D. A. Bautista

Immunopathology and Cytokine Responses in Broiler Chickens Coinfected with Eimeria maxima and Clostridium perfringens with the Use of an Animal Model of Necrotic Enteritis

Abstract The incidence of necrotic enteritis (NE) due to Clostridium perfringens (CP) infection in commercial poultry has been increasing at an alarming rate. Although pre-exposure of chickens to coccidia infections is believed to be one of the major risk factors leading to NE, the underlying mechanisms of CP virulence remain undefined. The objectives of this study were to utilize an experimental model of NE produced by Eimeria maxima (EM) and CP coinfection to investigate the pathologic and immunologic parameters of the disease. Broilers coinfected with EM plus CP exhibited more severe gut pathology compared with animals given EM or CP alone. Additionally, EM/CP coinfection increased the numbers of intestinal CP bacteria compared with chickens exposed to an identical challenge of CP alone. Coinfection with EM and CP repressed nitric oxide synthase gene expression that was induced by EM alone, leading to lower plasma NO levels. Intestinal expression of a panel of cytokine and chemokine genes following EM/CP coinfection showed a mixed response depending on the transcript analyzed and the time following infection. In general, IFN-α, IFN-γ, IL-1β, IL-2, IL-12, IL-13, IL-17, and TGF-β4 were repressed, whereas IL-8, IL-10, IL-15, and LITAF were increased during coinfection compared with challenge by EM or CP alone. These results are discussed in the context of EM and CP to act synergistically to create a more severe disease phenotype leading to an altered cytokine/chemokine response than that produced by infection with the individual pathogens.

An outbreak of gangrenous dermatitis in commercial broiler chickens

The present report describes an outbreak of gangrenous dermatitis (GD) infection in a commercial poultry farm in Delaware involving 34-day-old broiler chickens. In addition to obvious clinical signs, some GD-affected broilers also showed severe fibrino-necrotic enteritis and large numbers of Gram-positive rods in the necrotic tissue. Histopathological findings included haemorrhage, degeneration and necrosis of parenchymatous cells, especially of skin, muscle, and intestine. Immunofluorescence staining revealed Clostridium-like bacilli in the skin and the intestine. Both Clostridium perfringens and Clostridium septicum genomic sequences were identified by polymerase chain reaction in bacterial cultures isolated from the skin, muscle, and intestine, and in the frozen tissues from the GD-affected birds. Serological analysis demonstrated that both affected and clinically healthy birds from the same house had high serum antibody titres against C. perfringens, C. septicum, Eimeria, chick anaemia virus, and infectious bursal disease virus. These results are discussed in the context of the relationship between the different Clostridium spp. and the pathogenesis of GD.

Immunopathology and cytokine responses in commercial broiler chickens with gangrenous dermatitis

Gangrenous dermatitis (GD) is an emerging disease of increasing economic importance in poultry resulting from infection by Clostridium septicum and Clostridium perfringens type A. Lack of a reproducible disease model has been a major obstacle in understanding the immunopathology of GD. To gain better understanding of host–pathogen interactions in GD infection, we evaluated various immune parameters in two groups of birds from a recent commercial outbreak of GD, the first showing typical disease signs and pathological lesions (GD-like birds) and the second lacking clinical signs (GD-free birds). Our results revealed that GD-like birds showed: reduced T-cell and B-cell mitogen-stimulated lymphoproliferation; higher levels of serum nitric oxide and α-1-acid glycoprotein; greater numbers of K55+, K1+, CD8+, and MHC class II+ intradermal lymphocytes, and increased K55+, K1+, CD8+, TCR1+, TCR2+, Bu1+, and MHC class II+ intestinal intraepithelial lymphocytes; and increased levels of mRNAs encoding proinflammatory cytokines and chemokines in skin compared with GD-free chickens. These results provide the first evidence of altered systemic and local (skin and intestine) immune responses in GD pathogenesis in chickens.

Impact of Fresh or Used Litter on the Posthatch Immune System of Commercial Broilers

SUMMARY. This study was carried out to investigate the effects of exposure of growing broiler chickens of commercial origin to used poultry litter on intestinal and systemic immune responses. The litter types evaluated were fresh wood shavings or used litter obtained from commercial poultry farms with or without a history of gangrenous dermatitis (GD). Immune parameters measured were serum nitric oxide (NO) levels, serum antibody titers against Eimeria or Clostridium perfringens, mitogen-induced spleen cell proliferation, and intestinal intraepithelial lymphocyte or splenic lymphocyte subpopulations. At 43 days posthatch, birds raised on used litter from a GD farm had higher serum NO levels and greater Eimeria or C. perfringens antibody levels compared with chickens raised on fresh litter or used, non-GD litter. Birds raised on non-GD and GD used litter had greater spleen cell mitogenic responses compared with chickens raised on fresh litter. Finally, spleen and intestinal lymphocyte subpopulations were increased or decreased depending on the litter type and the surface marker analyzed. Although it is likely that the presence of Eimeria oocysts and endemic viruses varies qualitatively and quantitatively between flocks and, by extension, varies between different used litter types, we believe that these data provide evidence that exposure of growing chicks to used poultry litter stimulates humoral and cell-mediated immune responses, presumably due to contact with contaminating enteric pathogens.

Effects of anticoccidial and antibiotic growth promoter programs on broiler performance and immune status

This study investigated the effects of various coccidiosis control programs in combination with antibiotic growth promoters (AGPs) on growth performance and host immune responses in broiler chickens. The coccidiosis programs that were investigated included in ovo coccidiosis vaccination (CVAC) with Inovocox or in-feed medication with diclazuril as Clinacox (CLIN) or salinomycin (SAL). The AGPs were virginiamycin or bacitracin methylene disalicylate plus roxarsone. As a negative control, chickens were non-vaccinated and fed with non-supplemented diets (NONE). All animals were exposed to used litter from a commercial broiler farm with confirmed contamination by Eimeria parasites to simulate in-field exposure to avian coccidiosis. Broiler body weights in the CVAC group were greater at 14 and 32 days of age, but not at day 42, compared with the NONE, CLIN, and SAL groups. At day 14, the SAL group showed decreased body weight and reduced ConA-stimulated spleen cell proliferation compared with the CLIN and SAL groups. In contrast, at days 34 and 43, splenocyte proliferation was greater in the CVAC and CLIN groups compared with the NONE and SAL groups. Lymphocyte subpopulations and cytokine mRNA expression levels in the intestine and spleen were also altered by the denoted treatments. Collectively, these results suggest that in ovo coccidiosis vaccination or coccidiostat drug medication programs in combination with AGPs influences chicken growth and immune status in an Eimeria-contaminated environment.

Effects of in ovo vaccination and anticoccidials on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters

The present study reports the effects of various field anticoccidial programs on the distribution of Eimeria spp. in poultry litter and serum antibody titers against coccidia in broiler chickens raised on the used litters. The programs included in ovo vaccination and various medications with either chemicals, ionophores, or both. In general, serum samples from these chickens showed anticoccidial antibody titers when tested at days 7 and 14 post hatch with the peak response at day 43. Serum anticoccidial titers were highest in birds fed a non-medicated diet compared with those vaccinated or fed medicated diets. Total number of Eimeria oocysts and the composition of Eimeria spp. present in the litter samples from different treatment groups varied depending on the type of anticoccidial program. Oocyst counts in general ranged from 3.7×10(3) to 7.0×10(4) per g of litter. Importantly, both morphological and molecular typing studies revealed four major predominant Eimeria spp., E. acervulina, E. maxima, E. praecox, and E. tenella in the litter samples. Collectively, these results indicate that the field anticoccidial programs influenced the type and abundance of Eimeria spp. present in the litter samples and also modulated host immune response to Eimeria.

Massachusetts Live Vaccination Protects Against a Novel Infectious Bronchitis Virus S1 Genotype DMV/5642/06

Abstract Four infectious bronchitis virus (IBV) isolates were recovered from commercial broiler chicken flocks located on the Delmarva Peninsula (east coast of the United States) in the spring of 2006. Sequence analysis of the S1 subunit of the spike glycoprotein gene showed the four isolates were highly related to each other (≥99.6% nucleotide identity; ≥98.9% amino acid identity). Basic local alignment search tool analysis indicated the highest S1 amino acid identity of isolate DMV/5642/06, typical of the four Delmarva (DMV) isolates, was to CA/1737/04, an isolate obtained from broilers in California in 2004. A pathogenicity study conducted, using two-week-old commercial broilers, showed that DMV/5642/06 caused respiratory but not renal (kidney) disease. A vaccination–challenge study in three-week-old specific-pathogen-free leghorn chickens demonstrated that a commercial live attenuated IBV vaccine containing the Massachusetts strain conferred protection against challenge with DMV/5642/06 based on virus reisolation attempts and microscopic pathology.

Comparison of live Eimeria vaccination with in-feed salinomycin on growth and immune status in broiler chickens.

Coccidiosis vaccines and anticoccidial drugs are commonly used to control Eimeria infection during commercial poultry production. The present study was conducted to compare the relative effectiveness of these two disease control strategies in broiler chickens in an experimental research facility. Birds were orally vaccinated with a live, attenuated vaccine (Inovocox), or were provided with in-feed salinomycin (Bio-Cox), and body weights, serum levels of nitric oxide (NO) and antibodies against Eimeria profilin and Clostridium perfringens PFO proteins, and intestinal levels of cytokine gene transcripts were measured. Vaccinated chickens had increased body weights, greater NO levels, and higher profilin and PFO antibody levels compared with salinomycin-fed birds. Transcripts for interleukin-6 (IL-6), tumor necrosis factor superfamily 15, and interferon-γ were increased, while mRNAs for IL-4 and IL-10 were decreased, in immunized chickens compared with salinomycin-treated chickens. In conclusion, vaccination against avian coccidiosis may be more effective compared with dietary salinomycin for increasing body weight and augmenting pro-inflammatory immune status during commercial poultry production.

Effect of Bacillus Subtilis-based Direct-fed Microbials on Immune Status in Broiler Chickens Raised on Fresh or Used Litter.

Type of dietary direct-fed microbials (DFMs) or poultry litter could directly influence the composition of gut microbiota. Gut microbiota plays an important role in shaping the developing immune system and maintaining the homeostasis of the mature immune system in mammal and chickens. The present study was carried out to investigate the interaction among litter, DFMs and immunity in broiler chickens exposed to a field-simulated environment. Immune status of broiler chickens was assessed by serum antibodies against Eimeria spp. and Clostridium spp. and intestinal cytokine mRNA expression. The current experimental design had a 3 ×2 factorial arrangement of treatments with three types of litter, i.e., fresh litter or used litter that was obtained from a farm with no disease outbreak (used litter) or a farm with history of a gangrenous dermatitis outbreak (GD litter), and two dietary treatments with or without DFMs. It was found that either DFM addition or type of litter significantly affected anticoccidial antibody levels of broiler chickens at d 42. In general, dietary DFMs increased the anticoccidial antibodies in the fresh-litter raised chickens, but lowered the levels in the GD-litter raised chickens. Serum antibodies against Clostridium perfringens α-toxin were significantly (p<0.05) higher in chickens raised on GD litter compared with those raised on fresh litter. Cytokine mRNA expression was significantly (p<0.05) altered by either the type of litter or DFMs. Of interest, dietary DFMs lowered interferon-γ, interleukin 1beta, and CXCLi2 cytokine mRNA expression in chickens raised on fresh litter but increased them in GD-litter raised chickens. In conclusion, dietary DFMs modulate various immune parameters of broiler chickens, but the DFM-mediated effects were dependent upon the type of litter on which chickens were raised.

Characterization of Nephropathogenic Infectious Bronchitis Virus DMV/1639/11 Recovered from Delmarva Broiler Chickens in 2011

SUMMARY. A limited outbreak of nephropathogenic infectious bronchitis (NIB) occurred in three Delmarva (DMV) commercial broiler chicken flocks in 2011. Isolates of NIB virus (NIBV)—DMV/1639/11, DMV/3432/11, and DMV/3902/11—were characterized by sequence analysis of the N-terminal subunit (S1) of the spike (S) gene. Findings indicated that the isolates were identical to each other and to PA/9579A/10, a 2010 isolate from poultry in Pennsylvania. The 2010 and 2011 isolates appear to have originated from a 1997–2000 NIB outbreak in Pennsylvania. DMV/1639/11 and PA/9579A/10 were determined to be nephropathogenic in susceptible chickens, yielding virus reisolations from kidney and inducing characteristic interstitial nephritis microscopic lesions. In a controlled laboratory study, 40% of chickens vaccinated with a combination live vaccine containing infectious bronchitis virus (IBV) strains Massachusetts (Mass) + Connecticut (Conn) were positive on virus isolation attempts after challenge with DMV/1639/11, compared with only 13% of Mass + Arkansas (Ark) vaccinates. Both combination vaccines gave partial protection against the development of DMV/1639/11-induced renal lesions. Although numerically fewer chickens vaccinated with Mass + Conn had interstitial nephritis compared with those vaccinated with Mass + Ark, neither vaccine combination offered greater protection (P < 0.05) than observed in unvaccinated chickens challenged with DMV/1639/11. Mass + Ark vaccinations, applied under commercial conditions in the hatchery (spray) and on-farm (spray), did not protect the trachea or kidney from DMV/1639/11 challenge. Serologic testing of broiler flocks found <3% (2 of 69) tested to possess specific antibodies to DMV/1639/11, indicating the virus had not become established in the region. RESUMEN. Caracterización del virus nefropatogénico de la bronquitis infecciosa DMV/1639/11 aislado de pollos de engorde en el área de Delmarva en el año 2011. Se presentó un brote limitado de bronquitis infecciosa nefropatogénica (NIB) en tres parvadas comerciales de pollos de engorde en el año 2011 en el área de Delmarva. Se caracterizaron los aislamientos de virus nefropatogénicos de bronquitis infecciosa DMV/1639/11, DMV/3432/11 y DMV/3902/11 se caracterizaron mediante el análisis de la secuencia del gene de la subunidad S1 de la espícula (S). Los resultados indicaron que los aislamientos eran idénticos entre sí y con el aislamiento PA/9579A/10, que es un aislamiento de aves comerciales en Pennsylvania del año 2010. Las cepas de los años 2010 y 2011 parecen haberse originado a partir de un brote de bronquitis infecciosa nefropatogénica que se presentó entre los años 1997 al 2000 en Pennsylvania. Se determinó que los aislamientos DMV/1639/11 y PA/9579A/10 eran nefropatogénicos en pollos susceptibles, que eran reaislados del riñón e inducían las lesiones de nefritis intersticial microscópica característica. En un estudio controlado de laboratorio, el 40% de los pollos vacunados con una vacuna viva que contenía la combinación cepas Massachussets (Mass) y Connecticut (Conn) fueron positivos a los intentos de aislamiento del virus después del desafío con el virus DMV/1639/11, en comparación con sólo el 13% de los pollos vacunados con la combinación Massachussets y Arkansas (Ark). Ambas combinaciones de vacunas confirieron una protección parcial contra el desarrollo de las lesiones renales inducidas por el virus DMV/1639/11. Aunque un número menor de los pollos vacunados con la combinación Massachussets y Connecticut mostraron nefritis intersticial en comparación con los pollos vacunados con Massachussets y Arkansas, ninguna combinación de vacunas ofreció una mayor protección (P < 0.05) que la observada en los pollos no vacunados y desafiados con el virus DMV/1639/11. Las vacunaciones con la combinación Massachussets y Arkansas, aplicadas en condiciones comerciales en la planta de incubación (aerosol) y en las explotaciones agrícolas (aerosol), no protegieron a la tráquea o a los riñones ante el desafío con el virus DMV/1639/11. Las pruebas serológicas demostraron que menos del 3% de las parvadas (2 de 69) poseían anticuerpos específicos contra el virus DMV/1639/11, lo que indica que el virus no se había establecido en la región.