Concurrent Histomonas meleagridis and Hemorrhagic Enteritis Virus Infection in a Turkey Flock with Recurrent History of Blackhead Disease.

Intestinal health is one of the key factors required for the growth and production of turkeys. Histomoniasis (blackhead disease), caused by a protozoan parasite, Histomonas meleagridis, is a reemerging threat to the turkey industry. Increased incidences of histomoniasis have been reported in recent years due to withdrawal of antihistomonas treatments. H. meleagridis affects ceca and causes cecal inflammation and necrosis. H. meleagridis migrates from ceca to the liver and causes liver necrosis, resulting in high mortalities. Ironically, field outbreaks of histomoniasis are not always associated with high mortalities, while low mortalities have also been documented. There are several exacerbating factors associated with high mortality rates in histomoniasis outbreaks, with concurrent infection being one of them. Recurrent histomoniasis outbreaks in a newly constructed barn were documented, and concurrent infection of H. meleagridis and hemorrhagic enteritis virus was confirmed. Currently, neither commercial vaccines nor prophylactic or therapeutic solutions are available to combat histomoniasis. However, there are treatments, vaccines, and solutions to minimize or prevent concurrent infections in turkeys. In addition to implementing biosecurity measures, measures to prevent concurrent infections are critical steps that the turkey industry can follow to reduce mortality rates and minimize the production and economic losses associated with histomoniasis outbreaks.

Infección simultánea por Histomonas meleagridis y el virus de la enteritis hemorrágica en una parvada de pavos con antecedentes recurrentes de enfermedad de la cabeza negra. La salud intestinal es uno de los factores clave necesarios para el crecimiento y producción de los pavos. La histomoniasis (enfermedad de la cabeza negra), causada por un parásito protozoario, Histomonas meleagridis, es una amenaza reemergente para la industria del pavo. En los últimos años se ha informado de un aumento de la incidencia de histomoniasis debido al retiro de los tratamientos con antihistomonas. Histomonas meleagridis afecta los ciegos y causa inflamación y necrosis cecal. Histomonas meleagridis migra desde los ciegos al hígado y causa necrosis hepática, lo que resulta en una alta mortalidad. Irónicamente, los brotes de histomoniasis en el campo no siempre se asocian con una mortalidad elevada, aunque también se han documentado mortalidades bajas. Hay varios factores exacerbantes asociados con altas tasas de mortalidad en los brotes de histomoniasis, siendo la infección concurrente uno de ellos. Se documentaron brotes recurrentes de histomoniasis en un alojamiento avícola recién construido y se confirmó la infección concurrente de H. meleagridis y el virus de la enteritis hemorrágica. Actualmente no se dis-pone de vacunas comerciales ni soluciones profilácticas o terapéuticas para combatir la histomoniasis. Sin embargo, existen tratamientos, vacunas y soluciones para minimizar o prevenir infecciones concurrentes en los pavos. Además de implementar medidas de bioseguridad, las medidas para prevenir infecciones concurrentes son pasos críticos que la industria del pavo puede seguir para reducir las tasas de mortalidad y minimizar las pérdidas económicas y de producción asociadas con los brotes de histomoniasis.

Early Detection of Histomoniasis in Blood Samples by PCR and Sequencing.

Histomoniasis is a deadly disease of turkeys causing devastating economic losses to the poultry industry. In field outbreaks, a presumptive diagnosis is made based on gross pathology lesions and confirmed by histopathology. An early detection tool with quick turnaround time is needed to prevent the spread of histomoniasis. With this objective, two studies were conducted in turkeys. In Study 1, 40 poults were housed in two pens (20 poults/pen) and challenged at 14 days of age with Histomonas meleagridis by intracloacal route. Blood samples were collected 4 days postchallenge. Fifty-five percent (22/40) of the blood samples tested positive for H. meleagridis based on PCR using primers targeted against the 18S rRNA gene and confirmed by sequencing. In Study 2, 40 poults were housed in two groups and raised in floor pens. Groups 1 and 2 served as negative and challenge controls, respectively. At 14 days of age, the birds in Group 2 were challenged with H. meleagridis by intracloacal route. Blood samples were collected 2 days postchallenge. Five percent (1/20) of the blood samples tested positive for H. meleagridis, based on PCR and confirmed by sequencing. The results from both studies indicate that H. meleagridis DNA can be detected in the blood samples by PCR and confirmed by sequencing as early as 4 days postchallenge. This early detection method could be applied in field outbreaks to detect and confirm histomoniasis as early as possible.

Detección temprana de histomoniasis en muestras de sangre mediante PCR y secuenciación La histomoniasis es una enfermedad mortal de los pavos que causa pérdidas económicas devastadoras a la industria avícola. En los brotes de campo, se realiza un diagnóstico presuntivo basado en lesiones patológicas macroscópicas y se confirma mediante histopatología. Se necesita una herramienta de detección temprana con un tiempo de respuesta rápido para prevenir la propagación de la histomoniasis. Con este objetivo, se realizaron dos estudios en pavos. En el Estudio 1, se alojaron 40 pavipollos en dos corrales (20 pavipollos/corral) y se desafiaron a los 14 días de edad con Histomonas meleagridis por vía intracloacal. Se recolectaron muestras de sangre a los cuatro días después del desafío. El cincuenta y cinco por ciento (22/40) de las muestras de sangre resultaron positivas para H. meleagridis según el método de PCR utilizando iniciadores dirigidos contra el gene 18S rRNA y confirmado mediante secuenciación. En el Estudio 2, se alojaron 40 pavipollos en dos grupos y se criaron en corrales en piso. Los grupos 1 y 2 sirvieron como controles negativos y de desafío, respectivamente. A los 14 días de edad, las aves del Grupo 2 fueron expuestas a H. meleagridis por vía intracloacal. Se recolectaron muestras de sangre dos días después del desafío. El cinco por ciento (1/20) de las muestras de sangre dieron positivo para H. meleagridis, según el método de PCR y confirmado mediante secuenciación. Los resultados de ambos estudios indican que el ADN de H. meleagridis puede detectarse en las muestras de sangre mediante PCR y confirmarse mediante secuenciación tan pronto como cuatro días después de la exposición. Este método de detección temprana podría aplicarse en brotes de campo para detectar y confirmar la histomoniasis lo antes posible.

Concurrent Infection of Histomonas meleagridis and Pentatrichomonas hominis in a Blackhead Disease Outbreak in Turkeys.

Intestinal health plays a major role in profitable and efficient turkey production. Blackhead disease (histomoniasis) is caused by Histomonas meleagridis, an anaerobic protozoan parasite. Histomonas meleagridis disrupts intestinal integrity and may cause systemic infection. Some field outbreaks of blackhead disease are associated with low morbidity and mortality, while in some instances, it may cause severe morbidity and mortality. In the current study, a presumptive diagnosis of blackhead disease was made based on the characteristic gross lesions in the liver and ceca. The cecal culture, PCR, and sequencing confirmed the presence of H. meleagridis and Pentatrichomonas hominis. Pentatrichomonas hominis has been reported in enteritis cases of several other species, such as dogs, cats, and cattle. The impact of P. hominis on intestinal health of turkeys has not previously been studied, and to the best of our knowledge, this is the first case report of concurrent H. meleagridis and P. hominis infection in turkeys.

Reporte de caso- Infección simultánea de Histomonas meleagridis y Pentatrichomonas hominis en un brote de enfermedad de la cabeza negra en pavos. La salud intestinal juega un papel importante en la producción rentable y eficiente de pavos. La enfermedad de la cabeza negra (histomoniasis) es causada por Histomonas meleagridis, que es un parásito protozoario anaeróbico. Histomonas meleagridis altera la integridad intestinal y puede causar una infección sistémica. Algunos brotes de campo de la enfermedad de la cabeza negra están asociados con una baja morbilidad y mortalidad, mientras que en algunos casos puede causar una morbilidad y mortalidad severas. En el presente estudio, se realizó un diagnóstico presuntivo de la enfermedad de la cabeza negra con base a las lesiones macroscópicas características en el hígado y el ciego. El cultivo cecal, un método de PCR y secuenciación confirmaron la presencia de H. meleagridis y Pentatrichomonas hominis. Se ha reportado la presencia de Pentatrichomonas hominis en casos de enteritis de varias otras especies, como perros, gatos y ganado. El impacto de P. hominis en la salud intestinal de los pavos no se había estudiado previamente y según nuestro conocimiento, este es el primer reporte de un caso de infección simultánea por H. meleagridis y P. hominis en pavos.

Chicken astrovirus as an aetiological agent of runting-stunting syndrome in broiler chickens.

Despite descriptions of runting-stunting syndrome (RSS) in broiler chickens dating back over 40 years, the aetiology has not yet been described. A novel chicken astrovirus (CkAstV) was isolated in an LMH liver cell line from the intestines of chickens affected with RSS. Clinical RSS is characterized by retarded growth and cystic crypt lesions in the small intestine. In 1-day-old broiler chickens infected with the CkAstV isolate, virus was only detected in the intestinal epithelial cells during the first few days after infection. Notably, the preferred host cells are the crypt epithelial cells following initial replication in the villous epithelial cells, thus implying viral preference for immature intestinal cells. Nevertheless, the CkAstV isolate did not induce remarkable pathological changes, despite the presence of the virus in situ. Serial chicken-to-chicken passages of the virus induced increased virulence, as displayed by decreased weight gain and the presence of cystic lesions in the small intestine reproducing clinical RSS in chickens. The analysis of the full-length genome sequences from the isolated CkAstV and the CkAstV from the bird-to-bird passages showed >99 % similarity. The data obtained in this study suggest that the CkAstV isolate is capable of inducing RSS following serial bird-to-bird passages in broilers and is as an aetiological agent of the disease.

An in vivo experimental model to determine antigenic variations among infectious bursal disease viruses.

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus causing infectious bursal disease in chickens. IBDV undergoes antigenic drift, so characterizing the antigenicity of IBDV plays an important role for identification and selection of vaccine candidates. In this study, an in vivo experimental model was developed to differentiate a new antigenic variant of IBDV. To this end, a hyper-immune serum to IBDV E/Del-type virus was generated in specific pathogen-free chickens and a standard volume of the hyper-immune serum was serially diluted and injected in specific pathogen-free birds via intravenous, subcutaneous, or intramuscular routes. The chickens were bled at different time points in order to evaluate the dynamics of virus neutralization titres. Based on the results, chickens were injected with different serum dilutions by the subcutaneous route. Twenty-four hours later, chickens were bled and then challenged with 100 median chicken infectious doses of the E/Del virus and a new IBDV variant. Chickens were euthanized at 7 days post infection and the bursa of Fabricius was removed for microscopic evaluation to determine the bursal lesion score. The determined virus neutralization titre along with the bursal lesion score was used to determine the breakthrough titre in the in vivo chicken model. Based on the data obtained, an antigenic subtype of IBDV was identified and determined to be different from E/Del. This model is a sensitive model for determination of IBDV antigenicity of non-tissue culture adapted IBDV.

Protective vaccination against infectious bursal disease virus with whole recombinant Kluyveromyces lactis yeast expressing the viral VP2 subunit.

Here we report on vaccination approaches against infectious bursal disease (IBD) of poultry that were performed with complete yeast of the species Kluyveromyces lactis (K. lactis). Employing a genetic system that enables the rapid production of stably transfected recombinant K. lactis, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV). Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant K. lactis was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied K. lactis that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine.

Investigation of the antigenic evolution of field isolates using the reverse genetics system of infectious bursal disease virus (IBDV).

The antigenic profiles of over 300 infectious bursal disease virus (IBDV) isolates were analyzed using a panel of monoclonal antibodies in a reverse genetics system. In addition, the sequences of a large portion of the neutralizing-antibody-inducing VP2 of IBDV were determined. Phylogenetic analysis of nucleotide and amino acid sequences in combination with the antigenic profiles obtained using the monoclonal antibody panel, revealed a lack of correlation between antigenicity and isolate's placement within the phylogenetic tree. In-depth analysis of amino acid exchanges revealed that changes within a certain region of the VP2 molecule resulted in differences in the antigenicity of the virus. This comprehensive analysis of VP2 sequences indicated a high selective pressure in the field that was likely due to vaccination programs, which increase the rate of evolution of the virus.