Blackhead disease (Histomonas meleagridis) aggravated in broiler chickens by concurrent infection with cecal coccidiosis (Eimeria tenella).

The effect of concurrent cecal coccidiosis infections on severity of Histomonas meleagridis (blackhead disease) in chickens was investigated in a series of experiments. Cecal lesions from H. meleagridis were severe in all inoculated control groups and did not appear to be affected by the introduction of Eimeria tenella infection. However, the severity of liver lesions and number of birds positive for liver lesions of H. meleagridis increased significantly with the presence of E. tenella. The increase was similar when 10(3) or 10(4) oocysts of E. tenella were given and was the same when oocysts were given at the same time as H. meleagridis or 4 days prior. The liver lesions increased directly as doses of H. meleagridis increased from 7.5 x 10(3) cells to 30, 100, or 300 x 10(3) when E. tenella was given along with H. melelagridis but not when H. meleagridis was given alone. Administration of a live coccidiosis vaccine containing very low levels of E. tenella also gave a significant boost to liver lesions but at a much lower level than that observed with larger doses of E. tenella. The positive relationship between infections of cecal coccidiosis and H. meleagridis in chickens suggests that such dual exposure may contribute to increased clinical outbreaks of blackhead disease in chickens under field conditions.     

Improved culture of Histomonas meleagridis in a modification of Dwyer medium

Dwyer medium is the most frequently employed culture medium for Histomonas meleagridis. Both for subculturing and for resuscitation of H. meleagridis from storage in liquid nitrogen, modified Dwyer medium with an increased rice powder concentration (0.8%) and no chicken embryo extract proved superior to Dwyer standard medium, with threefold (10(6.3) vs. 10(5.8) histomonads/ml) to 10-fold (10(6.7) vs. 10(5.8) histomonads/ml) higher concentrations of parasites after resuscitation or subculturing, respectively.     

The efficacy of some drugs with known antiprotozoal activity against Histomonas meleagridis in chickens

Nine drugs with known or suspected antiprotozoal activity were tested in vitro, and in vivo for activity against Histomonas meleagridis. The nitroimidazoles dimetridazole, metronidazole, ornidazole, and tinidazole suppressed growth of H. meleagridis in vitro at 10 microg/ml or higher. Paromomycin sulfate, and carbadox were weakly effective at high levels. Quinolinol, mebendazole, diloxanide furoate, and albendazole had no demonstrable efficacy in vitro. Drugs showing some activity in vitro were tested in young chickens inoculated intracloacally with 2 x 10(5) H. meleagridis/bird. Dimetridazole, metronidazole, ornidazole, and tinidazole were highly effective at 200 ppm in feed. Paromomycin sulfate, and carbadox were ineffective in vivo, with no improvement in liver or cecal lesion scores compared to that of infected controls. Thus, the only new entities with efficacy against blackhead disease in vivo were nitroimidazoles, related to the positive control dimetridazole.     

Comparison of the specificity and sensitivity of PCR, nested PCR, and real-time PCR for the diagnosis of histomoniasis

Blackhead, also known as enterohepatitis, is caused by a protozoan parasite called Histomonas meleagridis. Clinical symptoms are nonspecific. Until now, diagnosis has been mainly based on postmortem lesions and microscopical and histopathological examination. In many cases, especially in layer flocks, these conventional methods are not sufficient, as the lesions are sometimes not clear. The technique for isolation of histomonads in vitro offers many advantages, but the confirmation of histomonads growing in culture may require a time-consuming procedure of rectal inoculation of culture material into chickens or turkeys. The aim of our investigation was to establish a conventional polymerase chain reaction (PCR), a nested PCR, and a real-time PCR, and to examine their specificity as well as sensitivity in the diagnosis of histomoniasis. The obtained results have shown that the conventional PCR is more sensitive than the real-time PCR. Furthermore, the sensitivity of the PCR can be increased by adding the nested PCR. However, the real-time PCR is more specific.     

Progression of histomonosis in commercial chickens following experimental infection with an in vitro propagated clonal culture of Histomonas meleagridis

Four commercial strains of chickens, namely, ISA brown leghorn (ISA), TETRA-SL brown (TETRA-SL), Lohmann brown (LB), and Lohmann LSL (LSL), were infected with a well-defined clonal culture of Histomonas meleagridis (H. meleagridis/Turkey/Austria/2922-C6/04) to investigate their susceptibility to histomonosis. Each group included 16 chickens, which were housed under the same conditions in separate pens. All chickens were infected with 10(4) histomonads orally and intracloacally at 14 days of age. No mortality or clinical signs were observed during the experiment in all birds. Three birds of each chicken strain were euthanatized on days 4, 7, 10, 14, and 21 postinfection. Incidence of histomonosis on the basis of cecal lesions was found to be 64.00% in TETRA-SL, 62.50% in LB, 53.12% in LSL, and 43.75% in ISA chickens. Fewer lesions were noticed in livers than in ceca, with an incidence of 15.62% in TETRA-SL, 9.37% in LB, and 3.12% in ISA chickens. No liver lesions were found in the LSL chickens. Statistical analysis revealed that there was no significant difference (P > 0.05) in susceptibility to experimental H. meleagridis infection based on cecal and liver involvement. Polymerase chain reaction (PCR) and immunohistochemistry were found to be reliable tools to confirm the presence of histomonads and changes in the ceca. However, some negative PCR results were recorded from the livers despite the presence of macroscopic lesions. Additionally, DNA of H. meleagridis was detected by PCR in a few of the lungs, but immunohistochemistry was negative. Nucleic acid of the protozoan parasite was not detected in samples from kidney, brain, spleen, or bursa of Fabricius. Altogether, the high susceptibility of commercial chicken lines to histomonosis could be demonstrated and characterized by severe lesions in the ceca and insignificant involvement of the liver, approaching a maximum on days 7-14 postinfection.     

Reoccurrence of histomonosis in turkey breeder farm

Histomonosis is a severe disease caused by the protozoan parasite Histomonas (H.) meleagridis, which can lead to high losses in turkeys. The present report describes the reoccurrence of histomonosis in a turkey breeder farm. The first outbreak occurred in 2005 in 17 weeks old hens, the second in 2009 in 8 weeks old hens. The disease remained restricted in one house and one compartment, respectively. Mortality rose to 26 and 65% respectively within few days in spite of therapy with various compounds. Both flocks had to be euthanized. In both cases H. meleagridis belonging to genotype A was detected. The source of infection remained unclear in both cases.     

基于18S rRNA的鸡组织滴虫病PCR诊断方法的建立

河南某鸡场约4周龄鸡疑似发生鸡组织滴虫病.根据鸡组织滴虫18 S rRNA序列设计引物,提取肝脏、盲肠内客物寄生虫DNA,采用PCR方法检测.结果表明,PCR扩增到与预期大小一致的产物,经测序比对,证实为鸡组织滴虫感染.所建立的PCR方法具有灵敏、特异、快速等优点,不仅能用于鸡组织滴虫病临床诊断,还能用于开展流行病学研...     

Recurring histomonosis on an organic farm

This report describes outbreaks of histomonosis, a severe disease caused by the protozoan parasite Histomonas meleagridis, which occurred over a period of 3 yr on an organic farm in southern Germany. Among other species, the farm houses layers, broilers, and turkeys. In August 2005 one group of turkeys was naturally infected with H. meleagridis. The strain causing infection was typed by C-profiling as genotype B. A second outbreak occurred 3 yr later. Again, a group of turkeys was naturally infected. The strain causing the infection belonged to genotype A. Two months later one group of broilers became infected with H. meleagridis type B and a group of turkeys with H. meleagridis type A. Four weeks later two further groups of broilers showed symptoms. DNA of H. meleagridis was detected but genotyping was not possible. In conclusion, genotyping of the histomonal strains causing the disease showed that at least two different histomonal strains caused the outbreaks and that the strains circulated on the farm at the same time.     

Failure of Heterakis bonasae to transmit Histomonas meleagridis

Studies were conducted to determine whether Heterakis bonasae eggs from bobwhite quail infected with Histomonas meleagridis would transmit histomoniasis to turkeys. Fifteen helminth-free bobwhites were inoculated per os with embryonated H. bonasae eggs. Each bobwhite was then infected with H. meleagridis via rectal inoculation. Bobwhites that developed cecal lesions rarely retained mature H. bonasae. H. bonasae eggs recovered from bobwhites exposed to or known to have concurrent H. meleagridis infections were inoculated per os to eleven helminth-free turkeys. None of the turkeys developed H. meleagridis infections.     

10株火鸡组织滴虫18S rRNA基因的克隆及进化分析

应用聚合酶链反应(PCR)鉴定湖南娄底(简称HMLD1-3)、湘潭(HMXT1-2)、永州(HMYZ1-3)和宁乡(HMNX1-2)共10株火鸡组织滴虫,并用18S rRNA部分序列重构火鸡组织滴虫与其它相近原虫的种群遗传关系.作者通过PCR扩增火鸡组织滴虫虫株的18S rRNA部分序列,将PCR扩增出的片段纯化后克隆...     

Evaluation of dietary Natustat for control of Histomonas meleagridis in male turkeys on infected litter

Histomoniasis (histomonosis, infectious enterohepatitis, or blackhead) is a disease of turkeys on litter or range caused by the protozoan Histomonas meleagridis, a parasite of worms, primarily spread in feces, in Heterakis gallinarum (cecal worm) eggs, or in Eisenia foetida (earthworms). In this trial, Natustat (Alltech, Inc., Nicholasville, KY), a proprietary plant-derived product, was used at 1.925 kg/tonne and compared with nitarsone in male hybrid turkey diets to 42 days of age on histomonad infected litter (day 7) from broiler breeders. Infected nonsupplemented and uninfected nonsupplemented control groups were also included. Natustat and nitarsone significantly improved 28- and 42-day feed conversion ratios and lowered 28- and 35-day cecal and liver lesion scores compared with infected nonsupplemented turkeys. The body weight at 42 days was greater in the Natustat and nitarsone supplemented groups than in the infected nonsupplemented group.     

Persistence of Histomonas meleagridis in or on materials used in poultry houses

Histomonas meleagridis is the causative agent of blackhead disease or histomonosis in turkeys, and previous research suggests that this parasite survives poorly outside of hosts except within heterakid nematodes. However, we investigated the viability of H. meleagridis in or on several artificially contaminated materials kept at ambient room temperature (22 +/- 2 C) to mimic the situation in the field. The protozoan survived for up to 1 hr on wood, rubber, and metal; up to 3 hr on egg-tray cartons, egg shells, and bricks; up to 6 hr on straw, turkey feathers, and feed; and up to 9 hr in nonchlorinated tap water and fecal matter. Therefore, contaminated water, fresh fecal matter, or both could play a role in transmission of the parasite within and among poultry houses rather than other materials tested in this study.     

Specific detection of Histomonas meleagridis in turkeys by a PCR assay with an internal amplification control

Histomoniasis or blackhead is a disease of gallinaceous birds, caused by the protozoon Histomonas meleagridis. Since traditional diagnostics for the detection of this disease are complex and far less sensitive than molecular tools, a PCR would provide a more rapid and sensitive alternative. However, intestinal material and droppings, which are preferably used in epidemiological studies of histomoniasis, often contain PCR inhibitory substances. To detect these false negative results, the use of an internal amplification control is essential. Nevertheless, the recently developed PCR tests lack this internal control. Therefore, a new PCR assay with H. meleagridis specific primers was developed which does include an internal amplification control. The diagnostic value of the PCR assay was evaluated in comparison to three other conventional H. meleagridis specific PCR tests (HIS5, HM1 and HM2). None of the organ samples originating from uninfected turkeys, showed positive PCR results in any of the tests. Among the lesion-positive, inhibition-free samples, 95.4% were positive by our PCR assay, while only 50, 66.7 and 83.3% of the lesion-positive organs tested positive by the HM1, the HIS5 and the HM2 PCR respectively. In conclusion, our PCR offers the use of the internal control to detect false negative results and an increased sensitivity, and thus should be useful for routine diagnosis of H. meleagridis in poultry.     

Direct lateral transmission of Histomonas meleagridis in turkeys

The lateral transmission of Histomonas meleagridis in turkeys was studied in floor pens without the presence of Heterakis gallinarum. Battery-reared poults (120) were transferred at 2 wk of age to concrete-floored floor pens with fresh pine shavings litter (40/group). One group received no exposure. In other groups, either 10% or 25% of the birds were inoculated per cloaca with cultured H. meleagridis (200,000/bird) and placed in the pens as seeder birds. Inoculated birds died at 10-18 days postinfection (PI) showing typical liver and cecal lesions of histomoniasis. Birds in the high-exposure group died of histomoniasis beginning 16 days PI and continuing to 100% mortality by day 23 PI. Birds in the low-exposure (LE) group died beginning on day 19 PI and continuing through day 31 PI. All but one LE bird alive on day 31 PI had severe liver and cecal lesions of histomoniasis at necropsy. There was no evidence of histomoniasis in unexposed birds. No cecal worms (H. gallinarum) were found at necropsy of dead birds or in unexposed birds at the end of the experiment. Even though H. gallinarum is the only known reservoir for H. meleagridis, these results suggest that lateral transmission of histomoniasis through a flock can occur readily through normal contact between uninfected birds and infected birds and their droppings in the total absence of cecal worms.     

Blackhead disease in turkeys: direct transmission of Histomonas meleagridis from bird to bird in a laboratory model

The spread of Histomonas meleagridis infections through groups of turkeys in the absence of the cecal worm vector (Heterakis gallinarum) was studied in a battery cage model. Battery-reared poults were exposed at 2 wk of age by commingling with infected birds into cages that had the floor lined with paper. One treatment received no exposure, whereas other birds were commingled with two, three, or four birds/cage (25%, 37.5%, or 50%) inoculated per cloaca with cultured H. meleagridis (200,000/bird). Inoculated birds died at 7-13 days postinoculation (DPI) showing typical liver and cecal lesions of histomoniasis. By 14 DPI, 87.5% of the directly inoculated birds died or had severe lesions of histomoniasis. Turkeys commingled with two, three, or four infected birds became infected at the rate of 72%, 80%, or 75%, respectively. In another experiment, two birds/cage (25%) were inoculated with Histomonas from culture and allowed to commingle with other birds for 1, 2, 3, or 4 days. Two of 12 (16.7%) birds had minor cecal lesions after contact with inoculated birds for 1 day, but 87.5%-100% became infected if inoculated birds remained in the cage for 2-4 days. Contemporaneous inoculation with cecal coccidia (Eimeria adenoeides) as a predisposing factor in blackhead infections was studied using the model. Turkey poults directly inoculated with Histomonas were allowed to commingle for 5 days with uninoculated birds that had received inoculation with 0, 10(3), or 10(4) sporulated oocysts. The coccidian infection appeared to interfere with transmission of blackhead infection by 7 DPI, as suggested by lessened severity of cecal lesions and a lower percentage of infected birds. These studies confirm that histomoniasis is transmitted readily from directly exposed young turkeys to others in the absence of the cecal worm vector, and that this phenomenon can be reproduced in battery cages as an experimental model.     

Blackhead disease (histomoniasis) in poultry: a critical review

After its discovery in 1893 in Rhode Island, blackhead disease was reported across the continent and soon in many other countries. It decimated the turkey industry in New England and followed production like a faithful shadow. Blackhead disease causes high mortality in turkeys, sometimes approaching 100% of a flock. In chickens, the mortality may be 10%-20% with high morbidity, although many outbreaks pass unnoticed. Early workers identified Histomonas meleagridis, a protozoan related to Entamoeba histolytica, Giardia lamblia, and Trichomonas, as the causative agent. Like many other parasites, its life cycle is complex, involving as an intermediate host, the common cecal worm Heterakis gallinarum. The necessity for bacteria for Histomonas to become virulent in the turkey and chicken, notably Escherichia coli, Bacillus subtilis, and Clostridium spp., was discovered by research in gnotobiotic birds. Changes in management brought the disease under control, although it remained the first cause of mortality in turkeys until modern antihistomonal products were developed after WWII. The ban of nitroimidazole products in the United States and Europe was followed by an upsurge in reported cases in turkeys and chickens. Immunization is not an option for prevention, as birds do not reliably become resistant to reinfection after suffering a primary exposure. Recent research demonstrated that histomoniasis could spread rapidly through a flock of turkeys by direct contact, probably involving the phenomenon of cloacal drinking. Direct transmission was not demonstrated for chickens, stressing dependence on H. gallinarum as the source of infection. The lack of suitable treatment drugs or vaccines emphasizes the importance of prevention by worm control and management.     

Histomonas meleagridis in chickens: attempted transmission in the absence of vectors

The progress and transmission of blackhead disease in chickens was studied in battery cages and floor pens in the absence of vectors. Two-week-old chicks were inoculated intracloacally with Histomonas meleagridis and allowed to commingle with others in floor pens. There was no confirmed transmission of blackhead to other birds in the pen, whether stocked at 10% or 25% with infected birds. A second experiment evaluated the effects of feed restriction of chickens on spread of blackhead within floor pens. Inoculated seeder birds had severe cecal lesions of blackhead at necropsy, regardless of feed restriction. Uninoculated birds did not develop lesions by the time of necropsy at 42 days of age, regardless of whether full-fed or limited by skip-day feeding. Chickens inoculated intracloacally with H. meleagridis and placed in battery cages became infected and had cecal lesions of blackhead, but few liver lesions. Chickens allowed to commingle with the inoculated birds in batteries had no lesions of histomoniasis at necropsy 2 wk postinoculation. Coccidial oocysts from turkeys (Eimeria adenoeides) were inoculated along with H. meleagridis from cultures to test the effects of sporozoite penetration in the ceca on progress of blackhead disease. Histomoniasis was not worsened by the interaction with sporozoites, as shown by unchanged severity of cecal lesions, the number of birds showing liver lesions, or the overall number of positive birds. Overall, blackhead infections showed no inclination to spread from bird to bird under conditions of these studies, in contrast to what has been reported for turkeys. These results suggest that the dynamics of blackhead transmission in chickens differs significantly from that of turkeys, where transmission from bird to bird is rapid and effective in the absence of vectors.     

High seroprevalence of Histomonas meleagridis in Dutch layer chickens

Histomona meleagridis is a protozoan parasite that may cause outbreaks of histomonosis with high mortality, especially in turkey flocks. Chickens are less susceptible to the disease than are turkeys, but are considered to act as an important reservoir. To determine the seroprevalence of H. meleagridis in Dutch layer chicken flocks, a large scale seroepidemiologic study (3376 samples) was performed by sampling 12 organic flocks, 24 free-ranging flocks, 40 flocks with floor housing, and 40 flocks with cage housing. At the end of the laying period, approximately 30 blood samples per flock were collected for serology. The seroprevalence found was high. In every flock, at least one of the samples tested positive while in 87% of the flocks, at least one of the samples was strongly positive. There were no significant statistical differences in seropositivity between the housing types. To confirm the enzyme-linked immunosorbent assay (ELISA) results, a small-scale seroepidemiologic study (576 samples) was performed in 29 additional layer chicken flocks kept in different housing systems. Subsequently, a subset of five seropositive flocks was selected. Five birds were obtained from each of these flocks in order to detect the parasite using culture and PCR. In all five flocks, H. meleagridis was either isolated from (culture), detected in (PCR), or both, the birds sampled. Together with the previously performed validation studies, the latter results confirm that the positive ELISA serology found is genuine. We conclude that the seroprevalence of H. meleagridis in layers is, as anticipated, high.     

临床疑似病例中火鸡组织滴虫的检测及其在脏器分布规律

为了探明临床病鸡中火鸡组织滴虫的脏器分布规律,本研究收集21例临床疑似病鸡的321个组织器官,采用PCR技术对样品中的虫体DNA进行了检测。结果:组织脏器中肝脏和盲肠的阳性检出率最高,分别为57.1%和52.4%;其次为回肠、脾脏、肾脏、肌胃、胰腺和脑,阳性率分别为43.8%、40.0%、40.0%、37.5%、35.7%和33.3%;心、肺、法氏囊和其他肠道组织等检出率较低;胆囊未检测到。结论:火鸡组织滴虫主要感染肝脏和盲肠,其次为回肠、脾脏、肾脏、肌胃、胰腺和脑,其他脏器虫体分布较少。     

A novel multiplex polymerase chain reaction approach for detection of four human infective Cryptosporidium isolates: Cryptosporidium parvum, types H and C, Cryptosporidium canis, and Cryptosporidium felis in fecal and soil samples.

A nested multiplex polymerase chain reaction (PCR) approach was adopted for the simultaneous detection of 4 human infective genotypes of the protozoan parasite Cryptosporidium. Specific PCR primers were designed for the heat shock protein 70 gene of 2 genotypes of Cryptosporidium parvum (human and bovine types), Cryptosporidium canis, and Cryptosporidium felis. These 4 genotypes have all been found in human fecal samples. The primers amplified DNA fragments of specific sizes, each representing a unique genotype. The limit of detection of the method was found to vary between 10 and 100 oocysts per 1 ml fecal material. There appeared to be no cross-reactivity with other organisms commonly present in feces and soil, and the approach has a high specificity. The rapid identification of various human infective Cryptosporidium isolates is a part of the authors' long-term aim of determining the routes of infection with oocysts and thereby increase their epidemiological understanding of Cryptosporidium infection in humans and animals.