Phylogeny of sheep and goat<Emphasis Type="Italic"> Theileria</Emphasis> and<Emphasis Type="Italic"> Babesia</Emphasis> parasites

The phylogenetic relationship of Theileria and Babesia species infecting sheep and goats on the basis of their 18S RNA gene structure was addressed in the present study. For this purpose, the complete sequences of the small ribosomal RNA genes of a panel of sheep and goat piroplasm isolates, including T. lestoquardi, T. ovis, T. separata, B. ovis, B. motasi, B. crassa and several novel species, were sequenced and compared. The classification based on the established phylogenetic tree corresponded with traditional systematics and revealed that sheep/goat piroplasm species are of polyphyletic origin. The independent evolution of almost all sheep/goat piroplasms suggests that speciation may have occurred after transfer of the piroplasm-transmitting tick from a primal wild ruminant host to domestic sheep and goats. In accordance with recent reports, our study confirms the existence of at least two additional sheep/goat piroplasm species, designated Theileria sp. 1 (China) and Theileria sp. 2 (China). The recently reported pathogenic sheep/goat Theileria sp. 1 (China) seems to be identical with a Theileria sp. isolated from Japanese serow. Furthermore, our results suggest that T. ovis represents a single species.     

Babesia ovis as the main causative agent of sheep babesiosis in Iran

Babesiosis is a haemoparasitic disease with high economical losses in livestock industry worldwide. The early diagnosis and successful therapy of babesiosis belong to the key steps of control and health management of livestock. Ethanol-fixed blood samples of 400 sheep were analyzed for Babesia infection. Reverse line blot (RLB) was established specifically for Theileria lestoquardi, Theileria (China 1), Theileria (China 2), Theileria ovis, Theileria separata, Babesia ovis, Babesia motasi, Babesia crassa, and Babesia (Lintan). The DNA was extracted from the ethanol-fixed blood samples and amplified with a common primer pair derived from 18S rRNA gene, amplifying both Theileria spp. as well as Babesia spp. Regarding the differences in the length of nucleotide sequences of the polymerase chain reaction (PCR) products obtained from Theileria spp. and Babesia spp., the PCR products derived from Babesia spp. were out screened and analyzed by RLB. The RLB analysis showed that 28 samples within the 400 blood samples were B. ovis positive. No B. motasi, B. crassa, or Babesia (Lintan) could be detected. The sequence analysis of one PCR product as a representative for other B. ovis-positive PCR products confirmed the results of RLB. Our results and the results of other investigators showed that B. ovis could be considered as a main causative agent of sheep babesiosis in Iran. Furthermore, our results also showed that RLB can be used as a reliable method for a simultaneous differentiation of Theileria and Babesia species from each other.     

Simultaneous detection and differentiation of<Emphasis Type="Italic"> Theileria</Emphasis> and<Emphasis Type="Italic"> Babesia</Emphasis> parasites infecting small ruminants by reverse line blotting

Characteristic sequence signatures were identified within the hypervariable region 4 (V4 region) of the small ribosomal RNA gene of ovine/caprine piroplasm species including Theileria lestoquardi, T. ovis , T. separata , Babesia ovis , B. motasi , B. crassa [comprising strains B. crassa (Iran) and B. crassa (Turkey)] and several novel species: Theileria sp. 1 (China), Theileria sp. 2 (China) and Babesia sp. (China), [comprising strain Babesia sp. (Lintan), and Babesia sp. (Ningxian)] as defined previously. Based on the ascertained gene variations a reverse line blotting (RLB) assay was developed enabling direct, concurrent, highly specific and sensitive identification of virtually all presently known ovine/caprine piroplasm species. All probes bound to their respective target sequence only, therefore, no cross-reaction was observed resulting in clear recognition of either individual strains, species or groups. No signal was observed when ovine and caprine genomic DNA was used as the control, demonstrating that the signals are due to the presence of parasite DNA in investigated samples. Furthermore, the sensitivity of RLB could be considerably enhanced to detect a parasitemia level of at least 10^-12% by reamplification of PCR products (nested PCR) thereby substantially increasing the possibility of identifying carrier animals.     

Detection and differentiation of ovine Theileria and Babesia by reverse line blotting in China

A reverse line blot (RLB) assay was developed for detection and specific identification of the different ovine Theileria and Babesia parasites. In a polymerase chain reaction (PCR), the hypervariable region 4 (V4 region) of the 18S ribosomal DNA gene was amplified with a set of general primers specific for members of the genera Theileria and Babesia. Meanwhile, specific oligonucleotide probes were designed and bound on membrane. After one single-PCR amplification, the amplified fragment was hybridized against different generic and species-specific probes. It was able to detect four species, i.e., Babesia motasi (Chengde, Lintan, Ningxian, Tianzhu), Babesia sp. (Kashi), Theileria luwenshuni (Lintan, Madang, Ningxian), Theileria uilenbergi (Longde, Zhangjiachuan) as defined previously. All probes bound to their respective target sequence only; therefore, no cross-reaction was observed, resulting in clear recognition of either individual strains, species, or groups in normal positive tests. Meanwhile, no signal was observed when ovine genomic DNA and water were used as a control, demonstrating that the signals are due to the presence of parasite DNA in the samples. Furthermore, the sensitivity of RLB could be considerably enhanced to detect a parasitemia level between10⁻³% and 10⁻⁸%. Finally, 117 samples from field were tested with RLB, PCR, and enzyme-linked immunosorbent assay (ELISA). The positive rate of RLB was higher than that of PCR and ELISA, and furthermore, RLB could determinate the species of piroplasms, the samples were infected with. Samples, 1,117, from five areas in Gannan Tibet Autonomous Region have been examined with RLB assay and compared with ELISA assay for corresponding samples. The results showed that the positive rate of RLB was higher than that of ELISA test obviously, and both T. luwenshuni and T. uilenbergi were widely distributed in these areas. RLB developed here could be used for differentiation of Babesia and Theileria infection and for epidemiological survey, which was difficult to achieve by classical methods. In conclusion, the RLB is a versatile technique for simultaneous detection and identification of all ovine piroplasms.     

A study on ovine tick-borne hemoprotozoan parasites (Theileria and Babesia) in the East Black Sea Region of Turkey

In this study, the frequency of Theileria and Babesia species was assessed via reverse line blotting and blood smear-based diagnostic methods in small ruminants. A total of 201 apparently healthy animals from 26 randomly selected herds located in 4 locations (Artvin, Giresun, Gumushane, and Tokat) of East Black Sea Region of Turkey were investigated for the blood protozoans. In a polymerase chain reaction (PCR), the hypervariable V4 region of the 18S ribosomal RNA gene was amplified with a set of general primers specific for all Theileria and Babesia species. The PCR products were hybridized against catchall and species-specific (Theileria spp., Theileria lestoquardi, Theileria ovis, Theileria sp. OT1, Theileria sp., OT3, Theileria sp., MK, Theileria luwenshuni, Theileria uilenbergi, Babesia spp., Babesia ovis, Babesia motasi, and Babesia crassa) probes. Theileria piroplasms were identified in nine (4.47%) samples by microscopic examination. Reverse line blotting (RLB) detected the infection in 19.90% of the samples. The infection rate of sheep (28.90%) was higher than goats (4.10%). T. ovis, Theileria sp., MK, and Theileria sp. OT3 were detected by RLB. The most prevalent Theileria species was T. ovis (18.90%) followed by Theileria sp. MK (0.99%). Theileria sp. OT3 was detected in one sample (0.43%). A single animal was infected as mix with T. ovis and Theileria sp. MK. The other Theileria (T. lestoquardi, Theileria sp. OT1, T. luwenshuni, and T. uilenbergi) and Babesia (B. ovis, B. motasi, and B. crassa) species were not detected. This study is the first molecular survey on ovine tick-borne protozoans in East Black Sea Region of Turkey.     

Multiplex PCR for diagnosis of Theileria uilenbergi,Theileria luwenshuni,and Theileria ovis in small ruminants

Infections with Theileria sp. may cause significant economic losses to the sheep industry. Species identification based on microscopic examination is difficult, and more suitable methods are required for the rapid detection and identification of Theileria sp, in clinical specimens. In this study, a multiplex polymerase chain reaction (mPCR) assay was developed to simultaneously identify three individual Theileria species in small ruminants. Three pairs of specific, sensitive primers were designed on the basis of the 5.8S ribosomal RNA gene (Theileria luwenshuni and Theileria ovis) and the 18S ribosomal RNA gene (Theileria uilenbergi) to generate target products of 303, 884, and 530 bp, respectively. Standard DNA for each of the three species was extracted from blood recovered from infected sheep, and a preliminary study was conducted on 56 sheep to verify the reliability of the system. Optimal PCR conditions, including primer concentration, annealing time, and the number of amplification cycles, were established. The assay sensitivity under these conditions was 10^?3 % parasitemia, and its specificity was 100 %. The results of the study suggest that mPCR represents a simple, efficient test method as a practical alternative for the rapid detection and identification of Theileria species in small ruminants.     

Determination of prevalence and risk factors for infection with <Emphasis Type="Italic">Babesia ovis</Emphasis> in small ruminants from Turkey by polymerase chain reaction

In this study, PCR and thin blood smear-based diagnostic methods were used to assess the frequency of Babesia infection in small ruminants. A total of 300 sheep and 100 goats from 37 randomly selected herds located in eight locations of eastern Turkey were examined for the presence of Babesia infection and any tick species on the body of the animals. Of 400 blood samples examined, 6 (1.5%) were positive for Babesia spp. piroplasms upon microscopic examination, whereas 33 (8.25%) were positive for the presence of B. ovis by PCR. The prevalence of babesiosis in small ruminants detected by PCR was significantly higher than obtained in microscopic examination of thin blood smears (P < 0.05). Thirty-three animals produced the DNA fragment specific for Babesia ovis of which 32 (10.66%) were sheep and 1 (1%) was goat. The difference between the prevalence of Babesia infection in sheep and goats were statistically significant (P < 0.05). The prevalence of Babesia infection in different age groups of sheep were statistically non-significant (P > 0.05). The frequency of B. ovis infection was higher in herds with tick burden than no tick burden (P < 0.05). Seven amplicons (six from sheep and one from goat) were sequenced. The resulting sequences were identical to the recently reported nucleotide sequence of B. ovis. A total of 510 ticks belonging to the Rhipicephalus spp. were collected from sheep. Ticks were identified to be R. bursa and R. turanicus on the basis of morphological features. Nucleotide sequences data reported in this paper are available in EMBL, GenBank and DDJB databases under the accession numbers: DQ409332, DQ409333, DQ409334, DQ409335, DQ409336, DQ409337, DQ409338.     

Evaluation of a PCR and comparison with RLB for detection and differentiation of Theileria sp. MK and other Theileria and Babesia species of small ruminants

Theileria sp. MK in sheep and goats were detected first time by polymerase chain reaction (PCR) and detection limit of PCR and reverse line blotting (RLB) were compared. A part of 18S ssu rRNA gene was amplified from blood samples that were taken from sheep and goats naturally infected with Theileria sp. MK by PCR. Detection limit of both PCR and RLB methods was one infected cell in 10(7) sheep erythrocytes. Nine hundred twenty field samples that had been tested previously by RLB were evaluated by the PCR assay. As found by RLB previously, 12 of 920 (1.30%) samples were detected as positive by PCR. Two positive PCR products, one of which was from sheep and the other from goat, were sequenced. These sequences were identical to the reported nucleotide sequence of Theileria sp. MK. It is concluded that the PCR described in this study will be useful for epidemiological studies and for discrimination between Theileria sp. MK and other Theileria species. In addition, PCR has superiority over RLB because of its ease of use and time period required.     

Experimental transmission of Theileria sp. (China 1) infective for small ruminants by Haemaphysalis longicornis and Haemaphysalis qinghaiensis

The experimental transmission of a recently identified new Theileria (China 1) species pathogenic for sheep and goats in northern China is described. Haemaphysalis qinghaiensis nymphs and adults developed from larvae and nymph engorged on sheep infected with Theileria sp. (China 1) were able to respectively transmit the Theileria sp. to splenectomized sheep. Meanwhile, H. longicornis nymphs and adults developed from larvae and nymphs engorged on sheep infected with Theileria sp. (China 1) were also able to respectively transmit this new Theileria sp. (China 1) to splenectomized sheep. These experiments suggested that the Theileria sp. (China 1) could be transmitted by at least two species of Haemaphysalis sp. ticks, H. longicornis and H. qinghaiensis, and the mode of transmission is stage to stage.     

Relationship of faecal egg count with packed cell volume and anaemia in Sahel sheep and goats in semi-arid northeastern Nigeria

Sahel sheep and goats, under extensive or semi-intensive management in smallholder farms in semi-arid northeastern Nigeria (Maiduguri), were evaluated to determine the relationship between faecal egg count (FEC) and packed cell volume (PCV) with associated anaemia and whether concurrent haemoparasitic infections would confound the variables. Faecal and anticoagulated blood samples from Sahel sheep (n?=?227) and goats (n?=?415) were collected January–December 2006, after conjunctival colour was estimated as FAMACHA score (FS) as follows: red (1), red–pink (2), pink (3), pink–white (4) or white (5). Presumed level of helminth infection, based on FEC with modified McMaster technique, was graded as mild (<600/g), moderate (600–1,500/g) or severe (>1,500/g). Out of 642 animals, 276 (43.0 %) had helminth infection based on FEC. More goats (11.5 %) were severely infected than sheep (1.3 %). PCV was decreased by only severe helminth burden. FS correlated with PCV (r?=??0.69, ?0.86; p?Babesia, 0.8 %; Anaplasma, 5.6 %) was observed in 16 (2.5 %) animals and did not have confounding effects on FEC and PCV. This report has provided preliminary information needed for further evaluation of the diagnostic criteria for helminthiasis and the associated anaemia in a semi-arid environment.     

Serological survey of antibodies to Toxoplasma gondii in cats,goats, and sheep in Kerman,Iran

The present study was carried out to determine, the presence of antibodies to toxoplasmosis in cats as reservoirs and in sheep and goats as meat sources in this area. Serum samples from 108 stray cats, 114 goats, and 90 sheep were tested for antibody to Toxoplasma gondii by indirect fluorescent antibody test (IFAT). Antibodies were found in 2.7 % of cats, 1.7 % of goats, and 3.3 % of sheep, at a dilution of ≥1:16 and more. T. gondii was isolated by bioassay in mice in two (66.6 %) of three brain samples, of IFAT-positive cats. There were no statistically significant differences in the presence of antibodies based on the gender of the cats, goats, and sheep (p?>?0.05). In conclusion, the study revealed that cats can be significant reservoirs for the spread of the disease in this region.     

Babesia lengau sp. nov., a Novel Babesia Species in Cheetah (Acinonyx jubatus,Schreber, 1775) Populations in South Africa

In a previous paper, we reported on a large number of cheetah blood specimens that gave positive signals only for Babesia and/or Theileria genus-specific probes on the reverse line blot (RLB) assay, indicating the presence of a novel species or variant of an existing species. Some of these specimens were investigated further by microscopic, serological, sequencing, and phylogenetic analyses. The near-full-length 18S rRNA genes of 13 samples, as well as the second internal transcribed spacer (ITS2) region, were amplified, cloned, and sequenced. A species-specific RLB probe, designed to target the hypervariable V4 region of the 18S rRNA gene for detection of the novel Babesia sp., was used to screen an additional 137 cheetah blood specimens for the presence of the species. The prevalence of infection was 28.5%. Here we describe the morphology and phylogenetic relationships of the novel species, which we have named Babesia lengau sp. nov.The cheetah (Acinonyx jubatus, Schreber, 1775) is regarded as a vulnerable species according to the 2008 World Conservation Union (IUCN) Red List of Threatened Species (http://www.iucnredlist.org) and is listed in Appendix I (which includes species that are most threatened) of the Convention of International Trade in Endangered Species (CITES) (9). This is mainly because of loss of habitat in the wild and conflicts with farmers in remaining habitats (23). Between 12,000 and 15,000 cheetahs remain in the wild, mostly in isolated populations in 24 to 26 countries in Africa. Free-ranging cheetahs still inhabit a broad section of Africa, including areas of North Africa, the Sahel, and East and southern Africa. The farmlands of north-central Namibia have the largest free-ranging population, of about 300 animals (16, 24). The Asiatic cheetah (Acinonyx jubatus venaticus), found only in the Kavir Desert region of Iran, is critically endangered, with an estimated population of ca. 50 mature individuals (IUCN).Both large (>2.5 μm) and small (<1.5 μm) intraerythrocytic piroplasms have been reported from a variety of domestic and wild felid species from several continents (17). Small piroplasms reported from felids include Babesia felis (12), reported for African wild cats (Felis sylvestris ocreata) and caracals (Felis caracal) (38) in Africa; Babesia leo, reported for lions (Panthera leo) in South Africa (38) and Tanzania (32); and Cytauxzoon felis, reported for domestic cats and bobcats (Lynx rufus) (15, 45), mountain lions (Felis concolor), and Florida panthers (Felis concolor coryi) (7, 41) in North America.Records of Babesia parasites in wild cheetahs are surprisingly rare, with reports of occurrence of piroplasms in three animals in the Serengeti National Park in Tanzania (36) and South Africa (6). Theileria-like piroplasms have also been reported for cheetahs in the Serengeti National Park and Ngorongoro Crater, Tanzania (2). Babesia felis and B. leo have been reported from cheetahs in southern Africa, but not as mixed infections (6). Although the prevalence of infection with these two species in free-ranging cheetahs in Namibia was low (7.5%), a large number (52.9%) of cheetahs were infected with an as yet undescribed Babesia sp. (6). The aim of the current study was to characterize this undescribed Babesia sp. by phylogenetic analysis based on sequences from the 18S rRNA gene and the second internal transcribed spacer region (ITS2).     

Identification of different Theileria species (Theileria lestoquardi, Theileria ovis, and Theileria annulata) in naturally infected sheep using nested PCR-RFLP

Ovine theileriosis is an important hemoprotozoal disease of sheep and goats in tropical and subtropical regions that leads to economic losses in these animals. A nested PCR–restriction fragment length polymorphism (RFLP) was carried out to identification Theileria species in sheep in some area in western half of Iran (Sari, Rasht, Urmia, Ilam, and Ahvaz). Two hundred and fifty blood samples were taken from sheep during tick activating season (summer of 2008). Microscopic examination revealed that 9.2% (23/250) sheep were infected by Theileria spp. piroplasms. Parasitemia ranged from 0.011% to 0.015%. In nested PCR assessment of DNA samples, 32.8% (82/250) sheep were positive. The negative samples were confirmed by amplifying of ovine beta-actin gene as an internal control. The differentiation of Theileria species was based on RFLP patterns using three restriction enzymes: HpaII, Rsa1, and Bsh 1285I. Out of 82 positive samples, 54.8% (45/82) and 40.2% (33/82) were positive for Theileria lestoquardi and Theileria ovis respectively. Mixed infection was detected in 4.8% (4/82) cases. Based on their PCR product digestion pattern with HpaII (1178, 900, 278, and 106 bp), it seemed to be mixture of Theileria annulata and T. lestoquardi. The presence of T. annulata was supported by sequence analysis. This is the first report of naturally infected sheep with T. annulata in Iran. Geographical distribution of Theileria species in sheep is shown according to the result of microscopy and nested PCR and RFLP data.     

The role of acute phase cytokines in the recovery and disease progress of Theileria annulata-infected cattle

The present study was conducted to evaluate the cytokine response following natural infection of Theileria annulata in cattle. Initial survey included 173 crossbred cattle which were examined for the presence of Theileria piroplasms. The investigated cattle were clinically and parasitologically examined. Blood samples were collected from all examined cattle for microscopic examination, PCR assays (using primers of Theileria spp., Babesia spp., and T. annulata), and cytokines measurement. It was found that 38 cattle were positive for the presence of at least one species of Theileria; meanwhile Babesia piroplasms were not detected either by microscopy or PCR assay. When T. Annulata-specific primers were used, 33 gave positive results. Twenty-two out of 33 T. annulata-infected cattle were only included in this study together with contemporaneous controls (n?=?10). According to the severity of clinical signs, T. annulata-infected cattle were categorized into two groups; group 1 included ten cattle with mild clinical signs and group 2 included 12 cattle with overt clinical signs. Biochemically, tumor necrosis factor alpha, interleukin (IL)-1β, IL-6, IL-12, haptoglobin, and Fb were significantly higher (p?T. annulata infection provide a valuable and quantitative assessment of the response to infection. It seems likely that the pro-inflammatory cytokines play an important role in the host response to T. annulata. Our findings suggest that the pro-inflammatory cytokines are suitable markers of inflammatory reactions in T. annulata-infected cattle.     

Phylogenetic analysis of<Emphasis Type="Italic"> Theileria</Emphasis> species transmitted by<Emphasis Type="Italic"> Haemaphysalis qinghaiensis</Emphasis>

The phylogenetic relationships between six isolates of Theileria spp. infective to small ruminants, and two isolates of Theileria spp. infective to yak, all transmitted by Haemaphysalis qinghaiensis, together with the Theileria orientalis/sergenti/buffeli group and T. sinensis, were analyzed using the 18S ssrRNA gene sequence. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was used either for direct sequencing or was ligated to the PCR II vector for sequencing. The length of the 18S ssrRNA gene of all Theileria spp. involved in this study was around 1,740 bp. Two phylogenetic trees were inferred based on the 18S ssrRNA gene sequence of the Chinese isolates only, and Chinese isolates and other species of Theileria available in GenBank. In the first tree, the Theileria sp. infective to yaks was found to be T. sinensis. The Theileria sp. infective to small ruminants was found to be composed of two separate species of Theileria. Theileria sp. from Qinghai, Madang, Ningxian and Lintan, which was identical to the unidentified Theileria sp. described previously, is designated Theileria sp (China 1). The Theileria sp. from Longde, Zhangjiachuan and Lintan, which has not been described previously, is designated Theileria sp. (China 2) in order to avoid confusion. In the second tree, Theileria sp. (China 1) was closely related to benign Theileria, such as T. buffeli and T. sergenti, while Theileria sp. (China 2) was separated from other Theileria spp. The results indicate that H. qinghaiensis transmit at least three species of Theileria, two which are infective to sheep and goats, but not yak and one which is infective to yaks and cattle, but not to sheep and goats.     

Prevalence and diversity of Babesia,Hepatozoon, Ehrlichia,and Bartonella in wild and domestic carnivores from Zambia,Africa

A molecular survey was conducted for several hemoparasites of domestic dogs and three species of wild carnivores from two sites in Zambia. Three Babesia spp. were detected including Babesia felis and Babesia leo in lions (Panthera leo) and a Babesia sp. (similar to Babesia lengau) in spotted hyenas (Crocuta crocuta) and a single lion. All wild dogs (Lycaon pictus) and domestic dogs were negative for Babesia. High prevalences for Hepatozoon were noted in all three wild carnivores (38–61 %) and in domestic dogs (13 %). Significantly higher prevalences were noted in hyenas and wild dogs compared with domestic dogs and lions. All carnivores were PCR negative for Ehrlichia canis, Ehrlichia ewingii, and Bartonella spp. Overall, high prevalences and diversity of Babesia and Hepatozoon were noted in wild carnivores from Zambia. This study is the first molecular characterization of Babesia from any hyena species and is the first report of a Babesia sp. closely related to B. lengau, a parasite previously only reported from cheetahs (Acinonyx jubatus), in lions and hyenas. Although usually benign in wild carnivores, these hemoparasites can be pathogenic under certain circumstances. Importantly, data on vectors for these parasites are lacking, so studies are needed to identify vectors as well as determine transmission routes, infection dynamics, and host specificity of these hemoparasites in wildlife in Africa and also the risk of transmission between domestic animals and wildlife.     

Intramuscular and subcutaneous coenurosis in goats and sheep in south-east Iran

A 1-year study was conducted in a Kerman slaughterhouse over four seasons between 2009 and 2010. In this study, 66,114 slaughtered sheep and 47,805 slaughtered goats were carefully inspected for the presence of intramuscular or subcutaneous coenurosis cysts. Additionally, the skull of infected animals was also inspected for the presence of brain cysts. Suspected samples were transported in formalin to the food hygiene controlling lab. For each sample, characteristics of the coenurosis cyst such as number, size and scolex were determined. According to the results, 36 (0.03 %) carcasses were infected, 32 goats (0.07 %) and four sheep (0.01 %). There was a significant difference in size (length and width) of the cyst and number of scolex between goats and sheep (p?<?0.05). There was no significant difference in the prevalence rate of infected animals according to age and season (p?>?0.05). This is the first report of non-cerebral coenurosis in sheep in Iran.     

Ribosomal small-subunit RNA gene-sequence analysis of Theileria lestoquardi and a Theileria species highly pathogenic for small ruminants in China

A fatal disease of sheep and goats in the northwestern part of China has been reported to be due to Theileria lestoquardi (syn. T. hirci). However, some characteristics of the causative agent are not in accordance with attributes ascribed to this parasite. We therefore determined the nucleotide sequence of the small-subunit ribosomal RNA (srRNA) gene of T. lestoquardi and the parasite identified in China and compared it with that of other Theileria and Babesia species. In the inferred phylogenetic tree the srRNA sequence of the Chinese parasite was found to be most closely related to T. buffeli and clearly divergent from T. lestoquardi, suggesting that it is an as yet unrecognized Theileria species. Extensive structural similarities were observed between the srRNA sequences of T. lestoquardi and T. annulata, revealing a close phylogenetic relationship between these two Theileria species. On the basis of the srRNA nucleotide sequence, polymerase chain reaction (PCR) primers were designed that specifically amplified genomic DNA of the Chinese Theileria species. These primers may be valuable tools in future epidemiology studies. Received: 15 July 1999 / Accepted: 6 August 1999     

Molecular detection and characterization of Theileria species in the Philippines

Theileriosis is a tick-borne disease of domestic and wild animals that cause devastating economic loss in livestock in tropical and subtropical regions. Theileriosis is not yet documented in the Philippines as compared to babesiosis and anaplasmosis which are considered major tick-borne diseases that infect livestock in the country and contribute major losses to the livestock industry. The study was aimed to detect Theileria sp. at genus level in blood samples of cattle using polymerase chain reaction (PCR) assay. Specifically, it determined the phylogenetic relationship of Theileria species affecting cattle in the Philippines to other Theileria sp. registered in the GenBank. A total of 292 blood samples of cattle that were collected from various provinces were used. Theileria sp. was detected in 43/292 from the cattle blood samples using PCR assay targeting the major piroplasm surface protein (MPSP) gene. DNA sequence showed high similarity (90–99%) among the reported Theileria sp. isolates in the GenBank and the Philippine isolates of Theileria. Phylogenetic tree construction using nucleotide sequence classified the Philippine isolates of Theileria as benign. However, nucleotide polymorphism was observed in the new isolate based on nucleotide sequence alignment. It revealed that the new isolate can be a new species of Theileria.     

Development of a recombinant indirect ELISA for the diagnosis of Theileria sp. (China) infection in small ruminants

Theileria sp. (China) causes severe limitations on the development of the livestock industry in the north-west of China. An enzyme-linked immunosorbent assay (ELISA) based on merozoite homogenate of the parasite for diagnosis of infection has been established; however, cross-reactivity with other small ruminant-infecting piroplasms could not be excluded. Thus, a prerequisite for epidemiological surveys and diagnosis was the establishment of a recombinant protein-based ELISA. To this end, serum from Theileria sp. (China)-infected sheep was used to screen a Theileria lestoquardi expression library, resulting in the identification of a specifically reacting clone with a high identity to the heat shock protein 70 (HSP70) of Theileria parva and Theileria annulata and thus named TlHSP70. An HSP70 homologue was also confirmed to be expressed by Theileria sp. (China) merozoites (TcHSP70). A part of the TlHSP70 protein, found to be conserved in TcHSP70, was recombinantly expressed and used to establish an ELISA. A total of 260 field serum samples tested resulted in a sensitivity and specificity of 94.3 and 89.5%, respectively, in comparison with the merozoite homogenate ELISA. The potentials of the application of the test in epidemiological surveys to map out the prevalence of the disease and for routine diagnostics are described.