Guiquan Guan

Phylogenetic analysis of Theileria species transmitted by Haemaphysalis qinghaiensis

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 1,740xa0bp. 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.

Transmission of Babesia sp to sheep with field-collected Haemaphysalis qinghaiensis

Abstract.Haemaphysalis qinghaiensis ticks collected in the Gannan Tibet Autonomous Region were infested onto a sheep from a Babesia-free area. A strain of small Babesia (1.8–2.1xa0µm in length) was isolated from the sheep. Most of the Babesia in erythrocytes were round, oval, single pyriform, double pyriform, budding or elongated in form. Measurements were made of 100xa0single sides of the double-pyriform Babesia and compared with those for B. motasi and B. ovis from Holland, using Students t-test. The Gannan small Babesia was similar to the B. ovis from Holland, but differed significantly from the Dutch B. motasi.

Transmission of an unidentified Theileria species to small ruminants by Haemaphysalis qinghaiensis ticks collected in the field

Abstract. The transmission of a recently identified Theileria species pathogenic for sheep and goats in northern China is described. Haemaphysalis qinghaiensis nymphs which had been collected from grass in epidemic areas were able to transmit this Theileria species to sheep. The pathogen was also transmitted to sheep and goats by three batches of adult ticks collected from grass, ticks collected when moving about on sheep and ticks which were found partially engorged on sheep or goats.

Detecting and differentiating Theileria sergenti and Theileria sinensis in cattle and yaks by PCR based on major piroplasm surface protein (MPSP).

Theileria sergenti and Theileria sinensis are closely related members of benign Theileria species found in cattle and yaks in China. They are morphologically indistinguishable. A polymerase chain reaction (PCR) targeting major piroplasm surface protein of T. sergenti and T. sinensis was developed in this study. The newly developed oligonucleotide primer set was able to specifically amplify the DNA of T. sinensis and in conjunction with primers for T. sergenti and these two species could be detected and distinguished. Specificity testing also revealed that there was no cross-reaction with the other tick-borne diseases Theileria annulata, Babesia ovata, Anaplasma marginale as well as bovine white blood cells. Phylogenetic analysis based on the MPSP gene sequences confirmed the specificity of PCR assays. The sensitivity of the methods was 0.1pg DNA for the T. sergenti PCR and 1pg DNA for T. sinensis PCR. Two hundred and thirty-six field blood samples from of cattle and yaks were collected from five different geographical regions in China where benign Theileria species have been found. T. sergenti was found in all five provinces but was absent from one county in Gansu Province. T. sinensis was only found in Gansu Province. In both counties in Gansu where the parasites co-existed, mixed infections were detected. Our results indicate that the PCR methods developed in this study are suitable for the detection and differentiation of T. sergenti and T. sinensis.

A new ovine Babesia species transmitted by Hyalomma anatolicum anatolicum

The pathogenicity and morphology of a large Babesia species, Babesia sp. Xinjiang, are described here. The parasite has very low virulence for sheep, and caused no detectable clinical symptoms. Splenectomized sheep infected with the parasite showed mild fever and low parasitemia and would recover gradually. If splenectomized sheep were immuno-suppressed with dexamethasone, the parasitemia could reach 8.5%, and death occurred. A splenectomized calf could not be infected with the Babesia species. Paired parasites were the typical form of the Babesia species in erythrocytes and the average size of a pair of parasites was 2.42 (+/-0.35) microm x 1.06 (+/-0.22) microm. Merozoites were found in the gut, salivary gland, haemolymph, ovary and eggs of female Hyalomma anatolicum anatolicum engorged on sheep infected with the parasites. The results of experimental transmission showed that the larval, nymph and adult stages of H. a. anatolicum could transmit the Babesia species to sheep.

Babesia sp. BQ1 (Lintan): Molecular evidence of experimental transmission to sheep by Haemaphysalis qinghaiensis and Haemaphysalis longicornis

Ovine babesiosis is an economically important disease induced by tick transmitted haemoparasites throughout the world. In China, several ovine Babesia strains have been isolated from field-collected ticks or sheep blood during the last two decades but little is known about the vector ticks and transmission pattern. Babesia sp. BQ1 (Lintan) is a Babesia strain infective for sheep and goats, isolated from blood of sheep experimentally infested with Haemaphysalis qinghaiensis collected in field. In the present study, we explored the experimental transmission of Babesia sp. BQ1 (Lintan) to sheep by H. qinghaiensis and Haemaphysalis longicornis. Based on the evidence from nested PCR, it suggested that H. qinghaiensis and H. longicornis are the potential vector ticks of Babesia sp. BQ1 (Lintan) and that larvae, nymphs and adults of both tick species were able to transmit Babesia sp. BQ1 (Lintan) to sheep. Parasites could be detected in the blood, by specific nested PCR, for one month post-infestation.

Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences.

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene for six Babesia spp. isolated from different geographic origins were characterized. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among these isolates. Phylogenetic analysis of the ITS1-5.8S gene-ITS2 region clearly separated the isolates into two clusters. One held an unidentified Babesia sp. transmitted by Hyalomma anatolicum anatolicum. The second held five other isolates, which were considered to be Babesia motasi. Each Babesia species cluster possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. The results showed that ITS1, ITS2 and the complete ITS1-5.8S-ITS2 region could be used to discriminate these ovine Babesia spp. effectively.

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.

Cloning and characterization of a cDNA clone encoding calreticulin from Haemaphysalis qinghaiensis (Acari: Ixodidae)

The application of anti-tick vaccine has been shown to be the most promising alternative strategy compared to the current use of acaricides that suffer from a number of serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. Previously, we have cloned 21 positive clones (named from Hq02 to Hq22) by immunoscreening complimentary DNA (cDNA) libraries of Haemaphysalis qinghaiensis; however, some of those clones did not contain open reading frames (ORF). In this study, we amplified the entire sequence of Hq07 by using rapid amplification of the cDNA ends. Hq07 contains an ORF of 1,233xa0bp that encodes for 410 amino acid residues with a coding capacity of 47xa0kDa. Search of the cloned sequences against GenBank revealed that Hq07 is a calreticulin (CRT)-similar clone and designated HqCRT. Expression analysis by reverse transcription–polymerase chain reaction showed that this gene is ubiquitously expressed at different developmental stages and in different tissues of H. qinghaiensis. The gene was expressed as glutathione S-transferase-fused proteins in a prokaryotic system. Western blot analysis revealed that native HqCRT was secreted into their hosts by ticks during blood sucking. Vaccination of sheep with rHqCRT conferred protective immunity against ticks, resulting in 54.3% mortality in adult ticks, compared to the 38.7% death rate in the control group. These results demonstrated that rHqCRT might be a useful vaccine candidate antigen for biological control of H. qinghaiensis.

Development of an enzyme-linked immunosorbent assay for the diagnosis of Theileria sp. infection in sheep

Abstract. A rapid, sensitive and specific diagnostic method, an enzyme-linked immunosorbent assay (ELISA), was developed for the diagnosis of Theileria sp. infection in sheep; and optimal conditions were established, such as antigen concentration, serum dilution, coating time, Tween-20 concentration and conjugate. The results were analyzed by measuring the coefficient of variation (CV). Three sera titers (high, middle, low) were analyzed over the measurement range, resulting in a CV of around 10%, whereas a 30% variation is the maximum acceptable. The cut-off value was determined by the mean of a negative control plus three standard deviations. Cross-reaction was found only with Babesia ovis. However, this result may be questionable, because it cannot be excluded that these sheep were already infected with both Theileria sp. and B. ovis. The ELISA described in the present study proved to be a useful tool for studying the epidemiology of Theileria sp.