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Simple Summary: Histomonas meleagridis is a protozoan parasite that causes histomonosis in gallinaceous birds. This study identified α-actinin 1 of H. meleagridis. α-actinin 1 was predicted as a virulence factor involved in cell adhesion in our previous differential proteomic analysis. It localized in different cellular regions as detected by an indirect immunofluorescent assay between virulent histomonads and attenuated histomonads JSYZ. Administration of recombinant expressed rHma-actinin 1 reduced lesion scores in the cecum and liver, improved weight gain, and induced humoral and cellular immune responses against H. meleagridis in chickens. 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. [ABSTRACT FROM AUTHOR]

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. A 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Histomonosis has become a serious disease not only for turkeys but for the entire poultry sector. Despite a focus on research on histomonosis over recent years, possible disease-favouring conditions are still poorly understood. For this reason, an interview-based case series study was initiated on 31 affected turkey farms in Germany. Despite a generally good risk awareness among participating farmers for disease-favouring conditions, an insufficient attitude towards certain biosecurity aspects was detected. Furthermore, hypotheses concerning various possibilities of pathogen introduction via animate or inanimate vectors could be generated, which need to be tested in follow-up studies. Overall, this study demonstrates that improvements in biosecurity, flock, and health management are necessary to ensure animal welfare in the future. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The aim of the current study was to evaluate the influence of stressors on histomoniasis development and lateral transmission of Histomonas meleagridis. In the following experiments, half of the birds in each pen were inoculated with H. meleagridis to study disease transmission and progression. Birds were infected at 5 wk of age (experiments 1 and 3) or at 2 wk of age (experiment 2). Disease progression was evaluated by infection rate, mortality rate, and pathologic lesions in the ceca and liver. Reported results were applicable for directly infected birds as lateral transmission was not induced in these experiments. In experiment 1, the results showed high electrolyte, low-density diet (HE + LD), feed withdrawal (FW), caused higher infection rates and increase lesion scores in the liver and ceca compared to con. Experiment 2 further investigated the influence of low-density diet (LD) in conjunction with coccidiosis (LD + C) or feed withdrawal (LD + FW). All treatments had higher infection rates, mortality rates, ceca and liver scores compared to PC. In experiment 3, birds were fed diets containing naturally occurring aflatoxin at 0 ppb (AFLB1), 6.26 ppb (AFLB1 Low), or 19.82 ppb (AFLB1 High). No significant differences among treatments were observed. Though lateral transmission was not induced by theses stressors, the results of these experiments demonstrated that low-density diets, feed withdrawal and/or coccidial infection facilitated more severe histomoniasis infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • First report on miRNAs expression in chicken liver during H. meleagridis infection. • 120 and 118 miRNAs were found in chicken liver at 10 and 15 days post-infection. • MiRNAs on the 10 days post-infection regulate the balance of inflammatory response. • MiRNAs on the 15 days post-infection exacerbate inflammatory responses. • Liver miRNAs showed increased pro-inflammatory activity 10–15 days post-infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Histomoniasis is a disease caused by Histomonas meleagridis, a flagellated protozoan that can cause severe necrotizing hepatitis and typhlitis in several bird species. The disease has a cosmopolitan distribution. In experimental infection, peacocks (Pavo spp.) showed susceptibility to histomoniasis, however there are few reports on natural histomoniasis in this species. In northeastern Brazil, reports about its occurrence in avian species are scarce and nonexistent in peacocks. Therefore, this report aims to describe the epidemiological and clinicopathological aspects of a histomoniasis case in a peacock (Pavo cristatus) in the Brazilian semiarid region. Case: A 3-month-old male peacock with a history of apathy and anorexia was attended in the Veterinary Hospital of the Universidade Federal Rural do Semi-Árido (UFERSA), Mossoró, RN, Brazil. The animal was raised extensively in a farm without basic sanitary measures, also with a history of living with animals of different species. After clinical examination, in which intense apathy and weight loss were confirmed, the bird was submitted to emergency therapeutic measures, however there was no clinical improvement and the bird died. At necropsy, intense diffuse bilateral necrotizing typhlitis and multifocal to coalescent necrotizing hepatitis were observed. Fragments of the organs were collected in 10% neutral buffered formalin for histopathological analysis and cecal content were collected for microbiological analysis. Histopathology of the cecum revealed transmural necrotizing typhlitis associated with myriads of trophozoites morphologically compatible with Histomonas meleagridis. The same microorganisms were observed in association with necrotizing hepatitis lesions, which allowed the diagnosis of histomoniasis. Also, the bacterium Pantoea agglomerans was isolated from the cecal content. Discussion: The macroscopic and microscopic findings allowed the diagnosis and the first recording of histomoniasis in peacock in the semi-arid region of Rio Grande do Norte. The bird was raised in an extensive breeding without sanitary management, such as the use of anthelmintics, which may favor infection by the nematode Heterakis gallinarum, which transmits the protozoan Histomonas meleagridis. The contamination, which begins by the orofecal route, happens through the ingestion of eggs of the nematode contaminated with H. meleagridis that pass through the gastrointestinal tract, reaching the ceca and causing intense lesions in the organ, such as the bilateral transmural typhlitis that we observed in this case. From ceca, the protozoan has access to the bloodstream and reaches the liver, where it causes necrotic hepatitis, also present in the peacock. Both cecal and hepatic lesions were associated with myriads of microorganisms morphologically compatible with H. meleagridis, which allowed the diagnosis of the disease. The challenge in diagnosing this disease occurs mainly due to nonspecific clinical signs, such as apathy and weight loss, the only signs reported by the breeder and observed in this peacock. Confirmation of the occurrence of histomoniasis in any region is important to establish the disease among the differential diagnoses for the species, as in this case. Since this is the first report of peacock histomoniasis in the semi-arid region of Rio Grande do Norte, it is evident the need to consider the disease among possible diagnoses in cases of nonspecific symptoms and it also demonstrates the need to implement control and prophylaxis measures in peacock breeding aiming to avoid losses of birds and economic losses to the breeders and to promote animal welfare. [ABSTRACT FROM AUTHOR]

significance for many commercial turkey and broiler breeder production companies because of the unavailability of drugs or vaccines. The protozoa Histomonas meleagridis (HM) requires the presence of enteric microflora to promote the disease. The objectives of this research note were to evaluate the effect of dietary administration of sodiumchlorate (SC) and sodiumnitrate (SN) in vitro and in vivo for HM prophylaxis in poults. A total of 128 day-of-hatch female poults obtained from a commercial hatchery were wing-tagged and randomly assigned into 1 of 4 experimental groups: negative control (NC), positive control, dietary inclusion of SC (3,200 ppm) and SN (500 ppm). Poults from groups SC and SN started on their respective diets on day 12. All groups, except the NC, were challenged with 2x105 HMon day 19. Controls were fed a basal diet, identical to the treatment diets but not supplemented with SC or SN. Body weight gain (BWG)was determinedweekly, starting on day 1 until day 28, and postchallenge morbidity and mortality were recorded. On day 28 of age, all surviving poults were lesion scored for hepatic and cecal lesions. Ceca and distal ileum were collected on day 28 for bacterial recovery on selective media for total aerobic, lactic acid bacteria, or gramnegative bacteria. The addition of SC and SN in the in vitro growth of HM greatly reduced the growth of the protozoa after 20 h of incubation when compared with the control nontreated group (P <0.05). However, dietary supplementation of SC and SN had no effect against HM in vivo, as was demonstrated by BWG, the severity of lesions in the liver and ceca or bacterial recovery of treated poults when compared with the positive control group. [ABSTRACT FROM AUTHOR]

Histomoniasis, caused by the protozoan parasite Histomonas meleagridis, is a disease to which turkeys are especially susceptible. Currently, no chemoprophylaxis compounds are available to mitigate this disease. Boric acid (BA) exhibits antifungal, antiseptic, and antiviral properties and has been used in the treatment of yeast infections. Based on these characteristics, an experiment was conducted to evaluate whether BA might be an efficacious prophylaxis against challenge with wild-type H. meleagridis (WTH). On day-of-hatch, poults were randomly assigned to either a basal control diet or a BA diet (basal diet 1 0.2% BA). Groups consisted of a nonchallenged control (NC; basal diet), 0.2% BA1challenge (BA; 0.2% BA diet), and a positive-challenged control (PC; basal diet). On day 21, challenged groups were intracloacally inoculated with 2 × 105 WTH cells/turkey, and lesions were evaluated on day 14 postchallenge. Individual body weights were recorded on day 0, 21, and 35 to calculate the pre-challenge and postchallenge body weight gain (BWG). TheBA group resulted in lower prechallenge day 0 to day 21BWG(P50.0001) than the NC group. Postchallenge day 21 to day 35, BWG was also lower (P 5 0.0503) in the BA group than the PC group. No differences between the BA and PC groups were detected for mortalities associated with histomoniasis. Moreover, liver and cecal lesions were not statistically different between the BA and PC groups. Taken together, these data suggest that BA was not efficacious in the prevention or reduction of histomoniasis disease severity when provided at 0.2% dietary concentration under these experimental conditions. [ABSTRACT FROM AUTHOR]

Histomonas meleagridis is an extracellular protozoan parasite and the aetiological agent of histomonosis, an important poultry disease whose impact is greatly accentuated by inaccessibility of any treatment. A special feature of the parasite is its intricate interplay with bacteria in vitro and in vivo , the focus of this article. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Blackhead disease, caused by the protozoan Histomonas meleagridis , is commonly found in layer pullets raised on the floor. We examined the effects of blackhead disease during the pullet-rearing period and on subsequent productivity during the first 8 wk of the laying cycle. Treatments were (1) uninfected controls and (2) H. meleagridis -infected pullets, with 4 replicate pens/treatment, 32 pullets/pen (Hy-LineW-36). Pullets in the challenge treatment were infected with H. meleagridis on day 18. Four birds/pen were necropsied on days 23 and 28 for lesion scores and day 176 for detection of H. meleagridis. Hens were moved to individual layer cages on day 120 and observed daily for feed consumption, date of first lay and egg production parameters. Pullets were positive for signs of blackhead disease in 83%–90% of infected birds necropsied on days 23 and 28, with average cecal lesion scores of 2.5 and 2.9. No liver lesions were observed. On day 176, 40% of infected birds were positive for H. meleagridis in the ceca. During the laying cycle, there were no significant differences (P ≤ 0.05) between treatments in terms of date of first lay, hen-day egg production, egg weight, feed conversion, egg mass/hen, or other reproduction measurements. These results showed that while there was no long-term effect of blackhead infection on layer productivity under laboratory conditions, H. meleagridis persisted in the flock, providing a reservoir for infection. [ABSTRACT FROM AUTHOR]

Environmental dust samples obtained from 65 turkey flocks in France, of which six suffered from histomonosis whereas the rest remained apparent healthy until the end of production, were tested for the presence of Histomonas meleagridis DNA by recently developed real-time PCR based on the 18S rRNA locus. In order to determine the genotype of detected histomonads, positive samples were further subjected to conventional 18S PCR and sequencing. Additionally, production data of all tested flocks, such as average daily gain, feed conversion ratio and production index, were statistically evaluated and compared to see the effect of positive dust samples in apparently healthy flocks. Histomonad DNA was detected in the dust obtained from all six clinically affected and, surprisingly, in nine apparent healthy flocks. Sequencing of the 18S rRNA gene resulted in only one DNA sample homologous to H. meleagridis whereas 11 others revealed the presence of several other flagellates. Average daily gain and production index were negatively affected in flocks with clinical histomonosis, resulting in significant difference in comparison with the data obtained from clinically healthy flocks independent of the presence of histomonad DNA in the dust. Overall, there was no significant difference following statistical analysis of production parameters between the two last mentioned groups of tested flocks. Altogether, this is the first investigation demonstrating the presence of H. meleagridis DNA in environmental dust samples obtained from clinically unaffected turkey flocks. However, this finding could not be correlated with impact on production based on analysis and comparison of selected production data. RESEARCH HIGHLIGHTS Environmental dust obtained from clinically healthy turkey flocks, in addition to dust from flocks affected by histomonosis, was found positive for the presence of Histomonas meleagridis DNA. Histomonas-positive dust samples in clinically unaffected flocks did not have a negative effect on production parameters. The results demonstrate a wider spread of H. meleagridis DNA in flocks of commercial meat turkeys than previously thought. [ABSTRACT FROM AUTHOR]

• Three diagnostic PCR primers sets were designed to detect Heterakis gallinarum DNA. • H. gallinarum diagnostic primers can be used to investigated vectors of the nematode. • H. gallinarum DNA could be detected in earthworms collected from poultry facilities. • Darling beetles may act as a carrier of H. gallinarum. Heterakis gallinarum is a widely distributed cecal nematode that parasitizes gallinaceous birds including chickens and turkeys. H. gallinarum infection poses a problem for the poultry industry as the nematode egg serves as a vector for the protozoan parasite, Histomonas meleagridis , the causative agent of histomonosis. The only means of detecting H. gallinarum in the environment is microscopic identification of the eggs in soil or feces; however, H. gallinarum eggs are often mistaken for those of Ascaridia galli. Three primer sets were designed from sequences cloned from the H. gallinarum genome to develop a diagnostic PCR. Each of these primer sets amplified a single product from H. gallinarum , but were unable to amplify DNA from H. meleagridis , Ascaridia galli , or Cestode sp. H. gallinarum DNA was amplified from Lumbricus sp. (earthworms) and Alphitobius diaperinus (darkling beetles), confirming that the earthworm acts as a paratenic host for H. gallinarum and suggesting that the darkling beetle may be a carrier for this nematode. [ABSTRACT FROM AUTHOR]

The exoproteome of parasitic protists constitutes extracellular proteins that play a fundamental role in host-parasite interactions. Lytic factors, especially secreted proteases, are capable of modulating tissue invasion, thereby aggravating host susceptibility. Despite the important role of exoproteins during infection, the exoproteomic data on Histomonas meleagridis are non-existent. The present study employed traditional 1D-in-gel-zymography (1D-IGZ) and micro-LC-ESI-MS/MS (shotgun proteomics), to investigate H. meleagridis exoproteomes, obtained from a clonal virulent and an attenuated strain. Both strains were maintained as mono-eukaryotic monoxenic cultures with Escherichia coli. We demonstrated active in vitro secretion kinetics of proteases by both parasite strains, with a widespread proteolytic activity ranging from 17 kDa to 120 kDa. Based on protease inhibitor susceptibility assay, the majority of proteases present in both exoproteomes belonged to the family of cysteine proteases and showed stronger activity in the exoproteome of a virulent H. meleagridis. Shotgun proteomics, aided by customized database search, identified 176 proteins including actin, potential moonlighting glycolytic enzymes, lytic molecules such as pore-forming proteins (PFPs) and proteases like cathepsin-L like cysteine protease. To quantify the exoproteomic differences between the virulent and the attenuated H. meleagridis cultures, a sequential window acquisition of all theoretical spectra mass spectrometric (SWATH-MS) approach was applied. Surprisingly, results showed most of the exoproteomic differences to be of bacterial origin, especially targeting metabolism and locomotion. By deciphering such molecular signatures, novel insights into a complex in vitro protozoan- bacteria relationship were elucidated. [ABSTRACT FROM AUTHOR]

The current study focused on Histomonas meleagridis , a unicellular protozoan, responsible for histomonosis in poultry. Recently, the occurrence of the disease increased due to the ban of effective chemotherapeutic drugs. Basic questions regarding the molecular biology, virulence mechanisms or even life cycle of the flagellate are still puzzling. In order to address some of these issues, we conducted a comparative proteomic analysis of a virulent and an attenuated H. meleagridis strain traced back to a single cell and propagated in vitro as monoxenic mono-eukaryotic cultures. Using two-dimensional electrophoresis (2-DE) for proteome visualization with computational 2-DE gel image and statistical analysis, upregulated proteins in either of the two H. meleagridis strains were detected. Statistical analysis fulfilling two criteria (≥threefold upregulation and P  < 0.05) revealed 119 differentially expressed protein spots out of which 62 spots were noticed in gels with proteins from the virulent and 57 spots in gels with proteins from the attenuated culture. Mass spectrometric analysis of 32 protein spots upregulated in gels of the virulent strain identified 17 as H. meleagridis -specific. The identification revealed that these spots belonged to eight different proteins, with the majority related to cellular stress management. Two ubiquitous cellular proteins, actin and enolase, were upregulated in multiple gel positions in this strain, indicating either post-translational modification or truncation, or even both. Additionally, a known virulence factor named legumain cysteine peptidase was also detected. In contrast to this, mass spectrometric analysis of 49 protein spots, upregulated in gels of the attenuated strain, singled out 32 spots as specific for the flagellate. These spots were shown to correspond to 24 different proteins that reflect the increased metabolism, in vitro adaptation of the parasite, and amoeboid morphology. In addition to H. meleagridis proteins, the analysis identified differential expression of Escherichia coli DH5α proteins that could have been influenced by the co-cultivated H. meleagridis strain, indicating a reciprocal interaction of these two organisms during monoxenic cultivation. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

The protozoan parasite Histomonas meleagridis is the causative agent of histomonosis in gallinaceous birds. In turkeys, the disease can result in high mortality due to severe inflammation and necrosis in caecum and liver, whereas in chickens the disease is less severe. Recently, experimental vaccination was shown to protect chickens and turkeys against histomonosis but dynamics in the cellular immune response are not yet demonstrated. In the present work, different groups of birds of both species were vaccinated with attenuated, and/or infected with virulent histomonads. Flow cytometry was applied at different days post inoculation to analyse the absolute number of T-cell subsets and B cells in caecum, liver, spleen and blood, in order to monitor changes in these major lymphocyte subsets. In addition, in chicken samples total white blood cells were investigated. Infected turkeys showed a significant decrease of T cells in the caecum within one week post infection compared to control birds, whereas vaccination showed delayed changes. The challenge of vaccinated turkeys led to a significant increase of all investigated lymphocytes in the blood already at 4 DPI, indicating an effective and fast recall response of the primed immune system. In the caecum of chickens, changes of B cells, CD4 + and CD8α + T cells were much less pronounced than in turkeys, however, mostly caused by virulent histomonads. Analyses of whole blood in non-vaccinated but infected chickens revealed increasing numbers of monocytes/macrophages on all sampling days, whereas a decrease of heterophils was observed directly after challenge, suggesting recruitment of this cell population to the local site of infection. Our results showed that virulent histomonads caused more severe changes in the distribution of lymphocyte subsets in turkeys compared to chickens. Moreover, vaccination with attenuated histomonads resulted in less pronounced alterations in both species, even after challenge. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Histomonas meleagridis (H. meleagridis) is a protozoan parasite that may cause histomoniasis, a disease of special importance to the poultry industry and public health. The molecular characterization of H. meleagridis in China has not been established. The 5.8S and flanking ITS regions were amplified by polymerase chain reaction from 15 liver samples of chickens which were preliminarily diagnosed with H. meleagridis infection by observing clinical symptoms and macroscopic changes in the organs in Eastern China between 2012 and 2013. The obtained sequences were aligned and compared with other known sequences of H. meleagridis and related protozoan species based on ITS1-5.8S rRNA-ITS2 or 5.8S rRNA region alone. Out of the 15 obtained sequences, 8 sequences were identified as H. meleagridis and were grouped into five clades, suggesting the possibility of multiple genotypes within the samples. Among the remaining 7 sequences, 4 sequences were more related to Trichomonas and 3 sequences were more related to Tetratrichomonas, which suggests the possibility of misdiagnosis or coinfection with other protozoans. Therefore, there is obvious genetic diversity of H. meleagridis based on the 5.8S and flanking ITS regions, which suggests the presence of different genotypes in chickens from Eastern China. [ABSTRACT FROM AUTHOR]

Histomoniasis is caused by the protozoa Histomonas meleagridis (HM) that are laterally transmitted among birds leading to high mortality in commercial flocks. This study tested an HM infection model assessing the lateral transmission of HM in turkey poults raised on floor pens. Day (d)-old female turkey poults (n = 320) were individually wing-tagged and allocated to one of four treatment groups (4 floor pens/group and 20 poults/pen) based on the percentage of poults inoculated with HM: 1) 10% (HM10); 2) 20% (HM20); 3) 30% (HM30); and 4) 40% (HM40). On d 9, seeder poults intracloacally received a 1 mL inoculum/bird containing ∼80,000 histomonads. Poults were individually weighed on d 0, 9, and 25 and feed intake recorded on per pen basis. On d 25, all birds were euthanized by cervical dislocation and ceca and liver were evaluated for HM lesions. Data were analyzed using JMP (Pro16) and significance (P ≤ 0.05) between treatments were determined by LSD test. Mortality was 7.63%, 12.5%, 21.58%, and 20.59%, while transmission rates from inoculated to non-inoculated birds were 62.5%, 57.5%, 92.43%, and 78.75% in HM10, HM20, HM30, and HM40 groups, respectively. Average daily feed intake was proportionally reduced with the increasing number of inoculated poults from HM10 to HM40. Average daily gain was significantly lower in HM30 and HM40 poults compared to those in HM10 and HM20 during the postchallenge period (d 10–25). Therefore, we herein report the successful lateral transmission of HM among turkey poults raised on floor pens. This research model closely resembles commercial field conditions and affords a much-needed platform for conducting relevant basic and applied research on histomoniasis in poultry. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To gain more insight into the within flock transmission ofHistomonas meleagridis, the shedding of parasites was quantified by a newly developed real-time quantitative (q)PCR and the basic reproduction number (R0) and the mean number of secondary infections per infectious bird per day in a susceptible population (β) ofH. meleagridisin the absence of heterakis were assessed. Forty turkeys were divided into two groups of 10 and 30 birds at 14 days of age. Birds of the first group were inoculated with 200,000 histomonads each, the second group served as a susceptible contact group. Cloacal swabs were taken at −1, 1, 4, 7, 9, 11, 14, 18 and 21 days post inoculation (p.i.) to assess the shedding of the parasite by the qPCR (detection limit 330 histomonads/ml droppings). The experiment ended at 28 days p.i. Mortality was 100% in the inoculated birds and started at day 12 p.i., while in the contacts, it was 83% and started at 16 days p.i. Shedding started 1 day after the inoculation in both groups. The mean shedding levels (and 95% CI) expressed as parasite equivalents per gram cloacal content on a log10scale in the inoculated, contact birds that died and contact birds alive were 2.0 (1.6–2.4), 1.6 (1.4–1.9) and 1.2 (0.5–2.0), respectively. Birds that died shed histomonas more often and were infectious for 13.4 days; in contrast, those that recovered were infectious for 5.7 days.R0was estimated to be 8.4 andβ0.70. Simulations made with the parameters obtained were in agreement with the experimental results, confirming their validity. [ABSTRACT FROM AUTHOR]

Histomonas meleagridis, a protozoan parasite that can infect gallinaceous birds, affects mainly the liver and caeca of infected birds. As a consequence of the recent ban of chemotherapeuticals in Europe and the USA, histomonosis gained somewhat more attention due to its re-emergence and the fact that there is no effective treatment available. Therefore, special attention is now also given towards the diagnosis and the control of the disease. In the actual study we report the development of highly specific and sensitive real-time PCR methods for detection and quantification of the parasite, based on two protein coding genes, Fe-hydrogenase ( FeHYD ) and rpb1 . Both genes seem to be in a single copy in H. meleagridis as shown by southern blotting and absolute quantification using real-time PCRs on samples containing a known amount of the parasite. The real-time PCR assays based on FeHYD and rpb1 genes were found to be an efficient method for the quantification and detection of H. meleagridis in in vitro grown cultures, tissues of infected birds and in faecal samples. Both real-time PCRs were able to detect up to a single cell in in vitro cultures of H. meleagridis and in fecal samples spiked with H. meleagridis . Finally, qPCR assays were shown to be highly specific for H. meleagridis as samples containing either of the two H. meleagridis genotypes were positive, whereas samples containing other protozoa such as Tetratrichomonas gallinarum, Trichomonas gallinae, Simplicimonas sp., Tritrichomonas sp., Parahistomonas wenrichi , Dientamoebidae sp. and Blastocystis sp. were all negative. [ABSTRACT FROM AUTHOR]

The protozoan flagellate Histomonas meleagridis is the etiological agent of histomonosis, first described in 1893. It is a fastidious disease in turkeys, with pathological lesions in the caeca and liver, sometimes with high mortality. In chickens the disease is less fatal and lesions are often confined to the caeca. The disease was well controlled by applying nitroimidazoles and nitrofurans for therapy or prophylaxis. Since their introduction into the market in the middle of the previous century, research nearly ceased as outbreaks of histomonosis occurred only very rarely. With the ban of these drugs in the last two decades in North America, the European Union and elsewhere, in combination with the changes in animal husbandry, the disease re-emerged. Consequently, research programs were set up in various places focusing on different features of the parasite and the disease. For the first time studies were performed to elucidate the molecular repertoire of the parasite. In addition, research has been started to investigate the parasite's interaction with its host. New diagnostic methods and tools were developed and tested with samples obtained from field outbreaks or experimental infections. Some of these studies aimed to clarify the introduction of the protozoan parasite into a flock and the transmission between birds. Finally, a strong focus was placed on research concentrated on the development of new treatment and prophylactic strategies, urgently needed to combat the disease. This review aims to summarize recent research activities and place them into context with older literature. [ABSTRACT FROM AUTHOR]

Repeated serial in vitro passage of Histomonas meleagridis , the etiological agent of histomoniasis (blackhead) of turkeys, was demonstrated to markedly achieve attenuation and reduction of virulence as compared to the original wild-type isolate. Four experiments were performed to evaluate the route (oral vs. intracloacal) and age (day-of-hatch vs. d 14) for administration of attenuated H. meleagridis isolates as vaccine candidates against homologous or heterologous wild-type challenge. Attenuated H. meleagridis were developed from 2 different strains (Buford strain originating in Georgia; PHL2017 strain originating in Northwest Arkansas). Buford P80a (passage 80, assigned as isolate lineage "a" following repeated passage) was selected as the primary vaccine candidate and was evaluated in Experiments 1–3. Experiment 4 evaluated selected candidates of attenuated PHL2017 (P67, P129) and Buford (P80a, P200a, P138b, P198c) strains against Buford wild-type challenge. As has been demonstrated previously, wild-type H. meleagridis cultures administered orally after 1 day of age were not infective in the current studies, but infection with wild-type cultures could be induced orally at day-of-hatch. Infection was effectively achieved via the intracloacal route at day-of-hatch and in older turkeys (d 21, d 28–29, d 35). Intracloacal inoculation of turkeys with the attenuated passaged isolates as vaccine candidates at d 14 was shown to produce significant (P < 0.05) protection from mortality, reduction in body weight gain, as well as reduction in hepatic and cecal lesions in these experiments following challenge with either the homologous wild-type isolate or from a wild-type strain obtained years later from a geographically disparate area of the United States. Inoculation with the attenuated H. meleagridis isolates at day-of-hatch, either orally or cloacally, did not produce significant protection against subsequent wild-type challenge. While offering significant protection with minimal vaccine-related negative effects, the protection from cloacal vaccine administration was neither significantly robust nor encouraging for industry application using the methods evaluated in the present manuscript since mortalities and lesions were not completely reduced which could thereby potentially allow transmission from residual infection and shedding within a flock. [ABSTRACT FROM AUTHOR]

The article presents a case study of a male and female turkey who were presented to veterinary care for necropsy. Topics discussed include diagnosis of Histomoniasis, a protozoal infection of the cecal pouches and liver caused by Histomonas meleagridis; biosecurity measures including prevention of meleagridis for entering property on contaminated vehicles, shoes, clothing, and animals and preventing exposure to coccidia to prevent histomoniasis in chickens.

Histomonas meleagridis is the aetiological agent of histomonosis or “blackhead disease”. Histomonosis is of special importance today, because there is no effective treatment to prevent its occurrence with considerable losses for the poultry industry. Despite its importance only a few molecular studies have yet been performed to investigate the degree of genetic diversity between different isolates of this parasite. In the present study a collection of well defined samples, previously shown positive for the DNA of H. meleagridis, was used to investigate genetic relatedness of the parasite. Samples originated from 25 turkey flocks collected in France between 2007 and 2010. Additionally, diagnostic samples, collected at our Clinic in Vienna, from different European countries and Azerbaijan, during 2010 to 2013 were included in the analyses. For the analysis three different genetic loci were analyzed: 18S rRNA, α-actinin1 and rpb1 genes. To amplify partial sequences of α-actinin1 and rpb1 genes, primers specifically targeting H. meleagridis were designed. Following PCR, the sequences of 18S rRNA, α-actinin1 and rpb1 loci were analyzed. Phylogenetic analyses demonstrated separation of H. meleagridis isolates in two different clusters. The majority of isolates grouped within the cluster 1 and originated from different European countries. The cluster 2 was rare and predominantly found in samples originating from France. Considering that the genetic variability of clusters can be seen as two distinct genetic types we propose the term genotype instead of cluster. [ABSTRACT FROM AUTHOR]

Histomonas meleagridisis the causative agent of histomonosis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis, and high mortality. To develop a rapid and sensitive method for specific detection ofH. meleagridis, an assay based on loop-mediated isothermal amplification (LAMP) targeting the 18S rRNA gene was established. The detection limit of the LAMP assay was 10 copies for standard plasmids containing an 18S rRNA gene fragment, which was superior to that of a classical PCR method. Specificity tests revealed that there was no cross-reaction with other protozoa such asTrichomonas gallinae,Blastocytissp,Tetratrichomonas gallinarum,Plasmodium gallinaceum,Toxoplasma gondii,Eimeria tenella,Leucocytozoon caulleryiandLeucocytozoon sabrazesi. The assay was evaluated for its diagnostic utility using liver and caeca samples collected from suspected field cases, the detection rate was 100 and 97.92%, respectively. These results indicate that the LAMP assay may be a useful tool for rapid detection and identification ofH. meleagridisin poultry. [ABSTRACT FROM AUTHOR]

Highlights: [•] Reversion to virulence and residual pathogenicity of in vitro attenuated Histomonas meleagridis were investigated. [•] No adverse clinical signs or mortality in chickens and turkeys following infection with attenuated histomonads. [•] No increase in number and severity of lesions within 5 cycles of backpassaging. [•] Lesion scores caused by the virulent strain were statistically different to those induced by attenuated histomonads. [•] In vivo passaging of attenuated Histomonas meleagridis does not foster reversion to virulence in chickens or turkeys. [Copyright &y& Elsevier]

Abstract: Histomonosis (blackhead disease or infectious enterohepatitis) caused by the extracellular protozoon parasite Histomonas meleagridis is an important disease of turkeys and a threat to the poultry industry. Due to recent legislation on drug restrictions, research to find new alternatives is an urgent matter in the battle against histomonosis. In the present study, intracloacal inoculation of a low-virulent H. meleagridis strain isolated after serial passages in turkeys clearly demonstrated a reduction of virulence and hence its effectiveness as a vaccine against histomonosis. The low-virulent isolate has been evaluated in a comparative experimental infection study. No mortality nor predominant caecal or liver lesions could be observed in the groups inoculated with 103, 104 or 105 histomonads per bird. Only dilated caeca with a yellow and foamy content could be noticed. Groups inoculated with similar doses of a virulent strain displayed a dose-related pathology and mortality up to 100%. The protective capacity of the strain with reduced virulence could be demonstrated as none of the birds cloacally inoculated with 103, 104 or 105 histomonads died upon challenge with 105 H. meleagridis of the virulent strain. Hereby, 71% of the challenge control group died. Interestingly, no or only very minor pathological lesions in the caeca and liver could be detected after challenge of the birds inoculated with the passaged histomonads. In conclusion, cloacal inoculation of the low-virulent strain obtained after serial backpassages was able to induce protection of turkeys against challenge with a virulent H. meleagridis strain. [Copyright &y& Elsevier]

Histomoniasis, also commonly referred to as blackhead disease, is caused by the protozoan parasite Histomonas meleagridis. Since the removal of nitarsone in 2015, no approved prophylactics are available for mitigating histomoniasis. Disease incidence and high mortalities are frequently associated with turkey flocks, although infection of broiler breeders also occurs. Quinine is a naturally occurring alkaloid with antimalarial properties. In vitro assays have shown strong antihistomonal properties of quinine, leading to our hypothesis that quinine inclusion within the feed could prevent histomoniasis in turkeys. Selected concentrations of quinine were included within a turkey starter diet to evaluate effects on body weight gain (BWG), liver lesions, cecal lesions, and mortality of H. meleagridis- challenged turkeys. On day-of-hatch, poults were randomly assigned to either the basal diet or a quinine diet. Groups consisted of a non-challenged control (NC ; basal diet), 0.022% quinine + challenge, 0.067% quinine + challenge, 0.2% quinine + challenge, or a positive-challenged control (PC ; basal diet). On d 10, challenged groups were intracloacally inoculated with 105 H. meleagridis cells/turkey, and lesions were evaluated on d 21 post-infection. Individual body weights were recorded on d 0, d 10, and d 31 to calculate the pre-challenge and post-challenge BWG. No significant differences (P > 0.05) were observed between the d 0 to 10 pre-challenged BWG between quinine treatment diets and the basal diet. Similarly, no differences (P > 0.05) were observed in post-challenge d10-31 BWG of the quinine dietary treatments as compared to the PC. Cumulative mortalities, liver lesions, and cecal lesions related to histomoniasis were not reduced (P > 0.05) in any of the quinine treatment groups as compared to the PC. Although quinine successfully reduced H. meleagridis cells in vitro , results from the in vivo experiment indicated no reduction in histomoniasis severity as evidenced by similar lesions and mortality as the PC. Taken together, these data indicate that quinine inclusion within the feed at these concentrations and under these experimental conditions was not efficacious in the prevention or treatment of histomoniasis. [ABSTRACT FROM AUTHOR]

In recent years, a number of studies about Histomonas meleagridis, and more specifically about experiments in vivo involving H. meleagridis to investigate the pathogenicity and efficacy of drugs or vaccines, have been published. Together with older publications, a considerable amount of information about experimental infections with H. meleagridis exist, which is helpful for planning future animal studies and can reduce the number of birds used in such studies toward better animal welfare. One hundred sixty-seven publications describing experimental infections with H. meleagridis were published in scientific journals between 1920 and 2012. One hundred forty-two of these publications describe infections of turkeys ( Meleagris gallopavo) and 52 infections of chickens ( Gallus gallus). In 18 studies, experiments involving other species were done. The most popular routes of infection were the intracloacal application of histomonal trophozoites from culture material, from lesions or from feces of infected birds, or using larvae of the cecal worm Heterakis gallinarum (83 studies) and the oral application of eggs or other stages of the cecal worm containing histomonal stages (83 studies). During the last 10 years, intracloacal application of trophozoites has become the most popular way to experimentally infect birds with H. meleagridis due to its high reproducibility and reliability. In most studies, infection doses of several 10,000 or 100,000 histomonal trophozoites were used for infection, and the resulting mortality in turkeys was more than 70 %. First mortality can occur as early as 6 days p.i.; peak mortality usually is 13-15 days p.i. Lower infection doses may delay mortality about 2 days. In chickens infected by the intracloacal route, mortality and clinical signs are rare, but infection rates are similar. Cecal lesions can be observed from 3 to 4 days p.i., lesions up to 3 weeks p.i.; liver lesions may be lacking completely or be present only in a small number of birds. In most studies infecting birds with Heterakis eggs containing histomonal stages, several 100 to 1,000 Heterakis eggs were used. However, lower doses might be sufficient, as infection with as few as 58 eggs per bird caused a mortality up to 90 % in turkeys. Clinical symptoms start 9 days p.i., and first mortality occurs after 12 days, while most of the infected birds die between 19 and 21 days p.i. The infectivity of Heterakis eggs containing histomonal stages for chickens is similar as for turkeys, but mortality and clinical signs are rare. Further infection was done by oral application of histomonal trophozoites either grown in culture or using lesions or feces of infected birds (26 studies). These yielded very mixed results, with infection rates between 0 and more than 80 % in turkeys and chickens. After successful oral infection of turkeys, mortality occurs at roughly the same time as after intracloacal infection. Further 18 studies employed seeder birds to infect in-contact birds. Other means of infection were exposure to contaminated soil or litter (22 studies), feeding contaminated earthworms (7 studies), intracecal inoculation (4 studies), or parenteral injection (4 studies). Main methods to assess the course of the infection were mortality, observation of clinical signs and pathological lesions, monitoring of the weight of the infected birds, and detection of the parasite by various methods. [ABSTRACT FROM AUTHOR]

Five different Artemisia annua-derived materials (i.e. dry leaves, pure artemisinin, and hexane, dichloromethane or methanol extracts of leaves) were screened for their in vitro activities against six clonal cultures of Histomonas meleagridis. Except for the methanol extract, all tested materials displayed in vitro activity against all tested protozoal clones. Neither the dry plant material, extracts nor artemisinin showed any antibacterial activity against the xenic bacteria accompanying the six H. meleagridis clones at concentration levels identical to the antihistomonal setting. The dichloromethane extract of dry leaves (Ext-DCM) (minimal lethal concentration=1.0 mg/ml) and artemisinin (half-maximal inhibitory concentration=1.295 mg/ml) had the most promising antihistomonal properties and were therefore subsequently tested in a standardized experimental infection model in both turkeys and chickens infected with clonal H. meleagridis. There were no differences between treatment groups, where all infected turkeys showed severe clinical histomonosis and demonstrated severe typhlohepatitis typical for histomonosis. Consistent with the infection model used, the infected chickens did not show any adverse clinical signs but contracted severe lesions in their caeca 7 and 10 days post infection (d.p.i.), liver lesions were absent to mild after 7 d.p.i. and progressed to severe lesions at 10 d.p.i.; thus no differences between treatment groups were observed. In conclusion, neither artemisinin nor Ext-DCM was able to prevent experimental histomonosis in turkeys and chickens at the given concentrations, which is contrary to the antihistomonal effect noticed in vitro even though the same clonal culture was used. The results of this study therefore clearly demonstrate the importance of defined in vivo experimentation in order to assess and verify in vitro results. [ABSTRACT FROM AUTHOR]

Histomonas meleagridis is a trichomonad parasite, which is capable to cause severe inflammations of ceca and livers in gallinaceous birds. It rarely can be satisfactorily explained, how the parasite was introduced into the flock. The role of wild birds as possible reservoir or as vectors has not been explored sufficiently. In the present study two experiments were done to determine if pigeons are susceptible to intracloacal infections with H. meleagridis and have the potential to act as vectors. In a first experiment nine racing pigeons (Columba livia forma domestica) were infected intracloacally with 400,000 living histomonads. Three further pigeons were kept as contact birds. Histomonal DNA was detected in cloacal swabs until one week after infection, but reisolation was not possible. In a second experiment 24 racing pigeons were either infected intracloacally with 250,000 viable histomonads or with the same culture, which had been inactivated. Histomonal DNA was detected in cloacal swabs of both groups until five days after infection, but reisolation was not possible. In both experiments neither clinical signs nor gross lesions were observed in any bird and in the ceca no histomonal DNA was detected. It was concluded that pigeons do not act as vectors for H. meleagridis after intracloacal infection. [ABSTRACT FROM AUTHOR]

Abstract: Based on clonal cultures of Histomonas meleagridis, monoxenic cultures have, to our knowledge for the first time, been established in a liquid medium. The faecal flora was exchanged for defined bacterial strains by selective destruction of the initial bacteria with a variety of antibiotics, keeping the flagellate alive. The growth of the protozoan parasite was found to depend on the bacteria, especially on their energy metabolism. Escherichia coli was found to strongly support the growth of the parasite, whereas Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa were less efficient. Confocal laser microscopy showed that H. meleagridis could take up green fluorescent protein-tagged E. coli DH5α, suggesting that bacteria serve as a food supply for the protozoa. By exchanging the bacterial flora for E. coli strain DH5α in H. meleagridis cultures that underwent continuous in vitro passages, it was possible to show that the in vivo attenuation process was independent of the bacteria. Furthermore, the gut flora in infected turkeys had no negative effect on the protozoan’s virulence. Consequently, attenuation depends not on the bacteria in the culture but on the in vitro passages. Finally, the experiments provided evidence that the infection of turkeys with H. meleagridis enabled infection of the liver with E. coli. [Copyright &y& Elsevier]

Avian pathogenic Escherichia coli strains originating from 10 free-range layer flocks were characterized by multi-locus sequence typing and plasmid profile analysis to investigate their phylogenetic relationship and diversity, respectively. In addition to colibacillosis, all flocks tested positive for antibodies against avian metapneumovirus (aMPV) during production, and six of the flocks were concurrently affected by histomonosis. Accumulated average mortality for flocks concurrently affected by colibacillosis and histomonosis made up 17.4%, while the average mortality for E. coli-infected flocks was 16.5%. A total of eight different sequence types (STs) and 47 different plasmid profiles were demonstrated among the E. coli isolates. Within each flock between one and four different STs and between three and 13 different plasmid profiles were demonstrated. A statistical significant difference in STs and plasmid profile diversity of the population of E. coli was not demonstrated between flocks affected by histomonosis compared with histomonosis-free flocks. Only minor clonal diversity was demonstrated for each flock, and in all but one flock colibacillosis started before antibodies against aMPV were detected. All isolates, except two, carried plasmids greater than 100 kb, but only a single plasmid replicon type, IncFIB, was demonstrated, suggesting plasmids representing this type might represent a common pathogenicity factor for the different STs of E. coli. Within each flock a clonal tendency was observed, indicating that only certain clones of E. coli possess a significant pathogenic potential. These clones act as primary rather than secondary pathogens, resulting in colibacillosis without predisposing factors, including histomonosis and aMPV. [ABSTRACT FROM AUTHOR]

Little is known about the induction and modulation of gut-associated immune reactions after nematode infection in the chicken. The objective of the present study was to compare the pathogenesis, induction of immune reactions and electrophysiological changes of the gut after mono-infection with Heterakis gallinarum and after dual infection with H. gallinarum and Histomonas meleagridis in layer chickens. In two experiments 3-week-old chickens were inoculated with embryonated H. gallinarum eggs, which were positive for H. meleagridis. While birds of the first experiment were left untreated, those of the second experiment were treated with dimetridazol to prevent H. meleagridis co-infection. Mild to moderate histological lesions and local immune reactions with a significant increase in CD4+, CD8α+, TCRαβ+ and TCRδγ+ cells in the lamina propria and induction of the T-helper type 2 (Th2) cytokine interleukin-13 dominated the H. gallinarum immune response at 2 weeks post infection. Co-infection with H. gallinarum and H. meleagridis induced an increase in mRNA expression of the T-helper type 1 (Th1) cytokine interferon-γ, a decrease in splenic CD4+ cells and severe destruction of the caecal mucosa in association with strong T-cell infiltration in the caecal lamina propria. There was no obvious effect on the chloride secretion of the caecal epithelium, which was investigated once the mucosa had almost recovered from the infection, in either experiment. These results suggest that the local T-cell reactions to nematode infections in chickens may be comparable with mammals and may be shifted from a Th2-dominated to a Th1-dominated response when accompanied by a protozoan infection. [ABSTRACT FROM AUTHOR]

Abstract: It was hypothesized that the establishment and fecundity of Histomonas meleagridis free Heterakis gallinarum may be affected by dietary non-starch polysaccharides (NSPs). One-day-old female layer chicks (N =670) were fed ad libitum for 11wk one of the following diets in a three-times repeated experiment: basal diet (CON), basal diet plus pea bran rich in insoluble NSP (I-NSP), basal diet plus chicory root meal as a source of inulin rich soluble NSP (S-NSP). At the end of wk three, each feeding group was subdivided into an uninfected and an infected group of birds each being inoculated with a placebo or with 200 H. meleagridis free eggs of H. gallinarum. The birds were slaughtered 8wk post infection and their worm burdens, the nematode egg excretion, caeca sizes and weights as well as intracaecal pH and volatile fatty acid (VFA) concentrations were determined. The NSP supplemented diets and also infection led to reduced body weights (BWs) of birds and impaired the feed conversion rate (P <0.001). The NSP supplemented diets increased average length of caecum (P <0.001) with S-NSP exerting a stronger effect than I-NSP (P <0.05). Full caeca weight was increased by S-NSP (P <0.001). Feeding S-NSP lowered intracaecal pH and molar proportion of acetate and increased that of butyrate compared to CON and I-NSP (P <0.001). Caecal pool of VFA was increased with S-NSP (P <0.001). The NSP-diets elevated incidence of infection (P <0.01), average number of larvae (P <0.009) and total worm burden (P <0.001) compared to CON. The daily amount of faeces increased in NSP-fed birds (P <0.001). Number of eggs per gram of faeces (EPG), number of eggs excreted per worm population of a bird within 24h (EPD) and female worm fecundity (EPD/female worm) were elevated after feeding S-NSP (P ≤0.002), whereas I-NSP led to lower EPG/female worm (P <0.05). The EPD increased in the sequence of CON

In the present work, chickens and turkeys were infected with virulent or attenuated Histomonas rneleagridis to investigate and compare the effect of both isolates on birds. Thereby, histomonads of a clonal culture were propagated in vitro either for a short period of time (21 passages) to preserve virulence or for 295 passages to achieve attenuation. On the first day of life birds of each species were infected with either virulent or attenuated parasites. Throughout the experiment, all birds were examined daily for clinical signs attributable to the infection. Furthermore, the excretion of viable parasites was determined after in vitro reisolation from cloacal swabs. For the investigation of pathological changes of organs a defined number of infected birds were killed on d 4, 7, 10, 14, and 21 postinfection (PT) and necropsy was performed. By this routine, changes in livers and ceca were classified by a scoring system to evaluate the severity of lesions. Samples of cedum, liver, and lung were generated and screened for the presence of parasites by PCR and immunohistochemistry. Turkeys infected with virulent histomonads showed first clinical manifestation of histomonosis on d 10 PT, whereas the remaining birds did not express clinical signs. Positive reisolations of virulent and attenuated histomonads were obtained intermittently from individual chickens and turkeys from d 2 PT until the end of the experiment. Both species of birds displayed lesions in the ceca and the liver following infection with virulent parasites, whereas no changes occurred in birds inoculated with attenuated histomonads. The PCR revealed the dissemination of virulent histomonads in ceca, livers, and lungs of some chickens and turkeys in contrast to attenuated parasites, which were exclusively found in cecal samples. The attenuated isolate of H. meleagridis did not induce clinical signs or pathological changes and offers high safety after infection of chickens and turkeys. Therefore, the in vitro attenuation and the use of avirulent histomonads represent a viable tool for vaccination against histomonosis. [ABSTRACT FROM AUTHOR]

Abstract: Nutrition of the host animal may not only influence interactions between the host and its parasites, but also relations between different parasites species residing on the same host. We investigated effects of insoluble and soluble non-starch polysaccharides (NSP) on establishment and development of Heterakis gallinarum in chicken being treated or left untreated against Histomonas meleagridis. Six groups of one-day-old birds were allocated to three diets, two on each diet. The birds were fed ad libitum either a basal diet (CON), or CON+insoluble NSP (I-NSP) or CON+soluble NSP (S-NSP) until an age of 11wk. At an age of 19d, one of each diet groups was prophylactically treated for 9d with dimetridazole (0.05%, w/v) via drinking water against histomonas. The remaining three groups were left un-treated. Two days after starting dimetridazole treatment (at 3wk), each of the 6 groups was divided into two sub-groups. One dimetridazole treated and one dimetridazole un-treated groups of birds on each diet (6 groups) were infected with 200 embryonated eggs of H. gallinarum that were previously harvested from histomonas-carrying H. gallinarum infected chickens. The remaining 6 groups of uninfected birds, either treated or left un-treated against H. meleagridis, served as controls. Worm burdens of infected birds were determined 8wk p.i. Treatment against H. meleagridis significantly increased incidence of H. gallinarum infection and average worm length in all infected groups independent of the diet consumed (p <0.001). An interaction between effects of diet and dimetridazole treatment on worm burden (p <0.001) indicated that the S-NSP diet resulted in lowest worm burden in dimetridazole un-treated birds, whereas it caused the highest worm burden in the treated birds (p <0.05). Furthermore, the treatment resulted in higher worm burdens when compared to un-treated birds on the corresponding diets (p <0.05). Infection with H. gallinarum impaired body weight (BW) of the chicks (p <0.05) and H. meleagridis aggravated this effect (p <0.05). Dimetridazole treated and un-treated uninfected birds developed similar BW (p >0.05). Both NSP supplemented diets resulted in lower (p <0.05) BW when compared with the CON diet, S-NSP being inferior to I-NSP (p <0.05). It is concluded that H. meleagridis harms the definitive host as well as H. gallinarum. Both insoluble and soluble NSP supplemented diets favor H. gallinarum infection while S-NSP additionally intensifies histomonas infection, which then impairs establishment and development of H. gallinarum. [Copyright &y& Elsevier]

One-day-old turkey poults were vaccinated against histomonosis (syn. histomoniasis) via the oral route by application of in vitro attenuated Histomonas meleagridis. Subsequently, two different groups composed of 14 birds each were challenged cloacally with highly virulent histomonads after 2 or 4 weeks. Two additional groups of non-vaccinated birds were infected with the challenge inoculum at the same time points. In addition, a group of 19 birds, of which 14 were vaccinated but not challenged, were kept for clinical and serological examinations. Non-vaccinated and non-challenged birds (n=10) represented the negative control group. All non-vaccinated but infected birds and 10 out of 14 vaccinated turkeys challenged 2 weeks post vaccination (w.p.v.) contracted severe histomonosis. Turkeys challenged 4 w.p.v. and all remaining birds used in this experiment did not show any pathognomonic clinical signs. In addition, no adverse effect regarding the weight gain could be observed in birds that were vaccinated but not challenged. The excretion of attenuated and virulent live histomonads was observed very infrequently by re-isolation, but transmission to in-contact birds was very efficient. Presence of antibodies was first noticed 3 w.p.v. and antibody levels remained above the cut-off value until termination of the experiment at 16 w.p.v. The present experiment demonstrates for the first time the potential efficacy of in vitro attenuated histomonads used as an orally applied vaccine to 1-day-old turkeys for protection against fatal histomonosis without affecting performance. [ABSTRACT FROM AUTHOR]

Abstract: Histomonosis is an infectious disease of mainly galliform birds that can cause high mortality, especially in commercial turkey flocks. Several diagnostic tools were available to detect its causative agent, the flagellated protozoan Histomonas meleagridis. However, serological tools were largely lacking. Recently, an indirect ELISA has been described, but this test was not validated for specificity and might suffer from cross-reactivity with related protozoa. Therefore, a specific blocking-ELISA for the detection of antibodies against H. meleagridis in chicken and turkey sera was developed. For this purpose, monoclonal antibodies were raised against a detergent extracted protein of H. meleagridis. These MAbs bound to morphologically identified histomonads in liver tissue of an infected turkey. The MAbs were conjugated with horseradish peroxidase, after which the most reactive conjugate was used to set-up the blocking-ELISA. Experimentally infected turkeys (n =9) and chickens (n =10) seroconverted in the blocking-ELISA within 2–4 weeks following inoculation with a H. meleagridis field strain. The MAb did not bind to Tetratrichomonas gallinarum antigen and experimentally inoculated T. gallinarum seropositive layer chickens (n =18) showed the same inhibition percentages in the blocking-ELISA as negative control birds did. Therefore, it was concluded that H. meleagridis blocking-ELISA does not cross-react with T. gallinarum, which is a closely related protozoan frequently occurring in poultry. The repeatability and reproducibility of the H. meleagridis blocking-ELISA were high. The new blocking-ELISA is a promising tool for experimental studies; however, further validation of this test with field samples is necessary before it can be used for diagnostic purposes. [Copyright &y& Elsevier]

The study deals with the pleomorphic zooflagellate Histomonas meleagridis, which was cultivated under different stress conditions to induce a possible encystation. In the present paper, the morphological changes were analyzed by light and electron microscopy. The determination of the proliferation under different adverse conditions led to conclusions on the tenacity of the flagellate. H. meleagridis parasitizes in the intestinal tract of galliform birds and may cause enormous losses in poultry farming. For the development of new therapy approaches, clarification of the transmission pathways will be helpful. Different clonal cultures of H. meleagridis established by micromanipulation and exposed to media lacking different ingredients, inappropriate temperatures, and/or distinct reagents were investigated. Lowering of temperature was proven to have adverse effects on the survival of H. meleagridis. The flagellate could not survive in a frozen medium, and survival in a temperature of 4°C lasted no longer than 23 h. An addition of sodium chloride induced an increased proliferation; pH values between 2 and 8 set limits for the survival of the parasite in different ways. H. meleagridis was able to survive under high acidic conditions for only 1 h. The major amount of cells, which could be discovered in the controls, measured 8-12 µm appeared amoebic (stage 1) and were filled with enclosures of rice starch. A rounding of most cells was noted 4 h at 4°C after incubation in minimal essential medium in the absence of rice starch and fetal calf serum. A higher osmolarity of the medium, which was initiated by the addition of sodium chloride or magnesium chloride, did not induce an encystation process. After addition of hypochlorite base and cultivating at pH values between 7 and 8, spherical stages without a flagellum were formed (stage 2) measuring about 8-12 µm in diameter. Their interior consisted of a central and a peripheral region when studied by transmission electron microscopy. This aspect was due to the location of the glycogen granules. The central zone was described as totally filled with the carbohydrates, which made totally invisible the other organelles. The solidity of the amorphous layer below the cell membrane seemed to hinder the invasion of the glycogen granules. The amorphous layer below the cell membrane made it apparently possible that the cell might survive under adverse conditions--at least for a short time. This special structure might enable H. meleagridis to proceed a fast transmission and to infect many birds in a rather short time, which was shown in the past by several studies. Double-membraned cells, which were guessed to be cyst-like structures of the parasite, were also detected (stage 3). The size of these cells, however, was much smaller than that of the amoebic stages or the above-described spherical forms of H. meleagridis. Furthermore, the small cells were characterized by other granula structures. These findings might be interpreted that the small stages are possibly long-term (true) cysts and that the spherical stages with the amorphous layer beneath the cell membrane might be short-term cysts. Both, however, should be able to survive situations outside of a body and thus might be transmitted from feces to another animal. [ABSTRACT FROM AUTHOR]

Paromomycin is an aminoglycoside antibiotic with activity against protozoa. Currently, paromomycin is registered for food producing animal species. In a pilot study we evaluated the efficacy of different doses of paromomycin in the feed against Histomonas meleagridis in experimentally challenged turkey poults. Groups consisting of 30 birds each were given feed with 100, 200 and 400 ppm paromomycin, respectively, starting on day 1 through to day 42. One group of 30 birds was left untreated. On day 21 all birds were infected intracloacally with H. meleagridis. Additionally, 10 birds were kept as a non-infected and non-treated control group. Before the challenge there was no significant difference between untreated and treated groups with regards to feed consumption and feed conversion rate. After the challenge, mortality was 80% in the infected non-treated birds. In the treated groups the mortality rate was 73.3%, 43.3% and 20%, respectively. No histomonal DNA was found in caeca and livers of the surviving birds. In addition, higher doses of paromomycin (200 and 400 ppm) led to reduced counts of Clostridium perfringens in the droppings. In conclusion, a prophylactic application of paromomycin as a feed additive was effective against the challenge with H. meleagridis under experimental conditions. [ABSTRACT FROM AUTHOR]

Abstract: Histomonas meleagridis is a protozoan parasite known to cause histomonosis (syn. typhlohepatitis) in poultry. Due to the economic losses which the disease entails, there is an urgency to understanding its pathogenesis. In the present investigation, three partial cDNA sequences encoding heterogenic α-actinins previously identified for H. meleagridis were completed and characterized. These three H. meleagridis α-actinin isoforms were named α-actinin1, α-actinin2, and α-actinin3. Through mRNA analysis, it was possible to estimate their expected complete coding lengths to ca. 1.9kb, 3.5kb, and 3kb, respectively. Protein structure predictions of completed cDNA sequences revealed that the three H. meleagridis α-actinins contain two N-terminal calponin homology (CH) domains that bind actin, a central rod domain of varying lengths, and only one C-terminal EF-Hand. The well conserved N- and C-termini of the H. meleagridis α-actinins show amino acid identities between 54% and 82.5% to those of the related trichomonad Trichomonas vaginalis. Additionally, all three α-actinins were shown to immune-react with turkey antiserum, while only the immune reaction of α-actinin2 and α-actinin3 was visible using chicken serum. This demonstrates the antigenic potential of α-actinins in host animals. The identification of α-actinins with varying rod domain lengths within one organism is a relatively new concept, described as yet only for Entamoeba histolytica. [Copyright &y& Elsevier]