Histomonosis in German turkey flocks: possible ways of pathogen introduction.

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.

MicroRNA expression profile of chicken cecum in different stages during Histomonas meleagridis infection.

BACKGROUND: Histomonas meleagridis is an anaerobic, intercellular parasite, which infects gallinaceous birds such as turkeys and chickens. In recent years, the reemergence of Histomoniasis has caused serious economic losses as drugs to treat the disease have been banned. At present, H. meleagridis research focuses on virulence, gene expression analysis, and the innate immunity of the host. However, there are no studies on the differentially expressed miRNAs (DEMs) associated with the host inflammatory and immune responses induced by H. meleagridis. In this research, high-throughput sequencing was used to analyze the expression profile of cecum miRNA at 10 and 15 days post-infection (DPI) in chickens infected with Chinese JSYZ-F strain H. meleagridis. RESULTS: Compared with the controls, 94 and 127 DEMs were found in cecum of infected chickens at 10 DPI (CE vs CC) and 15 DPI (CEH vs CCH), respectively, of which 60 DEMs were shared at two-time points. Gene Ontology (GO) functional enrichment analysis of the target genes of DEMs indicated that 881 and 1027 GO terms were significantly enriched at 10 and 15 DPI, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG, www.kegg.jp/kegg/kegg1.html ) pathway enrichment analysis of the target genes of DEMs demonstrated that 5 and 3 KEGG pathways were significantly enriched at 10 and 15 DPI, respectively. For previous uses, the Kanehisa laboratory have happily provided permission. The integrated analysis of miRNA-gene network revealed that the DEMs played important roles in the host inflammatory and immune responses to H. meleagridis infection by dynamically regulating expression levels of inflammation and immune-related cytokines. CONCLUSION: This article not only suggested that host miRNA expression was dynamically altered by H. meleagridis and host but also revealed differences in the regulation of T cell involved in host responses to different times H. meleagridis infection.

Complete genomes of the eukaryotic poultry parasite Histomonas meleagridis: linking sequence analysis with virulence / attenuation.

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.

Typhlitis induced by Histomonas meleagridis affects relative but not the absolute Escherichia coli counts and invasion in the gut in turkeys.

Unlike in chickens, dynamics of the gut microbiome in turkeys is limitedly understood and no data were yet published in context of pathological changes following experimental infection. Thus, the impact of Histomonas meleagridis-associated inflammatory changes in the caecal microbiome, especially the Escherichia coli population and their caecal wall invasion in turkeys was investigated. Birds experimentally inoculated with attenuated and/or virulent H. meleagridis and non-inoculated negative controls were divided based on the severity of macroscopic caecal lesions. The high throughput amplicon sequencing of 16SrRNA showed that the species richness and diversity of microbial community significantly decreased in severely affected caeca. The relative abundances of operational taxonomic units belonging to Anaerotignum lactatifermentans, E. coli, and Faecalibacterium prausnitzii were higher and paralleled with a decreased abundances of those belonging to Alistipes putredinis, Streptococcus alactolyticus, Lactobacillus salivarius and Lactobacillus reuteri in birds with the highest lesion scores. Although the relative abundance of E. coli was higher, the absolute count was not affected by the severity of pathological lesions. Immunohistochemistry showed that E. coli was only present in the luminal content of caecum and did not penetrate even severely inflamed and necrotized caecal wall. Overall, it was demonstrated that the fundamental shift in caecal microbiota of turkeys infected with H. meleagridis was attributed to the pathology induced by the parasite, which only led to relative but not absolute changes in E. coli population. Furthermore, E. coli cells did not show tendency to penetrate the caecal tissue even when the intestinal mucosal barriers were severely compromised.

Spotlight on Histomonosis (blackhead disease): a re-emerging disease in turkeys and chickens.

Histomonosis, or blackhead disease, is a well-known disease in turkeys that can cause high mortality, but outbreaks with lower losses are also observed. The disease is less fatal in chickens but is economically important due to reduced performance and its co-appearance with colibacillosis. The lack of specific prophylactic and therapeutic interventions has led to a re-emergence of the disease in recent years, mainly in turkeys, free-range layers and chicken parent stock.

Cholesterol supplementation improves growth rates of Histomonas meleagridis in vitro.

Research on the energy metabolism of various protozoan parasites showed the essentiality and benefits of cholesterol in the cultivation of these organisms. However, not much is known about the energy metabolism of Histomonas meleagridis, although such information is of high importance to improve cultivation of the parasite for advancements in diagnostics, research and vaccine development. By supplementing a serum enriched cultivation medium with cholesterol, numbers of parasites could be doubled in comparison to unsupplemented negative controls. This effect was demonstrated for two different strains of the parasite, at different levels of in vitro-passages and for histomonads under xenic or monoxenic settings. Supplementing medium free of serum with cholesterol, resulted in significant growth of the parasite over 72 h. However, there were differences in growth behaviour in serum free medium between the different histomonad cultures and continuous passaging of the cultures without serum was not possible. Monitoring the bacterial growth of two different co-cultivated E. coli strains in monoxenic histomonad cultures during these experiments showed that there was no significant impact of cholesterol on the bacteria. Therefore, a direct effect of cholesterol on the parasite itself could be demonstrated. The results of these experiments supply new insights into the metabolism of H. meleagridis and it can be concluded that cholesterol is an important component to enhance parasite growth in vitro.

An Outbreak of Histomoniasis in Backyard Sanhuang Chickens.

Histomonas meleagridis is a facultative anaerobic parasite, which can cause a common poultry disease known as histomoniasis. The species and age of the birds impacts on the susceptibility, with turkey being the most susceptible species. Chickens are less susceptible to H. meleagridis than turkeys and usually serve as reservoir hosts. Here, the diagnosis of an outbreak of histomoniasis in backyard Sanhuang chickens is described. The primary diagnosis was made based on clinical symptoms, general changes at necropsy, histopathology, and the isolation and cultivation of parasites. The pathogen was further confirmed by cloning, PCR identification, and animal inoculation tests. A strain of H. meleagri- dis, named HM-JSYZ-C, with a higher pathogenicity level in chickens was obtained. The study lays a foundation for further investigations into H. meleagridis and histomoniasis in chickens.

Histomonosis in poultry: previous and current strategies for prevention and therapy.

Histomonosis is a parasitic disease of poultry with worldwide prevalence. The disease can cause morbidity and mortality in chicken and turkey flocks entailing severe economic losses. In the first half of the last century, there was a high demand to control histomonosis as the turkey industry was severely affected by the disease. Consequently, numerous chemical compounds were tested for their efficacy against Histomonas meleagridis with varying outcomes, that are summarized and specified in this review. At the same time, preliminary attempts to protect birds with cultured histomonads indicated the possibility of vaccination. Several years ago antihistomonal drugs were banned in countries with tight regulations on pharmaceuticals in order to comply with the demand of consumer protection. As a consequence, outbreaks of histomonosis in poultry flocks increased and the disease became endemic again. New approaches to prevent and treat histomonosis are, therefore, needed and recently performed studies focused on various areas to combat the disease, from alternative chemotherapeutic substances to plant-derived compounds until vaccination, altogether reviewed here. Considering existing regulations and the overall outcome of experimental studies, it can be concluded that vaccination is very promising, despite the fact that various challenges need to be addressed until the first ever developed vaccine based upon live flagellates in human or bird medicine can be marketed.

Long-term in vitro cultivation of Histomonas meleagridis coincides with the dominance of a very distinct phenotype of the parasite exhibiting increased tenacity and improved cell yields.

The majority of research on Histomonas meleagridis was performed in the first half of the last century, especially those on morphological aspects. In the present study identical monoxenic settings for cultures of the same H. meleagridis clonal strain in its virulent low passage and attenuated high passage form enabled a comparative analysis of parasite characteristics. For the first time, it could be shown that long-term in vitro cultivation led to a severe shift in cell morphology, with the occurrence of a very distinct phenotype expressing a flagellated and highly amoebic cell morphology. Furthermore, the attenuated parasites showed better growth rates and a higher tenacity when confronted with adverse conditions. During these experiments up to 100% of the parasites, both virulent and attenuated, assumed a completely rounded morphology elucidated by electron microscopy. The findings indicate that such previously reported cyst-like stages are a defence strategy of H. meleagridis, independent of the passage level in vitro and pathogenicity in vivo. In conclusion, long-term in vitro passaging of H. meleagridis led not only to an attenuation of the parasite, as previously demonstrated, but also to a shift in the parasite's phenotype regarding morphology, growth behaviour and a higher level of tenacity.

An in vitro attenuated strain of Histomonas meleagridis provides cross-protective immunity in turkeys against heterologous virulent isolates.

In the current study, cross-protective immunity induced by a well-defined clonal strain of Histomonas meleagridis, attenuated by prolonged in vitro cultivation against different clonal heterologous isolates of the same parasite was investigated. For this purpose, 86 turkey poults were assigned to groups consisting of 9-10 birds. Birds of four groups were vaccinated on their 1st day of life followed by re-vaccination on their 14th day of life when the remaining turkeys were left untreated. The challenge was performed using four strains of H. meleagridis that were isolated from chickens or turkeys from different outbreaks of histomonosis in Europe and three of them showed diversities in their genome. Hence, every strain used for the challenge was applied to a group of vaccinated and a group of non-vaccinated birds while birds of the negative control group were sham inoculated. Non-vaccinated birds suffered from severe histomonosis due to the challenge with fatalities reaching from 5 to 10 turkeys per group. Vaccinated birds did not contract clinical signs of the disease following challenge and the increase in weight was unaffected compared to birds of the negative control group. A significant difference in lesion scores was recorded between vaccinated and non-vaccinated groups, with very few instances of liver involvement in the former groups. Livers of vaccinated birds that were without recordable macroscopic lesions were also found negative by immunohistochemical investigation. According to the data obtained, the present study demonstrates, for the first time, the cross-protective capability of a tentative vaccine strain of H. meleagridis attenuated in vitro against heterologous virulent isolates of different origin.

Prevalence and Genetic Characterization of Histomonas meleagridis in Chickens in Vietnam.

This study was performed to investigate the prevalence and to characterize the genetic diversity of Histomonas meleagridis isolates in chickens in southern Vietnam. A total of 194 chickens, randomly selected from 18 backyard and 18 commercial flocks, were screened for H. meleagridis infection using both macroscopic diagnosis and an 18S rRNA gene-based PCR method. Overall, 12.9% of birds, representing 19 flocks, showed gross lesions typical for histomonosis whereas 25.3% of the birds from 29 flocks were positive by PCR assay. Following initial diagnostic approaches, H. meleagridis-positive samples were further analyzed by sequencing three different genomic loci; the 18S rRNA, alpha-actinin1, and rpb1. Thirteen samples from 12 flocks were genetically identified as H. meleagridis, demonstrating a flock and sample prevalence of 33.3% and 6.7%, respectively. There was no significant difference in prevalence between different farm types, age groups, and seasonality. Genetic analysis demonstrated minor heterogeneity of Vietnamese isolates with 99% homology to H. meleagridis sequences from the database. This is the first survey of the prevalence and genetic characterization of H. meleagridis in chickens in Vietnam.

MicroRNA expression profile of chicken liver at different times after Histomonas meleagridis infection.

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.

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.

HISTOMONAS MELEAGRIDIS INFECTIONS IN TURKEYS IN THE USA: A CENTURY OF PROGRESS, RESURGENCE, AND TRIBUTE TO ITS EARLY INVESTIGATORS, THEOBALD SMITH, ERNST TYZZER, AND EVERETT LUND.

Histomoniasis, caused by the protozoan, Histomonas meleagridis, is an economically important disease of turkeys, and it also affects several other species of domesticated and wild Galliformes, including chickens. Under natural conditions, the parasite is transmitted through eggs of a nematode, Heterakis gallinarum, that shares its hosts with Hi. meleagridis. The protozoan infects tissues of both male and female He. gallinarum and eventually is carried within the worm egg. Histomonas meleagridis more readily infects and develops in chickens, and the proximity of chicken farms is a major risk factor for outbreaks in turkeys. Chemoprophylaxis had controlled Hi. meleagridis in turkeys very successfully, but histomoniasis has recently reemerged in turkeys because anti-histomonal drugs are no longer permitted by the United States Food and Drug Administration because of the concerns for residual toxins in poultry meat. Horizontal transmission of the protozoan in the absence of worm eggs remains a mystery because the flagellate trophozoite excreted in the feces of turkeys is not viable for any length of time. A proposed resistant stage of the protozoan has not yet been conclusively demonstrated. Here we review the discovery of the protozoan and the current status of the disease and its control.

Investigations of Histomonosis-Favouring Conditions: A Hypotheses-Generating Case-Series-Study.

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.

Tissue cytokines in chickens from lines selected for high or low humoral antibody responses, given supplemental Limosilactobacillus reuteri and challenged with Histomonas meleagridis.

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.

Identification and Characterization of α-Actinin 1 of Histomonas meleagridis and Its Potential Vaccine Candidates against Histomonosis.

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-ß-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.

Tandem mass tag-based quantitative proteomics analyses of a chicken-original virulent and its attenuated Histomonas meleagridis strain in China.

Introduction: 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. Methods: 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. Results: 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. Discussion: 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.

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.