北京市房山区羊梨形虫病流行病学调查

为了解北京郊区羊梨形虫病的流行情况,本研究采用形态学检测,同时结合使用梨形虫18S rRNA基因通用引物进行PCR与直接测序法,对来自北京市房山郊区某养殖场的120只山羊及15只绵羊全血样本进行检测。结果发现血涂片中梨形虫检测阳性率为95．0％,其中吕氏泰勒虫Theileria luwenshuni于山羊中检出115例阳性,阳性率95．8％;于绵羊中检出6只阳性;未发现其他泰勒虫以及巴贝斯虫的感染。本研究首次对北京周边地区进行羊梨形虫分子流行病学研究,发现房山地区为吕氏泰勒虫流行区,并且发现山羊对吕氏泰勒虫可呈现长期带虫感染。     

Ovine theileriosis in China: a new look at an old story

A fatal disease of sheep and goats in the northwestern part of China has in the past been reported to be due to Theileria lestoquardi. 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 18 small subunit ribosomal RNA (18S rRNA) 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 18S rRNA sequence of the Chinese parasite falls inside the clade consisting of Theileria species evidencing that it belongs to this genus. The 18S rRNA sequence of the Chinese parasite was found to be most closely related to Theileria buffeli and clearly divergent to T. lestoquardi, suggesting that it was a yet unrecognized Theileria species. The phylogenetic relationship of Theileria species infecting sheep and goats on the basis of their 18S rRNA gene structure was addressed. We report on the existence of at least two additional ovine and caprine piroplasm species, designated T. luwenshuni and T. uilenbergi.     

Development and evaluation of a loop-mediated isothermal amplification method for rapid detection of Anaplasma ovis

Anaplasma ovis is an intraerythrocytic rickettsial pathogen of small ruminants. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection method in which the target DNA can be efficiently amplified with high specificity and sensitivity under isothermal conditions. In this study, a LAMP method was developed for the specific detection of A. ovis, using LAMP primers designed on the basis of the major surface protein 4 gene. LAMP was performed at 65 °C for 30 min. Its specificity was confirmed by successful amplification of several A. ovis isolates and through EcoRI restriction analysis of LAMP products. No cross-reactivity with the A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi, or the Babesia sp. Xinjiang isolate was observed. Detection using the LAMP method was compared with that using conventional PCR in 227 field samples; LAMP demonstrated a sensitivity of 95.45%. In summary, LAMP is a specific, sensitive, and rapid test for the diagnosis of A. ovis infection, with the potential to be standardized as a detection method for A. ovis in areas of endemicity.     

Molecular identification of Theileria and Babesia in sheep and goats in the Black Sea Region in Turkey

This study was carried out to investigate presence and distribution of Theileria and Babesia species via microscopic examination and reverse line blotting (RLB) techniques in sheep and goats in the Black Sea region of Turkey. For this purpose, 1,128 blood samples (869 sheep and 259 goats) were collected by active surveillance from sheep and goats in different provinces of various cities in the region in the years 2010 and 2011. Smears were prepared from the blood samples, stained with Giemsa, and examined under the light microscope for Theileria and Babesia piroplasms. The genomic DNAs were extracted from blood samples. The length of 360–430-bp fragment in the variable V4 region of 18S SSU rRNA gene of Theileria and Babesia species was amplified using the gDNAs. The polymerase chain reaction products were hybridized to the membrane-connected species-specific probes. A total of 38 animals (3.37 %) including 34 sheep (3.91 %) and 4 goats (1.54 %) were found to be positive for Theileria spp. piroplasms in microscopic examination of smears while Babesia spp. piroplasm could not detected. Infection rates were 34.64 % in sheep, 10.04 % in goats, and totally 28.99 % for Theileria ovis while 0.58 % in sheep and totally 0.44 % for Babesia ovis. However, Theileria sp. OT3 was detected in 2.65 % of sheep and 2.04 % of all animals; besides Theileria sp., MK had 0.58 % prevalence in sheep and 0.77 % in goats, with a total 0.62 % with RLB. Although T. ovis and Theileria sp. MK were determined in both sheep and goats, B. ovis and Theileria sp. OT3 were observed only in the sheep. These results provide the first detailed molecular data for sheep and goat theileriosis and babesiosis in the region.     

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.     

Ticks of small ruminants in China

The importance of ticks and tick-borne diseases of small ruminants in China is discussed. Of the 109 species of ticks identified to date in China, 45 species infest small ruminants. Five species have been proved to be involved, or possibly involved, in the transmission of tick-borne diseases. Anaplasma ovis, Babesia motasi, Babesia ovis and two unidentified species of Theileria, have been recorded in small ruminants in China. The diseases caused by these organisms are widespread in China, causing great economic losses, estimated at approximately 70 million USD per annum. Anaplasmosis occurs from September to March in Inner Mongolia and during spring in other areas. Babesiosis and theileriosis occur in March to June in northwestern China. The vectors of A. ovis are Dermacentor nuttalli, Hyalomma asiaticum and Rhipicephalus pumilio. These three species of ticks do not appear to transmit A. ovis transstadially or transovarially, but rather through movement of partially engorged, infected adult ticks from A. ovis carrier animals. The vector ticks of the two species of Babesia have not been very well documented, but at least two species of Haemaphysalis are thought to transmit them. Haemaphysalis qinghaiensis transmits the two as yet unidentified species of Theileria transstadially. Priorities for future research on these diseases are summarised.     

Identification of a novel Babesia sp. from a sable antelope (Hippotragus niger Harris, 1838)

Babesiosis in a sable antelope (Hippotragus niger Harris, 1838) was first reported in 1930; the parasite was named Babesia irvinesmithi. Recently, specimens from an adult sable that presented with a sudden onset of disease and that subsequently died during immobilization were submitted for molecular characterization. Microscopic examination of thin blood smears revealed the presence of small piroplasms. DNA was extracted from blood samples; the V4 variable region of the 18S rRNA gene was amplified and analyzed using the reverse line blot (RLB) assay. Amplicons did not hybridize with any of the Babesia or Theileria species-specific probes present on the blot and hybridized only with a Babesia or Theileria genus-specific probe, suggesting the presence of a novel species. The full-length 18S rRNA gene sequence was obtained and aligned with published sequences of related genera, and phylogenetic trees were constructed. Sequence similarity analyses indicated that a Babesia species, designated Babesia sp. (sable), was present. The sequence showed its highest similarity to B. orientalis and to an unnamed Babesia species previously detected in bovine samples. The latter was later established to be Babesia occultans. A Babesia sp. (sable)-specific RLB oligonucleotide probe was designed and used to screen 200 South African sable samples, but so far, no other sample has been found to be positive for the presence of Babesia sp. (sable) DNA. In summary, we identified a novel piroplasm parasite from a sable antelope that died from an unknown illness. While the parasite was observed in blood smears, there is no direct evidence that it was the cause of death.     

Simultaneous Detection of Bovine Theileria and Babesia Species by Reverse Line Blot Hybridization

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of Theileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to the T. sergenti-T. buffeli-T. orientalis group. The Babesia species included were Babesia bovis, B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria and Babesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from Bos taurus or other hemoparasites (Trypanosoma species, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigemina were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.     

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.     

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.     

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.     

Detection of Theileria annulata in blood samples of carrier cattle by PCR.

We report the detection of Theileria annulata, the causative agent of tropical theileriosis, by PCR in blood samples obtained from carrier cattle. The assay employs primers specific for the gene encoding the 30-kDa major merozoite surface antigen of T. annulata. A 721-bp fragment was amplified from blood samples taken monthly from calves experimentally infected with one of four different stocks of T. annulata originating in either Mauritania, Portugal, Spain, or Turkey. At the end of the experiment, five animals carried the infection for 12 months and two animals remained infected for 15 months. DNAs from six other Theileria species, T. parva, T. mutans, T. sergenti, T. buffeli, T. velifera, and T. taurotragi, were not amplified. Moreover, DNAs from four other hemoparasites (Anaplasma centrale, Anaplasma marginale, Babesia bovis, and Babesia bigemina) were also not amplified. As a control, primers derived from the small subunit rRNA gene of Theileria spp. amplified a 1.1-kb DNA fragment from all Theileria species examined but not from the other four hemoparasites. As few as two to three parasites per microliter of infected blood in a 50-microliters sample volume were detected by Southern or microplate hybridization with a T. annulata-specific cDNA probe. In addition, 92 field samples obtained from cattle in Spain were tested; 22% were positive in blood smears, 40% were positive by immunofluorescent antibody test, and 75% were positive for T. annulata by PCR. The method provides a useful diagnostic tool for detecting T. annulata carrier cattle.     

Babesia bicornis sp. nov. and Theileria bicornis sp. nov.: tick-borne parasites associated with mortality in the black rhinoceros (Diceros bicornis)

A novel Babesia species, designated Babesia bicornis sp. nov., was identified in three black rhinoceroses (Diceros bicornis) that died in wildlife areas in Tanzania and South Africa. Screening of black rhinoceroses in South Africa revealed, in addition to B. bicornis, a second parasite, designated Theileria bicornis sp. nov.     

The use of different diagnostic tools for Babesia and Theileria parasites in cattle in Menofia, Egypt

Bovine piroplasmosis is caused by tick-borne hemoprotozoans of the genera Babesia and Theileria and is the most prevalent in tropical and subtropical countries, causing a major economic impact worldwide. In the current study, a total of 405 cattle of different ages, sexes, and breeds were randomly sampled for surveying and diagnosis of babesiosis and theileriosis using three methods: direct microscopy (blood smears), indirect fluorescent antibody test (IFAT) and polymerase chain reaction (PCR). Giemsa-stained blood smears revealed that, out of 405 examined cattle, 33 (8.15?%) were infected with Babesia sp. and 65 (16.05?%) with Theileria sp. (total number of infected cattle was 98). Mixed infection was seen in 11 (2.72?%) animals. Moreover, application of the three diagnostic assays on 158 randomly sampled cattle indicated that 17 (10.76?%) and 33 (20.89?%) were positive for Babesia and Theileria spp. by the direct smear technique, 25 (15.82?%) and 33 (20.89?%) by IFAT (fluorescence was greenish yellow for Babesia and yellowish for Theileria), and 20 (12.66?%) and 38 (24.05?%) by PCR. Using primers specific for Babesia and Theileria spp., we found that diagnostic bands appeared at ~350 and ~370?bp, respectively indicating the presence of these piroplasms. Statistically, there was a non-significant difference of the positivity in response to the three techniques; thus, any of these methods can be described as useful for diagnosing blood parasites in both domesticated animals and birds. On the basis of the obtained results, it could be concluded that direct microscopy can be used in acute infections, whereas IFAT and PCR are useful in chronicity.     

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.     

Comparative studies on surface phenotypes of Theileria lestoquardi and T. annulata schizont-infected cells

Phenotypes of sheep cell lines infected with Theileria lestoquardi or T. annulata were studied by flow cytometric analysis, following immunolabelling with a panel of monoclonal antibodies reacting to leukocyte differentiation antigens. Cell surface phenotypes of Theileria-infected sheep cell lines derived ex vivo and in vitro were compared, both with each other and with cell lines from cattle undergoing acute T. annulata infection. Besides the non-lineage specific markers CD45, MHC class I and MHC class II, myeloid lineage-associated antigens and B cell-specific markers were expressed in all five different types of line, suggesting that both T. lestoquardi and T. annulata had infected the same cell types in sheep as T. annulata in cattle, notably monocytes/macrophages and B cells. Lineage-specific markers were generally expressed at low frequency and intensity; any differences between the five types of cell lines were quantitative, rather than qualitative. Thus, relative rather than absolute differences in cell preference of sporozoites of T. lestoquardi and T. annulata may contribute to the differences observed in previous studies in the course of the infection of sheep with each of these two parasites and in the infection of cattle with T. annulata.     

Development of specific immunoglobulin Ga (IgGa) and IgGb antibodies correlates with control of parasitemia in Babesia equi Infection

In this study, the kinetics of specific immunoglobulin G (IgG) isotypes were characterized in Babesia equi (Theileria equi)-infected horses. IgGa and IgGb developed during acute infection, whereas IgG(T) was detected only after resolution of acute parasitemia. The same IgG isotype profile induced during acute infection was obtained by equi merozoite antigen 1/saponin immunization.     

A molecular survey of <Emphasis Type="Italic">Theileria</Emphasis> and <Emphasis Type="Italic">Babesia</Emphasis> parasites in cattle,with a note on the distribution of ticks in Tunisia

Between October and November 2006, a total of 278 bovine blood samples were examined, and 104 (37.4%) were positive for piroplasms by microscopy. A reverse line blot hybridisation with polymerase chain reaction detected Theileria annulata, T. buffeli, Babesia bovis and B. bigemina in cattle accounting for 48.6% of positive samples. The most frequently found species was T. buffeli, which was present in 39.2% of the samples. T. annulata was found in 48 samples (17.3%). Babesia infections were less frequently detected: B. bovis was found in 6.8% of the samples and B. bigemina in 4.3%. Mixed infections were detected in 45 samples, accounting for seven different combinations of species. Seven Ixodid tick species (Boophilus annulatus, Ixodes ricinus, Hyalomma marginatum, Hyalomma excavatum, Hyalomma detritum, Haemaphysalis punctata and Haemaphysalis sulcata) were collected from examined cattle in the 23 visited farms. I. ricinus was the dominant species (36%), mainly collected in the humid zone, while it seemed to be very rare in the semi-arid zone (where only 15 specimens were collected), whereas B. annulatus was the most commonly collected species in the sub-humid area (68.5% of ticks collected in this zone).     

Epidemiological studies on tick-borne diseases of cattle in Central Equatoria State,Southern Sudan

A herd-based study was carried out in Central Equatoria State, Southern Sudan, to study epidemiological aspects of tick-borne diseases. Six herds of cattle situated in three different locations were selected and investigated every 3 months during the year 2005. Blood smears for Giemsa staining and blood spots on filter paper for deoxyribonucleic acid extraction were collected from 600 apparently healthy indigenous cattle. A total of 69 (11.5%) samples showed the presence of piroplasms in Giemsa-stained blood smears, and polymerase chain reaction increased the detection limit to 297 (49.5%). Using reverse line blot, it was possible to detect and differentiate eight different piroplasms namely, Theileria parva (71.2%), Theileria mutans (73%), Theileria velifera (45.3%), Theileria taurotragi (2.7%), Theileria buffeli (0.5%), Theileria annulata (0.2%), Babesia bovis (1.7%), and Babesia bigemina (0.3%). Mixed infections were detected in 406 samples (67.7%) accounting for 17 different combinations. High infection of Theileria parva was reported among young calves compared to older cattle. The highest prevalence of Theileria parva was reported in the rainy season (October). The implications of these results on the epidemiology of tick-borne diseases are discussed with emphasis on East Coast fever. Nucleotide sequences data reported in this paper are available in GenBank™ database under the accession numbers EF469603, EF469604, EF469605, EF469606, and EF469607.