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Reducing Campylobacter jejuni Colonization of Poultry via Vaccination

Campylobacter jejuni is a leading bacterial cause of human gastrointestinal disease worldwide. While C. jejuni is a commensal organism in chickens, case-studies have demonstrated a link between infection with C. jejuni and the consumption of foods that have been cross-contaminated with raw or undercooked poultry. We hypothesized that vaccination of chickens with C. jejuni surface-exposed colonization proteins (SECPs) would reduce the ability of C. jejuni to colonize chickens, thereby reducing the contamination of poultry products at the retail level and potentially providing a safer food product for consumers. To test our hypothesis, we injected chickens with recombinant C. jejuni peptides from CadF, FlaA, FlpA, CmeC, and a CadF-FlaA-FlpA fusion protein. Seven days following challenge, chickens were necropsied and cecal contents were serially diluted and plated to determine the number of C. jejuni per gram of material. The sera from the chickens were also analyzed to determine the concentration and specificity of antibodies reactive against the C. jejuni SECPs. Vaccination of chickens with the CadF, FlaA, and FlpA peptides resulted in a reduction in the number of C. jejuni in the ceca compared to the non-vaccinated C. jejuni-challenged group. The greatest reduction in C. jejuni colonization was observed in chickens injected with the FlaA, FlpA, or CadF-FlaA-FlpA fusion proteins. Vaccination of chickens with different SECPs resulted in the production of C. jejuni-specific IgY antibodies. In summary, we show that the vaccination of poultry with individual C. jejuni SECPs or a combination of SECPs provides protection of chickens from C. jejuni colonization.

Coxiella-Like Infection in Psittacines and a Toucan

Abstract Seven psittacine birds and a toucan (Ramphastos toco) were diagnosed as infected with Coxiella-like bacteria, based on polymerase chain reaction and bacterial 16S rRNA gene sequence obtained from each birds liver tissue. Most of the birds exhibited lethargy and weakness for several days prior to death. Gross lesions included mild to moderate emaciation and severely enlarged and mottled pale livers and spleens. Microscopically, there was multifocal necrosis of hepatocytes with infiltration of a mixed population of inflammatory cells, including lymphocytes, heterophils, plasma cells, and macrophages randomly scattered throughout in most birds. In several birds within the macrophages there were vacuoles containing basophilic small cocco-bacilli organisms measuring about 0.5–1 µm. The spleens had increased numbers of mononuclear phagocytic system cells, some of which had vacuoles that contained similar organisms, as observed in the liver. There was inflammation in the epicardium and endocardium, interstitium of the lungs, kidney, adrenal and thyroid glands, lamina propria of the intestine, and in occasional birds in the brain, bursa of Fabricius, and bone marrow associated with similar organisms in the macrophages. Transmission electron microscopy of the liver and lungs in most birds and in the thyroid glands of one bird revealed pleomorphic round to elongated bacteria measuring about 0.45 µm in diameter and more than 1.0 µm in length. Most of these organisms contained a peripheral zone of loosely arranged electron dense material that was located immediately beneath a trilaminar membrane. Occasional organisms contained nucleoids. This is the first documentation of disease presumptively associated with Coxiella-like bacteria in birds. Infección con bacterias parecidas a Coxiella en psitácidos y un tucán. Se diagnosticó la infección con una bacteria parecida a Coxiella en siete aves psitácidas y en un tucán (Ramphastos toco), basándose en una prueba de reacción en cadena por la polimerasa y la secuenciación del gen 16S rRNA obtenido de tejido hepático de cada una de las aves. La mayoría de las aves mostraron letargia y debilidad por varios días antes de la muerte. Las lesiones macroscópicas incluyeron emaciación de leve a moderada, así como hígados y bazos pálidos, moteados y severamente aumentados de tamaño. Microscópicamente, se observó necrosis multifocal de los hepatocitos con infiltración de una mezcla de células inflamatorias incluyendo linfocitos, heterófilos, células plasmáticas y macrófagos distribuidos aleatoriamente en la mayoría de las aves. En varias de las aves, se observaron dentro de los macrófagos hepáticos vacuolas con pequeños organismos basofílicos del tipo cocobacilos que medían entre 0.5 y 1 µm. Los bazos tenían un número aumentado de células del sistema fagocítico, algunas de las cuales mostraban vacuolas que contenían organismos similares a los observados en el hígado. Se observó inflamación asociada a organismos similares en los macrófagos, en el epicardio y endocardio, en el intersticio de los pulmones, riñones, glándulas adrenal y tiroides, en la lámina propia del intestino y en algunas de las aves en el cerebro, bolsa de Fabricio y medula ósea. La microscopía electrónica del hígado y los pulmones en la mayoría de las aves y de la glándula tiroides de un ave, reveló la presencia de bacterias de redondas a alargadas de aproximadamente 0.45 µm de diámetro y más de 1 µm de longitud. La mayoría de estos organismos contenían una zona periférica de material denso localizado inmediatamente por debajo de la membrana trilaminar. Algunos organismos contenían nucléolos. Este es el primer reporte de enfermedad presumiblemente asociada con bacterias parecidas a Coxiella en aves. Abbreviations: bp = base pair; CAHFS = California Animal Health and Food Safety Laboratory System; FA = fluorescent antibody; H&E = hematoxylin and eosin; IHC = immunohistochemistry; NBF = neutral buffered formalin; PAS = periodic acid Schiff; RT-PCR = reverse transcriptase–polymerase chain reaction

Exotic Newcastle disease in a game chicken flock.

A sudden increase in mortality, preceded by a short history of respiratory signs and diarrhea, occurred in a backyard flock of 48 game chickens in the Central Valley of California. Necropsy findings included severe generalized linear hemorrhages and/or ulcers in the digestive tract, larynx, and trachea. Histology revealed severe multifocal hemorrhages and necrosis in the mucosa of the respiratory and digestive tracts, vasculitis, and necrosis of lymphoid tissue. The birds were serologically negative to Newcastle disease virus; this was consistent with an acute infection. The avian paramyxovirus type 1 isolated was characterized as velogenic viscerotropic Newcastle disease virus. A thorough epidemiologic investigation was carried out, and no other premises were found to have birds with clinical signs or evidence of exposure. The entire outbreak was limited to the original backyard flock and resolved within 14 days of the onset of clinical signs.

Histomoniasis in the Bursa of Fabricius of Chickens

Abstract Histomoniasis was diagnosed in a flock of 6-wk-old commercial chickens. Clinical signs included depression, stilted gait, inappetence, and a slight increase in mortality. At necropsy, there were pale-yellow to dark-gray circular and depressed necrotic lesions in the liver. The ceca were enlarged and impacted with caseous cores. Cecal worms were not observed either at necropsy or on histopathology. Histomonads were demonstrated microscopically within the bursa of Fabricius in addition to the liver, ceca, and spleen. This is the first report of the presence of histomonads in the bursa of Fabricius in commercial chickens.

COMPARISON OF DIAGNOSTICS TECHNIQUES IN AN OUTBREAK OF INFECTIOUS LARYNGOTRACHEITIS FROM MEAT CHICKENS

Abstract Various diagnostics techniques were compared for their ability to detect infectious laryngotracheitis (ILT) during an outbreak in chickens aged between 4 and 21 wk. Gross lesions ranged from excess mucus to accumulation of fibrinonecrotic exudate in the larynx and trachea. Syncytial cells with intranuclear inclusion bodies were found in sinus, conjunctiva, larynx, trachea, lung, and air sac. Virus isolation in chicken embryos was attempted in every case. Negative-stain electron microscopy detected herpesvirus in only 6% of the cases. Yet, isolation of ILT virus in the chorioallantoic membrane was presumed by histology in >20% of the samples and confirmed by fluorescent antibody (FA) in 35% of the embryos inoculated with conjunctivas or tracheas from affected birds. Overall, results from histology and FA tests were highly correlated. FA test has the advantage over histology of being diagnostically specific for ILT virus. Polymerase chain reaction was the most sensitive test and detected the viral DNA even in cases where histology and FA were negative. ILT virus DNA was quantified by real-time polymerase chain reaction (Re-Ti ILTV). Histologic and FA results from larynx and trachea were negative if the concentration of the viral DNA was ≤4 of log10. A viral DNA concentration higher than log10 4, as determined by Re-Ti ILTV, was required for clinical ILT to be manifested.

Femoral Fractures in a Young Male Turkey Breeder Flock

Twenty-two 32-to-35-wk-old male turkeys from a commercial breeding flock were presented to the California Veterinary Diagnostic Laboratory System, Fresno Branch, with a clinical complaint of lameness and increased mortality. Necropsy findings included a unilateral, closed oblique fracture involving the femur in 12 birds. Ten complete femoral fractures had periosteal new bone adjacent to and bridging the fracture. Periosteal callus formation, in this case, suggested that preexisting lesions preceded complete fracture of the femur. Factors such as selection for heavy body weight, lack of exercise, and handling might have contributed to the development and promotion of complete fractures.

Phenotypic and Genotypic Characterization of Salmonella arizonae from an Integrated Turkey Operation

Abstract Fifty cases submitted between 2000 and 2002 were selected for retrospective analysis to evaluate possible relationships between Salmonella arizonae isolated from breeder flocks, hatching eggs, and meat bird flocks belonging to a single turkey integrator. In all the meat bird cases selected for this study, arizonosis was the primary diagnosis. In birds under 1 month of age, clinical signs and pathologic changes were observed in older birds. The Salmonella arizonae isolates were analyzed by antibiotic resistance pattern and serotype and genotyped by pulsed-field gel electrophoresis (PFGE). Serotyping and PFGE yielded similar results, but the antibiotic resistance patterns did not correspond to either serotyping or PFGE typing. The presence of common pulsed-field patterns in breeder flocks, eggs, and meat bird flocks suggested that S. arizonae was being transmitted vertically from the breeder flock.

Salpingitis in Pekin ducks associated with concurrent infection with Tetratrichomonas sp. and Escherichia coli

Increased mortality (1.5% per week) and low egg production (5–10% lower than normal) were observed in a flock of domestic breeding Pekin ducks (Anas platyrhynchos). At necropsy, salpingitis and peritonitis were the most significant findings. Histologically, there was accumulation of necrotic debris in the lumen of the oviduct. Numerous bacteria and trichomonads were observed histologically in the lumen of the vagina and occasionally in the shell gland. Escherichia coli and a trichomonad were isolated from the oviduct. The trichomonads were oval (6–8 μm long, 4.5–6 μm wide) and had 4 anterior flagella and an undulating membrane extending over the entire length of the body, finishing in a long posterior flagellum. Morphology was consistent with trichomonads of the genus Tetratrichomonas. Comparative sequence analysis of the 5.8S ribosomal RNA gene and the flanking internal transcribed space regions of the trichomonad isolate did not closely match with available sequences of the same region of other trichomonadid protozoa.

Intervention Strategies for Laryngotracheitis: Impact of Extended Downtime and Enhanced Biosecurity Auditing

Abstract An outbreak of vaccinal infectious laryngotracheitis (LT) began in 2005 involving 57 ranches of two broiler companies in California. Standard biosecurity, and cleaning and disinfection programs along with vaccination, did not stop the outbreak. Due to the close proximity and number of birds in the same geographic area, the decision was made by both companies to attempt a joint regional and zonal depopulation strategy. The strategy involved extended downtime between flock placements on ranches located within close proximity to one another. This extended downtime on each ranch ranged from 30 to 91 days. An extensive biosecurity audit, with more than 70 items, was implemented. Briefly, this included heating all houses to 37 C for 100 hr, removing the litter, cleaning and disinfecting everything on the ranches, then again heating the houses to 37 C for 100 hr. Used litter was spread on crops away from poultry, or was sent to a litter processor for pasteurization. Extensive surveillance for LT at 28, 35, and 42 days of age was performed on the initial flocks, which had been placed immediately after the extended downtime. Since completion of this plan in early 2008, LT was diagnosed on only two of the previously 57 affected ranches. Those two ranches, and those within close proximity, went through the extended downtime program and biosecurity audit a second time. Currently, both companies are free of LT. This program lends credence to the importance of cooperation between companies to consider all the ranches within close proximity as the population at risk. In the control of LT in broilers, the program also highlights the necessity for extended downtime and enhanced biosecurity auditing of all flocks.

Production and Evaluation of Chicken Egg-Yolk-Derived Antibodies Against Campylobacter jejuni Colonization-Associated Proteins

Campylobacter jejuni is one of the most important causes of foodborne gastroenteritis. Chickens are considered a reservoir host of C. jejuni, and epidemiological studies have shown that contaminated chicken meat is a primary source of human infection. The objective of this study was to produce chicken egg-yolk-derived antibody (IgY) against the five C. jejuni colonization-associated proteins or CAPs (CadF, FlaA, MOMP, FlpA, and CmeC). Recombinant C. jejuni CAPs were expressed in Escherichia coli and were purified by affinity chromatography. Specific-pathogen-free laying hens were hyperimmunized with each recombinant CAP to induce production of α-CAP-specific IgY. Egg yolks were collected from immunized and nonimmunized hens and were lyophilized to obtain egg-yolk powder (EYP) with or without α-C. jejuni CAP-specific IgY. IgY was purified from EYP, and the antibody response in serum and egg yolk was tested by indirect enzyme-linked immunosorbent assay. The α-C. jejuni CAP-specific IgY levels were significantly (p<0.05) higher in both serum and EYP obtained from immunized hens as compared with the nonimmunized hens. Each α-C. jejuni CAP-specific IgY reacted with the C. jejuni cells and recombinant CAPs as detected by immunofluorescence microscopy and Western blot assays, respectively. We also show that α-CadF, α-MOMP, and α-CmeC IgY significantly reduced adherence of C. jejuni to the chicken hepatocellular carcinoma (LMH) cells, suggesting that these α-C. jejuni CAP-specific IgY may be useful as a passive immunotherapeutic to reduce C. jejuni colonization in chickens.