Pamela J. Ferro

Differential cytokine mRNA expression in heterophils isolated from Salmonella-resistant and -susceptible chickens

We recently showed that increased in vitro heterophil functional efficiency translates to increased in vivo resistance to a systemic Salmonella enteritidis (SE) infection utilizing a parental pair of broiler chickens (lines A and B) and the F1 reciprocal crosses (C and D). Heterophils produce cytokines and modulate acute protection against Salmonella in young poultry. Therefore, we hypothesize that heterophils from SE‐resistant chickens (A and D) have the ability to produce an up‐regulated pro‐inflammatory cytokine response compared to that of heterophils from SE‐susceptible chickens (B and C). In this study, heterophils were isolated from day‐old chickens and treated with either RPMI‐1640 (as the control), or phagocytic agonists (SE, or SE opsonized with either normal chicken serum or immune serum against SE) and cytokine mRNA expression assessed using real‐time quantitative reverse transcription–polymerase chain reaction. Heterophils from SE‐resistant chickens (A and D) had significantly higher levels of pro‐inflammatory cytokine (interleukin (IL)‐6, IL‐8, and IL‐18) mRNA expression upon treatment with all agonists compared to heterophils from SE‐susceptible lines (B and C). Further, heterophils from SE‐resistant chickens had significantly decreased mRNA expression levels of transforming growth factor‐β4, an anti‐inflammatory cytokine, when compared to heterophils from SE‐susceptible chickens. These data indicate cytokine gene expression in heterophils may be a useful parameter in determining resistance to Salmonella, as indicated by our previous in vivo SE studies. Therefore, heterophil functional efficiency and cytokine production may be useful biomarkers for poultry breeders to consider when developing new immunocompetent lines of birds.

Heterophils isolated from chickens resistant to extra-intestinal Salmonella enteritidis infection express higher levels of pro-inflammatory cytokine mRNA following infection than heterophils from susceptible chickens

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.

Detection and Characterization of a Distinct Bornavirus Lineage from Healthy Canada Geese (Branta canadensis)

ABSTRACT Avian bornaviruses (ABV), identified in 2008, infect captive parrots and macaws worldwide. The natural reservoirs of these viruses are unknown. Reverse transcription-PCR (RT-PCR) was used to screen oropharyngeal/cloacal swab and brain samples from wild Canada geese (Branta canadensis) for ABV. Approximately 2.9% of swab samples were positive for bornavirus sequences. Fifty-two percent of brain samples from 2 urban flocks also tested positive, and brain isolates were cultured in duck embryo fibroblasts. Phylogenetic analyses placed goose isolates in an independent cluster, and more notably, important regulatory sequences present in Borna disease virus but lacking in psittacine ABVs were present in goose isolates.

Multiyear surveillance for avian influenza virus in waterfowl from wintering grounds, Texas coast, USA.

This surveillance can help in assessments of the prevalence of wild animal-to-human transmission.

Avian Influenza Surveillance in Hunter-harvested Waterfowl from the Gulf Coast of Texas (November 2005–January 2006)

The objectives of our study were to determine prevalence of avian influenza viruses (AIV) on wintering grounds on the Texas Gulf Coast, USA, and to compare real-time reversetranscriptase polymerase chain reaction (rRT-PCR) and virus isolation for detection of AIV in cloacal swabs from wild waterfowl. Cloacal swabs were collected from hunter-harvested waterfowl from November 2005 to January 2006 at four wildlife management areas. Seven AIV were isolated from four species of ducks: Green-winged Teal (Anas crecca) in November; Blue-winged Teal (Anas discors) in November; Mottled Duck (Anas fulvigula) in December, and Northern Shoveler (Anas clypeata) in January. Prevalence of AIV for each of these species during the sampling period was 1.4, 2, 6, and 0.6%, respectively. The AIV subtypes detected were H1N2, H1N4, H4N6, H6N2, and H10N7, all previously reported in North American waterfowl. Our study identified AIV subtypes not previously reported on the Texas Gulf Coast and provides baseline data for a multiyear surveillance project.

Association between in vitro heterophil function and the feathering gene in commercial broiler chickens.

We recently showed that in vitro heterophil functional efficiency in commercial broiler chickens is genetically controlled and may be a sex-associated trait. To further characterize the genetic mechanism(s) of heterophil functional efficiency, we wanted to determine whether the feathering gene, present on the Z sex chromosome, contributes to heterophil functional efficiency. Heterophils from two pairs of broiler lines were evaluated; eachpair contained a fast feather (FF) (lines A and X) and a slow feather (SF) line (lines B and Y). On days 1 and 4 post-hatch, heterophils isolated from two sets of pure line broilers (A and B, and X and Y) were evaluated for their ability to (1) phagocytize Salmonella enteritidis, and (2) exhibit bactericidal activity against S. enteritidis. On days 1 and 4 post-hatch, heterophils isolated from the FF lines were statistically (P < 0.02) more proficient at phagocytizing S. enteritidis than heterophils from SF lines. Bactericidal activity was also statistically (p ≤ 0.02) greater on day 1 post-hatch in the heterophils isolated from FF lines compared to heterophils isolated from SF lines. These data indicate that the presence of the FF gene locus on the Z sex chromosome contributes to heterophil function and may contribute to the early innate immune competence of a flock.

Disparity in susceptibility to vancomycin-resistant Enterococcus organ invasion in commercial broiler chickens that differ in innate immune responsiveness

Enterococci, gram-positive bacterium, are found on virtually all consumer-ready retail meats. Recently, we characterized the innate immune response phenotype of parental lines of broilers (A and B) and F1 reciprocal crosses (C and D). In vitro heterophil functional analyses and in vivo challenge trials showed line A was more immunologically responsive and more resistant to Salmonella (Gram-negative bacteria) and Eimeria tenella (parasitic protozoan) infections than line B. Further, cross D was more responsive with increased resistance compared to cross C. We hypothesized that line A and D chickens were also more resistant to gram-positive bacteria. Day-old chickens were challenged with vancomycin-resistant Enterococcus gallinarum (VREG), euthanized, and hearts, livers, and spleens cultured for VREG. One-day post challenge, there were significantly fewer VREG-positive hearts, livers, and spleens in line A than line B. Likewise, cross D chickens had significantly fewer VREG-positive hearts, livers, and spleens than cross C chickens. To determine if VREG was cleared, chickens were necropsied seven days post-challenge. Again, significantly fewer organ cultures from line A and D were VREG-positive compared to line B and C. Further, this study showed the hematologic profile reflected changes associated with protection against VREG; there were significantly more circulating heterophils and monocytes in VREG-resistant lines A and D with no changes in VREG-susceptible lines B and C. Collectively, line A and D chickens are immunologically more responsive and more resistant to pathogens, including VREG, compared to line B and C chickens.

Infectious disease survey of Rio Grande wild turkeys in the Edwards Plateau of Texas

State wildlife agencies have translocated thousands of wild turkeys (Meleagris gallopavo) since the 1930s to reestablish this species. Because of threats to the domestic poultry industry and wild birds, screening for selected infectious agents has become routine since the early 1980s. One of the principal sources for Rio Grande wild turkeys (M. gallopavo intermedia) for translocation purposes was the Edwards Plateau of Texas (USA). Unfortunately, turkey abundance has declined in the southern Edwards Plateau since the late 1970s. Surprisingly few studies have addressed wild turkeys in this region, perhaps reflecting its status as the heart of Rio Grande turkey range. We surveyed 70 free-living Rio Grande wild turkeys from Bandera and Kerr counties, Texas, for evidence of exposure to Salmonella typhimurium, S. pullorum, Mycoplasma gallisepticum, M. meleagridis, M. synoviae, Chlamydophila psittaci, and the avian influenza, Newcastle disease, turkey corona, and reticuloendotheliosis viruses. Of these, 80% (56) were seropositive for both M. gallisepticum and M. synoviae on the serum plate antigen test. Ten of these individuals (14% of total) were positive for M. synoviae by hemagglutination inhibition testing. All other serologic tests were negative. Two adult females sampled in Kerr County, whose body mass was significantly less than that of other adult females trapped in the area, tested positive for reticuloendotheliosis virus (REV) proviral DNA on polymerase chain reaction. Reticuloendotheliosis virus was isolated from one of these individuals. The pathogenesis, transmission, and/or population-level influences of M. gallisepticum, M. synoviae, and REV in Rio Grande wild turkeys deserves further study.

Infectious disease survey of lesser prairie chickens in north Texas.

Lesser prairie chicken (Tympanuchus pallidicinctus) abundance, like that of most grassland birds, has declined rangewide for decades. Although habitat loss and degradation are likely ultimate causes for this decline, infectious agents, particularly microparasites, could be proximate contributors. No surveys of pathogenic bacteria or viruses have been published for this species. We surveyed 24 free-living lesser prairie chickens from Hemphill County, Texas (USA), for evidence of exposure to Salmonella typhimurium, S. pullorum, Mycoplasma gallisepticum, M. synoviae, Chlamydophila psittaci, and the avian influenza, Newcastle disease, infectious bronchitis, and reticuloendotheliosis viruses. Two of 18, and eight of 17 samples were seropositive for the Massachusetts and Arkansas serotypes of infectious bronchitis virus, respectively. Five of the eight positive individuals were juveniles, two of which were seropositive for both serotypes. All other serologic and genetic tests were negative. Because the ecological significance of these results is unknown, the pathogenesis, transmission, and/or population-level influences of infectious bronchitis and related avian coronaviruses for lesser prairie chickens deserves further study.

Use of FTA® Sampling Cards for Molecular Detection of Avian Influenza Virus in Wild Birds

SUMMARY. 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.