Qingli Niu

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.

Identification of 12 Piroplasms Infecting Ten Tick Species in China Using Reverse Line Blot Hybridization

Abstract Piroplasmosis, a disease of domestic and wild animals, is caused by tick-borne protozoa in the genera of Theileria and Babesia. There is limited information available about the prevalence of piroplasmosis in ticks in China, and to assess the potential threat of piroplasmosis in China, we investigated the infections of ovine and bovine Babesia and Theileria species in ticks collected from cattle, yaks, sheep, horses, and camels in several regions of China where tick-borne diseases have been reported. In total, 652 ticks were collected from the animals in 6 provinces of China. Babesia spp. and Theileria spp. were detected with a PCR-RLB method and identified by sequencing. Overall, 157 ticks (24.1%) were infected with 5 Babesia and 4 Theileria species. Among tested tick samples, 134 (20.6%) were single infections with 1 of 7 piroplasm species, with Theileria annulata (118/652, 18.1%) being dominant. Only 23 (3.5%) tick samples were double or triple infected, Theileria luwenshuni and Theileria sinensis (18/652, 2.8%) were frequently observed in co-infections. Some piroplasm species were carried by ticks that were not previously reported to be vectors.

Identification of piroplasm infection in questing ticks by RLB: a broad range extension of tick-borne piroplasm in China?

Sensitive and specific diagnostic method for rapid and simultaneous detection and discrimination of the different species is needed for an effective control of piroplasmosis. Here, a reverse line blot (RLB) assay was developed for piroplasm detection. A general pair of primer based on 18S ribosomal RNA (rRNA) gene was used to amplify V4 region of 18S rRNA gene. General and specific probes for 13 piroplasm species were cited from previous publications or designed according to the alignment of 18S rRNA gene sequences. For sensitivity test of RLB assay, serially diluted plasmids of the different species were used to access the sensitivity of the RLB. Four hundred and fifty tick samples collected from grass from different provinces of China were then detected. The result indicated that the RLB assay is highly specific and sensitive, detecting up to 102xa0copies/μl of recombinant plasmid DNA. Multiple piroplasms were detected as single or mixed infection from tick species. Eight piroplasm species, most of which were Theileria annulata (33/450, 7.3xa0%) or Babesia sp. Xinjiang (30/450, 6.7xa0%), were found to infect with 89 tick samples in four tick species; no infections with Babesia major, Babesia ovata, Babesia bigemina, Theileria sergenti, or Theileria equi were detected. The piroplasms species-specific RLB assay may have potential clinical application in the simultaneous detection and differentiation of Babesia and Theileria species.

Infection of small ruminants and their red blood cells with Theileria annulata schizonts.

Theileria annulata, the causative agent of tropical theileriosis, is a protozoan parasite that also causes lymphoproliferative diseases in cattle. In vivo, parasitized cells undergo clonal expansion and infiltrate both the lymphoid and non-lymphoid tissues of the infected host. To determine whether the small ruminants and their red blood cells (RBCs) were invaded by T. annulata schizonts or not, T. annulata schizonts were used to infect bovine, ovine and caprine RBCs in vitro, and sheep and goats in vivo. The results showed that the schizonts infected bovine, ovine and caprine RBCs in vitro, but not sheep and goats, which showed only an increase in body temperature and no development of piroplasms. To our knowledge, this is the first report of infection of small ruminants and their RBCs by T. annulata schizonts.

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 108xa0conidiaxa0mL−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.

A novel zoonotic Anaplasma species is prevalent in small ruminants: potential public health implications

BackgroundTick-borne diseases currently represent an important issue for global health. A number of emerging tick-transmitted microbes continue to be discovered, and some of these are already identified as the cause of human infections. Over the past two decades, Anaplasma phagocytophilum is considered to be mainly responsible for human anaplasmosis. However, a novel zoonotic pathogen provisionally named “Anaplasma capra” has recently been identified in China. In this study, we did an active surveillance of A. capra in goats and sheep in different geographical regions of China.MethodsThe presence of A. capra was determined by nested PCR in 547 blood samples collected from goats and sheep from 24 counties distributed in 12 provinces in China. The molecular characterization of A. capra isolates in sheep and goats was achieved based on four conventional genetic markers (16S rRNA, gltA, groEL and msp4 genes).ResultsAnaplasma capra was identified in 75 of 547 animals, with an overall prevalence of 13.7%. The infection rates in the survey sites ranged from 0 to 78.6%, and were significantly different (Pu2009<u20090.01). Phylogenetic analysis revealed that the isolates obtained from goats, sheep, Ixodes persulcatus ticks and humans create a separate clade within the genus Anaplasma and distinct from other recognized Anaplasma species. These findings indicated that these A. capra isolates possess the same molecular characteristics, suggesting that this organism could be a substantial health threat to both animals and humans.ConclusionsAnaplasma capra is an emerging tick-transmitted zoonotic pathogen. This novel Anaplasna species is widespread across China with an overall prevalence of 13.7% in goats and sheep with isolates indistinguishable from those found in humans. These findings warrant increased public health awareness for human anaplasmosis.

The efficacies of 5 insecticides against hard ticks Hyalomma asiaticum, Haemaphysalis longicornis and Rhipicephalus sanguineus

At present, chemical-based tick control strategies are still the most efficient and widely used methods in control of ticks and tick-borne diseases. In this study, the efficacies of lambda-cyhalothrin, beta-cypermethrin, emamectin benzoate, spirotetramat and hexaflumuron in vitro were evaluated against Hyalomma asiaticum, Haemaphysalis longicornis and Rhipicephalus sanguineus that are widespread and able to transmit a variety of human and animal diseases in China. The results showed that the LC (lethal concentration) 50 of lambda-cyhalothrin, beta-cypermethrin, emamectin benzoate, spirotetramat and hexaflumuron were 22.05, 107.35, 287.62, 432.25 and over 6250 mg/L to Hy. asiaticum engorged nymphs, respectively. The LC50 of lambda-cyhalothrin and beta-cypermethrin were each to 100.69 mg/L and 340.05 mg/L against Hy. asiaticum unfed adults. In addition, 50 mg/L of lambda-cyhalothrin could completely inhibit engorged females of the 3 tick species to lay eggs. These results indicate that lambda-cyhalothrin has the highest efficacy and broadest spectrum for against the 3 tick species. The present study provides some information for selecting chemical acaricides in control ticks and tick-borne-diseases, as well for preparing acaricide mixtures to improve killing efficacy, and retard the advent of tick-resistance of acaricides in China.

Molecular evidence of tick-borne pathogens in Hyalomma anatolicum ticks infesting cattle in Xinjiang Uygur Autonomous Region, Northwestern China

Although tick-borne pathogens have been widely reported in ticks in China, there is little information available on the prevalence of information in Hyalomma ticks from cattle. This study aims to determine the occurrence of pathogens in Hyalomma anatolicum collected from cattle in Xinjiang Uygur Autonomous Region, China, by PCR, sequencing and phylogenetic analysis. Borrelia burgdorferi s.s., Rickettsia massiliae and Anaplasma bovis were identified, whereas DNA of Ehrlichia species and an Anaplasma platys-like pathogen were also detected. Our findings highlight the risk of infection of animals and humans with these pathogens in north-western China.

Additional file 1: of Genetic characterization and molecular survey of Babesia bovis, Babesia bigemina and Babesia ovata in cattle, dairy cattle and yaks in China

a. Alignment of partial nucleotide sequences of rap-1a gene from different Chinese isolates. FJ2: B. bovis rap-1a isolate Fujian 2; GS: B. bovis rap-1a isolate Guansu; FJ1: B. bovis rap-1a isolate Fujian 1;GX: B. bovis rap-1a isolate Guangxi; HAN: B. bovis rap-1a isolate Hainan; YN1: B. bovis rap-1a isolate Yunnan 1; YN2: B. bovis rap-1a isolate Yunnan 2; CQ: B. bovis rap-1a isolate Chongqing; HEN2: B. bovis rap-1a isolate Henan 2; HEN 1: B. bovis rap-1a isolate Henan 1; HEN3: B. bovis rap-1a isolate Henan3. Nucleotide substitutions indicated with yellow background. Among these substitutions, nucleotide substitutions affected amino acid modifications indicated with red background. b. Alignment of partial amino acid sequences of rap-1a gene from different Chinese isolates. FJ2: B. bovis RAP-1a isolate Fujian 2; GS: B. bovis RAP-1a isolate Guansu; GX: B. bovis RAP-1a isolate Guangxi; CQ: B. bovis RAP-1a isolate Chongqing; HAN: B. bovis RAP-1a isolate Hainan; YN1: B. bovis RAP-1a isolate Yunnan 1; FJ1: B. bovis RAP-1a isolate Fujian 1; YN2: B. bovis RAP-1a isolate Yunnan 2; HEN2: B. bovis RAP-1a isolate Henan 2; HEN 1: B. bovis RAP-1a isolate Henan 1; HEN3: B. bovis RAP-1a isolate Henan3. Repeats in RAP-1a gene from different isolates are underlined. (DOC 1086Âxa0kb)