H. John Barnes

Restriction Endonuclease Analysis of Infectious Laryngotracheitis Viruses: Comparison of Modified-Live Vaccine Viruses and North Carolina Field Isolates

Six modified-live (ML) infectious laryngotracheitis (ILT) vaccine viruses, three reference strains, and 18 field isolates were compared by restriction endonuclease analysis of their DNA. Viral DNA digestion patterns were established for vaccine viruses using restriction endonucleases PstI, BamHI, KpnI, and HindIII. Using these enzymes, five of six ML vaccine viruses had identical restriction endonuclease cleavage patterns. Vaccine viruses had distinct patterns compared with ILT virus reference strains Illinois-N71851, Cover, and NVSL. Restriction endonuclease cleavage patterns of 18 field isolates of ILT virus, obtained from ILT outbreaks in North Carolina, were indistinguishable from vaccine viruses. These results suggest a possible role of vaccine or vaccine-like viruses in recent ILT outbreaks.

Associations Between Multidrug Resistance, Plasmid Content, and Virulence Potential Among Extraintestinal Pathogenic and Commensal Escherichia coli from Humans and Poultry

The emergence of plasmid-mediated multidrug resistance (MDR) among enteric bacteria presents a serious challenge to the treatment of bacterial infections in humans and animals. Recent studies suggest that avian Escherichia coli commonly possess the ability to resist multiple antimicrobial agents, and might serve as reservoirs of MDR for human extraintestinal pathogenic Escherichia coli (ExPEC) and commensal E. coli populations. We determined antimicrobial susceptibility profiles for 2202 human and avian E. coli isolates, then sought for associations among resistance profile, plasmid content, virulence factor profile, and phylogenetic group. Avian-source isolates harbored greater proportions of MDR than their human counterparts, and avian ExPEC had higher proportions of MDR than did avian commensal E. coli. MDR was significantly associated with possession of the IncA/C, IncP1-α, IncF, and IncI1 plasmid types. Overall, inferred virulence potential did not correlate with drug susceptibility phenotype. However, certain virulence genes were positively associated with MDR, including ireA, ibeA, fyuA, cvaC, iss, iutA, iha, and afa. According to the total dataset, isolates segregated significantly according to host species and clinical status, thus suggesting that avian and human ExPEC and commensal E. coli represent four distinct populations with limited overlap. These findings suggest that in extraintestinal E. coli, MDR is most commonly associated with plasmids, and that these plasmids are frequently found among avian-source E. coli from poultry production systems.

Campylobacter colonization of sibling turkey flocks reared under different management conditions

Uncertainty exists concerning the key factors contributing to Campylobacter colonization of poultry, especially the possible role of vertical transmission from breeder hens to young birds. A longitudinal study of Campylobacter colonization was performed in two sibling pairs of turkey flocks (four flocks total). Each pair of sibling flocks shared breeder hen populations and was obtained from the same hatchery. One flock of each pair was grown on a commercial farm, and the other was grown in an instructional demonstration unit (Teaching Animal Unit [TAU]). Flocks were located within a 60-mi (96.8-km) radius. The time of placement, feed formulations, stocking density, and general husbandry were the same for both flocks, and each flock was processed at a commercial processing plant following standard feed withdrawal and transport protocols. Both flocks grown on the commercial farms became colonized with Campylobacter between weeks 2 and 3 and remained colonized until processing. Between 80 and 90% of isolates were Campylobacter coli, and the remainder were Campylobacter jejuni. In contrast, neither C. coli nor C. jejuni were isolated from either of the TAU flocks at any time during the production cycle. None of the fla types of Campylobacter from the breeders that provided poults to one of the commercial flocks matched those from the progeny. These results failed to provide evidence for vertical transmission and indicate that this type of transmission either did not occur or was not sufficient to render the TAU turkey flocks Campylobacter positive. Management practices such as proper litter maintenance, controlled traffic between the TAU farm and other turkey flocks, and other less well-defined aspects of turkey production were likely responsible for the absence of Campylobacter in the TAU flocks before harvest.

Experimental Reproduction of Enterococcal Spondylitis in Male Broiler Breeder Chickens

Abstract There has been a recent emergence of epidemic spinal infections with necrosis causing lameness and mortality in male broilers and broiler breeders. Mortality in affected flocks may be as high as 15%. The disease has been called enterococcal spondylitis (ES), based on the frequent isolation of Enterococcus cecorum from the lesions and necrosis and inflammation observed in the free thoracic vertebrae (FTV) of affected birds. Male broiler breeders in an experimental setting were challenged with pure E. cecorum isolates obtained from ES-affected commercial flocks. Challenge routes included oral gavage (108), intravenous (IV; 103), and air sac (AS; 103). Half the study birds in each group were chemically immunosuppressed with dexamethasone. Spinal lesions were observed grossly in birds challenged intravenously (2.9%) and birds challenged orally (6.1%). Microscopic spinal lesions consistent with ES were more frequently identified compared with gross lesions in the orally challenged group (30.3%). Chemical immunosuppression with dexamethasone was not associated with a greater incidence of ES in this study. By recreating the disease experimentally, the study design reported here may help in the further development of an experimental challenge model for future studies on risk factors, prevention, and therapeutic intervention of ES.

Experimental Infection of Young Turkeys with Eastern Equine Encephalitis Virus and Highlands J Virus

Depression, somnolence, and increased mortality were observed in 2-week-old turkeys inoculated intramuscularly with either eastern equine encephalitis (EEE) virus or Highlands J (HJ) virus. Mortality rates in EEE virus- and HJ virus-inoculated turkeys were 7/30 (23%) and 9/30 (27%), respectively; no sham-inoculated controls died. Both EEE virus- and HJ virus-inoculated turkeys developed viremia that lasted 2 days; peak mean titers were 5.5 and 3.2 log10 plaque-forming units per ml of blood, respectively. Pathologic changes in both EEE virus- and HJ virus-inoculated turkeys consisted primarily of multifocal necrosis in the heart, kidney, and pancreas, and lymphoid necrosis and depletion in the thymus, spleen, and bursa of Fabricius. The findings indicate that EEE virus and HJ virus are pathogenic for young turkeys.

An Outbreak and Source Investigation of Enterococcal Spondylitis in Broilers Caused by Enterococcus cecorum

SUMMARY. Enterococcus cecorum was isolated from spondylitis lesions in broilers from two flocks in North Carolina that were experiencing increased mortality. Affected birds showed paresis and paralysis, clinical signs characteristic of enterococcal spondylitis (ES). Affected birds rested on their hocks and caudal abdomens with legs extended forward and were unable to stand or walk. Necropsy examination of affected birds revealed firm to hard inflammatory masses involving the vertebral bodies at the level of the free thoracic vertebra that bulged dorsally and compressed the spinal cord. When opened, lesions contained pale, tan to yellow caseonecrotic material. Microscopically, necrosis and fibrinoheterophilic spondylitis with intralesional gram-positive bacteria were seen. Heavy growth of E. cecorum recovered from vertebral lesions confirmed the diagnosis of ES. To investigate possible sources of the organism for one of the flocks bacterial cultures were made from the environment, water lines, mice trapped on the farm, cecal/cloacal swabs from one of the parent broiler breeder flocks, egg residue, hatching eggs, and the hatchery environment. Except for cecal/cloacal swabs from the breeders, E. cecorum was not isolated from any of these samples. When compared phenotypically and genotypically, cecal/cloacal isolates of E. cecorum from the breeders differed from isolates from spondylitis lesions in the broilers. The source of E. cecorum for the broiler flocks was not determined, but vertical transmission appears unlikely.

Selected Blood Chemistry and Gas Reference Ranges for Broiler Breeders Using the i-STAT® Handheld Clinical Analyzer

Abstract Selected blood chemistry and gas reference ranges for clinically healthy broiler breeder hens were established using CG8+ cartridges in an i-STAT® handheld point-of-care clinical analyzer. Samples from 165 hens (25–36 wk of age), representing three broiler breeder strains reared by four integrators, were evaluated. A standardized sampling technique was developed to minimize instrument error readings. The following reference ranges and means, respectively, were determined: sodium (141.6–152.6, 147.1 [mmol/L]), potassium (4.1–5.7, 4.9 [mmol/L]), ionized calcium (1.20–1.73, 1.47 [mmol/L]), glucose (207.2–260.7, 234.0 [mg/dl]), hematocrit (21.3–30.8, 26.1 [% packed cell volume]), hemoglobin (7.3–10.5, 8.9 [g/dl]), pH (7.28–7.57, 7.42), carbon dioxide partial pressure (25.9–49.5, 37.7 [mm Hg]), oxygen partial pressure (32.0–60.5, 46.2 [mm Hg]), bicarbonate (18.9–30.3, 24.6 [mmol/L]), total carbon dioxide (19.9–31.5, 25.7 [mmol/L]), base excess (−6.8 to 7.2, 0.2), and oxygen saturation (70.6–93.3, 82.0 [%]). Wide ranges in blood gases and base excess occurred in all strains. Cobb strain hens had significantly lower glucose and higher partial and saturated oxygen values compared with two Ross strains. Significant differences in several blood parameters were found among different integrators and in older postpeak production birds. The i-STAT handheld point-of-care clinical analyzer provides rapid, relatively low cost, blood chemistry values that are useful for investigating broiler breeder flock diseases of unknown or uncertain etiology, especially those suspected of having a metabolic cause.

Molecular epidemiology of Enterococcus cecorum isolates recovered from enterococcal spondylitis outbreaks in the southeastern United States

Enterococcus cecorum, a normal intestinal inhabitant, is increasingly responsible for outbreaks of arthritis and osteomyelitis in chickens worldwide. Enterococcal spondylitis (ES) is a specific manifestation of E. cecorum-associated disease in which increased flock morbidity and mortality result from chronic infection involving the free thoracic vertebra. In this study the genetic relatedness and antimicrobial resistance of isolates recovered from ES-affected flocks in the southeastern United States were determined. ES outbreaks from 2007 to 2011 were investigated in North Carolina (15 flocks, 13 farms, four integrators), South Carolina (one flock, one farm, one integrator) and Alabama (six flocks, six farms, one integrator). From these 22 epidemiologically distinct outbreaks, 326 isolates of E. cecorum were recovered. Isolates from spinal lesions and caeca of affected birds (cases) and caeca of unaffected birds (controls) were genotyped using pulsed-field gel electrophoresis; phenotyped using both GenIII MicroPlate™ (Biolog; Hayward, CA, USA) microbial identification plates and antimicrobial sensitivity testing; and compared with each other. Isolates from spinal lesions were incapable of mannitol metabolism and the majority of these isolates were genetically clonal. In contrast, caecal isolates from control birds varied in their ability to metabolize mannitol and were genetically diverse. Isolates from both case and control birds had high levels of antimicrobial resistance. These findings indicate that the increase in E. cecorum-associated disease in the southeast United States is due to the emergence of new clones with increased pathogenicity and multidrug resistance.

Disseminated Aspergillus flavus Infection in Broiler Breeder Pullets

Abstract Increased morbidity and mortality occurred in a 5-wk-old broiler breeder replacement pullet flock. The affected broiler pullet flock was housed on the first floor of a two-story confinement building. Mortality increased to 0.1%/day compared to the flock on the second floor, which had mortality levels of less than 0.01%/day. Clinical signs in the affected chickens included inactivity, decreased response to stimuli, and anorexia. No respiratory or neurologic signs were observed. On necropsy, affected pullets were dehydrated and emaciated and had disseminated variably sized single or multiple heterophilic granulomas that contained intralesional septate and branching fungal hyphae. Lesions were extensive around the base of the heart in the thoracic inlet and in the kidneys. Other affected organs included eyelid, muscle, proventriculus, ventriculus, intestine, liver, spleen, lung, and heart. Aspergillus flavus was cultured from the visceral granulomas. The source of flock exposure to the organism was not determined.

Comparison of gene expression patterns between avian and human ovarian cancers

OBJECTIVES A putative model of spontaneous cancer has been described in the laying hen that bears significant similarities to human ovarian cancer. Our objective was to characterize and compare the patterns of gene expression in chicken and human forms of this disease. METHODS RNA from 20 localized and metastatic ovarian and oviductal chicken tumor samples was isolated, amplified using in vitro transcription, and hybridized against normal ovarian epithelium to a customized cDNA microarray constructed for these studies. Differentially expressed genes were identified for localized ovarian, metastatic ovarian, and oviductal (or tubal) cancer by class comparison using BRB-ArrayTools. Results were validated with semi-quantitative PCR. A gene list (prediction model) constructed with the class prediction tool was used in a human ovarian cancer microarray obtained from the GEO datasets (GSE6008) in order to compare these results across species. RESULTS Class comparison analysis between localized ovarian, metastatic ovarian and oviductal cancer yielded 41 different informative probes that coded for 27 unique genes. Localized ovarian samples clustered between metastatic ovarian and oviductal cancer samples. Using our chicken data as a training set and leaving oviductal samples out of the analysis, we created a prediction model that classified early stage and advanced stage human ovarian cancer gene expression arrays with 78% overall accuracy. CONCLUSIONS Gene expression of spontaneous ovarian cancer in the chicken is comparable to gene expression patterns of human ovarian cancer.