Detection and identification of Theileria infection in sika deer ( Cervus nippon ) in China

The sika deer ( Cervus nippon ) is a first-grade state-protected animal in China and designated a threatened species by the World Conservation Union. To detect hemoparasite infection of sika deer, blood samples were collected from 24 animals in the Hubei Province Deer Center. Genomic DNA was extracted, and the V4 hypervariable region encoding 18S rRNA was analyzed by reverse line blot hybridization assay. PCR products hybridized with Babesia / Theileria genus-specific probes but failed to hybridize with any of the Babesia or Theileria species-specific probes, suggesting the presence of a novel, or variant, species. Here 18S rRNA and internal transcribed spacer (ITS) genes were amplified, cloned, and sequenced from 7 isolates. Alignment and BlastN of the cloned sequences revealed high similarities to the homologous 18S rRNA genes and ITS genes of Theileria cervi (AY735122), Theileria sp. CNY1A (AB012194), and Theileria sp. ex Yamaguchi (AF529272). Phylogenetic analysis based on the 18S rRNA gene and ITS sequences showed that all cloned sequences were grouped within the Theileria clade. Phylogeny based on the 18S rRNA gene divided the organisms into 2 groups. Group 1 was closest to Theileria sp. ex Yamaguchi (AF529272), and group 2 was distinct from all other identified Theileria and Babesia species. These results suggest the existence of Theileria sp. infection in sika deer in China. To our knowledge, this is the first report of cervine Theileria sp. in China.     

Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms

The morphologic, ultrastructural and genotypic characteristics of Babesia duncani n.sp. are described based on the characterization of two isolates (WA1, CA5) obtained from infected human patients in Washington and California. The intraerythrocytic stages of the parasite are morphologically indistinguishable from Babesia microti, which is the most commonly identified cause of human babesiosis in the USA. Intraerythrocytic trophozoites of B. duncani n.sp. are round to oval, with some piriform, ring and ameboid forms. Division occurs by intraerythrocytic schizogony, which results in the formation of merozoites in tetrads (syn. Maltese cross or quadruplet forms). The ultrastructural features of trophozoites and merozoites are similar to those described for B. microti and Theileria spp. However, intralymphocytic schizont stages characteristic of Theileria spp. have not been observed in infected humans. In phylogenetic analyses based on sequence data for the complete18S ribosomal RNA gene, B. duncani n.sp. lies in a distinct clade that includes isolates from humans, dogs and wildlife in the western United States but separate from Babesia sensu stricto, Theileria spp. and B. microti. ITS2 sequence analysis of the B. duncani n.sp. isolates (WA1, CA5) show that they are phylogenetically indistinguishable from each other and from two other human B. duncani-type parasites (CA6, WA2 clone1) but distinct from other Babesia and Theileria species sequenced. This analysis provides robust molecular support that the B. duncani n.sp. isolates are monophyletic and the same species. The morphologic characteristics together with the phylogenetic analysis of two genetic loci support the assertion that B. duncani n.sp. is a distinct species from other known Babesia spp. for which morphologic and sequence information are available.     

Identification of novel Babesia and Theileria genotypes in the endangered marsupials, the woylie (Bettongia penicillata ogilbyi) and boodie (Bettongia lesueur)

Piroplasms, which include the genera Theileria and Babesia, are blood-borne parasites transmitted mainly by tick vectors. Relatively little is known about their prevalence and clinical impact in Australian marsupials. In the present study the occurrence and molecular phylogeny of these parasites were studied in both wild and captive marsupials from Western Australia (WA) and Queensland (QLD). Blood samples were screened by microscopy and molecular methods, using PCR and DNA sequencing of the 18S ribosomal RNA gene (18S rDNA). Overall, 7.1% of the blood samples (8/113) were positive for piroplasm 18S rDNA. Theileria and Babesia rDNA was detected in 0.9% (1/113) and 6.2% (7/113) of the animals, respectively. The single Theileria positive was identified in one of three boodies (Bettongia lesueur) screened from a wildlife rehabilitation centre in WA, while all seven Babesia positives were detected in WA in wild captured woylies (Bettongia penicillata ogilbyi). Small intraerythrocytic inclusions were observed in blood films made from six of these individuals. This is the first report of a Babesia sp. in woylies, and Theileria sp. in boodies. Phylogenetic analysis indicated that the woylie-derived Babesia was genetically distinct and most closely related to Babesia occultans, the causative agent of a benign form of cattle babesiosis (genetic similarity 98.4%). The Theileria identified was most closely related to the marsupial-derived species Theileria penicillata from the woylie, Theileria brachyuri from the quokka (Setonix brachyurus), and Theileria sp. from the long-nosed potoroo (Potorous tridactylus).     

Molecular survey of Babesia,Theileria, Trypanosoma,and Anaplasma infections in camels (Camelus dromedaries) in Egypt

The one-humped camel (Camelus dromedarius) or dromedary is an economically important domestic animal. However, infectious diseases, including those caused by vector-borne hemopathogens, frequently compromise the health and production of camels. In this study, we examined infections caused by Babesia, Theileria, Trypanosoma, and Anaplasma species in camels in Egypt. We analyzed blood DNA samples from 148 camels reared in six Egyptian governorates (Giza, Asyut, Sohag, Qena, Luxor, and the Red Sea) using pathogen-specific Polymerase Chain Reaction (PCR) assays. Our results indicated that 29 (19.6%), 22 (14.9%), 1 (0.7%), 2 (1.4%), 1 (0.7%), 2 (1.4%), and 28 (18.9%) of the surveyed animals were infected with Babesia bovis, B. bigemina, Babesia sp. Mymensingh, Theileria sp. Yokoyama, Theileria equi, Trypanosoma evansi, and Anaplasma marginale, respectively. We found that a total of 68 (45.9%) animals were infected with at least one of the detected hemopathogens. Sequencing analyses of PCR amplicons confirmed our diagnostic results. This study is the first to report Theileria sp. Yokoyama and Babesia sp. Mymensingh in Egypt. This is also the first report of infection with these two species in one-humped camel. In conclusion, this study found that camels in Egypt are infected with several vector-borne hemopathogens, including novel parasite species.     

Phylogenetic relationships of the benign Theileria species in cattle and Asian buffalo based on the major piroplasm surface protein (p33/34) gene sequences.

In order to examine the taxonomic relationship of Theileria sp. of Asian buffalo to the benign Theileria spp. of cattle, we sequenced and compared the major piroplasm protein (p33/34) genes of these parasites. The two consensus sequences determined for the buffalo parasite were of the same length (852 bp) and showed >80% identity with the sequences of the homologous genes (849 bp) in the cattle parasites. Alignment of the inferred aa sequences with those of Theileria sergenti and Theileria buffeli predicted that there is an insertion of a single residue at the N-terminus in the inferred polypeptide of the buffalo parasite. Phylogenetic analyses based on the aa sequences suggested that Theileria sp. of the Asian buffalo should be classified within the benign Theileria parasite group as a separate species from the cattle parasites. Based on this, we propose a rearrangement of the currently used classification for the benign Theileria species in cattle and Asian buffalo.     

Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain

The genetic diversity and prevalence of virtually all Theileria and Babesia species in a sheep population were studied using a specifically designed reverse line blot macroarray. The amplified hypervariable V4 region of the 18S rRNA gene was hybridised against generic and species-specific probes. In a first screening (Study I), 320 apparently healthy animals corresponding to 32 flocks located in the Basque Country (Northern Spain) were analysed. The survey demonstrated a high prevalence of subclinical infections (64.7%). Three Theileria genotypes were identified, sharing 96.7-97.0% similarity between their 18S rRNA gene sequences: Theileria ovis, Theileria sp. OT1 (99.6% similarity with the recently described pathogenic piroplasm Theileria sp. China 1), and Theileria sp. OT3. Two Babesia species sharing 91.5% similarity were also detected: Babesia ovis and Babesia motasi. The complete 18S rRNA gene sequences of these and other piroplasm species were phylogenetically analysed. Prevalence of piroplasms was also investigated in a second group of 80 sheep from 16 flocks reared in mountain areas that had been heavily exposed to ticks and had suffered a recent abortion episode (Study II). The screening revealed a significantly higher (P < 0.05) prevalence (78.7%) of piroplasm infections compared to Study I. Although the prevalence rates for some piroplasm species were significantly related to abortion (e.g. Theileria sp. OT3), decreases in the red cell parameters were not significant. The widespread distribution of Theileria spp. in the studied sheep population suggests that the parasites involved are of relatively low pathogenicity, in contrast to what has been reported for Theileria sp. China 1 in other countries.     

Theileria gilberti n. sp. (Apicomplexa: Theileriidae) in the Gilbert's Potoroo (Potorous gilbertii)

ABSTRACT. The morphology and genetic characterisation of a new species of piroplasm identified in the blood of the Gilbert's potoroo ( Potorous gilbertii ) from the Two Peoples Bay Nature Reserve near Albany, Western Australia, is described from blood and tissue samples from 16 Gilbert's potoroos. Microscopy of blood showed these parasites are highly pleomorphic with a mean length of 1.8 μm and mean width of 0.85 μm. Phylogenetic analysis of 18S rRNA sequence data identified the piroplasm as a new species of Theileria that is closely related to other Australian marsupial piroplasm species. Based on biological and molecular data, it is proposed that the parasite from Gilbert's potoroo be given the name Theileria gilberti n. sp.     

Multiple Cryptosporidium genotypes detected in wild black rats (Rattus rattus) from northern Australia

As part of a broader investigation into the potential role of black rats (Rattus rattus) as disease vectors into native small mammal populations of northern Australia, blood and faecal samples from wild black rats were screened by molecular methods, for piroplasms (Babesia and Theileria), trypanosomes and the enteric parasite Cryptosporidium. While piroplasms and trypanosomes were not detected in the blood of these animals, the overall prevalence of Cryptosporidium 18S rDNA in faecal samples was 8.2% (7/85). Co-occurrence of multiple genotypes was observed in 57.1% of the infected individuals (4/7); cloning and re-sequencing resulted in 14 sequences which broadly grouped with Cryptosporidium sp. rat-genotypes II and III. A novel rat-derived Cryptosporidium sp. genotype at the actin locus was also obtained from five animals. The relatively low infection rate detected, and the epidemiological data on cryptosporidiosis, do not conclusively support a current threat to native Australian mammals from black rats carrying Cryptosporidium. However, this observation is based on sampling limited isolates, in limited regions. Further studies, also including sampling of native mammals, are required on larger sample sizes and from wider geographic areas, to determine the significance of these findings, including the public health importance of Cryptosporidium spp. from rodents.     

Evaluation of a Real-Time PCR Test for the Detection and Discrimination of Theileria Species in the African Buffalo (Syncerus caffer)

A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed. Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its identification in some samples by the qPCR assay was unexpected.Because of these discrepancies in the results, cox III qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection. The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the mildly pathogenic and benign Theileria spp. of buffalo and cattle.     

Helminths of California quail (Callipepla californica) and mountain quail (Oreortyx pictus) in western Oregon

Eighty California quail (Callipepla californica), collected from the E. E. Wilson Wildlife Area near Monmouth, Oregon (USA) during a 22 mo period, were examined for gastrointestinal helminths. Eight birds were infected with three species of nematodes, Heterakis isolonche, Dispharynx nasuta, and Capillaria sp., and two species of cestodes, Rhabdometra odiosa and Davainea sp. Except for D. nasuta, prevalence did not exceed 5% despite mesic conditions in the collection area. Two mountain quail (Oreortyx pictus) were collected from Lane County, Oregon (USA), near Blue River Reservoir; both were infected with the nematode Trichostrongylus tenuis.     

Strongyloidiasis in cotton rats (Sigmodon hispidus) from central Oklahoma

Thirty-one of 40 cotton rats (Sigmodon hispidus) collected from central Oklahoma were infected with Strongyloides sp. (78% prevalence). Larvae of Strongyloides sp. (rhabditiform or filariform) were not demonstrable in intestinal contents and scrapings. Female nematodes recovered from intestinal contents and scrapings had morphological similarities with Strongyloides sigmodontis. Cotton rats infected with Strongyloides sp. were indistinguishable clinically from non-infected hosts. Infected animals had no significant gross lesions, but the presence of Strongyloides sp. in the intestinal mucosa was associated with villus atrophy and mild to moderate infiltration of the lamina propria by lymphocytes, plasma cells and occasional eosinophils. Other organs or tissues examined were free from lesions induced by Strongyloides sp.     

Development of infectivity in Hyalomma detritum (Schulze, 1919) ticks infected with Theileria annulata (Dchunkowsky and Luhs, 1904).

The time-course for the development of infectivity was studied in Hyalomma detritum ticks fed as pre-imagoes on calves infected with Theileria annulata. Unfed adults derived from infected nymphs were non-infectious when inoculated into susceptible calves, whereas ticks of both sexes that had fed for 2-3 days or longer on calves or rabbits were always infectious. Some adults fed for only 1-2 days were infectious, whereas others were not. As few as 2 ticks were capable of infecting susceptible calves.     

Acanthocephala in amphibians (Anura) and reptiles (Squamata) from Brazil and Paraguay with description of a new species

In a survey of 1,732 amphibians and reptiles collected across S?o Paulo Province, Brazil, and 7 provinces in Paraguay, 26 species were found infected with acanthocephalans. Of 1,510 anurans, 14 anurans, representing 11 species, were infected with cystacanths of Centrorhynchus spp. and 1 anuran with cystacanths of Oligacanthorhynchus sp. Of 107 lizards, 1 lizard was infected with cystacanths of Centrorhynchus sp. and 1 lizard with cystacanths of Oligacanthorhynchus sp. Acanthocephalus caspanensis was found in 3 anurans (3 species) and Acanthocephalus lutzi in 3 anurans (2 species) and 2 snakes (2 species). The systematic position of A. lutzi cannot be resolved using presently available morphological data. Acanthocephalus saopaulensis n. sp. was found in a single individual of Bufo ictericus. The new species can be differentiated from all its congeners except A. caspanensis in having a sigmoid-shaped male posterior end and from A. caspanensis in having a proboscis armature of 16 rows of 5-7 hooks rather than 18-19 rows of 6-7 hooks and larger eggs. The status of Acanthocephalus and Pseudoacanthocephalus continues to be problematic.     

Tick-borne diseases in ruminants of Central and Southern Italy: epidemiology and case reports

Sera and blood from cattle and sheep were examined for the presence of Babesia and Theileria spp by microscopy and serology at the Parasitology Department of the Istituto Zooprofilattico Sperimentale of Abruzzo and Molise (IZSAM). Of the 47 bovine herds (323 animals) tested, 15 were found positive for Babesia bigemina and 1 for Babesia bovis. Two outbreaks occurred, one caused by B. bigemina and one by B. bovis. The B. bigemina outbreak occurred in Abruzzo and has been followed for two years. The isolate of B. bigemina was very pathogenic leading to the death of two cows out of 57. The vector responsible of the transmission appeared to be Rhipicephalus bursa. Parasites were observed in the erythrocytes for 30 days whereas sera were positive to indirect immunofluorescence (IFA) for at least one year. The B. bovis outbreak occurred in the province of Mantova (Northern Italy) in a group of 70 beef cattle imported from France. The infection resulted in the death of 5 animals and severe illness in another 6. In contrast with what occurred for Babesia infection, no clinical cases were recorded in cattle when species of Theileria were detected by microscopy. Of the 24 bovine herds (252 animals) tested for Theileria, 21 were found positive for the T. "sergenti"/buffeli/orientalis group. Single and mixed infection of T. "sergenti" and T. buffeli/orientalis were detected in herds of cross-bred cattle from Abruzzo and Marche. The parasites were identified by using a polymerase chain reaction which amplified DNA encoding p32/34. Most of the collected ticks (90%) were adults of R. bursa whereas the others were adults of Hyalomma detritum. During the period the animals have been observed (18 months), no clinical cases have been recorded and no associations have been found between blood abnormalities and animals found infected with Theileria. Prevalences of subclinically infected carriers increased from February till December (95.4%) even if the animals were indoors and no ticks were present. The prevalence then dropped dramatically six months later (76.7%). In calves less than 1 year old, the prevalence of infection significantly (p<0.05) increased with age, however intraerythrocytic stages of Theileria were found in the blood of three newborn calves (<7 days of age). Of the 18 ovine flocks tested for Babesia spp. (150 animals examined), 1 was positive for B. ovis and 2 for B. motasi. B. motasi infection was not associated with symptoms, while an outbreak of babesiosis caused by B. ovis occurred in Abruzzo. The infection resulted in the death of 3 animals (0.75% of the flock), two rams (20% of the total number) and a ewe, and severe illness in another 5 ewes (2% of the flock). Specimens of R. bursa and R. turanicus were collected from the infected animals. Of the 18 flocks (150 animals) examined, 12 were microscopically positive for Theileria spp. No clinical cases were recorded and identification at species level was not possible on the basis of morphological criteria. The prevalence distribution of infected herds and infected animals within herds and flocks have been calculated by a Monte Carlo simulation model, running 10,000 iterations. The most likely levels of prevalence of infected herds and infected animals within herds found for the species observed were as follows: 20% for B. bigemina with a prevalence within herd of 27%, 11% for B. bovis (18% within herd), 10% for Babesia ovis (19% within herd), 10% for B. motasi (17.5% within herd), 63% for Theileria in cattle (66% within herd) and 51% for Theileria in sheep (55% within herd).     

Assessment of Theileria infections in Rhipicephalus appendiculatus ticks collected from the field

Collections of adult Rhipicephalus appendiculatus ticks were made from bait cattle and vegetation at two field sites in areas of Kenya in which East Coast fever caused by Theileria parva is endemic. These ticks, together with two experimentally infected batches of ticks, were examined for infection with Theileria by four methods. Whole salivary glands were stained with methyl green pyronin or Feulgen's stain. Whole ticks were ground in medium, the suspensions were filtered and centrifuged and the treated material was examined microscopically and tested for infectivity by inoculation into cattle. All field collections and experimental batches of ticks were infected with Theileria and all four methods detected the infections. Approximately 1.5% of the ticks in the field collections were found to be infected with Theileria and the treated material from these ticks transmitted T. parva to cattle. It is considered that it will be feasible to survey field infection rates quantitatively by collecting ticks from bait cattle and vegetation for examination by a combination of salivary gland staining and preparation of tick suspensions for microscopy and infectivity tests.     

Detection of Theileria annulata by the PCR in ticks (Acari: Ixodidae) collected from cattle in Mauritania

We report on the detection of Theileria annulata in infected Hyalomma ticks by the PCR using primers derived from the gene encoding the 30 kDa major merozoite surface antigen (Tams1–1). No inhibition of the PCR was observed and as little as 0.1 pg of parasite DNA, corresponding to 12 sporozoites, could be detected in non-infected tick DNA samples, spiked with T. annulata genomic DNA. Hyalomma dromedarii ticks, fed on a calf experimentally infected with T. annulata, were used to validate the PCR further. The infection rate in the adult ticks, fed as nymphs during the febrile reaction, was high (62%), dropped to zero for 1 day in tick batches that engorged after treatment with ButalexTM and increased to 30% 2 days later and 38% of the ticks acquired the infection after feeding as nymphs during a carrier state piroplasm parasitaemia of less than 0.1%. As an internal control, 16S tick rDNA sequences could be amplified from T. annulata-negative tick samples. Finally, 202 adult ticks from Mauritania, collected from zebu cattle carrying low levels of Theileria piroplasms, were tested by the PCR. Thirty-eight out of 52 (73%) and 17 out of 30 (57%) H. dromedarii from the Gorgol and Trarza regions, respectively and two out of 30 (7%) Hyalomma marginatum rufipes from the Gorgol region were positive. Hyalomma marginatum rufipes, Rhipicephalus evertsi evertsi and Rhipicephalus guilhoni from the Trarza region were negative. These findings confirm that H. dromedarii is the main vector of T. annulata in Mauritania and that the PCR is a useful method of determining the infection rates in ticks collected from cattle carrying low levels of T. annulata piroplasms.     

Giardia in beaver (Castor canadensis) and nutria (Myocastor coypus) from east Texas

We examined the prevalence of Giardia sp. infection in nuisance beaver (Castor canadensis) and nutria (Myocastor coypus) in east Texas. From October 1992 through September 1993, 100 beaver and 30 nutria were collected in routine wildlife management activities conducted by the U.S. Department of Agriculture-Animal and Plant Health Inspection Service-Texas Animal Damage Control Service. Fecal and duodenal mucoid samples were preserved from each animal. Fecal samples were examined for the occurrence of Giardia sp. cysts using the Merifluor immunoassay detection kit: 30 beaver (30.0%) and 20 nutria (66.7%) were positive for Giardia sp. Duodenal mucoid samples were examined for Giardia sp. trophozoites using trichrome staining. with 26 beaver (26.0%) and 20 nutria (73.3%) testing positive. Combining both techniques resulted in 33 beaver (33.0%) and 22 nutria (73.3%) testing positive for Giardia sp. We found no relationship between Giardia sp. and host age, sex, river system, habitat, county, or season in beaver. However, a relationship was found when season and habitat were considered together. No relationship was found between Giardia sp. and age, river system, habitat, county, or season in nutria: however, more males (87.5%) were infected than females (46.4%).     

Helminth parasites of unisexual and bisexual whiptail lizards (Teiidae) in North America. V. Mesocestoides sp. tetrathyridia (Cestoidea: Cyclophyllidea) from four species of Cnemidophorus

Two hundred and one whiptail lizards, Cnemidophorus spp., from Texas and Colorado (USA), were examined for Mesocestoides sp. tetrathyridia. Eleven (5%) were infected, including three of 58 (5%) C. dixoni, six of 70 (9%) C. gularis septemvittatus, one of 35 (3%) C. marmoratus, and one of 34 (3%) C. tesselatus; four C. inornatus heptagrammus were not infected. In addition, 41 non-cnemidophorine lizards from the same study area were not infected. Free tetrathyridia were found in the body cavity of lizards and encapsulated tetrathyridia were observed in the heart, liver, stomach, mesenteries, ovaries, intestines, and lungs. None of the Mesocestoides sp. exhibited any evidence of asexual proliferation such as multiple scoleces or buds. This note, the fifth in a series of reports on helminths of Cnemidophorus spp., represents the first time Mesocestoides sp. has been reported from these four taxa, and Colorado is a new geographic locality record for this parasite.