Biochemical characterization of a flavodiiron protein from bird parasite Histomonas meleagridis: superoxide as a reaction intermediate.

Ascarid parasites, such as Ascaridia galli and Heterakis gallinarum, are nearly ubiquitous in poultry and can cause serious production losses. H. gallinarum is of particular concern because of its role as a vector for the protozoan Histomonas meleagridis, the cause of blackhead disease. Currently, only the benzimidazole anthelmintic, fenbendazole (FBZ), is approved for use in poultry, and recently, FBZ resistance has been discovered and validated in populations of the turkey ascarid Ascaridia dissimilis and in ascarid of gallinaceous birds H. gallinarum. Here, we further explore the prevalence of resistance in poultry ascarids by testing FBZ efficacy against thirteen isolates of A. galli and eight isolates of H. gallinarum. Isolates were used to infect day-old naive chickens. Four weeks after infection, animals to be treated received the label-recommended dosage of FBZ (SafeGuard Aquasol) for five days, per the manufacturer’s directions. One week after the fifth day of treatment, animals were euthanized and parasite burdens were counted to determine treatment efficacy between the untreated and treated groups. Resistance was identified and validated in a single isolate of A. galli, marking the first confirmed case in the species. All isolates of H. gallinarum were found to be resistant. The emergence of resistance in A. galli and the high prevalence of resistance in H. gallinarum highlight the growing concern of resistance in parasites of poultry. Without approved alternative treatments, the detrimental effects of infections cannot be mitigated in resistant populations, significantly impacting profit margins. Diagnostics that enable broader surveys are necessary to understand the full scope of the problem. However, we show that resistance is present across production species and should act as an impetus for the discovery of new treatments and the adoption of new management strategies.

ABSTRACTBackground and Aim:Heterakis gallinarum is a parasitic nematode that commonly infects the ceca of domestic and wild gallinaceous birds, acting as a vector for Histomonas meleagridis, the causative agent of blackhead disease. Despite its significance in poultry health, molecular data from Iraq, particularly Sulaymaniyah province, remain limited. This study aimed to characterize the morphological and genetic features of H. gallinarum isolated from free-range chickens in Sulaymaniyah using mitochondrial cytochrome c oxidase subunit 1 (COX1) gene sequencing.Materials and Methods:A total of 140 free-range adult chickens were purchased from local markets in Sulaimani city between January 2023 and October 2024. Birds were euthanized, and cecal contents were examined for Heterakis spp. Adult worms were morphologically identified under light microscopy. DNA was extracted from representative isolates, and the COX1 gene was amplified using polymerase chain reaction (PCR) and sequenced. Phylogenetic analysis was conducted using the neighbor-joining method based on the Kimura-3 parameter model.Results:Out of 140 chickens examined, 51 (36.43%) were infected with H. gallinarum. Morphological analysis revealed typical features, including unequal spicules in males and barrel-shaped eggs. PCR confirmed a 1325-bp COX1 gene fragment. Basic Local Alignment Search Tool analysis showed 98%–99% similarity with known H. gallinarum sequences. Phylogenetic reconstruction clustered the Iraqi isolate (GenBank: PQ395216.1) with isolates from China, South Africa, Tunisia, and Bangladesh, indicating low genetic divergence across geographic regions.Conclusion:This is the first molecular documentation of H. gallinarum in free-range chickens from Sulaymaniyah, Iraq. The findings highlight the widespread genetic conservation of this parasite and underscore the need for further epidemiological studies to determine risk factors and potential impacts on poultry productivity.

Histomonas meleagridis, a protozoan parasite responsible for histomonosis (syn. Blackhead disease, histomoniasis), presents an increasing challenge for poultry health, particularly with the ban of licensed prophylactic and treatment options. Recent studies have explored H. meleagridis proteome, exoproteome, and surfaceome, linking molecular data to virulence and in vitro attenuation. Nevertheless, proteins involved in interactions with hosts remain largely unknown. In this study, we conducted immunoproteome analyses to identify key antigens involved in the humoral immune response of the parasite’s main hosts, turkeys and chickens. Immunogenic proteins were isolated via immunoprecipitation using sera from chickens and turkeys that were vaccinated with a single attenuated strain and challenged with virulent strains of the protozoan, respectively. Mass spectrometry identified 155 putative H. meleagridis immunogenic proteins, of which 43 were recognized by sera from both hosts. In silico antigenicity screening (VaxElan) identified 33 pan-reactive antigens, with VaxiDL further highlighting 10 as potential vaccine candidates. Comparative analysis revealed host-specific immune responses, with 16 differential immunogenic proteins in chickens (6 specific to virulent and 10 to attenuated preparations) and 19 unique proteins in turkeys, all associated with virulent strains. These results enhance our understanding of H. meleagridis immunogenic protein dynamics and host-pathogen specificities, supporting the development of improved diagnostic tools and potential protective measures against the infection.

Histomonas meleagridis, the causative agent of histomonosis (i.e., blackhead disease), threatens the poultry industry with serious economic losses due to its high mortality and morbidity in turkey and chicken flocks. In vitro studies are complicated by the inability to culture the parasite axenically. Histomonas meleagridis has been propagated in Dwyer's media, which contains a starch source and serum, for over 50 years. The presence of insoluble starch component in Dwyer's media represents an obstacle for the commercialization of such media, and the role of starch in media is poorly understood. To investigate the intracellular metabolomic differences in H. meleagridis and undefined bacteria grown in Dwyer's media with rice starch (SD) and without rice starch (NR), we conducted a global metabolomics analysis using ultra-high-performance liquid chromatography-high-resolution mass spectrometry. SD significantly supported the growth of H. meleagridis compared to NR. There was no significant difference in bacterial growth between SD and NR media at various timepoints. From the intracellular metabolic analysis of samples collected from the SD and NR media, a total of 170 known metabolites were identified. H. meleagridis appears to be the major contributor to global metabolic differences. We found that riboflavin had the highest variable importance in the projection score, and metabolites involved in riboflavin biosynthesis significantly contributed to the differences between SD and NR in the media immediately after the inoculation of H. meleagridis and undefined bacteria, warranting further investigations into the role of riboflavin biosynthesis in H. meleagridis growth.

Phenylpropanoid pathway mediated the defense response of ‘Korla’ fragrant pear against Alternaria alternata infection

Dimetridazole (DMZ) is a broad-spectrum anti-anaerobic and antiprotozoal drug extensively used for the control of blackhead disease in poultry (especially turkeys). The presence of DMZ and its metabolites in animal food poses potential risks to human health. In this study, we developed a high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) method for the precise detection of DMZ and its metabolite 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI). Our results demonstrate a strong linear relationship (r2 > 0.99) between the concentrations of DMZ and HMMNI in tissues and egg within the range of 1~100 ng/g. The limits of detection (LOD) were determined to be 0.5 ng/g, with corresponding limits of quantification (LOQ) at 1.0 ng/g. Furthermore, average recoveries in tissues and egg fell within the range of 84.90% to 103.01%, with coefficients of variation below 15% for both intra-day and inter-day analyses. To investigate the residue elimination pattern of DMZ and HMMNI, diets containing 500 mg/kg DMZ were fed to healthy SanHuang chicken and Hy-line Gray laying hens for 10 consecutive days. The results indicated that the concentration of HMMNI consistently exceeded that of DMZ during the same period, in both broiler tissues and egg. Sebum showed the slowest elimination of DMZ and HMMNI, becoming undetectable after 168 h of withdrawal. In egg, residues of both substances peaked on the first day after drug withdrawal, followed by slow elimination with half-lives of 0.45 days for DMZ and 0.66 days for HMMNI. Based on these findings, WT1.4 software was used to calculate a withdrawal time of 11 days for broilers and an egg abandonment period of 14 days after withdrawal for laying hens, providing a scientific basis for the safe and rational use of DMZ in poultry farming.

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.

Development of defense system and secondary metabolites of Korla fragrant pear during Alternaria alternata infection

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.

Alternaria alternata stimulates blackhead disease development of ‘Korla’ fragrant pear (Pyrus bretschneideri Rehd) by regulating energy status and respiratory metabolism

Parasites are highly prevalent in poultry; thus, the management of parasites is a key component in the profitable production of poultry. The most common nematode parasite of poultry, Heterakis gallinarum, typically causes no direct pathology but is the vector of Histomonas meleagridis, a highly pathogenic protozoan parasite that causes blackhead disease. There are no approved treatments for H. meleagridis, making control reliant on controlling the helminth vector. In the United States, the benzimidazole anthelmintic fenbendazole (FBZ) is the only approved treatment for H. gallinarum. We were contacted by an industry veterinarian regarding clinical problems with histomoniasis despite frequent anthelmintic treatments. Given that we had recently diagnosed FBZ resistance in the closely related parasite Ascaridia dissimilis, we were interested to determine if H. gallinarum had also evolved resistance. An initial on-farm pilot study using 20 birds suggested that FBZ was poorly effective, therefore a larger controlled study was initiated. Heterakis gallinarum eggs were isolated from litter at the farm and used to infect 118 chicks. Treatment groups included a non-treated control, a label-, and a 2×-label dose of FBZ, with 36 birds per group divided into two replicates of 18 birds. Three weeks post-hatch, birds were infected with 150 embryonated eggs. Two weeks post-infection treated birds were administered either a label- or 2× label-dose of FBZ in water for five days (SafeGuard® Aquasol, 1mg/kg BW). To increase the likelihood that all birds consumed the full intended dose, the dosage was calculated using 1.25 times the average body weight. One-week post-treatment, birds were euthanized, and parasites enumerated. There were no significant differences in worm numbers recovered from any of the three groups (p-value=0.3426), indicating that both dosages of FBZ failed to provide the expected levels of efficacy. These data provide strong evidence that H. gallinarum has developed resistance to FBZ on this farm. Consequently, on this farm, or any farm with FBZ-resistant H. gallinarum, H. meleagridis will continue to cycle in an unrestricted manner despite administration of anthelmintic treatments. Given recent evidence of increasing problems with histomoniasis, and the fact that resistance was documented on the first farm we investigated, further investigations are needed to determine the prevalence of resistance in H. gallinarum on poultry farms. These data, when viewed together with our recent findings of FBZ resistance in A. dissimilis on multiple farms, suggest that drug resistance in ascarid nematodes may be an emerging problem in the US poultry industry.

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.

Radopholus Similis (R. Similis) or burrowing nematode, is one of the most damaging and widespread nematodes attacking bananas, causing toppling or blackhead disease. A mathematical model for the population dynamics of R. Similis is considered, with the aim of investigating the impact of climatic factors on the growth of R. Similis. In this paper, based on the life cycle of R. Similis, we first propose a mathematical model to study and control the population dynamics of this banana pest. We show also how control terms based on biological and chemical controls can be integrated to reduce the population of R. Similis within banana-plantain roots. Sensitivity analysis was performed to show the most important parameters of the model. We present the theoretical analysis of the model. More precisely, we derive a threshold parameter [Formula: see text], called the basic offspring number and show that the trivial equilibrium is globally asymptotically stable whenever [Formula: see text], while when [Formula: see text], the non trivial equilibrium is globally asymptotically stable. After, we extend the proposed model by taking account climatic factors that influence the growth of this pest. Biological and chemical controls are now introduced through impulsive equations. Threshold and equilibria are obtained and global stabilities have been studied. The theoretical results are supported by numerical simulations. Numerical results of model with biological and chemical controls reveal that biological methods are more effective than chemical methods. We also found that the month February is the best time to apply these controls.

The aim of the present review is to demonstrate the possibility of limiting antibiotic therapy in intensive poultry production through the use of various immunomodulators. In an era of increasing resistance of microorganisms to antimicrobial agents, it is particularly important to improve the efficiency of the immune system, which is indispensable for birds to survive in an environment where this system is constantly exposed to a number of stimuli that directly or indirectly affect its functioning. This is all the more important as the health of birds kept under intensive systems depends mainly on the functional status of their immune system, which determines the level of postvaccination immunity and the effectiveness of treatment. That is because its proper function is needed for the elimination of pathogenic microorganisms, stimulation of repair processes in damaged tissues and protection against reinfection. This paper focuses on the possibilities of modulating immune mechanisms and correcting the reactivity of the immunological system in poultry by using natural or synthetic immunomodulators. The results presented here suggest that methisoprinol and β-glucans are effective in modulating immune processes in turkeys and may find practical application in mass rearing of these birds: β-glucans mainly in prophylactic programmes, whereas methisoprinol both in prevention and treatment of diseases of viral etiology occurring with immunosuppression. It has also been shown that phytoncides contained in well-composed complementary feeds show immunomodulatory effects in broiler chickens and turkeys and are effective in the prevention and treatment of black head disease in turkeys. Considering the key role of the immune system in fighting pathogens, it should be concluded that synthetic and natural immunomodulators, including phytoncides used in the prevention and treatment of poultry diseases, can effectively reduce and, in some cases, replace or support antibiotic therapy.

Postharvest UV-C irradiation inhibits blackhead disease by inducing disease resistance and reducing mycotoxin production in ‘Korla’ fragrant pear (Pyrus sinkiangensis)

Research Note: Evaluation of quinine as a chemoprophylactic candidate against histomoniasis in turkeys

Blackhead caused by Alternaria alternata is a fatal necrotrophic fungal that affects Korla fragrant pear. To date, little is known at the molecular level about the defense response of pear to blackhead disease and the pathogenic mechanism of A. alternata infection. To investigate the specific host-pathogen interaction between A. alternata and pear, we examined the accumulation of host-responsive mRNAs using RNA-seq technology. A total of 25,877 differentially expressed genes (DEGs) were identified. Further analysis revealed that the DEGs mainly participate in plant cell wall integrity, plant hormone pathways, plant-pathogen interactions and the defense response (transcription factors, defense-related proteins). Most of the DEGs involved in the plant hormone, PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) pathways, as well as defense-related proteins, were significantly up-regulated. In addition, DEGs encoding enzymes involved in cutin and wax synthesis and most transcription factors are significantly down-regulated. Based on these results, we speculate that these pathways play important roles in the response of pear to A. alternata. This study has presented new insights into the molecular mechanisms that regulate the response of pear fruits to A. alternata infection. © 2021 Friends Science Publishers

Nitarsone is an organoarsenic antiprotozoal drug widely used to treat blackhead disease in turkeys and chickens. However, since its biological conversion into inorganic arsenic, a carcinogen was known, its residue in foods should be regulated. Thus, here, a novel method to determine residual nitarsone in various food commodities (pork, milk, egg, halibut, eel, and shrimp) using QuEChERS and LC-MRM was developed. The developed method was successfully validated through specificity, linearity (coefficient of determination, at least 0.991), recovery (R, 63.6 - 85.6%), precision (the relative standard deviation of R, 0.5 - 10.6%), and sensitivity (the lower limit of quantitation, 5 ppb) by following the Ministry of food and drug safety (MFDS) guidelines. The present method is the first mean to quantitate nitarsone using LC-MRM, and it was designed to be conveniently merged into a new method to quantitate multiple veterinary drugs for the positive list system (PLS). Therefore, the present method could contribute to fortify the food safety system in South Korea.

IntroductionTurkey histomonosis poses a serious threat to poultry production due to the ban on the use of effective drugs. The aim of the study was to evaluate the influence of a phytoncidal feed supplement on the course of histomonosis. The preparation was also analysed for immunomodulatory properties.Material and MethodsClinical observations and production monitoring were conducted in a flock of turkeys with histomonosis from their 11th to 56th weeks of life which were treated with the adiCoxSOLPF soluble supplement in a dose of 2.5 mL/L water. Later the preparation was used in a preventive dose (1 mL/L). The influence on the immune system was evaluated in broiler turkeys having been given adiCoxSOLPF for 3 days in doses of 1 or 3 mL/L. The T and B lymphocyte percentages in turkey blood and spleen tissue were analysed with flow cytometry. ELISA was implemented to evaluate antibody titres after Ornithobacterium rhinotracheale vaccination, and biochemical analyses were performed to evaluate the supplement’s safety.ResultsAdiCoxSOLPF was found effective in therapy and prevention of histomonosis. Additionally, adiCoxSOLPF stimulated both humoral and cell-mediated immune mechanisms, without impairing the functions of internal organs. The treated turkeys also yielded better production results (eggs/hen, fertility, and hatchability).ConclusionAdiCoxSOLPF possesses immunomodulatory properties and it can be used successfully in the prevention and therapy of histomonosis in turkeys.