Infectivity of Histomonas meleagridis of Cecal and Liver Origins Compared

SYNOPSIS. Turkey poults were inoculated rectally with 100, 1000, 10,000, or 100,000 Histomonas meleagridis from the ceca of a group of experimentally infected turkeys. Other poults were given the same numbers of histomonads from an infected liver from the same group of source birds, Comparisons of the incidence of infection, liver involvement, mortality, and average survival time following these inoculations showed that organisms of cecal origin were about 100 times more effective in producing histomoniasis than were organisms of liver origin. It is suggested that this difference in infectivity resulted from heavy losses of histomonads of liver origin that were due to various selective processes.     

Histomonas wenrichi n. sp. (Mastigophora: Mastigamoebidae), a Nonpathogenic Parasite of Gallinaceous Birds

SYNOPSIS. A nonpathogenic histomonad, morphologically identical to that found in pheasants by Wenrich in 1943, is described as Histomonas wenrichi n. sp. Its dimensions are about 1.5 × those of H. meleagridis. It has 4 flagella instead of the 1 or 2 that characterize the latter species. It does not multiply in the host tissues nor produce visible responses to its presence there, and it does not exist as the clear "tissue form" that is common in active H. meleagridis infections.
Histomonas wenrichi n. sp. can be transmitted to turkeys, chickens, and pheasants by either rectal inoculation or by feeding embryonated eggs of Heterakis gallinarum grown in birds harboring the nonpathogenic histomonad in the ceca. Usually fewer than half the female heterakids so grown produce eggs capable of transmitting this histomonad, and a total of 200 to several thousand eggs from worms recovered from Histomonas -infected birds may be required to produce one Histomonas infection.
Histomonas wenrichi n. sp. has been propagated in more than 2000 chickens and turkeys over an 8-year period, during which time it has been transmitted by 18 generations of Heterakis. It has frequently been grown in birds in which H. meleagridis was also present, and upon reisolation it maintained all of its original characteristics. It has not yet been cultivated in vitro.     

Indirect Fluorescent Antibody Tests Comparing Two Strains of Histomonas meleagridis and H. wenrichi

SYNOPSIS. Histomonas meleagridis cultured in vitro for 8 years, H. meleagridis freshly isolated from cecal contents of turkeys, and freshly isolated, nonpathogenic H. wenrichi were compared by indirect fluorescent antibody tests. The Histomonas antisera were produced in rabbits in response to cultured and freshly isolated H. meleagridis . Using fluorescein-conjugated antirabbit globulin, fluorescent reactions of cultured and freshly isolated H. meleagridis with either H. meleagridis antisera were indistinguishable. Such results suggest that the antigens involved in these reactions remained relatively unchanged during prolonged cultivation. H. wenrichi reacted feebly with both H. meleagridis antisera.     

Effect of dimetridazole on transmission of Histomonas meleagridis by Heterakis gallinarum

The administration of an antihistomonal drug, dimetridazole, at a dose of 0.08% in feed, controlled experimental infections with Histomonas meleagridis in chickens. The treated birds developed no lesions and the duration of infection with H. meleagridis was reduced. This drug regimen, however, did not always prevent incorporation of H. meleagridis into eggs of Heterakis gallinarium; heterakid eggs pooled from medicated chickens in which H. meleagridis had never been detected transmitted the protozoan to 1 of 10 turkeys fed the eggs. Thus, therapeutic treatment of chickens with dimetridazole may reduce, but not eliminate, transmission of H. meleagridis by eggs of H. gallinarum from medicated birds.     

The wild turkey as a host for Heterakis gallinarum and Histomonas meleagridis.

Freshly embryonated eggs of Heterakis gallinarum gathered from naturally infected domestic turkeys and chickens developed the first 4 weeks essentially as well in young wild turkeys as in domestic poults, but then became progressively retarded and failed in most birds to result in females with fertile eggs. There was no significant difference in the prevalence or progress of infections with Histomonas meleagridis in the two kinds of turkeys, both of which differed from chickens only in that the latter had neither liver involvement nor mortality. In a second test, heterakids hatched from eggs stored 5-6 months at 4 C (comparable to overwintering) sustained very heavy losses in all birds, with greatly accelerated liberations of H. meleagridis, Few worms reached maturity and still fewer produced fertile eggs. In turkeys, and especially in wild turkeys, replacement of infective stages was so poor, that these birds were of no importance in contaminating the soil.     

Effect of Bile on Infectivity of Histomonas meleagridis of Liver Origin

SYNOPSIS After 30 min exposure to fresh turkey bile at 99 F, 100,000 Histomonas meleagridis of turkey liver origin failed to infect any of 20 poults inoculated rectally. Only one of 20 poults similarly inoculated with histomonads exposed to fresh turkey bile for only 5 min at 99 F became infected. Fifteen of 20 poults became infected following rectal inoculation with 100,000 H. meleagridis of liver origin held in physiologic saline for 30 min at 99 F. All infected birds developed liver lesions and died. Altho H. meleagridis was very abundant in the livers of all potential donor poults and all infected recipient poults, histomonads were never found in the gall bladders of any of these birds. In some such birds, a few apparently degenerating histomonads were detected in the duodenum near the entrance of the bile ducts. Histomonads of liver origin are regarded as of little or no consequence in the transmission of this protozoon.     

Immunizing Action of a Nonpathogenic Strain of Histomonas against Blackhead in Turkeys

SYNOPSIS. A nonpathogenic strain of Histomonas was used in an attempt to immunize young turkeys against blackhead. Rectal inoculation of several thousand nonpathogenic histomonads on 2 or 3 consecutive days afforded considerable protection against modest rectal challenges with pathogenic histomonads 3 to 6 weeks later, but was much less effective against pathogenic histomonads introduced by feeding eggs of Heterakis gallinae. It is believed that an immune barrier limited to the surface of the cecal mucosa was established, and that the larvae of the cecal worms often penetrated this barrier before liberating their histomonads, thus permitting blackhead to develop. Immunization by the introduction of nonpathogenic histomonads with Heterakis eggs was not satisfactory. Apparently, the nonpathogenic organisms introduced in this way were too few to assure formation of an intact barrier.     

Efficacy of a herbal product against Histomonas meleagridis after experimental infection of turkey poults

Histomoniasis (infectious enterohepatitis, blackhead) is caused by the protozoan parasite Histomonas meleagridis (H. meleagridis). After the ban of all prophylactic and therapeutic drugs in the European Union, histomoniasis is increasingly responsible for considerable economic problems to the poultry industry. The aim of this study was to investigate the effect of a herbal product with extracts from cinnamon, garlic, lemon, and rosemary on H. meleagridis in turkey poults in vivo. For this purpose, 60 two-week-old poults were divided into three groups. Group 1 received the herbal product in the feed six days before infection and in water three days before infection, then in feed and drinking water until the end of the experiment. Groups 2 and 3 were left untreated. At week 3 of age, Groups 1 and 2 were infected intracloacally with H. meleagridis. Three weeks after infection the surviving birds were euthanized and examined for pathological lesions. Mortality was 20% in Group 1 and 50% in Group 2. There were no deaths in Group 3. DNA of histomonads was detected in all examined caeca and livers of the dead birds, but was not detected in any examined organ of the surviving birds of all groups. There was no noticeable difference in the lesion scores of the dead birds between the groups. The surviving birds of all groups did not show lesions post mortem. Since all effective prophylactic and therapeutic drugs against histomoniasis were banned in the EU, under given conditions the investigated herbal product seems to be an effective alternative for the reduction of mortality in turkeys caused by histomoniasis.     

Partial sequence of the alpha-tubulin gene from Histomonas meleagridis isolates from the United States

Histomonas meleagridis , the causative agent of histomoniasis, is a protozoan parasite classified in the Dientamoebidae (order Tritrichomonadida). The α-tubulin gene of 7 H. meleagridis isolates originating from either domestic chickens or turkeys from the United States was amplified by nested PCR and sequenced. A 91.4-99.8% nucleotide identity was shared among the 7 different sequences, and phylogenetic analysis disclosed that the 7 isolates were divided into at least 3 clades. These sequences had a 91-99% nucleotide identity and a 96-100% amino acid identity compared with 3 H. meleagridis α-tubulin sequences obtained from isolates originating from turkeys in Germany. Further α-tubulin gene analysis from species in the Dientamoebidae will be useful in elucidating the evolutionary relationship of these protozoans.     

Histomonas meleagridis: immunohistochemical localization of parasitic cells in formalin-fixed, paraffin-embedded tissue sections of experimentally infected turkeys demonstrates the wide spread of the parasite in its host

In the present investigation, a polyclonal antibody-based immunohistochemical technique was developed to localize Histomonas meleagridis in formalin-fixed, paraffin-embedded tissues of experimentally infected turkeys. The developed technique was highly specific for histomonads as no immunohistochemical reaction was observed with cultures of Tetratrichomonas gallinarum, Trichomonas gallinae and Blastocystis sp. In addition, tissues positive for various other protozoan parasites and fungi were also tested to evaluate the specificity of the technique. It was possible to detect immunohistochemically histomonad antigens in all the tested samples (n=5) of caecum, liver, spleen and lung from infected turkeys, 3 out of 5 bursa of Fabricius, 1 out of 2 bone marrow, 2 out of 5 heart and 1 out of 5 each of proventriculus, pancreas and cerebellum. An immunohistochemical reaction indicative of presence of histomonads was also detected in blood vessels of various organs that indicated a possible hematogenous route of spread of the parasite in the host. A comparative study with routine diagnostic staining techniques indicated a high sensitivity and specificity of the newly developed immunohistochemical technique. Altogether, the technique developed can be used to study the sequential pathogenesis of histomonosis in turkeys and to obtain new insights into the mechanisms of interaction with the host tissues.     

Morphologic, host specificity, and molecular characterization of a Hungarian Cryptosporidium meleagridis isolate

This study was undertaken in order to characterize Cryptosporidium meleagridis isolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and other Cryptosporidium species. The phenotypic differences between C. meleagridis and Cryptosporidium parvum Hungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts of C. meleagridis were successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location of C. meleagridis was the small intestine, like the location of C. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene of C. meleagridis isolated from turkeys with other Cryptosporidium sequence data in the GenBank database revealed that the Hungarian C. meleagridis sequence is identical to a C. meleagridis sequence recently described for a North Carolina isolate. Thus, C. meleagridis is a distinct species that occurs worldwide and has a broad host range, like the C. parvum zoonotic strain (also called the calf or bovine strain) and Cryptosporidium felis. Because birds are susceptible to C. meleagridis and to some zoonotic strains of C. parvum, these animals may play an active role in contamination of surface waters not only with Cryptosporidium baileyi but also with C. parvum-like parasites.     

Immunologic Analysis by Gel Diffusion of Effects of Prolonged Cultivation on Histomonas meleagridis (Smith)*

SYNOPSIS. Antigens were prepared from each of 4 lines of Histomonas meleagridis: Hm-L1, a strain highly virulent for both turkeys and chickens; Hm-L1 /C12, Hm-L1 /C24, Hm-L1 /C52, 3 avirulent substrains derived from Hm-L1 after 12, 24, 52 weeks of in vitro cultivation, respectively. Hm-L1 strain and the 3 substrains were maintained in liquid nitrogen. Antisera were developed in rabbits against Hm-L1 and Hm-L1 /C24 parasites. Both antisera were reacted on gel diffusion plates with homologous and heterologous antigens. Two groups of precipitin lines and/or bands designated arbitrarily as A and B, were observed on the slides. Analysis of these bands revealed the common antigenic composition of the 4 histomonads with respect to some of the group A and group B antigens. The concentrations and numbers of precipitin lines in both groups increased, however, with the length of cultivation. These antigenic differences are discussed in the light of their possible relationship to pathogenicity.     

Prevalence and pathology of the nematode Heterakis gallinarum, the trematode Paratanaisia bragai, and the protozoan Histomonas meleagridis in the turkey, Meleagris gallopavo

The prevalence of infection and associated pathology induced by two helminth and one protozoan species infecting Brazilian turkeys are reported. The intestinal nematode Heterakis gallinarum appeared with a prevalence of 70% in the infected birds, without gross lesions when not associated to the protozoan Histomonas meleagridis. Histological findings in the ceca were represented by the presence of H. gallinarum worms, intense chronic diffuse inflammatory processes with mononuclear and polymorphonuclear (heterophils) leucocyte infiltrations. The prevalence of the protozoan H. meleagridis associated to H. gallinarum was of 2.5% and microscopic examination revealed a severe inflammatory process in the liver and cecum with the presence of small clear areas with round eosinophilic parasites. Gross lesions were absent in turkeys infected with the renal digenetic trematode Paratanaisia bragai; the parasite was prevalent in 20% of the cases and cross-sections of the kidneys showed a remarkable distension of the collecting ducts with several worms in the lumen. The walls of the ducts presented a discrete heterophilic infiltrate among mononuclear cells.     

Occurrence and seasonal transmission of hematozoa in wild turkeys

The occurrence and seasonal patterns of transmission of the blood protozoa of wild turkeys (Meleagris gallopavo silvestris) were studied at Tallahala Wildlife Management Area (TWMA) (Jasper County, Mississippi, USA). Blood smears obtained from wild turkeys in winter, spring and summer, and from sentinel domestic turkeys throughout the year were examined for Haemoproteus meleagridis and Leucocytozoon smithi. Whole blood from wild turkeys captured in summer was subinoculated into malaria-free domestic turkey poults and recipient birds were examined for Plasmodium spp. The prevalence of H. meleagridis and L. smithi were not different (P greater than 0.05) between adults and juveniles or between male and female turkeys in any season. Leucocytozoon smithi was not detected in poults in summer or in juveniles examined in winter. Sentinel studies and information from wild birds revealed that transmission of H. meleagridis and L. smithi did not overlap. Haemoproteus meleagridis was transmitted from May through November, while L. smithi was transmitted only from January through April. The onset of transmission of H. meleagridis coincided with peak hatching (mid-May) and brood-rearing (May-November) of turkey poults. Plasmodium spp. were not found in turkeys from TWMA (n = 27) nor in birds from three widely separated counties (n = 28) in Mississippi.     

Hematozoan parasites of Rio Grande wild turkeys from southern Texas

One hundred twenty-three of 300 blood samples (41%) taken from Rio Grande wild turkeys (Meleagris gallopavo intermedia) from three locations in southern Texas (Welder Wildlife Refuge, Chaparrosa Ranch, and Campo Alegre Ranch) and subinoculated into domestic broad-breasted white turkey poults were positive for a Plasmodium (Novyella) sp. Analysis of blood films from 350 turkeys revealed Haemoproteus meleagridis in 76% of the birds. A significantly greater mean parasite intensity was observed in birds from Welder Wildlife Refuge. Birds from the Campo Alegre Ranch exhibited a significantly higher prevalence of H. meleagridis than birds from Chaparrosa. The Plasmodium sp. was infective for canaries (Serinus canaria), bobwhites (Colinus virginianus), and ring-necked pheasants (Phasianus colchicus), but would not produce infection in white leghorn chickens (Gallus gallus) or Coturnix quail (Coturnix coturnix). Attempts to infect Culex tarsalis and C. pipiens were unsuccessful. Asexual erythrocytic synchrony was not observed when blood-induced infections were monitored in two domestic turkey poults every 4 hr for 72 hr. Exoerythrocytic stages were not found upon examination of impression smears and tissue samples taken from brain, liver, spleen, kidney, lung, and bone marrow. The Plasmodium sp. is most similar morphologically to three species in the subgenus Novyella, P. hexamerium, P. vaughani, and P. kempi. The most striking similarities are to P. hexamerium, and involve mean merozoite number, erythrocytic schizont location, and vertebrate host susceptibility. It differs from P. vaughani in being able to infect turkeys and in type of parasitized erythrocytes. Differences to P. kempi include mean merozoite number, and ability to infect pheasants, and its inability to develop in C. pipiens and C. tarsalis.     

Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence.

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G + C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonasfoetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.     

Cryptosporidiosis in zoo and pet birds.

Cryptosporidiosis is recognized as a primary disease in commercially raised chickens, turkeys, and bobwhite quail. Little is known about cryptosporidial infections in zoo and pet birds, although, infections of the small intestines, proventriculus, respiratory tract, and kidneys have been reported. In the present study, we reviewed cases of cryptosporidial infections in zoo and pet birds submitted to the State Veterinary Diagnostic Laboratory, Auburn, Alabama for necropsy or histopathologic examination. We identified infections in cockatiels, white-lored euphonias, bronze mannikin finches, and Australian diamond firetailed finches. Infections in the cockatiels occurred mostly in the small intestine, but parasites were also observed in the esophageal glands, air sacs, and proventriculus of some birds. Separate cases of small intestinal and proventricular infections were identified in the white-lored euphonias. Cryptosporidial parasites were found only in the proventriculus of the bronze mannikin finches and Australian diamond firetail finches. No cases of renal cryptosporidiosis were observed. Co-pathogens or other disease conditions were present in all birds. The Cryptosporidium species responsible for causing proventricular infection in zoo and pet birds may be different from C. meleagridis and C. baileyi.     

Heterakis gallinarum in the guniea fowl, Numida meleagris: survival and comparative potential for transmitting Histomonas meleagridis

As measured by survival to maturity, size, and reproductive potential, Heterakis gallinarum thrived in young guinea fowl, unless concurrent infections with Histomonas meleagridis caused severe cecal lesions. Eggs of heterakids recovered from guinea fowl transmitted H. meleagridis to young turkeys. The young guinea fowl seems to be somewhat less important than the young New Hampshire chicken in spreading histomoniasis, but considerably more important than the young turkey.     

Isolation of Mycoplasma gallopavonis from free-ranging wild turkeys in coastal North Carolina seropositive and culture-negative for Mycoplasma gallisepticum.

Serum samples and choanal cleft swabs were collected from livetrapped and hunter killed wild turkeys (Meleagris gallopavo) from Martin and Bertie counties, North Carolina (USA). Sera were tested for antibodies to Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma meleagridis by hemagglutination inhibition (HI). Sera from 33% (five of 15) of livetrapped turkeys were positive for antibodies to M. gallisepticum by HI, and all were negative for antibodies to M. synoviae and M. meleagridis. Choanal cleft swabs from 22 livertrapped and five hunter killed wild turkeys cultured in Frey's broth medium resulted in 23 mycoplasma isolations. Using direct immunofluorescence, 74% (17/23) were M. gallopavonis, and 26% (six of 23) were unidentified; no isolate was identified as M. gallisepticum, M. synoviae or M. meleagridis.     

16S rRNA-based analysis of microbiota from the cecum of broiler chickens.

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.