

Newcastle disease, caused by virulent strains of avian paramyxovirus 1 (APMV-1), occurs globally and has significant social and economic impact. APMV-1 is a rapidly evolving RNA virus and is genetically divided into class I and class II with almost all virulent viruses being of class II. The considerable genetic diversity of the virus adds complexity to maintaining the high sensitivity and specificity of molecular detection assays. The current USDA’s fusion gene rRT-PCR assay was designed for class II APMV-1 isolates with an emphasis on early-2000s US strains. Assessment with globally circulating genotypes confirmed previously described lower sensitivity (sub-genotypes VII.1.1, VII.2) and identified absence of detection (genotype XIV). An additional forward primer and two probes were designed using a comprehensive complete fusion gene sequence database. The optimized multiplex assay detected genotype XIV and improved sensitivity for sub-genotypes VII.1.1 and VII.2, with maintained sensitivity for the remaining genotypes. No near-neighbors or APMV-1 of low virulence were detected. Using field and experimental clinical samples, both the specificity and sensitivity were determined to be 100%, compared to the current assay with 100% and 93%, respectively. The new assay identifies all known chicken virulent APMV-1 genotypes with the benefit of using an exogenous internal positive control, which monitors extraction efficiency and inhibitors.

The geographic expansion of chronic wasting disease (CWD) in U.S. white-tailed deer (Odocoileus virginianus) has been largely unabated by best management practices, diagnostic surveillance, and depopulation of positive herds. Using a custom Affymetrix Axiom single nucleotide polymorphism (SNP) array, we demonstrate that both differential susceptibility to CWD, and natural variation in disease progression, are moderately to highly heritable (h2=0.337±0.079─0.637±0.070) among farmed U.S. white-tailed deer, and that loci other than PRNP are involved. Genome-wide association analyses using 123,987 quality filtered SNPs for a geographically diverse cohort of 807 farmed U.S. white-tailed deer (n = 284 CWD positive; n = 523 CWD non-detect) confirmed the prion gene (PRNP; G96S) as a large-effect risk locus (P-value < 6.3E-11), as evidenced by the estimated proportion of phenotypic variance explained (PVE ≥ 0.05), but also demonstrated that more phenotypic variance was collectively explained by loci other than PRNP. Genomic best linear unbiased prediction (GBLUP; n = 123,987 SNPs) with k-fold cross validation (k = 3; k = 5) and random sampling (n = 50 iterations) for the same cohort of 807 farmed U.S. white-tailed deer produced mean genomic prediction accuracies ≥ 0.81; thereby providing the necessary foundation for exploring a genomically-estimated CWD eradication program.

Efforts to breed Attwater's prairie chickens (APC; Tympanuchus cupido attwateri) in captivity to supplement wild populations of this endangered bird have been negatively affected by infections with Avipoxvirus and reticuloendotheliosis virus (REV). Because REV can be integrated into the genome of fowlpox virus (FPV) and may be transmitted in that manner, identifying the source of avipox disease in APC is important to mitigate the impact of this virus. Tissue samples from APC were collected from breeding programs in Texas from 2016 to 2020. These samples consisted of 11 skin lesions and three internal organs from a total of 14 different birds that died of unknown causes or were euthanized. Avipoxvirus was detected by PCR and isolation in embryonating chicken eggs in all skin lesion samples but was not detected in internal organs. Using sequence analysis of FPV polymerase and 4b genes, we determined that 10 out of 11 Avipoxvirus detections resided within the fowlpox clade and a single sample resided within the canarypox clade. REV sequences were detected in all FPV positive samples and in all internal organ tissues but were not detected in the sample matching the canarypox clade. Analysis of REV sequences and PCR detection showed the REV infecting APC was consistent with REV-A and had little variability on analysis of the U3 region of the long terminal repeat. The results of this study indicate control of REV in APC breeding colonies may benefit by a vaccination program targeting FPV and REV. However, a commercially available vaccine for REV is not available at this time.

Previous studies showed differences in in vitro heterophil function between parental (A>B) broilers and F1 reciprocal crosses (D>C). Our objectives were to (1) determine if in vitro variations translate to differences in resistance to Salmonella enteritidis (SE) and (2) quantitate cytokine mRNA in heterophils from SE-infected chicks. One-day-old chicks were challenged and organs were cultured for SE. Chicks with efficient heterophils (A and D) were less susceptible to SE compared to chicks with inefficient heterophils (B and C). Heterophils were isolated from SE-infected chicks and cytokine mRNA expression was evaluated using quantitative real-time RT–PCR. Pro-inflammatory cytokine mRNA was up-regulated in heterophils from SE-resistant chicks compared to susceptible chicks. This is the first report to quantitate cytokine mRNA in heterophils from SE-infected chicks. These data show a relationship between in vitro heterophil function, increased pro-inflammatory cytokine mRNA expression, and increased resistance to SE in 1-day-old chicks.

Tick-borne diseases (TBD) are common across the United States and can result in critical and chronic diseases in a variety of veterinary patients. Moreover, borreliosis, anaplasmosis, rickettsiosis, ehrlichiosis, and babesiosis are zoonotic and have been cited as the most common TBDs. Molecular diagnostic methodologies utilized for screening domestic dogs for these causative agents include real-time PCR (qPCR) assays in both singleplex and multiplex formats. However, current limitations of qPCR instruments restrict the number of fluorogenic labels that can be differentiated by the instrument for a given reaction. This study describes the development of the TickPath Layerplex, a diagnostic assay based on qPCR methodology that was adapted for the simultaneous detection and characterization of 11 pathogens responsible for causing 5 common TBDs in domestic dogs. The analytical and diagnostic performance of the layerplex assay was evaluated and shown to be compatible with common instruments utilized in molecular diagnostic laboratories. Test results revealed no inhibition or reduction in sensitivity during validation of the layerplex assay, and the limit of detection was determined to be near 16 genome copy equivalents per microliter. Overall, the high sensitivity, specificity, and screening capability of the assay demonstrate its utility for broadly screening dogs for common TBDs.

Current avian influenza (AI) virus surveillance programs involving wild birds rely on sample collection methods that require refrigeration or low temperature freezing to maintain sample integrity for virus isolation and/or reverse-transcriptase (RT) PCR. Maintaining the cold chain is critical for the success of these diagnostic assays but is not always possible under field conditions. The aim of this study was to test the utility of Finders Technology Associates (FTA®) cards for reliable detection of AI virus from cloacal and oropharyngeal swabs of wild birds. The minimum detectable titer was determined, and the effect of room temperature storage was evaluated experimentally using multiple egg-propagated stock viruses (n  =  6). Using real time RT-PCR, we compared results from paired cloacal swab and samples collected on FTA cards from both experimentally infected mallards (Anas platyrhynchos) and hunter-harvested waterfowl sampled along the Texas Gulf Coast. Based on the laboratory trials, the average minimal detectable viral titer was determined to be 1 × 104.7 median embryo infectious dose (EID50)/ml (range: 1 × 104.3 to 1 × 105.4 EID50/ml), and viral RNA was consistently detectable on the FTA cards for a minimum of 20 days and up to 30 days for most subtypes at room temperature (23 C) storage. Real-time RT-PCR of samples collected using the FTA cards showed fair to good agreement in live birds when compared with both real-time RT-PCR and virus isolation of swabs. AI virus detection rates in samples from several wild bird species were higher when samples were collected using the FTA cards compared with cloacal swabs. These results suggest that FTA cards can be used as an alternative sample collection method when traditional surveillance methods are not possible, especially in avian populations that have historically received limited testing or situations in which field conditions limit the ability to properly store or ship swab samples.

Wild waterfowl are considered the natural reservoir of type A influenza viruses, and the migratory nature of many waterfowl species presents a possible vehicle for global dissemination of these infectious agents. In order to fully understand the ecology of influenza viruses, multiyear surveillance efforts are critical, particularly in understudied areas, such as waterfowl wintering areas. Herein we report results obtained during the fifth year of a 5-yr avian influenza virus (AIV) surveillance project conducted on waterfowl wintering grounds of the Texas Coast. During year 5, the 2009–2010 hunting season (September, November–January), 655 cloacal swabs were collected from hunter-harvested waterfowl and screened for AIV by real-time RT-PCR (rRT-PCR) followed by virus isolation on all positive samples. Molecular methods were used for subtyping all AIV isolates. Sixty-five (9.5%) samples were positive for AIV by rRT-PCR, and 24 (3.7%) AIVs were isolated. Eight different hemagglutinin (H3, 4, 5, 6, 8, 9, 10, and 11) and seven different neuraminidase (N1, 2, 3, 4, 6, 8, and 9) subtypes were identified. This was the first year H8 and H9 were isolated throughout the 5-yr survey. Our results support the fact that continued multiyear surveillance of natural reservoirs, particularly in understudied areas, is needed in order to better understand the ecology of AIVs in nature.

The objective of this study was to determine the prevalence of avian influenza viruses (AIV) in bobwhite quail (Colinus virginianus) populations from the rolling plains of Texas, U. S. A. A total of 1320 swab samples (652 tracheal swabs and 668 cloacal swabs) and 44 serum samples were collected from wild-captured or hunter-harvested bobwhite quail from November 2009 to April 2011 at the Rolling Planes Quail Research Ranch, Fisher County, Texas, U. S. A. The presence of AIV in the swabs was determined by real-time reverse-transcription–PCR (rRT-PCR) and all samples positive or suspicious by rRT-PCR were further processed for virus isolation in embryonated chicken eggs. A total of 18 (1.4%) swab samples tested positive for AIV by rRT-PCR (cycle threshold [Ct] values <35): 13 cloacal swabs (1.9%) and 5 tracheal swabs (0.8%). In addition, 100 (7.6%) swab samples were considered suspicious (Ct values 35.1–40): 69 cloacal swabs (10.3%) and 31 tracheal swabs (4.7%). No virus was isolated from any of the rRT-PCR–positive or suspicious samples tested. Additionally, 44 serum samples were screened for AIV antibodies and were negative. The results presented here indicate low prevalence of AIV in wild populations of bobwhite quail.

Historically, virus isolation has been the method of choice for conducting surveillance for avian influenza virus (AIV) in avian species. More recently, the primary screening method has become real-time reverse transcription–polymerase chain reaction (RRT-PCR). We wanted to determine how these two testing methods (virus isolation and RRT-PCR) affected AIV prevalence estimation, particularly in an understudied, low-prevalence region—the waterfowl wintering grounds along the Texas mid–Gulf Coast. Cloacal swabs were collected from hunter-harvested waterfowl and other wetland-associated game birds during four consecutive hunting seasons (2005–2006 through 2008–2009). Overall prevalence by RRT-PCR (5.9%, 6.5%, 11.2%, and 5.5%) was approximately an order of magnitude higher than prevalence by virus isolation (0.5%, 1.3%, 3.9%, and 0.7%) for the four hunting seasons, respectively. Apparent AIV prevalence by virus isolation conducted only on RRT-PCR–positive samples resulted in estimates nearly identical in magnitude to those derived from parallel testing (0.5% vs. 0.6%, 1.3% vs. 1.7%, and 3.9% vs. 4.0% for 2005–2006, 2006–2007, and 2007–2008, respectively). Unlike most reports of seasonal variation in AIV prevalence, we documented differences in prevalence estimates among months by RRT-PCR only during 2008–2009 and by virus isolation only during 2006–2007 and 2007–2008. Our data indicate that screening samples by RRT-PCR followed by virus isolation only on RRT-PCR–positive samples provides a reasonable means to generate prevalence estimates close to the true prevalence as determined by virus isolation. We also confirmed the low prevalence of AIV in waterfowl wintering grounds along the Texas mid–Gulf Coast and demonstrated little variation in prevalence among months during the four hunting seasons sampled.