Parimal Roy

Results of Salmonella Isolation from Poultry Products, Poultry, Poultry Environment, and Other Characteristics

SUMMARY. Five hundred sixty-nine Salmonella were isolated out of 4745 samples from poultry products, poultry, and poultry environment in 1999 and 2000 from the Pacific northwest. These Salmonella were identified to their exact source, and some were serogrouped, serotyped, phage typed, and tested for antibiotic sensitivity. Food product samples tested included rinse water of spent hens and broilers and chicken ground meat. Poultry environment samples were hatchery fluff from the hatcheries where eggs of grandparent broiler breeders or parent broiler breeder eggs were hatched and drag swabs from poultry houses. Diagnostic samples were of liver or yolk sac contents collected at necropsy from the young chicks received in the laboratory. Of these samples tested, 569 were Salmonella positive (11.99%). Ninety-two Salmonella were serogrouped with polyvalent somatic antisera A-I and the polymerase chain reaction. Somatic serogroups B and C comprised 95.25% of all the Salmonella. Out of a total of 569 positive samples, 97 isolates of Salmonella were serotyped. A total of 16 serotypes and an unnamed Salmonella belonging to serogroup C1 were identified. The Salmonella serotypes were heidelberg (25.77%); kentucky (21.64%); montevideo (11.34%); hadar and enteritidis (5.15% each); infantis, typhimurium, ohio, and thompson (4.12% each); mbandaka and cerro (3.09% each); senftenberg (2.06%); berta, istanbul, indiana, and saintpaul (1.03% each); and an unnamed monomorphic Salmonella (2.06%). Ninety-two Salmonella were tested for drug sensitivity with nine different antimicrobials. All of the 92 Salmonella were resistant to erythromycin, lincomycin, and penicillin except one sample (S. berta), which was moderately sensitive to penicillin. All of the tested Salmonella were susceptible to sarafloxacin and ceftiofur. The percentages of Salmonella susceptible to sulfamethoxazole-trimethoprim, gentamicin, triple sulfa, and tetracycline were 97.83%, 92.39%, 86.96%, and 82.61%, respectively.

Detection of Pigeon Circovirus by Polymerase Chain Reaction

SUMMARY. Pigeon circovirus was identified by polymerase chain reaction (PCR) in young pigeons belonging to 12 different lofts. Viral DNA was extracted from formalin-fixed, paraffin-imbedded tissues containing primarily bursa and occasionally liver and spleen with a commercial kit. PCR primers were selected from a published sequence for columbid circovirus and evaluated in a PCR assay. The histopathologic examination of various tissues revealed basophilic globular intracytoplasmic inclusions in the mononuclear cells of the bursa of Fabricius and occasionally in the spleen characteristic for a circovirus. Transmission electron microscopy of a few bursas of Fabricius revealed virus particles measuring 18–21 nm. All the samples were negative by PCR for psittacine beak and feather disease (PBFD) virus and chicken infectious anemia virus. The primers for both pigeon circovirus and PBFD virus did not react in PCR with the chicken anemia virus DNA. Most of the circovirus-infected pigeons had concurrent infections of Escherichia coli, Salmonella, Pasteurella, Aspergillus, candidiasis, nematodiasis, or capillariasis.

Pathogenicity of Different Serogroups of Avian Salmonellae in Specific-Pathogen-Free Chickens

The pathogenicity of one isolate of Salmonella typhimurium, four isolates of Salmonella heidelberg, three isolates of Salmonella kentucky, two isolates of Salmonella montevideo, one isolate of Salmonella hadar, and two isolates of Salmonella enteritidis (SE), one belonging to phage type (PT)13a and the other to PT34, was investigated in specific-pathogen-free chicks. Three hundred eighty-four chicks were separated into 16 equal groups of 24 chicks. Thirteen groups were inoculated individually with 0.5 ml of broth culture containing 1 x 10(7) colony-forming units (CFU) of either S. typhimurium (one source), S. heidelberg (four sources), S. montevideo (two sources), S. hadar (one source), S. kentucky (three sources), SE PT 13a (one source) or SE PT 34 (one source) by crop gavage. Two groups of 24 chicks were inoculated in the same way with 1 x 10(7) CFU of SE PT4 (chicken-CA) and Salmonella pullorum. Another group of 24 chicks was kept as an uninoculated control group. The chicks were observed daily for clinical signs and mortality. Isolation of salmonella was done from different organs at 7 and 28 days postinoculation (DPI). All the chicks were weighed individually at 7, 14, 21, and 28 DPI. Two chicks chosen at random from each group were euthanatized and necropsied at 7 and 14 DPI and all the remaining live chickens, at 28 DPI. Selected tissues were taken for histopathology at 7 and 14 DPI. Dead chicks were examined for gross lesions and tissues were collected for histopathology. Chicks inoculated with S. pullorum had the highest mortality (66.66%), followed by S. typhimurium (33.33%). Chicks inoculated with S. heidelberg (00-1105-2) and SE PT4 (chicken-CA) had 12.5% mortality and 8.3% mortality, respectively, with SE PT 13a. Ceca were 100% positive for salmonellae at acute or chronic infection compared with other organs. Mean body weight reduction ranged from 0.67% (inoculated with S. kentucky 00-926-2) to 33.23% (inoculated with S. typhimurium 00-372) in the inoculated groups at different weeks compared with uninoculated controls. Gross and microscopic lesions included peritonitis, perihepatitis, yolk sac infection, typhilitis, pneumonia, and enteritis in some groups, especially those inoculated with S. typhimurium, S. heidelberg (00-1 105-2), SE PT4 (chicken-CA), and S. pullorum.

High mortality in egg layers as a result of necrotic enteritis.

Abstract A new facility was designed to hold 1.8 million birds in 10 houses; chickens were placed in five of the houses, and the remaining five houses were under construction when this outbreak occurred. An increase in mortality was reported in five houses; however, mortality in house 7 was quite high. Well-fleshed birds were suddenly found dead without a significant drop in egg production. The middle and distal intestines were distended with gas, congested, thin walled, atonic, and bluish or pale in color with sloughed mucosa in some places. Necrotic enteritis was diagnosed as the cause of increased mortality. The ingesta in the crop occasionally contained flies. The 4-wk mortality in house 7 was 6.55% with a loss of 10,898 chickens. The 4-wk mortality rate in the other houses ranged from 0.54% to 1.98%. The houses affected with necrotic enteritis were treated for coccidiosis with amprolium because low numbers of the oocysts were present in the intestinal specimens of some of the chickens. Household bleach was added to the water at a dilution of one part bleach to 1040 parts water to control bacterial contamination. The fly (Musca domestica) population was out of control. Clostridium perfringens was isolated from the alcohol-washed macerated flies caught from houses 4 and 7. Dead flies were often seen in the feed troughs. The chickens may possibly have had C. perfringens infection as a result of consumption of dead flies or their secretions/excretions. The alcohol-washed, macerated, clarified fly extract from the affected houses caused death in 11 inoculated mice and paralysis in one mouse. Similarly, illness and mortality were present in four mice inoculated with clarified intestinal contents. The bacterium isolated on anaerobic culture was identified as C. perfringens by polymerase chain reaction. The disease was brought under control after straw was added and mixed in with the litter. As a result, the litter temperature increased, causing a decrease in the fly population. This study suggests that flies in the poultry houses acted as mechanical transmitters of C. perfringens and that the development of necrotic enteritis was by ingestion of bacteria present in the flies and their secretions/excretions.

Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens.

Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease and poses a huge threat to poultry industry. The risks associated with conventional attenuated viral vaccines make it indispensable to probe into the development of novel and rationally designed subunit vaccines which are safer as well as effective. VP2 is the major host-protective antigen found in IBDV capsid. It encompasses different independent epitopes responsible for the induction of neutralizing antibody. Here, we report the efficacy of the immunodominant fragment of VP2 which induces both humoral and cellular immunity against infectious bursal disease. A 366 bp fragment (52-417 bp) of the VP2 gene from an IBDV field isolate was amplified and expressed in Escherichia coli as a 21 kDa recombinant protein. The efficacy of rVP2(52-417) antigen was compared with two commercial IBDV whole virus vaccine strains. The rVP2(52-417) induced significantly high antibody titres in chicken compared to commercial vaccines and the anti-rVP2(52-417) sera showed reactivity with viral antigens from both commercial strains (P<0.0001) and field isolates. Also, the chicken splenocytes from rVP2(52-417) immunized group showed a significantly high proliferation (P<0.01) compared to other groups, which implies that the rVP2(52-417) fragment contains immunogenic epitopes capable of eliciting both B and T cell responses. Further, rVP2(52-417) conferred 100% protection against vIBDV challenge in the immunized chickens which was significantly higher (P<0.001) compared to 55-60% protection by commercial vaccine strains. Hence, the study confirms the efficacy of the immunodominant VP2 fragment that could be used as a potent vaccine against IBDV infection in chicken.

Efficacy of an inactivated oil emulsion vaccine against hydropericardium syndrome in broilers.

Two inactivated vaccines were prepared against hydropericardium syndrome. The vaccine prepared from liver homogenate extracted with chloroform, inactivated with formalin and adjuvanted with liquid paraffin was highly effective against challenge in chickens aged three, five and seven weeks. Seroconversion following vaccination and challenge was assessed by the agar gel immunodiffusion test. The inactivated oil emulsion vaccine was highly effective against the syndrome in both experimental trials and field trials.

DNA vaccination with VP2 gene fragment confers protection against Infectious Bursal Disease Virus in chickens.

Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens by destruction of antibody producing B cells in the Bursa of Fabricius and poses a potential threat to the poultry industry. We have examined the protective efficacy of a subunit DNA vaccine against IBDV infection in chickens in this study. An immunodominant VP2 gene fragment (VP252-417) was cloned into CMV promoter based DNA vaccine vector pVAX1 and in vitro expression of the DNA encoded antigens was confirmed by transfection of CHO cells with vaccine constructs followed by RT-PCR and western blot analysis using IBDV-antiserum. Two weeks old chickens were immunized intramuscularly with pVAXVP252-417 and the in vivo transcription of the plasmid DNA was confirmed by RT-PCR analysis of DNA injected muscle tissue at different intervals of post immunization. Tissue distribution analysis revealed that the plasmid DNA was extensively distributed in muscle, spleen, kidney, liver, and bursa tissues. Chickens immunized with pVAXVP252-417 developed high titer (1:12,000) of anti-VP252-417 antibodies. Further, chicken splenocytes from pVAXVP252-417 immunized group showed a significantly high proliferation to the whole viral and recombinant antigen (P<0.01) compared to control groups, which implies that pVAXVP252-417 codes for immunogenic fragment which has epitopes capable of eliciting both B and T cell responses. This is evident by the fact that, pVAXVP252-417 immunized chicken conferred 75% protection against virulent IBDV (vIBDV) challenge compared to the control group. Thus, the present study confirms that the immunodominant VP2 fragment can be used as a potential DNA vaccine against IBDV infection in chickens.

Antigenetically Unusual Newcastle Disease Virus from Racing Pigeons in India

Newcastle disease virus isolated from an outbreak in racing pigeons in India was found to be velogenic, based on the mean time to death in 10-day-old embryonated hens eggs, the intravenous pathogenicity index in 6-week-old chickens and the pathogenesis in chickens and pigeons. The virus induced disease in chickens without prior adaptation in chickens. The virus was antigenically unusual since it could not be grouped with the available panel of monoclonal antibodies at the World Reference Laboratory for Newcastle disease, UK. However, commercially available lentogenic and mesogenic vaccines provided 100% protection to chickens against this antigenically unusual NDV.

A modified filter paper technique for serosurveillance of Newcastle disease.

Newcastle disease (ND) poses a continuous and unresolved threat to poultry farmers. In tropical countries, with a dense population of poultry in open houses near farms, eradication of ND is difficult. Hence, vaccination is necessary to control the disease. Assessment of the post-vaccinal immune response is essential to monitor the efficacy of a vaccination schedule and of the vaccines used. This can be done by the haemagglutination-inhibition (HI) test, which is easy to perform and rapid, but large-scale serosurveillance presents some problems. These include collection of the sera, their storage and transportation to a laboratory. To overcome some of these difficulties, the use of filter paper to collect blood samples for serosurveillance has been recommended (Beard and Brugh, 1977; Brugh and Beard, 1980; Giambrone, 1981; Rivetz et al., 1985). An attempt was made to reduce the test time by modifying the fdter paper technique using Whatman No. I filter paper for collection of blood samples, Brij-35 solution for quick elution of antibody, and a micro-HI procedure to evaluate the antibody content.

Postvaccinal immune response to regimens of Newcastle disease vaccination by filter paper sampling technique.

SummarySeven hundred and ten blood samples were collected at random from commercial layers in Tamil Nadu on Whatman filter paper No. 1 instead of the conventional method of serum collection. The birds were subjected to different Newcastle disease (ND) vaccination schedules and samples were collected to study the vaccinal response to ND at field level. Eluates were obtained from sample areas of filter paper using Brij-35 solution [detergent] and subjected to the micro haemagglutination inhibition (HI) test for ND antibodies. The HI titre ranged from less than 24 to 29. The possible causes of poor immune response to ND vaccinations are discussed.Résumé710 échantillons de sang furent collectés au hasard dans des élevages industriels dans le Tamil Nadu utilisant des filtres Whatman No. 1 au lieu de la méthode conventionnelle d’échantillonnage des serums. Les oiseaux furent soumis à différents traitements de vaccination contre la maladie de Newcastle et les échantillons furent recueillis pour étudier la réponse vaccinale contre la maladie de Newcastle au miveau environnemental. Les éluats furent obtenus à partir de la surface des filtres utilisant une solution de Brij-35 (détergent) et soumis à un test d’inhibition par micro-hémagglutination des anticorps contre la maladie Newcastle. Les titres d’inhibition par hémagglutination furent compris entre 24 et 29, les causes probables de cette faible réponse immunitaire face à la vaccination contre la maladie de Newcastle sont discutées.ResumenSe obtuvieron 710 muestras de sangre de ponedoras seleccionadas al azar en Tamil Nadu. En lugar de utilizar el método tradicional de obtención de suero, las muestras de sangre se recognieron sobre papel de filtro Waltham no. 1. Los animales habían recibido diferentes protocolos de vacunación frente a la enfermedad de Newcastle y las muestras se recogieron para estudiar la respuesta vacunal a nivel de campo. El papel de filtro se lavó con solución Brij-35 y después se realizó el test de inhibición de la microhemaglutinación para detectar anticuerpos frente a Newcastle. Los títulos de anticuerpos variaron entre 24 y 29. Se discuten las posibles causas de la baja respuesta observada.