Biological Parameters of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Fed on Rabbits, Sheep, and Cattle.

In order to determine the effect of various hosts on feeding performance of Rhipicephalus (Boophilus) microplus, we used 3 mammalian species as hosts, cattle (Qinchuan), sheep (T an), and rabbits (Japanese white rabbit) for infest-ing ticks. Five hundreds of R. microplus larvae were exposed to each animal (3 animals/host species). Tick recoveries were 11.0%, 0.47%, and 5.5% from cattle, sheep, and rabbits, respectively. The averages of tick feeding periods were not significantly different on cattle, sheep, and rabbits, 28.8, 25.3, and 26.7 days, respectively. The average weights of individual engorged female from cattle, sheep, and rabbits were 312.5, 219.1, and 130.2 mg, respectively and those of egg mass weights each to 85.0, 96.6, and 17.8 mg. The highest egg hatching rate was in the ticks from cattle (96.0%), fol-lowed by those from rabbits (83.0%) and sheep (19.2%). These data suggest that rabbits could be as an alternative host to cultivate R. microplus for evaluating vaccines and chemical and biological medicines against the tick in the laboratory, although the biological parameters of ticks were less than those from cattle.

Scanning electron microscopy of all parasitic stages of Haemaphysalis qinghaiensis Teng, 1980 (Acari: Ixodidae).

Haemaphysalis qinghaiensis Teng (Acta Zootaxon Sin 5:144-149, 1980) is an endemic species in China. This tick species was first described based on engorged or semi-engorged specimens, and the drawings and description in words of morphological characteristics were poor. Therefore, the present study aims to redescribe morphological characteristics of all active stages of this tick species in detail by scanning electron microscopy. Additionally, a comparison between H. qinghaiensis and other sympatric Haemaphysalis species was also analyzed. Males of H. qinghaiensis can be distinguished from sympatric Haemaphysalis species by the following characters: palpi less salient laterally and curved in contour; ventrointernal setae of palpal segment II thin, number <7; the tips of palpal segment III not so strongly recurved inward to become "pincerlike" and lacking dorsal spur; dental formula 5/5; lateral grooves enclose first festoon; coxa IV with a short, broadly triangular spur; tarsi somewhat humped; and spiracular plates long comma-shaped. Females of H. qinghaiensis can be distinguished by palpi less salient laterally and curved in contour; ventrointernal setae of palpal segment II thin, number <7; segment III of palpi lacking dorsal spur; dental formula 4/4; scutum subcircula; and tarsi somewhat humped. Nymphs of H. qinghaiensis can be distinguished from those of other species by palpi less salient laterally and curved in contour; dental formula 2/2; basis capituli rectangular, with distinct dorsal cornua, without ventral cornua; and spiracular plates with short and narrow dorsal prolongation. Larvae of H. qinghaiensis can be distinguished by palpi less salient laterally and curved in contour; basis capituli rectangular, without distinct cornua.

Susceptibility of the tick Haemaphysalis qinghaiensis to isolates of the fungus Metarhizium anisopliae in China.

Haemaphysalis qinghaiensis, a prevalent tick species in China, causes severe economic losses. In this study, we investigated the pathogenicity of six isolates of the fungus Metarhizium anisopliae to engorged female H. qinghaiensis using concentrations of 10(6), 10(7) and 10(8) conidia ml(-1). The results indicated that M.aAT08 and M.aAT13 isolates were highly virulent against the ticks. Metarhizium anisopliae has potential for biocontrol of H. qinghaiensis.

Additional data for a new Theileria sp. from China based on the sequences of ribosomal RNA internal transcribed spacers.

Theileria sinensis was recently isolated and named as an independent Theileria species that infects cattle in China. To date, this parasite has been described based on its morphology, transmission and molecular studies, indicating that it should be classified as a distinct species. To test the validity of this taxon, the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were cloned and sequenced from three T. sinensis isolates. The complete ITS sequences were compared with those of other Theileria sp. available in GenBank. Phylogenetic analyses based on sequence data for the complete ITS sequences indicate that T. sinensis lies in a distinct clade that is separate from that of T. buffeli/orientalis and T. annulata. Sequence comparisons indicate that different T. sinensis isolates possess unique sizes of ITS1 and ITS2 as well as species-specific nucleotide sequences. This analysis provides new molecular data to support the classification of T. sinensis as a distinct species from other known Theileria spp. based on ITS sequences.

The in vitro efficacy of deltamethrin and alpha-cypermethrin against engorged female Haemaphysalis qinghaiensis ticks (Acari: Ixodidae).

Currently, the most efficient and widely used method for tick control is the application of acaricides, especially deltamethrin and alpha-cypermethrin, two pyrethroids with neurotoxic action. In this study, the in vitro efficacy of deltamethrin and alpha-cypermethrin was assessed on engorged female Haemaphysalis qinghaiensis ticks. An in vitro bioassay (adult immersion test) was carried out to determine the LC (lethal concentration) 50 and LC90 of both compounds, calculated by probit analysis. The LC50 and LC90 values of deltamethrin and alpha-cypermethrin were 5.67 (LC50) and 51.72ppm (LC90), and 166.56 (LC50) and 1366.69ppm (LC90), respectively. This study provides important information on the efficacy of deltamethrin and alpha-cypermethrin for the control of H. qinghaiensis.

Phylogenetic analysis based on 28S rRNA of Babesia spp. in ruminants in China.

Molecular phylogenetic analyses are mainly based on the small ribosomal RNA subunit (18S rRNA), internal transcribed spacer regions, and other molecular markers. We compared the phylogenetic relationships of Babesia spp. using large subunit ribosomal RNA, i.e., 28S rRNA, and the united 28S + 18S rRNA sequence fragments from 11 isolates of Babesia spp. collected in China. Due to sequence length and variability, the 28S rRNA gene contained more information than the 18S rRNA gene and could be used to elucidate the phlyogenetic relationships of B. motasi, B. major, and B. bovis. Thus, 28S rRNA is another candidate marker that can be used for the phylogenetic analysis of Babesia spp. However, the united fragment (28S + 18S) analysis provided better supported phylogenetic relationships than single genes for Babesia spp. in China.

The life cycle of Haemaphysalis qinghaiensis (Acari: Ixodidae) ticks under laboratory conditions.

The developmental stages in the life cycle of Haemaphysalis qinghaiensis were investigated under laboratory conditions. The larval, nymphal and adult ticks were fed on sheep at 25-27 °C, 50 % relative humidity (RH) and exposed to daylight. All free-living stages were maintained in an incubator (28 °C with 90 % RH and a 12-h photoperiod). The whole life cycle of H. qinghaiensis was completed in an average of 176 days (range 118-247 days). The average developmental periods were 34.44 days for egg incubation; 5.83, 4.20 and 33.70 days for larval pre-feeding, feeding and pre-molting; and 3.88, 5.30 and 46.50 days for nymphal pre-feeding, feeding and pre-molting. The average times for pre-feeding, feeding, pre-oviposition and oviposition of female adult ticks were 2.60, 11.40, 8.50, and 19.35 days, respectively. The results confirmed the positive correlation between the weight of the engorged female and the egg mass laid (r = 0.557, P < 0.05). The reproductive efficiency index and reproductive fitness index in females were 5.49 and 4.98, respectively. Engorged nymphs moulting to females (4.53 ± 0.16 mg) were significantly heavier (P < 0.001) than those moulting to males (3.45 ± 0.19 mg). The overall sex ratio of the adult ticks was 1:1.1 (M:F).

Phylogenetic analysis of ruminant Theileria spp. from China based on 28S ribosomal RNA gene.

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.

Molecular survey and genetic identification of Anaplasma species in goats from central and southern China.

Anaplasma species are obligate intracellular rickettsial pathogens that impact the health of humans and animals. Few studies have been carried out on Anaplasma infections in central and southern China. This study was conducted to determine the coinfection rates of Anaplasma ovis, A. bovis, and A. phagocytophilum from 262 field blood samples of goats in these regions. The average prevalences of single infection of A. ovis, A. bovis, and A. phagocytophilum were 15.3, 16.0, and 6.1%, respectively. Coinfection of A. ovis and A. bovis was dominant, with an infection rate of 27.1%. Coinfection of A. ovis and A. phagocytophilum was 1.9% and that of A. bovis and A. phagocytophilum was 4.2%. Three-pathogen coinfection was found in three of four investigated provinces with a prevalence between 0 and 5.3%. The accuracy of the PCR results was corroborated by sequencing. Analysis of the 16S rRNA gene sequences of A. bovis and A. phagocytophilum confirmed the presence of these pathogens at the investigated sites and indicated the possible genetic diversity of A. phagocytophilum. Field blood inoculation of experimental animals led to successful identification and observation of the morphological shapes of A. bovis in the infected monocytes of sheep. Phylogenetic study with msp4 sequences of A. ovis indicated that the A. ovis genotypes from sheep in the north differed from the genotypes of goats in the investigated sites.

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.

Detection and differentiation of Borrelia burgdorferi sensu lato in ticks collected from sheep and cattle in China.

BACKGROUND: Lyme disease caused by Borrelia burgdorferi sensu lato complex is an important endemic zoonosis whose distribution is closely related to the main ixodid tick vectors. In China, isolated cases of Lyme disease infection of humans have been reported in 29 provinces. Ticks, especially ixodid ticks are abundant and a wide arrange of Borrelia natural reservoirs are present. In this study, we developed a reverse line blot (RLB) to identify Borrelia spp. in ticks collected from sheep and cattle in 7 Provinces covering the main extensive livestock regions in China. RESULTS: Four species-specific RLB oligonucleotide probes were deduced from the spacer region between the 5S-23S rRNA gene, along with an oligonucleotide probe which was common to all. The species specific probes were shown to discriminate between four genomic groups of B. burgdorferi sensu lato i.e. B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana, and to bind only to their respective target sequences, with no cross reaction to non target DNA. Furthermore, the RLB could detect between 0.1 pg and 1 pg of Borrelia DNA.A total of 723 tick samples (Haemaphysalis, Boophilus, Rhipicephalus and Dermacentor) from sheep and cattle were examined with RLB, and a subset of 667 corresponding samples were examined with PCR as a comparison. The overall infection rate detected with RLB was higher than that of the PCR test.The infection rate of B. burgdoreri sensu stricto was 40% in south areas; while the B. garinii infection rate was 40% in north areas. The highest detection rates of B. afzelii and B. valaisiana were 28% and 22%, respectively. Mixed infections were also found in 7% of the ticks analyzed, mainly in the North. The proportion of B. garinii genotype in ticks was overall highest at 34% in the whole investigation area. CONCLUSION: In this study, the RLB assay was used to detect B. burgdorferi sensu lato in ticks collected from sheep and cattle in China. The results showed that B. burdorferi senso stricto and B. afzelii were mainly distributed in the South; while B. garinii and B. valaisiana were dominant in the North. Borrelia spirochaetes were detected in Rhipicephalus spp for the first time. It is suggested that the Rhipicephalus spps might play a role in transmitting Borrelia spirochaetes.

Theileria ovis discovered in China.

A polymerase chain reaction (PCR) using 989/990 primers was conducted to identify a newly isolated Theileria sp. in Xinjiang Province of China. The target DNA fragments of the complete 18S rRNA gene were cloned and sequenced. The phylogenetic relationship of newly isolated Theileria spp. was inferred based on the 18S rRNA gene. The results showed that the new Theileria sp. belonged to the cluster of Theileria ovis. Moreover, the findings were confirmed by T. ovis species-specific PCR. An expected 520 bp fragment of T. ovis DNA was obtained from 25 out of 320 (8%) field blood samples, and blood of an experimental sheep infested by Hyalomma anatolicum anatolicum collected in Xinjiang. The infection rate of T. ovis was 78% (25/32) in Xinjiang province. The investigation did not find T. ovis positive samples from the field samples collected from the other twelve provinces. This study indicates that T. ovis is prevalent in Xinjiang province of China and its transmission vector is H. anatolicum anatolicum.

Beauveria bassiana: Synergistic effect with acaricides against the tick Hyalomma anatolicum anatolicum (Acari: Ixodidae).

Owing to the need to combat the spread of chemical acaricide resistance in ticks, we evaluated the efficacy of a mixture of the entomopathogenic fungus Beauveria bassiana AT17 and acaricides for the control of Hyalomma anatolicum anatolicum in China. A mixture of B. bassiana AT17 at the concentration of 10(8)conidia/mL and the synthetic pyrethroid deltamethrin at concentrations of 2500, 250, 25, 5, 2.5, 0.5 and 0.25ppm was tested in vitro. The germination capability, vegetative growth, conidia production, and viability of B. bassiana AT17 were assessed and the efficacy of the mixture in killing engorged H. anatolicum anatolicum females was measured. High mortality rates were achieved when the entomopathogen was combined with different concentrations of deltamethrin. Neither B. bassiana AT17 nor deltamethrin alone at the same concentrations could cause the higher mortality rates seen with the combination. In addition the combination killed the ticks more rapidly than did either agent alone (3-5days more rapidly). Our results indicate that the mixture of B. bassiana AT17 and deltamethrin has potential as a new type of reagent for integrated control of H. anatolicum anatolicum.

Biological control of engorged female Haemaphysalis qinghaiensis (Acari: Ixodidae) ticks with different Chinese isolates of Beauveria bassiana.

Seven isolates of the fungus Beauveria bassiana (B.b) were assessed for their lethality against Haemaphysalis qinghaiensis, a prevalent tick species in China. Fourteen days after exposure to the isolates B.bAT1, B.bAT5, and B.bAT7 (at 10(8) conidia mL(-1)), the mortality rate had reached 100%. The results indicated that these three B. bassiana isolates were highly virulent against the engorged female H. qinghaiensis ticks. The present study suggests that B. bassiana has potential for biocontrol applications to eradicate H. qinghaiensis.

Sequence analysis of Theileria annulata surface protein in Chinese isolates.

OBJECTIVE: To study the TaSP polymorphism in three Chinese isolates of Theileria annulata. METHODS: The isolates from Inner Mongolia Autonomous Region, Ningxia Hui Autonomous Region and Xinjiang Uygur Autonomous Region were cultured in RPMI 1640 medium. TaSP gene was amplified from genomic DNA extracted from schizonts using polymerase chain reaction (PCR) and sequenced. Its amino acid sequence comparison was carried out with Clustal W2 multiple sequence alignment program. Molecular component and motif prediction were performed with online servers. RESULTS: The comparison of TaSP amino acid sequences of the three isolates showed that the central region (aa position 38-161) predicted to be the highly immunogenetic domain was polymorphic both in size and amino acid sequence, while the N-terminal (first 37 aa) and C-terminal (last 154 aa) parts were strongly conserved. Phylogenetic analysis and percentage identity revealed that the Chinese isolates were closely related to the isolates from Turkey, but quite different from those of India, Morocco and Tunisia. More importantly, variability was noticed among Chinese isolates, which caused both the location and number's differences of motif (casein kinase II phosphorylation sites) among three TaSP sequences. CONCLUSION: TaSP polymorphism exists in the Chinese isolates of T. annulata.

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.

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.

Experimental transmission of Theileria uilenbergi infective for small ruminants by Haemaphysalis longicornis and Haemaphysalis qinghaiensis.

The experimental transmission of a recently designated Theileria uilenbergi pathogenic for sheep and goats in Northern China is described. Haemaphysalis qinghaiensis nymphs and adults developed from larvae and nymphs engorged on sheep infected with T. uilenbergi were able to respectively transmit the latter to sheep. However, when H. longicornis ticks picked up T. uilenbergi either at larval or nymphal, only the subsequent adult could transmit the parasites to sheep, the subsequent nymph could not transmit the parasites to sheep. This experiment suggested that the T. uilenbergi 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.

[Morphological comparison of two species of Theileria in sheep].

Two splenectomized sheep were infected respectively with Theileria luwenshuni and T. uilenbergi, 2 species identified by PCR. When piroplasms were found in blood smears from ears of the sheep, morphological observation on the Theileria spp. was carried out by optical microscopy. By Giemsa staining, the cytoplasm exhibited in slight blue and nucleus in purple. The two Theileria species displayed various shapes, but pyriform-shaped, round-shaped and needle-shaped parasites appeared in every stage of the infection. In fact, there is no significant morphological difference between the two species.