M.G.R. Matthijs

Ability of Massachusetts-type infectious bronchitis virus to increase colibacillosis susceptibility incommercial broilers: A comparison between vaccine and virulent field virus

The abilities of Massachusetts-type vaccine virus and virulent infectious bronchitis (IB) field virus to increase colibacillosis susceptibility were compared. In four experiments, 29-day-old female commercial broilers housed in isolators, were infected intratracheally and oculonasally with IB vaccine strains (HI20 and H52) or virulent IB field strains (D387 and M41) (4.8 or 6.8 log10 median embryo infective dose, per broiler). Five days later, Escherichia coli 506 strain was given intratracheally (5.6 to 8.8 log10 colony forming units/broiler). The incidence of nasal discharge at 3 and 5 days after IB virus infection was used to assess the clinical effect of the IB infection, while mortality, body weight uniformity and E. coli lesions at 7 days following E. coli inoculation were used as parameters for colibacillosis. Nasal discharge was observed in 61/117 (5%), 26/119 (22%), 35/119 (29%) and 115/120 (96%) of broilers infected with H120, H52, D387 and M41 virus, respectively. Apart from H52 and D387, differences between IBV strains were significant. IB vaccine and virulent IB viruses did not generally differ significantly in their ability to induce colibacillosis susceptibility. Mean colibacillosis lesion scores of H52-infected birds even significantly exceeded those of birds infected with the other IB viruses. The ability of H120 virus to induce colibacillosis susceptibility tended to be the weakest. The practical consequences of these findings are discussed.

The role of phagocytic cells in enhanced susceptibility of broilers to colibacillosis after Infectious Bronchitis Virus infection.

Abstract Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. We investigated whether IBV affects recruitment and function of phagocytic cells and examined NO production, phagocytic and bactericidal activity, and kinetics of peripheral blood mononuclear cells (PBMC) and splenocytes. Moreover, we measured cytokine mRNA expression in lung and spleen samples. Broilers were inoculated with IBV H120 vaccine or virulent M41 and challenged 5 days later with E. coli 506. A PBS control and E. coli group without previous virus inoculation were also included. Birds were sacrificed at various time points after inoculation (h/dpi). Inoculation with IBV induced extended and more severe colibacillosis than with E. coli alone. At 4dpi, the number of KUL-01+ PBMC in all E. coli-inoculated groups was significantly higher than in PBS-inoculated birds, which correlated with lesion scores. From 1 to 4dpi, NO production by PBMC from all E. coli-inoculated animals was elevated compared to PBS birds. Bactericidal activity of PBMC in IBV-inoculated animals at 7dpi was lower than in PBS- and E. coli-inoculated birds, but phagocytic capacity and recruitment were not severely impaired. In spleen samples of IBV-infected animals reduced expression of IL-1β, IL-6, IL-8, IL-10, IL-18 and IFN-γ mRNA was found 1dpi. Our results suggest that enhanced colibacillosis after IBV infection or vaccination is caused at least by altered innate immunity and less by impairment of phagocytic cell function.

Course of infection and immune responses in the respiratory tract of IBV infected broilers after superinfection with E. coli

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. The aim of this study was to investigate how IBV affects the course of events upon infection with E. coli. Broilers were inoculated with IBV H120 vaccine virus or virulent M41 and challenged 5 days later with E. coli 506. A PBS and E. coli group without previous virus inoculation were included. Sections of trachea, lung and airsacs were stained for CD4, CD8, gammadelta-TCR, alphabeta1-TCR, and for macrophages (KUL-01) and both pathogens. Changes in the mucociliary barrier of trachea, lung and airsacs did not predispose for bacterial superinfection. The disease in the lungs of the E. coli group and both IBV/E. coli groups was similar. Lesions in the airsacs were more pronounced and of longer duration in the IBV/E. coli groups. The immunocytological changes differed substantially between the E. coli group and both IBV/E. coli groups. In trachea, lungs and airsacs the CD4+ and CD8+ populations were significantly larger than in the E. coli and PBS groups. In the lungs and the airsacs the macrophages were more numerous in the IBV/E. coli and the E. coli groups than in the PBS group. The presence of high numbers of T cells and macrophages in IBV infected birds most likely induced an altered immune response, which is responsible for the enhanced clinical signs of colibacillosis.

Progression of lesions in the respiratory tract of broilers after single infection with Escherichia coli compared to superinfection with E. coli after infection with infectious bronchitis virus

The progression of Escherichia coli lesions was studied in the respiratory tract of 4-week-old commercial broilers. Lesions were induced after a single intratracheal E. coli infection, and after an infection with E. coli preceded 5 days earlier by an oculo-nasal and intratracheal infectious bronchitis virus (IBV) infection of either the virulent M41 strain or the H120 vaccine strain. Trachea, lung and thoracic airsac lesions were examined macroscopically and microscopically. Tissue samples were taken at 3h post-inoculation (hpi), and 1, 2, 4 and 7 days post-inoculation (dpi) with E. coli. The location of both pathogens was assessed by immunohistochemistry. Single E. coli inoculation induced pneumonia and airsacculitis; in case it was preceded by IBV infection, the same macroscopical lesions and also viral tracheitis were found. No clear difference existed between the single and dual infected birds with respect to inflammatory reactions in the lung, which had disappeared within 7 days, except for the presence of more follicles in dual infected birds. IBV antigen was detected in secondary bronchi and airsacs up to 2 dpi and in the trachea up to 4 dpi. E. coli bacteria were found in the tracheal lumen included in purulent material, the parabronchi and airsacs. In lung tissue E. coli antigen was found up to 4 dpi. No clear difference existed between single and dual inoculated birds regarding the presence of E. coli in the lung. In the airsacs, a few bacteria were found from 0.5 hpi up to 4 dpi in E. coli and IBV-E. coli inoculated birds. Although both pathogens were cleared beyond detection at 7 dpi, in IBV-E. coli inoculated birds lesions in the airsac persisted, in contrast to broilers inoculated with E. coli only. In the present study it is shown that 4-week-old broilers are not resistant to intratracheal E. coli inoculation, however, these birds can overcome the induced E. coli infection within a short time span. Moreover, a preceding infection with vaccine or virulent IBV does not seem to impair the clearance of E. coli in the respiratory tract of broilers, but rather induces an exaggerated inflammatory response in the airsacs only, which seems to be the mechanism behind the pattern of airsacculitis in commercial poultry in the field.

Outbreak of avian influenza H7N3 on a turkey farm in the Netherlands

This case report describes the course of an outbreak of avian influenza on a Dutch turkey farm. When clinical signs were observed their cause remained unclear. However, serum samples taken for the monitoring campaign launched during the epidemic of highly pathogenic avian influenza in 2003, showed that all the remaining turkeys were seropositive against an h7 strain of avian influenza virus, and the virus was subsequently isolated from stored carcases. The results of a reverse-transcriptase pcr showed that a h7n3 strain was involved, and it was characterised as of low pathogenicity. However, its intravenous pathogenicity index was 2·4, characterising it as of high pathogenicity, suggesting that a mixture of strains of low and high pathogenicity may have been present in the isolate. The outbreak remained limited to three farms.

Rapid NK-cell activation in chicken after infection with infectious bronchitis virus M41

Abstract Natural killer (NK) cells are cytotoxic lymphocytes and play an important role in the early defence against viruses. In this study we focussed on NK cell and interferon (IFN) responses after infection with infectious bronchitis virus (IBV). Based on surface expression of CD107+, enhanced activation of lung NK cells was observed at 1dpi, whereas in blood prolonged NK-cell activation was found. IFN-α and IFN-β mRNA and proteins were not rapidly induced whereas IFN-γ production in lung, measured by Elispot assay, increased over time at 2 and 4dpi. In contrast, IFN-γ production in blood was highest at 1dpi and decreased over time down to levels comparable to uninfected birds at 4dpi. Collectively, infection with IBV-M41 resulted in activation of NK cells in the lung and blood and rapid production of IFN-γ and not IFN-α and IFN-β compared to uninfected birds.

Transmissibility of Infectious Bronchitis Virus H120 Vaccine Strain among Broilers under Experimental Conditions

Abstract The aim of this study was to quantify transmission of infectious bronchitis virus (IBV) H120 vaccine strain among broilers, and to assess whether birds that have been exposed to vaccine strain-shedding birds were protected against clinical signs after infection with a virulent strain of the same serotype. A transmission experiment and a replicate were carried out, each with six groups of commercial broilers. At day of hatch (n  =  30) or at 15 days of age (n  =  20), half of each group was inoculated with either IBV H120 vaccine (H120 group), virulent IBV M41 (M41 group), or were mock-infected, thereby contact-exposing the other half of each group. Nasal discharge was recorded, and antibody response and virus shedding were measured. To measure clinical protection, four weeks after inoculation all birds, in all groups, were challenged with IBV M41. The reproduction ratio (R; the average number of contact infections caused by one infectious bird) was determined to quantify virus transmission. All contact-exposed birds, except for one in an H120 group, became infected with either IBV H120 or IBV M41. Almost all birds contact-infected with IBV H120 or IBV M41 were subsequently protected against clinical signs after challenge with IBV M41. The lower limits of the 95% confidence interval (CI) of the R of IBV H120 vaccine, and of IBV M41, were significantly <1. For both IBV H120 and IBV M41, the 95% CI was [2.1–∞] following inoculation at day of hatch and [1.8–∞] after inoculation at 15 days of age. This finding demonstrates that IBV H120 vaccine is able to spread extensively among broilers. This implies that this vaccine strain might be able to become endemically present in the poultry population. It also implies that, even if not all birds received vaccine during spray application, due to the ability of the vaccine to spread in the flock, they will most likely be protected against clinical signs after a subsequent field virus infection. Transmisibilidad de la cepa vacunal H120 de bronquitis infecciosa entre pollos de engorde bajo condiciones experimentales. El objetivo del presente trabajo fue cuantificar la transmisión de la cepa vacunal H120 de bronquitis infecciosa entre pollos de engorde y evaluar si las aves que han sido expuestas a aves diseminando la cepa vacunal del virus, estaban protegidas contra los signos clínicos luego de un desafío con una cepa virulenta perteneciente al mismo serotipo. Se realizó un experimento de transmisión viral y una réplica, cada uno con seis grupos de pollos de engorde comerciales. Al día de edad (n  =  30) o a los 15 días de edad (n  =  20) la mitad de cada grupo se inoculó con la vacuna H120 (grupo H120), con virus virulento de bronquitis infecciosa cepa M41 (grupo M41) o no fueron infectadas, en consecuencia exponiendo por contacto a la otra mitad de cada grupo. Se llevó registro de las descargas nasales y se midió la respuesta de anticuerpos y la diseminación del virus. Cuatro semanas después de la inoculación, todas las aves se desafiaron con la cepa virulenta de bronquitis infecciosa M41 para evaluar la protección clínica. Cuantificando la transmisión del virus, se determinó la tasa de reproducción (promedio de infecciones por contacto causadas por un ave infecciosa). Todas las aves expuestas por contacto, excepto una en el grupo H120, se infectaron con el virus H120 o M41. Casi todas las aves expuestas por contacto con el virus H120 o con M41mostraron protección contra signos clínicos luego de un desafío con la cepa M41 de bronquitis infecciosa. Los limites inferiores de los intervalos de confianza 95% de la tasa de reproducción de la vacuna H120 y del virus M41 fueron significantes <1. Para ambos virus el intervalo de confianza 95% fue [2.1–∞] luego de la inoculación al día de edad y [1.8–∞] después de la inoculación a los 15 días de edad. Este hallazgo demuestra que la vacuna de bronquitis infecciosa H120 es capaz de diseminarse ampliamente entre pollos de engorde. Esto implica que la vacuna puede tornarse endémica en la población avícola. Esto a su vez implica que si no todas las aves recibieron vacuna durante la aplicación por aspersión, debido a la capacidad de la vacuna de diseminarse en el parvada, estas aves probablemente esten protegidas contra signos clinicos luego de una infeccion viral en el campo. Abbreviations: CI = confidence interval; HI = hemagglutination inhibiting; IB = infectious bronchitis; IBV = infectious bronchitis virus; MLE = maximum likelihood estimate; R = reproduction ratio

Effect of IBV-H120 Vaccination in Broilers on Colibacillosis Susceptibility After Infection with a Virulent Massachusetts-Type IBV Strain

Abstract Vaccination against infectious bronchitis (IB) is aimed to protect against clinical IB. The question is, however, whether vaccinated birds are also protected against predisposure for colibacillosis after a subsequent IBV infection. We examined this research question in four experiments. One-day-old commercial broilers, housed in isolators, were vaccinated with IB vaccine strain H120 by coarse spray or ocularly. Twenty-eight days after vaccination, broilers were challenged with the virulent IBV strain M41. Five days later, broilers were inoculated with Escherichia coli strain 506. Body weight uniformity, severity of E. coli airsacculitis, and systemic E. coli infection at 7 days following E. coli inoculation were used as parameters for colibacillosis. IBV vaccination reduced both the number of broilers with E. coli airsacculitis as well as the severity of airsacculitis significantly after challenge with IBV-M41 and E. coli 506. However, in spray-vaccinated groups, no significant reduction of the number of birds with systemic colibacillosis or the severity of this infection was obtained, and body weight uniformity was not significantly improved compared with nonvaccinated, IBV-M41, and E. coli 506-challenged groups. Eye-drop vaccination resulted in conflicting results.

Protective effect of in ovo treatment with the chicken cathelicidin analog D-CATH-2 against avian pathogenic E. coli.

Increasing antibiotic resistance and ever stricter control on antibiotic use are a driving force to develop alternatives to antibiotics. One such strategy is the use of multifunctional Host Defense Peptides. Here we examined the protective effect of prophylactic treatment with the D analog of chicken cathelicidin-2 (D-CATH-2) against a respiratory E. coli infection. Chickens were treated with D-CATH-2 in ovo at day 18 of embryonic development or intramuscularly at days 1 and 4 after hatch. At 7 days of age, birds were challenged intratracheally with avian pathogenic E. coli. Protection was evaluated by recording mortality, morbidity (Mean Lesion Score) and bacterial swabs of air sacs at 7 days post-infection. In ovo D-CATH-2 treatment significantly reduced morbidity (63%) and respiratory bacterial load (>90%), while intramuscular treatment was less effective. D-CATH-2 increased the percentage of peripheral blood lymphocytes and heterophils by both administration routes. E. coli specific IgM levels were lower in in ovo treated animals compared to intramuscular D-CATH-2 treatment. In short, in ovo treatment with the Host Defense Peptide derived D-CATH-2 can partially protect chickens from E. coli infection, making this peptide an interesting starting point to develop alternatives to antibiotics for use in the poultry sector.

Effect of anti-inflammatory drugs on colibacillosis lesions in broilers after Infectious Bronchitis Virus and subsequent Escherichia coli infection

Background: In case of persistent and sterile inflammation, anti-inflammatory drugs should be considered as first choice treatment instead of antibiotics. Objective: To assess the effect of anti-inflammatory drugs on lesions due to colibacillosis. Animals & methods: Five groups of day-old broilers of 15 birds each were housed in isolators and were inoculated at 29 days of age with Infectious Bronchitis Virus strain M41 by the oculo-nasal and IT (intratracheal) route (105.4 EID50 (egg infectious dosis 50)/broiler) and at 33 days of age with Escherichia coli strain 506 by the IT route (107.6 colony forming units/broiler). Broilers of four groups were treated from day 28 up to and including day 39 orally on a daily basis with either carbasalate calcium (4 × 12.5 mg), meloxicam (2 × 0.5 mg), acetaminophen (4 × 2.5 mg), or dexamethasone (1 × 1.0 mg). The fifth group was placebo-medicated. At 40 days of age, the experiment was ended and at post-mortem examination, colibacillosis lesions were assessed. Results: All broilers in the dexamethasone group died. This mortality exceeded significantly (p < 0.05) that of the other groups in which mortality ranged from 2 to 5. Mean lesion score of surviving broilers of medicated groups ranged from 5.3 to 5.8 compared to 3.9 in the placebo group and did not differ significantly between groups. Conclusion: None of the anti-inflammatory drugs had a positive effect on colibacillosis lesions. Clinical importance: Anti-inflammatory drugs cannot be considered as an alternative for antibiotic treatment.