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.

Histomonas meleagridis is a protozoan parasite that causes histomonosis in gallinaceous birds such as turkeys and chickens. Since the banning and restricted usage of effective drugs such as nitarsone, 80-100% morbidity and mortality occur in turkeys and 20-30% mortality in chickens. New ideas are needed to resolve the re-emergence of histomonosis in poultry. In this study, the α-actinin encoding gene from H. meleagridis was cloned. The 1839-bp gene encoding 612 amnio acids showed close phylogenetic relationships with Trichomonas vaginalis and Tritrichomonas foetus. It was then inserted into the prokaryotic expression vector pET28a(+) and induced with isopropyl-β-D-thiogalactopyranoside. A 73 kDa recombinant protein rHmα-actinin 1 was obtained and purified with a Ni-NTA chromatography column. rHmα-actinin 1 was recognized by mouse anti-rHmα-actinin 1 polyclonal antibody, mouse anti-rHmα-actinin 1 monoclonal antibody, and rehabilitation sera from H. meleagridis infected chickens. Native α-actinin 1 in the total proteins of H. meleagridis can also be detected with mouse anti-rHmα-actinin monoclonal antibody. Immunolocalization assays showed that Hmα-actinin 1 was mainly distributed in the cytoplasm of virulent histomonads JSYZ-D9 and in the peripheral regions (near the plasma membrane) of attenuated histomonads JSYZ-D195. Based on in vivo experiment, when chickens were subcutaneously immunized with rHmα-actinin 1 at 5 and 12 days old and then challenged with H. meleagridis at 19 days old, rHmα-actinin 1 reduced the lesion scores 12 days after infection (31 days old) and increased the body weight gain during the challenged period (19-31 days old). Furthermore, it also strengthened the cellular and humoral immune responses 7 days after the second immunization (19 days old). In conclusion, Hmα-actinin 1 could be used as a candidate antigen to develop vaccines against chicken histomonosis.

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Since the ban of effective feed additives and therapeutics, histomonosis has become an important disease and, subsequently, a welfare issue for turkey production. We conducted an interview-based case series study to generate hypotheses about possible disease-favouring conditions in 31 H. meleagridis-infected flocks. The determined parameters were related to the general farm (flock management, biosecurity measures, etc.) as well as the histomonosis-specific disease management. Some inadequate biosecurity measures were observed. An inappropriate usage of the hygiene lock and cleaning as well as the disinfection frequency of equipment, clothes, and the hygiene lock could possibly be histomonosis-favouring conditions. These factors could increase the risk for the introduction of H. meleagridis and the risk of a pathogen spread on an affected farm. Insects, wild birds, litter materials, and contaminated dung could be potential vectors of H. meleagridis. Predisposing gastrointestinal diseases were observed in 71% of the affected flocks. Additionally, stress events related to higher temperature, movement of birds, and vaccination were documented in association with clinical histomonosis. The results emphasise the need for both good disease control and health management to ensure sustainable animal health and welfare.

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.

Histomonosis has become an important disease of turkeys since the ban of effective feed additives and therapeutics. Some critical risk factors for pathogen introduction into a farm have already been identified but open questions remain. Therefore, a retrospective case-control-study was used to identify the most significant risk factors for Histomonas (H.) meleagridis-introduction into a turkey farm. A total of 113 questionnaires were collected from 73 control-farms and 40 Histomonas-positive case-farms in Germany between 20 April 2021 and 31 January 2022. The data were analysed for possible risk factors by descriptive and univariate, single- and multi-factorial analysis. The presence of earthworms, snails and beetles, as vectors of H. meleagridis, as well as the proximity to other poultry-keeping farms in addition to a frequent observation of wild birds nearby the turkey farm, showed the highest risk potential for histomonosis outbreaks. Furthermore, poor biosecurity measures seem to have increased the probability for an outbreak. Insufficient climate management, straw as litter material and an inadequate litter refill frequency might have promoted a favourable humidity for vector- or pathogen survival providing important areas for improved disease control measures in the future.RESEARCH HIGHLIGHTSA retrospective case-control-study was conducted to identify impactful risk factors for a H. meleagridis introduction.The probability of a histomonosis outbreak was increased by the presence of vectors and reservoirs nearby a farm.Impactful risk factors concerning biosecurity measures, climate and litter management were identified.

The ban of effective feed additives and therapeutics in the European Union and in other parts of the world led to a dramatic increase of histomonosis in turkeys. Despite the impact of the disease on the health and welfare of poultry, many questions remain open regarding the epidemiology of the pathogen. In this study, we retrospectively monitored a farm with recurring cases of histomonosis to identify possible routes of pathogen introduction and predisposing factors that may influence the disease development. We included 32 consecutive turkey flocks, which were fattened between 2007 and 2021 on the same farm under the same management and housing conditions. During this period, Histomonas meleagridis was detected in eight flocks of toms and four flocks of hens with a high variability in disease development. Outbreaks in toms led to significantly (P ≤ 0.05) higher mortality rates (5.3%-98.3%) than in hens (2.6%-6.1%). Most of the outbreaks (9/12) were diagnosed between June and September with a peak in August, suggesting a possible impact of higher temperatures either on the host or on the pathogen and pathogen-transmitting vectors. Further investigation is necessary to determine why hens might cope better with histomonosis than toms. Continuous flock and hygiene management is important to prevent an introduction of the causative pathogen and to control potential vectors.
Investigaciones retrospectivas de histomoniasis recurrente en una granja de pavos. La prohibición de aditivos alimentarios y productos terapéuticos efectivos en la Unión Europea y en otras partes del mundo condujo a un aumento dramático de histomoniasis en pavos. A pesar del impacto de la enfermedad en la salud y el bienestar de las aves, quedan muchas interrogantes abiertas con respecto a la epidemiología del patógeno. En este estudio, se monitoreó retrospectivamente una granja con casos recurrentes de histomoniasis para identificar posibles rutas de introducción de patógenos y factores predisponentes que puedan influir en el desarrollo de la enfermedad. Se incluyeron 32 lotes de pavos consecutivos, que fueron engordados entre 2007 y 2021 en la misma granja bajo las mismas condiciones de manejo y alojamiento. Durante este período, se detectó Histomonas meleagridis en ocho parvadas de machos y cuatro parvadas de gallinas con una alta variabilidad en el desarrollo de la enfermedad. Los brotes en machos llevaron a tasas de mortalidad significativamente más altas (P ≤ 0.05) (5.3 %– - 98.3 %) que en gallinas (2.6 %– - 6.1 %). La mayoría de los brotes (9/12) se diagnosticaron entre junio y septiembre con un pico en agosto, lo que sugiere un posible impacto de las temperaturas más altas sobre el huésped o en el patógeno y los vectores transmisores del patógeno. Se necesita más investigación para determinar por qué las gallinas pueden sobrellevar mejor la histomoniasis que los machos. El manejo continuo de la parvada y la higiene es importante para prevenir la introducción del patógeno causante y controlar a los vectores potenciales.

Histomonosis (syn. blackhead disease) is caused by the protozoan parasite Histomonas meleagridis and can result in high mortality in turkey flocks, a situation driven by the limitation of prophylactic and therapeutic interventions. Multi-locus sequence typing confirmed the existence of two genotypes, with the vast majority of reported histomonosis outbreaks being caused by genotype 1 in contrast to only a few detections of genotype 2. For the first time, genotype 2 of H. meleagridis was successfully isolated from an outbreak of histomonosis in a flock of 5-week-old turkeys and a clonal culture was established. Using this culture, an experimental infection was performed in naïve turkeys. The animal trial reflected the observations from the field outbreak and coincided with a previously reported case of histomonosis caused by genotype 2, albeit no mortality was observed in the infected birds whereas 17.1% mortality was noticed in the field outbreak from appearance of disease until slaughter. Post mortem investigations demonstrated that lesions were restricted to the caeca in the field outbreak and the experimental trial. In parallel with the experimental reproduction of pathological changes, an oral vaccination of day-old turkeys with a monoxenic genotype 1 vaccine was carried out to determine efficacy against a genotype 2 challenge. Successful vaccine uptake was characterized by the presence of the vaccine in the caeca determined by qPCR and immunohistochemistry (IHC). Excretion of the vaccine strain was confirmed prior challenge, with the majority of birds developing antibodies. The new monoxenic vaccine was able to minimize lesions in the caeca demonstrating heterologous protection. No parasites were detected in the liver by IHC in any of the vaccinated birds, compared to non-vaccinated animals. However, in 6 out of 17 birds of the vaccinated group a positive signal was obtained by real time PCR from liver samples with 2 positives being typeable by conventional PCR as genotype 2. Overall, H. meleagridis genotype 2 infection was successfully reproduced. Experimental vaccination with a genetically distantly related genotype 1 was able to reduce lesions, supporting protection by a recently developed vaccine candidate as an efficacious prophylactic strategy.

Histomonas meleagridis, the etiological agent of histomonosis, is a poultry parasite primarily detrimental to turkeys. Characteristic lesions occur in the liver and ceca, with mortalities in turkey flocks often reaching 80-100%. Chickens and other gallinaceous birds can be susceptible but the disease was primarily considered sub-clinical until recent years. Treating and preventing H. meleagridis infection have become more difficult since 2015, when nitarsone was voluntarily removed from the market, leaving the poultry industry with no approved prophylactics, therapeutics, or vaccines to combat histomonosis. Phytogenic compounds evaluated for chemoprophylaxis of histomonosis have varied results with in vitro and in vivo experiments. Some recent research successes are encouraging for the pursuit of antihistomonal compounds derived from plants. Turkeys and chickens exhibit a level of resistance to re-infection when recovered from H. meleagridis infection, but no commercial vaccines are yet available, despite experimental successes. Safety and stability of live-attenuated isolates have been demonstrated; furthermore, highly efficacious protection has been conferred in experimental settings with administration of these isolates without harming performance. Taken together, these research advancements are encouraging for vaccine development, but further investigation is necessary to evaluate proper administration age, dose, and route. A summary of the published research is provided in this review.

BACKGROUND: Histomonas meleagridis is a protozoan parasite and the causative agent of histomonosis, an important poultry disease whose significance is underlined by the absence of any treatment and prophylaxis. The recent successful in vitro attenuation of the parasite urges questions about the underlying mechanisms.
RESULTS: Whole genome sequence data from a virulent and an attenuated strain originating from the same parental lineage of H. meleagridis were recruited using Oxford Nanopore Technology (ONT) and Illumina platforms, which were combined to generate megabase-sized contigs with high base-level accuracy. Inspecting the genomes for differences identified two substantial deletions within a coding sequence of the attenuated strain. Additionally, one single nucleotide polymorphism (SNP) and indel targeting coding sequences caused the formation of premature stop codons, which resulted in the truncation of two genes in the attenuated strain. Furthermore, the genome of H. meleagridis was used for characterizing protein classes of clinical relevance for parasitic protists. The comparative analysis with the genomes of Trichomonas vaginalis, Tritrichomonas foetus and Entamoeba histolytica identified ~ 2700 lineage-specific gene losses and 9 gene family expansions in the H. meleagridis lineage.
CONCLUSIONS: Taken as a whole, the obtained data provide the first hints to understand the molecular basis of attenuation in H. meleagridis and constitute a genomics platform for future research on this important poultry pathogen.