Peter J. Groves

Immunosuppressive effects of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) in broiler chickens and the protective effect of HVT vaccination against MDV challenge

Much of the impact of Mareks disease in broiler chickens is considered to be due to immunosuppression induced by Mareks disease virus (MDV). The present study evaluates the effects of an Australian isolate of pathogenic MDV (strain MPF 57) and a non-pathogenic vaccinal strain of herpesvirus of turkeys (HVT) (strain FC 126) on the immune system of commercial broiler chickens for 35 days following challenge at days 0 or 3 of age. It also investigates the extent of protection provided by HVT vaccine against MDV-induced immunosuppression. Immune system variables, including relative lymphoid organ weight, blood lymphocyte phenotype (CD45+/CD3+, putatively T, and CD45+/LC+, putatively B) and antibody production following vaccination against infectious bronchitis (IB) at hatch, were used to assess the immune status of chickens. Immunosuppression was also assessed by susceptibility to secondary challenge with pathogenic Escherichia coli on day 29 post-MDV challenge. MDV infection reduced the weight of the thymus and bursa of Fabricius, the numbers of circulating T lymphocytes and B lymphocytes, and IB antibody titre. The timing of these effects varied. MDV infection greatly increased susceptibility to E. coli infection. HVT alone caused mild depletion of T and B lymphocytes but no effect on immune organ weight or IB titre. Vaccination with HVT provided good protection against most of the immunosuppressive effects of MDV but not against MDV-induced growth impairment and reduced responsiveness to IB vaccination, suggesting that recent Australian strains of MDV may be evolving in virulence to overcome the protective effects of HVT.

Kinetics of Marek's disease virus (MDV) infection in broiler chickens 1: effect of varying vaccination to challenge interval on vaccinal protection and load of MDV and herpesvirus of turkey in the spleen and feather dander over time.

Two experiments in commercial broiler chickens vaccinated with herpesvirus of turkeys (HVT) and challenged with Mareks disease virus (MDV) investigated the effects of the vaccination-to-challenge interval (VCI) on vaccinal protection against Mareks disease, and the kinetics of MDV and HVT load in the spleen and feather dander determined using real-time quantitative polymerase chain reaction. Experiment 1 in isolators tested VCI of 2, 4 and 7 days, while Experiment 2 in floor pens tested VCI of 0, 2, 4, 7 and 10 days. MDV challenge induced gross Mareks disease lesions in 14% to 74% of chickens by 56 days post-challenge. Vaccinal protection increased from ∼40% to ∼80% with increasing VCI between days 2 and 7 in both experiments, but not thereafter. MDV was detected in both the spleen and dander at 7 days post-challenge and increased rapidly to approximately 21 days post-challenge, after which levels plateaued, rose or fell gradually depending on treatment. HVT was also shed in significant amounts, 1 to 2 logs lower than for MDV, with a clear peak around 14 to 21 days post-vaccination. Vaccination significantly reduced the log10MDV load in the spleen (vaccinated, 2.99±0.20/106 spleen cells; unvaccinated, 4.60±0.23/106 spleen cells) and dander (vaccinated, 5.28±0.13/mg; unvaccinated, 6.00±0.18/mg) from infected chickens. The MDV load had a significant negative association with the VCI and the level of vaccinal protection. Measurement of dander production in Experiment 1 and the dust content of air in Experiment 2, combined with determination of the MDV load in these, enabled estimation of total daily shedding rates of MDV per chicken and of the MDV load in air for the first time.

A validated miniaturized MPN method, based on ISO 6579:2002, for the enumeration of Salmonella from poultry matrices

Aims:  To validate the effectiveness of a miniaturized most probable number method (mMPN) in enumerating Salmonella from poultry matrices.

Pathotyping of Australian isolates of Marek's disease virus in commercial broiler chickens vaccinated with herpesvirus of turkeys (HVT) or bivalent (HVT/SB1) vaccine and association with viral load in the spleen and feather dander.

OBJECTIVE To pathotype Australian isolates of Mareks disease virus (MDV) in commercial broiler chickens using standard methods and to evaluate early markers of pathotype. METHODS A complete 3 × 4 factorial experiment with two replicates was conducted using 648 Cobb broiler chickens in 24 isolators. The experimental factors were vaccination (unvaccinated, herpesvirus of turkeys (HVT), bivalent (HVT + SB1 strain of serotype 2 MDV)) and MDV challenge (unchallenged or 500 plaque-forming units of isolates MFP57, 02LAR or FT158). Mortality, body weight, immune-organ weights and viral load were measured to 56 days post challenge (dpc). Vaccinal protective index (PI) and virulence rank (VR) were calculated based on gross Mareks disease (MD) pathology. RESULTS The PIs provided by the HVT and bivalent vaccines against challenge with MPF57, 02LAR, and FT158 were 84.6% 56%, 61.4% and 82.2%, 60.8%, 57.7%, respectively, leading to putative pathotypes of virulent MDV for MPF57 and very virulent MDV for 02LAR and FT158. Significantly more of the unvaccinated chickens (85.7%) had MD lesions than chickens vaccinated with either the HVT (26.8%) or bivalent vaccine (27.6%). Strong linear relationships were observed between the incidence of MD at 56 dpc and MDV load in the spleen at 7 dpc (R(2) = 0.71) and MDV load in the isolator exhaust dust at 14 dpc (R(2) = 0.57) and 21 dpc (R(2) = 0.51). Immune organ weights had a weaker association with subsequent MD incidence. CONCLUSION Pathotyping results in broiler chickens with maternal antibody broadly agreed with those in specific-pathogen-free chickens in other studies, with some important differences. MDV load in the spleen at 7 dpc and in isolator dust at both 14 and 21 dpc was a powerful early predictor of subsequent MD incidence.

Development, Application, and Results of Routine Monitoring of Marek's Disease Virus in Broiler House Dust Using Real-Time Quantitative PCR

SUMMARY.  Results are presented from four studies between 2002 and 2011 into the feasibility of routinely monitoring Mareks disease virus serotype 1 (MDV-1) in broiler house dust using real-time quantitative PCR (qPCR) measurement. Study 1 on two farms showed that detection of MDV-1 occurred earlier on average in dust samples tested using qPCR than standard PCR and in spleen samples from five birds per shed assayed for MDV-1 by qPCR or standard PCR. DNA quality following extraction from dust had no effect on detection of MDV-1. Study 2 demonstrated that herpesvirus of turkeys (HVT) and MDV serotype 2 (MDV-2) in addition to MDV-1 could be readily amplified from commercial farm dust samples, often in mixtures. MDV-2 was detected in 11 of 20 samples despite the absence of vaccination with this serotype. Study 3 investigated the reproducibility and sensitivity of the qPCR test and the presence of inhibitors in the samples. Samples extracted and amplified in triplicate showed a high level of reproducibility except at very low levels of virus near the limit of detection. Mixing of samples prior to extraction provided results consistent with the proportions in the mixture. Tests for inhibition showed that if the template contained DNA in the range 0.5–20 ng/µl no inhibition of the reaction was detectable. The sensitivity of the tests in terms of viral copy number (VCN) per milligram of dust was calculated to be in the range 24–600 VCN/mg for MDV-1, 48–1200 VCN/mg for MDV-2, and 182–4560 VCN/mg for HVT. In study 4 the results of 1976 commercial tests carried out for one company were analyzed. Overall 23.1% of samples were positive for MDV-1, 26.1% in unvaccinated and 16.4% in vaccinated chickens. There was marked regional and temporal variation in the proportion of positive samples and the MDV-1 load. The tests were useful in formulating Mareks disease vaccination strategies. The number of samples submitted has increased recently, as has the incidence of positive samples. These studies provide strong evidence that detection and quantitation of MDV-1, HVT, and MDV-2 in poultry house dust using qPCR is robust, sensitive, reproducible, and meaningful, both biologically and commercially. Tactical vaccination based on monitoring of MDV-1 rather than routine vaccination may reduce selection pressure for increased virulence in MDV-1. RESUMEN.  Desarrollo, aplicación y resultados de un muestreo rutinario del virus de la enfermedad de Marek en polvo de casetas avícolas mediante PCR cuantitativo en tiempo real. Se presentan los resultados de cuatro estudios entre los años 2002 y 2011 sobre la viabilidad de un muestreo para la enfermedad de Marek serotipo 1 (MDV-1) llevado a cabo en polvo de casetas avícolas de pollo de engorde mediante PCR cuantitativo en tiempo real (qPCR). El estudio 1 llevado a cabo en dos granjas mostró que la detección de MDV-1 en promedio ocurría antes en las muestras de polvo con el método cuantitativo de PCR en comparación con el convencional y en las muestras de bazo de cinco aves por caseta analizadas para detectar al virus de Marek 1 por PCR en tiempo real o por PCR convencional. La calidad del ADN después de la extracción a partir de polvo no tuvo ningún efecto sobre la detección del virus de Marek 1. El estudio 2 demostró que el herpesvirus de pavos (HVT) y el serotipo 2 del virus de Marek (MDV-2), además del serotipo1 podrían ser fácilmente amplificados a partir de muestras de polvo de granjas comerciales, a menudo en mezclas. El serotipo 2 se detectó en 11 de 20 muestras a pesar de la ausencia de la vacunación con este serotipo. El estudio 3 investigó la reproducibilidad y la sensibilidad de la prueba de PCR cuantitativo y la presencia de factores inhibidores en las muestras. Las muestras extraídas y amplificadas por triplicado mostraron un alto nivel de reproducibilidad, excepto a niveles muy bajos del virus que estaban cerca del límite de detección. El mezclado de muestras antes de la extracción proporcionó resultados acordes con las proporciones de la mezcla. Las pruebas de inhibición mostraron que si el ADN extraído estaba en un rango de 0.5–20 ng/µl no se detectó inhibición de la reacción. Se calculó que la sensibilidad de las pruebas en términos de número de copias virales (VCN) por miligramo de polvo está en el rango de 24 a 600 copias virales por mg para el serotipo 1, de 48 a 1200 copias virales por mg para el serotipo 2, y de 182 a 4560 copias virales por mg para el herpesvirus de los pavos. En el estudio 4 se analizaron los resultados de 1976 pruebas llevadas a cabo por una empresa. En general el 23.1% de las muestras fueron positivas para el serotipo 1, el 26.1% en aves no vacunadas y el 16.4% en los pollos vacunados. Hubo una marcada variación regional y temporal de la proporción de muestras positivas y en la carga del virus de Marek 1. Las pruebas fueron útiles en la formulación de estrategias de vacunación contra la enfermedad de Marek. El número de muestras presentadas ha aumentado recientemente, de igual manera que la incidencia de muestras positivas. Estos estudios proporcionan una fuerte evidencia de que la detección y cuantificación del virus de Marek 1, del Herpesvirus de los pavos, y del serotipo 2, en el polvo de casetas avícolas mediante PCR cuantitativo es robusto, sensible, reproducible y significativo, tanto biológica como comercialmente. La táctica de vacunación basada en el seguimiento del serotipo 1en lugar de la vacunación de rutina puede reducir la presión de selección para una mayor virulencia en el serotipo 1.

Marek's disease in broiler chickens: Effect of route of infection and herpesvirus of turkey-vaccination status on detection of virus from blood or spleen by polymerase chain reaction, and on weights of birds, bursa and spleen

The polymerase chain reaction (PCR) has recently emerged as an additional tool for the monitoring and diagnosis of Mareks disease. We investigated a number of factors that may influence the interpretation of PCR results in commercial broiler chickens including the effects of route of infection and herpesvirus of turkeys (HVT)-vaccination status. We also investigated the suitability of peripheral blood lymphocytes (PBL) and spleen as tissues for Mareks disease virus (MDV) detection. HVT-vaccinated and unvaccinated commercial broiler chickens were challenged or not challenged with virulent MDV either by intraperitoneal injection or inhalation of feather dust containing the virus. Blood and spleen samples were collected at weekly intervals to day 35 post-infection for PCR of spleen or PBL. Live weight and lymphoid organ weights were also measured. Spleen and PBL were found to provide similar sensitivity of detection of MDV with a small advantage in favour of spleen. In terms of the timing of detection of MDV, intraperitoneal challenge broadly mimicked natural challenge via inhalation, although infection of birds by inhalation of infective feather dust resulted in slightly later but more complete detection of MDV in challenged birds. Vaccination with HVT delayed the detection of MDV by approximately 10 to 14 days and did not protect against the reduced growth observed in challenged chickens at day 35 post-challenge.

Influence of vaccine deposition site on post-vaccinal viraemia and vaccine efficacy in broiler chickens following in ovo vaccination against Marek's disease

In ovo vaccination against Mareks disease is a widely used technology in the broiler industry.A series of experiments was carried out to determine the site of vaccine deposition in the egg during automated in ovo vaccination, and the effect of vaccine deposition site and dose on vaccine responses following vaccination with cell-associated herpesvirus of turkeys in commercial broiler chickens. Vaccine deposition site following automated in ovo vaccination was principally influenced by the age of embryo, with egg size having a smaller effect. The frequency of vaccine deposition inside the embryo body increased as incubation progressed from day 17.5 to 19.5. In experiments using manual vaccine deposition intra-embryonically (IE) or extra-embryonically (EE) at day 18.5, EE vaccine deposition resulted in a significantly delayed development of post-vaccinal viraemia relative to both IE vaccination and subcutaneous vaccination at hatch. There were no effects of vaccine dose (2000, 4000 or 8000 plaque forming units) on the timing of post-vaccinal viraemia. The timing of post-vaccinal viraemia was found to be a good indicator of the level of protection provided by the vaccine against challenge with earlier viraemia associated with better protection. IE vaccine deposition induced significantly greater protection than EE deposition against challenge with a virulent strain of Mareks disease virus. IE deposition consistently produced a high level of protection (68 to 84%) irrespective of vaccine dose or challenge day, while EE vaccine deposition produced no or low levels of protection (0 to 27%) depending on the vaccine dose and day of challenge. The growth of challenged chickens was also affected by site of vaccine deposition, with significantly higher live weights at day 56 of age in IE compared with EE vaccinated groups. These data suggest that the site of vaccine deposition within the embryo is an important determinant of the success of in ovo vaccination.

Utilization of a novel autologous killed tri-vaccine (serogroups B [Typhimurium], C [Mbandaka] and E [Orion]) for Salmonella control in commercial poultry breeders.

An autologous killed trivalent vaccine (3×108 colony-forming units [CFU]), based on three Salmonella serovars (Typhimurium – serogroup B, Mbandaka – serogroup C, and Orion – serogroup E) prevalent in the flocks of Australian poultry companies, was developed using Salenvac® techniques. At 20 weeks, hens vaccinated at 12 and 17 weeks as well as non-vaccinated hens were challenged (250 µl of 107 CFU) with autologous and heterologous serovars belonging to serogroup B (Typhimurium and Agona), serogroup C (Mbandaka and Infantis) and serogroup E (Orion and Zanzibar). Overall, vaccination resulted in a significant difference in carriage of Salmonella between non-vaccinated and vaccinated commercial Cobb hens (P <0.05) for serogroups B and C. However, due to low colonization rates in the non-vaccinated birds, no significant difference (P>0.05) could be determined for serogroup E. All vaccinated flocks produced a significant antibody response (P<0.001) to the S. Typhimurium vaccine strain, measured using a S. Typhimurium enzyme-linked immunosorbent assay (Guildhay), which peaked at 20 weeks of age, with 39% of the hens positive. Maternal antibodies were detected in 16% of the yolks from eggs produced by these flocks. There was a significant difference after challenge with Salmonella (P <0.05) among 1-day-old chicks from vaccinated versus non-vaccinated parents, when challenged using 104 CFU but not when challenged with 108 CFU. The success of this trial resulted in the incorporation of this vaccine into a Salmonella control system in commercial broiler breeder production.

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

Comparisons of management practices and farm design on Australian commercial layer and meat chicken farms: Cage, barn and free range

There are few published studies describing the unique management practices, farm design and housing characteristics of commercial meat chicken and layer farms in Australia. In particular, there has been a large expansion of free range poultry production in Australia in recent years, but limited information about this enterprise exists. This study aimed to describe features of Australian commercial chicken farms, with particular interest in free range farms, by conducting on-farm interviews of 25 free range layer farms, nine cage layer farms, nine barn layer farms, six free range meat chicken farms and 15 barn meat chicken farms in the Sydney basin bioregion and South East Queensland. Comparisons between the different enterprises (cage, barn and free range) were explored, including stocking densities, depopulation procedures, environmental control methods and sources of information for farmers. Additional information collected for free range farms include range size, range characteristics and range access. The median number of chickens per shed was greatest in free range meat chicken farms (31,058), followed by barn meat chicken (20,817), free range layer (10,713), barn layer (9,300) and cage layer farms (9,000). Sheds had cooling pads and tunnel ventilation in just over half of both barn and free range meat chicken farms (53%, n = 8) and was least common in free range layer farms (16%, n = 4). Range access in free range meat chicken farms was from sunrise to dark in the majority (93%, n = 14) of free range meat chicken farms. Over half of free range layer farms (56%, n = 14) granted range access at a set time each morning; most commonly between 9:00 to 10.00am (86%, n = 12), and chickens were placed back inside sheds when it was dusk.