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1.
Benign Theileria parasites of cattle distributed in the Okinawa prefecture were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. Using universal or allele-specific primer sets, parasite DNA was amplified in 31 out of 48 blood samples obtained from beef cattle. Among the positive cases, mixed infections involving various combinations of I-, C-, and B-type parasites were detected in 24 (77.4%) samples. Phylogenetic analysis based on the MPSP gene sequences revealed that parasites with the MPSP types 1-5 and 7, exist within the Okinawa prefecture.  相似文献   

2.
Theileria orientalis is a benign bovine protozoan parasite that occasionally causes serious economic loss in the livestock industry. We report the findings of a molecular epidemiological survey of T. orientalis in 94 Vietnamese yellow cattle, 43 water buffaloes, 21 sheep, 21 goats and 85 blood-sucking ticks of cattle in the Thua Thien Hue province of Vietnam. The major piroplasm surface protein (MPSP) gene of T. orientalis was detected using polymerase chain reaction from 13 cattle (13.8%), 11 water buffaloes (25.6%), 1 sheep (4.8%) and 9 ticks (10.6%). Phylogenetic analysis using MPSP gene sequences showed the presence of seven genotypes, four previously categorized genotypes (Types 1, 3, 5 and 7) and three new genotypes (Types N-1, N-2 and N-3).  相似文献   

3.
In the present study, we have surveyed the presence of a bovine Theileria protozoan, Theileria orientalis, in Mongolian cattle and engorging tick populations from selected provinces and districts in Mongolia. The percentages of infection in the cattle and ticks ranged from 8.8 to 66.6 and from 3.7 to 73.3, respectively, on a per district basis. The genetic diversity of T. orientalis isolates was also studied, based on the protozoan gene encoding a major piroplasm surface protein (MPSP). At least five genotypes (types 1, 3, 5, 7, and N-3) of T. orientalis were found to be circulating among the Mongolian cattle and tick populations. In particular, types 3 and N-3 were common in most of the districts examined, while a strong geographical relationship among the genotypes was not detected in the present study. This is the first epidemiological report describing the presence of T. orientalis infection in Mongolian cattle.  相似文献   

4.
During a year from 1999 to 2000, a total of 247 blood samples were collected from 214 cattle and 33 water buffaloes in 16 distinct geographical locations of Thailand and analyzed by allele-specific PCR amplification of major piroplasm surface protein (MPSP) genes of benign Theileria parasites. Four allelic MPSP gene types were determined namely C-type, I-type, B-type and Thai-type, which were originally designated from Japanese Theileria orientalis (Chitose, Ikeda), Australian T. buffeli (Warwick) and Thai T. sp. (Kamphaeng Saen), respectively. Only two allelic MPSP gene types were successively amplified from 204 (82.6%) blood samples. Among positive cases, 138 (67.6%) and 17 (8.3%) samples contained either Thai-type or C-type parasites, respectively, while 49 (24%) samples contained both types. However, nucleotide sequences of MPSP genes of Thai T. sp. amplified by C-type specific primers revealed higher (96.3%) similarity to Indonesian T. sp. rather than (87.8% similarity) to Japanese T. orientalis (Chitose) designated as C-type.  相似文献   

5.
Twenty-eight field isolated Theileria parasite DNAs obtained from dairy and beef cattle in distinct geographical areas of Thailand were characterized by using polymerase chain reaction (PCR) amplification with six sets of oligonucleotide primers. Three sets of them were modified from two genes of immunodominant major piroplasm surface protein (MPSP) coding for 32 kDa (p32) of T. sergenti and 33/34 kDa (p33/34) of T. buffeli, and MPSP of Theileria spp.(Thai-isolate). The other three sets of primers were basically generated from three alleles of MPSP which were specific for Japanese T. sergenti-Ikeda stock (I-type), Japanese T. sergenti-Chitose stock (C-type) and Australian T. buffeli-Warwick stock (B1-type), respectively. The results indicated that 14 out of 28 isolates were amplified by the Thai-specific primer whereas 6 isolates were amplified by the p32 specific primer and the other 5 isolates were amplified by the p32 and Thai-specific primers. In addition, by using the allele-specific PCR, 14 out of 28 isolates contained C-type MPSP whereas 3 isolates contained B1 type parasites. Interestingly, 20 out of 28 isolates could be amplified by the Thai-specific primer. The majority of Theileria parasites distributed in Thailand contained Thai type parasites, whereas C-type parasites showed the mixed population with B1 and Thai type parasites. No I type parasite was detected.  相似文献   

6.
Culicoides brevitarsis transmits important ruminant arboviruses, such as Akabane, Aino and bluetongue viruses. The presence of this species has so far been recognized primarily in Okinawa, the southernmost prefecture of Japan. In entomological surveys in 2008 and 2009, C. brevitarsis was collected at 8 sites throughout Nagasaki, Kumamoto and Kagoshima Prefectures. The collection sites are all located near pastures, where the larvae of C. brevitarsis can grow in cattle dung left in the field. C. brevitarsis was confirmed at the same sites in two consecutive years, suggesting that it overwinters in Kyushu. Given the risk of arbovirus transmission, the ecology of C. brevitarsis in Japan, such as its distribution range, seasonal abundance and larval breeding sites, should be investigated in more detail.  相似文献   

7.
Between September 2010 and November 2011, 350 EDTA blood samples were received from 73 Australian cattle herds, as cases suspected to be infected with Theileria orientalis. Beef cattle were predominantly affected, with Angus and Angus-crossbred cattle representing 48% of smear positive samples examined. DNA extracts were tested in conventional polymerase chain reaction (PCR) assays for genes encoding the p32, Ikeda, Chitose and Buffeli major piroplasm surface proteins (MPSP). PCR findings were compared with results of clinical pathology examinations of stained blood smears for parasitaemia and packed cell volume (PCV). PCR testing was much more sensitive than clinical pathology examinations in detecting T. orientalis infections, and concurrent testing of neat and diluted extracts gave significantly more PCR positive results than testing of neat extract alone. Significant associations and correlations were shown between PCR results of p32 and Ikeda assays with PCV levels indicative of anaemia, and with the level of parasitaemia estimated by smears. A high proportion of samples had concurrent Ikeda and Chitose infection, and significantly more clinical cases of theileriosis were associated with the Ikeda MPSP type as the sole infection, compared with sole infection with types Chitose or Buffeli. The findings indicate Ikeda type organisms were significantly associated with clinical parameters of theileriosis in cattle herds in eastern Australia, and that this type is most likely to be responsible for outbreaks of theileriosis experienced in affected Australian herds. In New South Wales, 11 of 14 regulatory districts yielded Ikeda positive samples, with five (Mid-Coast, Cumberland, Central North, Hume and Lachlan) containing 234/307 (76%) of the Ikeda positive samples.  相似文献   

8.
Theileria orientalis is usually a benign parasite but some genotypes cause infection and economic losses to the cattle industry. This study was carried out to determine T. orientalis genotypes in cattle. T. orientalis positive 63 sample were analyzed by amplifying the MPSP gene region by PCR. As a result of the SSCP analysis, samples with different band profiles were sent to the sequence analysis and genotypes were determined. T. orientalis genotype-specific PCR was performed to determine the mix genotypes. Type 1 (chitose), type 3 and type 1-type 3 mix were found positive 11.1%, 46%, and 17.5% respectively. In addition, phylogenetic analysis was performed to separate the chitose genotypes, and two samples were found in chitose A, one sample was found in chitose B. Although chitose A genotype is suggested to be more pathogenic than chitose B, but there is little evidence for this. As a result of this study, we showed the presence of pathogenic genotype T. orientalis in Turkey. Therefore, extensive epidemiological studies are required to understand the geographic distribution, different genotypes and clinical pathologies of T. orientalis.  相似文献   

9.
Benign Theileria species of cattle are found in most parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a maker for epidemiological and phylogenetical studies of benign Theileria species. Parasites with Ikeda- or Chitose-type MPSP genes are dominant in Japan, but we report here mixed infection cases of Theileria parasites with an additional MPSP type parasite infecting cattle in Abashiri District, Hokkaido. The MPSP gene sequence found in the additional type was closely related to MPSP genes of Theileria parasites found in Southeast Asian countries, including Thailand (Narathiwat) and Indonesia (Java). Theileria parasites from the blood sample were also distinguishable from the Ikeda or Chitose type parasites by the small subunit (SSU) rRNA gene sequence analysis, and they are grouped into the SSU rRNA types C/D found in Korea, North America, and Spain. The present finding of mixed infections of cattle with three different types of Theileria makes epidemiological feature of bovine theileriosis in Japan more complex. We have designed a set of primers specific to this MPSP type in order to conduct further epidemiological study.  相似文献   

10.
Complete major piroplasm surface protein (MPSP) gene sequences of benign Theileria parasites were isolated from ticks of grazing cattle in Korea. A total of 556 tick samples were collected in five provinces: Chungbuk, Jeonbuk, Jeonnam, Gyeongbuk, and Jeju during 2010-2011. Fifteen samples from Chungbuk and Jeonnam were positive for the Theileria MPSP gene by PCR amplification using a specific primer set. A phylogenetic tree was constructed with the amplified gene sequences and 26 additional sequences published in GenBank. The benign Theileria parasites were classified into eight types, those isolated from Korean cattle ticks belonged to Types 1 (Ikeda), 2 (Chitose), 4, and 8. Types 2 and 4 were the most common types, with the rate of 40%, followed by Types 1 and 8 (with the rate of 13% and 7%, respectively). Nucleotide sequence identities of 23 theilerial MPSP sequences (15 MPSP gene sequences amplified and 8 sequences published) ranged from 67.3 to 99.8%. Multiple alignments of the deduced amino acid sequences also showed that each type was characterized by specific amino acids: 7 for Type 1, 9 for Type 2, 4 for Type 4, and 3 for Type 8.  相似文献   

11.
In this study, we describe the first survey in Thailand of Trypanosoma theileri, a widespread and prevalent parasite of cattle that is transmitted by tabanid flies. Investigation of 210 bovine blood samples of Thai cattle from six farms by hematocrit centrifuge technique (HCT) revealed 14 samples with trypanosomes morphologically compatible to T. theileri. Additional animals were positive for T. theileri by PCR based on the Cathepsin L-like sequence (TthCATL-PCR) despite negative by HCT, indicating cryptic infections. Results revealed a prevalence of 26 ± 15% (95% CI) of T. theileri infection. Additionally, 12 samples positive for T. theileri were detected in cattle from other 11 farms. From a total of 30 blood samples positive by HCT and/or PCR from 17 farms, seven were characterized to evaluate the genetic polymorphism of T. theileri through sequence analysis of PCR-amplified CATL DNA sequences. All CATL sequences of T. theileri from Thai cattle clustered with sequences of the previously described phylogenetic lineages TthI and TthII, supporting only two major lineages of T. theileri in cattle around the world. However, 11 of the 29 CATL sequences analyzed showed to be different, disclosing an unexpectedly large polymorphic genetic repertoire, with multiple genotypes of T. theileri not previously described in other countries circulating in Thai cattle.  相似文献   

12.
An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.  相似文献   

13.
AIMS: To use quantitative PCR assays to detect Theileria orientalis Ikeda type in cattle presumed infected with T. orientalis, to examine the relationship between theilerial piroplasm count and haematocrit (HCT), and the relationship with quantification cycle threshold (Cq) values.

METHODS: Blood samples in EDTA (n=1,024), derived from herds affected by anaemia associated with T. orientalis infection (TABA) between April and October 2013, were submitted for testing using quantitative PCR (qPCR) assays for T. orientalis and Ikeda type. Nucleotide sequencing of the major piroplasm surface protein (MPSP) gene was performed on 16 samples to identify T. orientalis types. Blood smear and/or HCT results were supplied with most samples. For data analysis, the number of theilerial piroplasm per 1,000 erythrocytes counted was categorised as negative (0), low (1–9), moderate (10–100) or high (>100). HCT was categorised as severely anaemic (<0.15 L/L), mildly anaemic (0.15–0.24 L/L) or not anaemic (>0.24 L/L). Differences between categories in proportion of samples positive for Ikeda type or mean Cq value were examined using χ2 tests or analysis of variance, respectively.

RESULTS: Of 1,022 samples containing amplifiable DNA, 916 (90%) were positive for T. orientalis and 789 (77%) were positive for Ikeda type. Nucleotide sequencing of MPSP amplicons also identified the presence of Chitose and Buffeli types in 11 samples without Ikeda. Ikeda was detected in a greater proportion of severely anaemic (288/302; 95%) than mildly anaemic (227/252; 90%) cattle (p=0.02). In non-anaemic cattle, 344/406 (85%) were positive for T. orientalis and 247/406 (60%) were positive for Ikeda type. In samples from cattle that were piroplasm-positive, a greater proportion of anaemic (483/505, 96%) than non-anaemic (211/307; 69%) cattle were positive for Ikeda type (p<0.001). In piroplasm-negative cattle, 20/37 (54%) anaemic and 25/78 (32%) non-anaemic cattle were Ikeda-positive (p<0.05). The distributions of Cq values differed between piroplasm count and HCT categories (p<0.001). Mean Cq differed between high and negative, and low piroplasm categories (p<0.001), but not between high and moderate categories (p=0.81), and differed between severely anaemic and mildly anaemic (p<0.001), and non-anaemic categories (p<0.001).

CONCLUSIONS: The Ikeda type was found in a high proportion of cattle during outbreaks of TABA in New Zealand. Analysis of Cq values suggested a relationship of Ikeda parasitaemia with severity of anaemia, but further investigation is required to better understand the role of parasitaemia in the pathogenesis of TABA.  相似文献   


14.
Polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) probes were used to characterise trypanosomes from cattle in Morogoro region of Tanzania. Blood samples collected from 390 beef and dairy cattle in selected farms in Morogoro region were examined for presence of trypanosomes using the buffy coat technique (BCT) and blood smears (BSs). Fifty-two animals were found infected: 40 with Trypanosoma congolense, 10 with T. vivax and two with both T. congolense and T. vivax. DNA extracted from all the parasitologically positive and 62 randomly selected parasitologically negative samples were subjected to PCR amplification using primers specific for different trypanosome species. Using a set of seven specific-pairs of primers on the parasitologically positive samples, we detected only T. congolense, either the Savannah- or the Kilifi-type, as single or mixed infections. With the PCR, trypanosome DNA could be detected in 27 (43%) out of 62 samples that were parasitologically negative. DNA hybridisation using probes specific for Savannah- or Kilifi-types T. congolense, or T. vivax, confirmed the presence of these parasites in cattle kept on some farms in Morogoro region of Tanzania. From these studies, it is clear that there is a need to undertake molecular epidemiological studies to determine the distribution of trypanosome species and subspecies, and to assess the economic impact of these parasites in the productivity of livestock in Tanzania. In particular, it would be desirable to verify the assumed association between the different presentations of trypanosomosis on one hand and genotypes of T. congolense on the other.  相似文献   

15.
To investigate the prevalence of Cryptosporidium spp. and Enterocytozoon bieneusi from large-scale cattle farms in Anhui Province, 955 fecal samples were collected from 16 cattle farms from March to October 2018, which included six dairy farms (526), seven yellow cattle farms (323), and three water buffalo farms (106) in different regions of Anhui Province. PCR was conducted on all fecal samples using the 18S ribosomal RNA of Cryptosporidium spp. and internal transcribed spacer gene of E. bieneusi to detect these two pathogens, and the positive samples were sequenced and analyzed. The results showed that 23 (2.4%) and 40 (4.2%) out of the 955 samples were positive for Cryptosporidium spp. and E. bieneusi, respectively. There were 11 (2.1%), 10 (3.1%), and 2 (1.9%) positive samples of Cryptosporidium spp. and 16 (3.0%), 23 (7.1%), and 1 (0.9%) positive samples of E. bieneusi collected from dairy cattle, yellow cattle, and water buffalo, respectively, and no co-infection was identified in this study. All positive samples of Cryptosporidium spp. were C. andersoni with some variations. Ten E. bieneusi genotypes were obtained, including two known genotypes, J and CHN11, and eight new genotypes, named AHDC1 and AHYC1-7. The genotype CHN11 belonged to zoonotic Group 1, and the other nine genotypes belonged to Group 2, which is mainly documented in ruminants. These results indicated that Cryptosporidium spp. and E. bieneusi infections were present in large-scale cattle farms in Anhui Province. Therefore, attention should be paid to the development of containment strategies of these two pathogens in cattle.  相似文献   

16.
Bayesian Inference (BI) and Neighbor Joining (NJ) analyses of the phylogenetic relationships between the nucleotide sequences of the N gene of Akabane virus revealed an unclear topology among genogroups I–III, which was probably caused by genetic reassortment or recombination between these genogroups. In contrast, nucleotide and amino acid phylogenetic tree analyses of the M RNA segment agreed with the topologies obtained by using the BI and NJ methods. Therefore, distinct genogrouping of Akabane virus isolates should be performed using the M RNA segment. Four Korea isolates were classified into genogroup II together with Akabane virus strains isolated from all areas of Japan, including Okinawa Island. However, more nationwide isolates and more clinical data from Korean cattle farms will be required in the future to confirm the precise relationships between genotypes and pathogenicity.  相似文献   

17.
OBJECTIVE: To demonstrate the value of PCR assays to determine the genotypes of Babesia bovis in cattle with clinical signs of babesiosis within 3 weeks after vaccination against tick fever. DESIGN: Samples from 5 cases of babesiosis in cattle soon after vaccination against tick fever were analysed in two PCR assays. PROCEDURE: Parasite DNA was purified from blood taken from cattle with signs of babesiosis within 3 weeks of vaccination against tick fever. DNA was also prepared from the tissues of animals that died of babesiosis. Two PCR assays that amplify repeat sequences of DNA within the B bovis genes, Bv80 and BvVA1, were used to differentiate the genotypes of field isolates and vaccine strains of B bovis. RESULTS: One of the five cases of babesiosis was found to be caused by a vaccine strain, but PCR analyses showed that the predominant isolate in the other four cases was not the vaccine strain. CONCLUSIONS: PCR assays on the DNA of B bovis obtained from the blood or tissues of cattle clinically affected with tick fever within 3 weeks after vaccination are useful to distinguish between vaccine strains and field isolates as the source of infection.  相似文献   

18.
To determine the prevalence of the 3 primary clonal lineages of Toxoplasma gondii (strain types I, II, and III) in pigs in Okinawa Prefecture, we analyzed lymph node samples that had been collected at an abattoir by PCR analysis using primers specific for the Toxoplasma gondii SAG2 locus. This study revealed the presence of this parasite in 57 out of 101 samples examined. Restriction fragment length polymorphism (RFLP) in PCR-amplified SAG2 products was used to group strains into one of the three genotypes of T. gondii. Genotypes I and II were equally predominant, accounting for 22 (44.9%) and 23 (46.9%) of 49 SAG2-positive samples, respectively, while the type III strain was found in only 4 (8.2%) of the 49 samples. The other 8 samples were indistinguishable by PCR-RFLP analysis. Polymorphisms for the 3 genotypes were confirmed at the sequence level for several samples using the sequences from the RH strain, the Beverley strain, and the C56 strain as references. On the other hand, the dihydropteroate synthase gene, which is responsible for sulfonamide resistance, was amplified in 40 of 54 SAG2-positive samples by PCR with the specific primers, and further RFLP and sequence analysis revealed that none of them carried the drug-resistant form of the dhps gene. This is the first report of genotyping of T. gondii distributed in Japan.  相似文献   

19.
Toxoplasma gondii from pigs in Okinawa Prefecture was characterized by nested PCR-restriction fragment length polymorphism (RFLP) and DNA sequence analysis of the dense granule antigen GRA6 gene. By nested PCR, parasite DNA was detected in 33 out of 91 lymph node samples with lesions similar to those found in toxoplasmosis samples that had been collected from pigs at an abattoir. RFLP analysis with MseI was successfully conducted in 29 of 33 PCR-positive samples to group the isolates into one of the three genotypes of T. gondii. Genotyping of the 29 studied samples rendered the following results: 13 of type I (44.8%), 14 of type II (48.3%), and 2 of type III (6.9%). The GRA6 genes of 12 Okinawa isolates were cloned and sequenced. Nine new nucleotide sequences were found, and nucleotide substitutions specific for the Okinawa isolates were found at 13 positions. Phylogenetic analysis indicated that all GRA6 sequences were divided into one of the 3 main groups, and Okinawa isolates of GRA6 genotypes II and III seemed to be closely related to the Beverley strain and the NED strain, respectively. The results from this study may provide basic and useful information for the analysis of the molecular epidemiology of T. gondii infection within Japan.  相似文献   

20.
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