Propagation of an Avirulent Turkey Hemorrhagic Enteritis Virus Isolate in Chickens.

A series of studies were undertaken to optimize the propagation of hemorrhagic enteritis virus (HEV) in specific-pathogen-free (SPF) chickens. A total of 562 SPF chickens were orally inoculated with an Australian avirulent HEV isolate of turkey origin at 9, 14, 21, or 28 days of age with 5, 6, 7, or 8 log 10 genomic copies (GC), while 102 chickens served as uninfected controls. No clinical signs were observed in infected chickens. There was an inoculum-dose-dependent increase in the relative spleen and liver weight ( P < 0.01). Relative spleen weight 7 days post-HEV inoculation was up to 85% higher in chickens that were inoculated with 6 to 7 GC compared with controls, with no further increase at higher doses. Relative liver weight increased up to 14% in chickens inoculated with 6 GC, with no further increase. Birds inoculated with a 7 GC dose had a higher frequency of HEV DNA-positive birds (77% to 86%) than birds inoculated with lower doses (33% to 59%; P < 0.01). The most efficient dose for live passage propagation was 7 GC inoculated in 9-to-14-day-old birds, yielding an infection rate of 81%. Livers and spleens from infected birds at all doses were processed to produce a putative vaccine with a final GC recovery in the vaccine material of 8.6 GC/bird. In summary, HEV of turkey origin can be readily propagated in SPF chickens, and conditions to maximize viral retrieval were established.

Real-time PCR quantification of infectious laryngotracheitis virus in chicken tissues, faeces, isolator-dust and bedding material over 28 days following infection reveals high levels in faeces and dust.

Infectious laryngotracheitis (ILT) is an important disease of chickens caused by ILT virus (ILTV). We used the Australian SA2 and A20 vaccine strains of ILTV to determine tissue distribution and excretion characteristics of ILTV in specific-pathogen-free chickens and to determine whether ILTV is readily detectable in environmental samples such as faeces, bedding material and dust using real-time quantitative PCR. Three groups of 10 freshly hatched chicks were placed in isolators and infected orally with high doses of the two strains of vaccine virus or left unchallenged as controls. Over a 28-day post-infection (p.i.) period, faecal and serum samples were collected at frequent intervals from six individually identified chickens in each group. Dust and litter samples from the isolators were collected less frequently. Tissue samples were collected from three to four sacrificed or dead/euthanized birds at 6, 14 and 28 days p.i. Infection resulted in clinical ILT, a pronounced antibody response and sustained qPCR detection of the viral genome in the trachea, Harderian gland, lung and kidney up to 28 days p.i. A high level of the viral genome was also detected in faeces between 2 and 7 days p.i., declining by about approximately four orders of magnitude to low, but detectable, levels at 21 and 28 days p.i. The finding of high-level shedding of ILTV in faeces warrants further investigation into the epidemiological role of this, and the sustained high levels of ILTV observed in dust suggest that it may be a useful sample material for monitoring ILTV status in flocks.

Development, application, and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR.

Results are presented from four studies between 2002 and 2011 into the feasibility of routinely monitoring Marek's 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/microl 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 Marek's 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.

Insulin and IGF-I stimulated RNA synthesis in primary cultures of neuronal cells: involvement of cyclic AMP and protein kinase-c

La insulina y el IGF-I promueven el crecimiento de las células neuronales de rata en cultivo primario. Con el objeto de investigar el mecanismo de transducción de señales hormonales en este sistema biológico, estudiamos el efecto de agonistas de AMP cíclico y un estimulador de la proteína kinasa-C sobre la síntesis de ARN basal e inducida por hormonas. Los agentes que aumentan los níveles de AMP cíclico endógenos (foraskolina, dibutiril-AMP cíclico, toxina colérica) bloquearon los efectos estimuladores de la insulina y el factor de crecimiento; el dibutiril AMP cíclico, sin embargo, no alteró la unión de las hormonas a sus receptores. Aunque a diferencia de los agentes antes mencionados, el ester de forbol elevó significativamente la síntesis de ARN basal; este, no obstante, inhibió la estimulación por la insulina. Este último efecto probablemente fue mediado por un incremento en los niveles de AMP cíclico, como se ha encontrado en otros tipos de células. La estaurosporina, un inhibidor de la proteína kinasa-C, también bloqueó los efectos de la insulina sobre la síntesis de RNA (AU)

Insulin and IGF-I stimulated RNA synthesis in primary cultures of neuronal cells: involvement of cyclic AMP and protein kinase-c

La insulina y el IGF-I promueven el crecimiento de las células neuronales de rata en cultivo primario. Con el objeto de investigar el mecanismo de transducción de señales hormonales en este sistema biológico, estudiamos el efecto de agonistas de AMP cíclico y un estimulador de la proteína kinasa-C sobre la síntesis de ARN basal e inducida por hormonas. Los agentes que aumentan los níveles de AMP cíclico endógenos (foraskolina, dibutiril-AMP cíclico, toxina colérica) bloquearon los efectos estimuladores de la insulina y el factor de crecimiento; el dibutiril AMP cíclico, sin embargo, no alteró la unión de las hormonas a sus receptores. Aunque a diferencia de los agentes antes mencionados, el ester de forbol elevó significativamente la síntesis de ARN basal; este, no obstante, inhibió la estimulación por la insulina. Este último efecto probablemente fue mediado por un incremento en los niveles de AMP cíclico, como se ha encontrado en otros tipos de células. La estaurosporina, un inhibidor de la proteína kinasa-C, también bloqueó los efectos de la insulina sobre la síntesis de RNA