Histomonas meleagridis, an anaerobic intercellular parasite, is known to infect gallinaceous birds, particularly turkeys and chickens. The resurgence of histomonosis in recent times has resulted in significant financial setbacks due to the prohibition of drugs used for disease treatment. Currently, research on about H. meleagridis primarily concentrate on the examination of its virulence, gene expression analysis, and the innate immunity response of the host organism. However, there is a lack of research on differentially expressed miRNAs (DEMs) related to liver infection induced by H. meleagridis. In this study, the weight gain and pathological changes at various post-infection time points were evaluated through animal experiments to determine the peak and early stages of infection. Next, High-throughput sequencing was used to examine the expression profile of liver miRNA at 10 and 15 days post-infection (DPI) in chickens infected with the Chinese JSYZ-F strain of H. meleagridis. A comparison with uninfected controls revealed the presence of 120 and 118 DEMs in the liver of infected chickens at 10 DPI and 15 DPI, respectively, with 74 DEMs being shared between the two time points. Differentially expressed microRNAs (DEMs) were categorized into three groups based on the time post-infection. The first group (L1) includes 45 miRNAs that were differentially expressed only at 10 DPI and were predicted to target 1646 genes. The second group (L2) includes 43 miRNAs that were differentially expressed only at 15 DPI and were predicted to target 2257 genes. The third group (L3) includes 75 miRNAs that were differentially expressed at both 10 DPI and 15 DPI and were predicted to target 1623 genes. At L1, L2, and L3, there were 89, 87, and 41 significantly enriched Gene Ontology (GO) terms, respectively (p<0.05). The analysis of differentially expressed miRNA target genes using KEGG pathways revealed significant enrichment at L1, L2, and L3, with 3, 4, and 5 pathways identified, respectively (p<0.05). This article suggests that the expression of liver miRNA undergoes dynamic alterations due to H. meleagridis and the host. It showed that the expression pattern of L1 class DEMs was more conducive to regulating the development of the inflammatory response, while the L2 class DEMs were more conducive to augmenting the inflammatory response. The observed patterns of miRNA expression associated with inflammation were in line with the liver\'s inflammatory process following infection. The results of this study provide a basis for conducting a comprehensive analysis of the pathogenic mechanism of H. meleagridis from the perspective of host miRNAs.

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.

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, a protozoan parasite, induces blackhead disease (histomoniasis) in poultry. During hatching, chicks from lines divergently selected for high (HAS) and low (LAS) antibody responses to sheep red blood cells were divided into two groups, each of HAS and LAS, and placed in pens with wood shavings as litter. Feed and water were allowed ad libitum. Half of the chicks from each line had Limosilactobacillus reuteri (L. reuteri) inoculated to their drinking water. On day 18, all chicks were given a transcloacal inoculation of 100,000 H. meleagridis cells. Then, 10 days later, they were euthanized, followed by collection of tissues from the brain, cecal tonsil, ceca, liver, thymus, and spleen for qPCR analyses of cytokines involved in immunological development. Changes in cytokine expressions were most numerous in the cecal tonsil, ceca, and liver. In the absence of a functional medication for control of histomoniasis, L. reuteri and/or its secretory product, reuterin, might serve, in some genetic populations, as a means to reduce the impact of histomoniasis in chickens. The data demonstrate that L. reuteri treatment had tissue specificity between the two genetic lines, in which the effects were targeted primarily toward the cecal tonsil, ceca, and liver, which are the primary tissue targets of the parasite (H. meleagridis), as well as the thymus and spleen. However, interactions among main effects reflect that responses to inflammatory markers observed in tissues for one genetic line may not be observed in another.

Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic-nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic-nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen Leishmania donovanii and the gallinaceous bird pathogen Histomonas meleagridis) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that Xenorhabdus budapestensis and X. szentirmaii are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered \"easy-PACId\" research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential.

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.

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.

UNASSIGNED: Histomonas meleagridis can cause histomonosis in poultry. Due to the prohibition of effective drugs, the prevention and treatment of the disease requires new strategies. Questions about its pathogenic mechanisms and virulence factors remain puzzling.
UNASSIGNED: To address these issues, a tandem mass tag (TMT) comparative proteomic analysis of a virulent strain and its attenuated strain of Chinese chicken-origin was performed.
UNASSIGNED: A total of 3,494 proteins were identified in the experiment, of which 745 proteins were differentially expressed (fold change ≥1.2 or ≤0.83 and p < 0.05), with 192 up-regulated proteins and 553 down-regulated proteins in the virulent strain relative to the attenuated strain.
UNASSIGNED: Surface protein BspA like, digestive cysteine proteinase, actin, and GH family 25 lysozyme were noted among the proteins up regulated in virulent strains, and these several proteins may be directly related to the pathogenic capacity of the histomonad. Ferredoxin, 60S ribosomal protein L6, 40S ribosomal protein S3, and NADP-dependent malic enzyme which associated with biosynthesis and metabolism were also noted, which have the potential to be new drug targets. The up-regulation of alpha-amylase, ras-like protein 1, ras-like protein 2, and involucrin in attenuated strains helps to understand how it is adapted to the long-term in vitro culture environment. The above results provide some candidate protein-coding genes for further functional verification, which will help to understand the molecular mechanism of pathogenicity and attenuation of H. meleagridis more comprehensively.

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.