Diagnosis of equine piroplasmosis in Xinjiang province of China by the enzyme-linked immunosorbent assays using recombinant antigens

The prevalence of equine piroplasmosis in Xinjiang province, China, was examined by enzyme-linked immunosorbent assays (ELISAs). A total of 70 serum samples were taken from horses pastured on three farms in western Xinjiang, and examined for diagnosis of equine Babesia equi (B. equi) infection and B. caballi infection by ELISAs using recombinant equi merozoite antigen 1 (EMA-1) and recombinant P48 antigen, respectively. Of the 70 samples, 28 (40.0%) and 17 (24.3%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 11 (15.7%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in western Xinjiang. To our knowledge, this is the first report describing a survey on equine piroplasmosis in Xinjiang province, China.     

Seroepidemiological evidence for the possible presence of Babesia (Theileria) equi and Babesia caballi infections in donkeys in western Xinjiang, China

The prevalence of Babesia (Theileria) equi and B. caballi infections in donkeys in western Xinjiang China was investigated. In total, 93 serum samples were randomly taken from donkeys in the Kashi and Ili areas, and examined for B. equi and B. caballi infections by enzyme-linked immunosorbent assays using recombinant antigens. Of the 93 samples, 9 (9.6%) and 36 (38.7%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 2 (2.2%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine babesiosis might be extensively prevalent in donkeys in western Xinjiang.     

青海省海西地区马焦虫病的血清学诊断

用ELISA方法调查了青海省海西地区马焦虫病的流行情况。从海西地区的格尔木市和乌兰县的牧户中收集了199份马属动物的血清样品，用马巴贝斯原虫的重组抗原P蚰48（GST-BcSAGlt）蛋白作为ELISA诊断抗原。进行了马巴贝斯原虫的诊断检查。从199份血清样品检出61份马巴贝斯原虫阳性，阳性率为30，65％。结果显示马焦虫病已在该地区广泛传播，并已严重威胁该地区的马属动物。     

新疆昭苏地区马梨形虫病病原及抗体检测

马梨形虫病是由马驽巴贝斯虫和马泰勒虫寄生于马属动物的红细胞内所引起的一类血液原虫病,呈全球性分布,尤其在新疆发病率更高,处于逐年上升趋势,对区域性马产业的发展影响极大。为了解2018年新疆昭苏养马区域马梨形虫的感染情况,随机采集昭苏县18个乡镇的马全血及血清各858份,采用PCR和间接ELISA分别进行检测,对两种方法检测的18个地区、不同年龄阶段的马驽巴贝斯虫、马泰勒虫及混合感染情况进行统计学分析。结果显示,PCR检测马驽巴贝斯虫、马泰勒虫及混合感染的阳性率分别为12.12%、13.87%和2.80%;间接ELISA检测马驽巴贝斯虫、马泰勒虫及混合感染的抗体阳性率分别为15.50%、10.14%和2.56%;不同年龄阶段筛查结果显示,在6岁以下的马匹感染马驽巴贝斯虫、马泰勒虫及混合感染的阳性率较高,并且不同地区的不同年龄阶段马匹的马梨形虫感染率存在不同程度的差异。此次获得的昭苏县马梨形虫感染情况的一线数据,可为当地养马区域马梨形虫病的综合防控提供技术支撑。     

Epidemiological study of equine piroplasmosis in Mongolia

The purpose of this study was to demonstrate the occurrence of equine piroplasmosis in Mongolia, a country in which the disease occurs epidemically in different climatic conditions. Antibodies to Babesia equi and B. caballi were determined in serum samples of 254 pastured horses in different locations of Mongolia using an enzyme-linked immunosorbent assay with recombinant antigens. One hundred and eighty-five (72.8%) and 102 (40.1%) of all serum samples were positive for B. equi and B. caballi infections, respectively. In addition, 78 (30.7%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread in Mongolia. To our knowledge, this is the first report describing an epidemiological study on equine piroplasmosis in different geographic regions in Mongolia.     

Evaluation of enzyme-linked immunosorbent assays with recombinant antigens for the serodiagnosis of equine Babesia infections

Two enzyme-linked immunosorbent assays (ELISA) with recombinant protein as antigens were evaluated by comparison with the indirect fluorescent antibody tests (IFAT) for the detection of specific antibodies to Babesia caballi and Babesia equi, respectively in 380 sera from experimentally infected, uninfected, and field horses. The high concordances of 92.4% (351/380) and 98.2% (373/380) between ELISA and IFAT for B. caballi and B. equi, respectively suggest that ELISA, especially for B. equi infection, could be alternative to the corresponding IFAT for serodiagnoses of equine piroplasmosis, although some improvements are required in ELISA for B. caballi.     

Seroepidemiologic studies on Babesia caballi and Babesia equi infections in Japan

Antibodies to Babesia caballi and Babesia equi were examined on a total of 2,019 horse serum samples that had been collected in 1971-1973 by the National Institute of Animal Health by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins and by Western-blot analysis. Based on the criterion for positivity by ELISA, 5.4% (109/2,019) and 2.2% (44/2,019) had antibodies against B. caballi and B. equi, respectively. The ELISA-positive sera were further examined by Western blot; 30/109 for B. caballi and 2/ 44 for B. equi were positive for native B. caballi or B. equi, but none of them was seropositive for both infections. Based on the results of this study, further investigations should be required to survey horses that have arrived in Japan relatively recently and tick vectors of equine Babesia using ELISA with some recombinant protein, a parasite detection method in an in vitro culture of equine Babesia, and PCR testing.     

A comparative study on the prevalence of Theileria equi and Babesia caballi infections in horse sub-populations in Turkey

Blood and serum samples were taken from 481 horses, from a stud farm or a racecourse, and tested by microscopic examination of blood smears and cELISA for Theileria equi (T. equi) and Babesia caballi (B. caballi) infections. At the time of sampling, animals were also examined for tick infestations and clinical disease, which were not observed in any of the sampled horses. During the microscopic examination of thin blood smears, parasites were detected in the three horses from the racecourse. Overall seroprevalence of infection was detected as 18.50% (89 of 481 horses) by cELISA, with T. equi being significantly more prevalent than B. caballi. Of the 481 blood samples, 78 (16.21%) were serologically positive for T. equi and 4 (0.83%) were serologically positive for B. caballi. In addition, 7 (1.46%) samples were positive for both T. equi and B. caballi antibodies. Seropositivity rates in the racecourse horses were higher than those determined in the stud farm horses. The rates for T. equi, B. caballi and both species were 13.39, 0.52 and 0% in the horses from the stud farm and 27, 2 and 7% in the racecourse horses, respectively. These results indicate that equine piroplasmosis is more common in racehorses than studhorses and therefore it might be a serious concern in horses that participate to international races.     

Prevalence of Theileria equi and Babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern Free State Province, South Africa

The prevalence of Theileria equi and Babesia caballi infections in the north-eastern Free State Province of South Africa was determined by examination of thin and thick Giemsa-stained blood smears, IFAT and PCR. No parasites were detected by microscopy from any blood samples collected at five study sites, Qwaqwa, Kestell, Harrismith, Vrede and Warden. Of the tested serum samples, 28/29 (96.5%), 20/21 (95.2%) and 42/42 (100%) were positive by IFAT for T. equi infections in Harrismith, Kestell and Qwaqwa, respectively, and 5/29 (17.2%), 13/21 (61.9%) and 30/42 (71.4%) were sero-positive for B. caballi infections in Harrismith, Kestell and Qwaqwa, respectively. All DNA samples from the study sites were negative for B. caballi infections by PCR, but five samples, two from each of Kestell and Warden and one from Vrede, were PCR positive for T. equi infections. The high prevalence of antibodies against T. equi and B. caballi in the sampled horses indicates that the animals had been exposed to T. equi and B. caballi infections but the absence of parasitaemia and very low number of positive PCR samples, however, imply that T. equi and B. caballi are endemically stable in the north-eastern Free State Province.     

Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses.

Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses. Apparent test specificities for the B. equi, B. caballi, T. equiperdum, and B. mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively. Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B. equi, B. caballi, T. equiperdum, and B. mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively. The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use. Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera.     

Serological prevalence of Babesia caballi and Theileria equi in horses of Lara State, Venezuela

The main objective of this study was to demonstrate the occurrence of equine piroplasmosis (EP) in horses of Lara State, Venezuela, and to correlate it with the factors host's sex and age in order to know the epidemiology of this disease at the Venezuelan Centroccidental Region. Antibody levels to Babesia caballi and Theileria equi were assessed in 360 equine serum samples, collected from 9 municipalities of Lara State, using an ELISA technique with recombinant antigens and monoclonal antibodies (Mabs). Antibodies to B. caballi were found in 254 horses (70.6%), whereas 181 animals (50.3%) were detected as seropositives to T. equi. In addition, 128 samples (35.56%) were seropositives to both hemoparasites. There were no significant differences between the seropositivity to B. caballi and T. equi with the factors sex and age of the horses. These results show that Lara State is an enzootic area for equine piroplasmosis, and are a contribution to a partial knowledge of the dynamic of this disease in Venezuela.     

Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis.     

Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens.

Serum samples from horses in the States of Sao Paulo and Mato Grosso do Sul, Brazil were examined for diagnosis of equine piroplasmosis by both the latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens. Of the 47 samples analyzed, 38 (81%) and 42 (90%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 35 (75%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in the States of Sao Paulo and Mato Grosso do Sul, Brazil.     

Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene.

Babesia equi and Babesia caballi are tick-borne haemoparasites that may cause babesiosis of Equidae. In southern Europe B. equi is enzootic and infections may occur asymptomatically and more frequently than those due to B. caballi. Complement fixation test (CFT) is the official serological test for the diagnosis of equine babesiosis, but it has low sensitivity during early and latent stages of the disease. With the aim of developing more sensitive and rapid direct diagnostic alternatives, PCR systems that amplified DNA targets of 664 or 659 bp regions of the 16S rRNA genes were designed and demonstrated to specifically detect the genomes of B. equi and B. caballi, respectively. An approximated parasitaemia of 0.000083% was detected by the PCR system for B. equi compared with reported limits of 0.001% for microscopic examination of stained blood smears, and up to 0.00025% for DNA probes. Although the sensitivity of the PCR system for B. caballi could not be estimated, samples with microscopically undetectable parasitaemia as well as those with 0.017% parasitised red blood cells were detected. DNA extracts of blood collected with EDTA as an anticoagulant from 23 horses from Portugal were tested with both PCR systems. Of these samples, 22 were positive for B. equi and 8 were positive for B. caballi with PCR tests and intraerythrocytic parasites were seen in all samples. Antibodies against both parasites were not detected by CFT in several cases, but in these cases the presence of either or both parasites was apparent by PCR tests. The PCR systems may be useful in the diagnosis of equine babesiosis covering a wider range of clinical disease, as useful adjuncts to serological, microscopic, and cultural methods, especially for the import and export testing of horses.     

Prevalence of the causative agents of equine piroplasmosis in the South West of The Netherlands and the identification of two autochthonous clinical Theileria equi infections

Equine piroplasmosis (EP) has not been considered indigenous in The Netherlands. However, following the detection of an apparently indigenous subclinical Babesia caballi infection in a horse on Schouwen-Duiveland (an island in the Zeeland Province), a survey was undertaken between May and September 2010 to assess the prevalence of the causative agents of EP in the South-West of The Netherlands. Blood samples from 300 randomly selected horses were tested for specific antibodies against Theileria equi and B. caballi using an indirect fluorescence antibody test (IFAT), and for parasite DNA using a specific polymerase chain reaction combined with reverse line blotting (PCR-RLB). Twelve of the horses (4%) were seropositive for EP. Of these, nine (75%) were positive (titre?1:160) for B. caballi alone and three (25%) were also positive for T. equi. PCR-RLB detected T. equi DNA in five horses (1.6%), two of which were seronegative. Four (1.3%) of the positive horses (three positive for T. equi and one for both B. caballi and T. equi) were considered truly indigenous. During the study, two indigenous ponies from a farm situated outside the sampling area were diagnosed with acute clinical piroplasmosis characterized by severe anaemia and pyrexia. Blood smears showed T. equi - like inclusions in red blood cells, and T. equi infection was confirmed in both ponies by PCR-RLB. The initial subclinical B. caballi infection, the survey results and the two acute clinical EP cases confirmed the autochthonous transmission of B. caballi and T. equi infections in The Netherlands.     

Control of equine piroplasmosis in Brazil.

The importance of equine piroplasmosis control in endemic countries has increased in recent years and plays an important role to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Babesia equi or Theileria equi and Babesia caballi) in Brazil--a country where the disease occurs endemically--in different climatic conditions, and to evaluate the results of a strategy for tick control in order to decrease infection rates. Blood samples were taken from 720 horses on 28 farms from different regions and subjected for complement fixation testing. The strategy was based on the control of the tick population by spraying the horses with acaricides, treating positive horses and preventing cattle and horses from grazing together. A significant association was found in the prevalence of antibody titres in tropical and subtropical areas. A significantly lower prevalence rate occurred on those farms where measures to control tick population were established. Farms in endemic countries may significantly reduce the prevalence of equine piroplasmosis by establishing measures to control the tick population and treating chronic carrier horses. Additional measures for controlling ticks in tropical areas are also discussed.     

Seroprevalence of equine piroplasms in the Republic of Korea

Equine piroplasms include two tick-borne protozoan parasites, Babesia caballi and Theileria equi. Although no clinical equine piroplasmosis has been reported in the Republic of Korea, the possible existence of the disease has been proposed due to a nationwide distribution of the vector ticks. To determine if the antibodies against B. caballi and T. equi were present, 184 sera of horses (Equus caballus) raised in the Republic of Korea from 2007 to 2010 were assessed using cELISA kits. Two (1.1%) out of 184 sera were positive for T. equi, but none were seropositive for B. caballi. Both samples tested positive came from one region (Gyeonggi province). The accuracy of the cELISA was confirmed by PCR using primers specific to the 18S rRNA of T. equi. This study presents for the first time horses infected by T. equi in the Republic of Korea. Since the infection of T. equi occurred in horses raised in the Republic of Korea, further studies with continuous monitoring of the vector ticks for equine piroplasms and appropriate control programs need to be established.     

Isolation of pure Babesia equi and Babesia caballi organisms in splenectomized horses from endemic areas in South Africa

Both Babesia equi and Babesia caballi are endemic in large parts of South Africa. Attempts were made to obtain pure local isolates of both B. equi and B. caballi for the purpose of developing serological tests to study the epidemiology of equine babesiosis in this country. The indirect fluorescent antibody test was used to screen horses for B. equi and B. caballi in an endemic area. Seven horses and 3 donkeys between 3 and 36 months of age that tested negative were subsequently splenectomized. The splenectomy operation was performed through the abdominal approach. A 100% survival rate was achieved through this method, probably because it reduced the risk involved in the operation. Blood collected from naturally infected horses and passaged in fully susceptible splenectomized horses and a donkey, under laboratory conditions, produced 2 isolates of Babesia caballi and 1 of B. equi. Microscopical and serological examinations confirmed that these were pure isolates.     

An investigation into the clinical pathological changes and serological response in horses experimentally infected with Babesia equi and Babesia caballi

Serologically negative horses, as determined with the indirect fluorescent antibody test (IFA), were infected with Babesia equi and 60 days later with Babesia caballi. The only clinical signs of disease observed in these animals were a febrile reaction and slight icterus. Haematological changes included a drop in haematocrit and haemoglobin concentration, as well as lowered platelet counts. The serum concentrations of albumin, iron and phosphorus were lowered. Mildly elevated serum bilirubin and fibrinogen concentrations were observed. Antibody titres were determined with the IFA and complement fixation (CF) tests. Antibodies to B. equi were first detected between Days 10-19 and 12-38 with the IFA and CF test, respectively, while the corresponding IFA periods for B. caballi were 6-8 days after infection. The parasitaemia of both B. equi and B. caballi infections never reached the 1% level.     

A case of transplacental transmission of Theileria equi in a foal in Trinidad

Equine piroplasmosis due to Theileria equi and Babesia caballi is endemic in Trinidad. A case of equine piroplasmosis due to T. equi was diagnosed in a thoroughbred foal at 10h post-partum. A high parasitaemia (63%) of piroplasms was observed in a Wright-Giemsa(?) stained thin blood smear from the foal. In addition, the 18S rRNA gene for Babesia/Theileria was amplified from DNA extracted from the blood of the foal and the mare. Amplified products were subjected to a reverse line blot hybridization assay (RLB), which confirmed the presence of T. equi DNA in the foal. The mare was negative by RLB but was positive for T. equi using a nested PCR and sequence analysis. In areas where equine piroplasmosis is endemic, severe jaundice in a post-partum foal may be easily misdiagnosed as neonatal isoerythrolysis. Foals with post-partum jaundice should be screened for equine piroplasmosis, which may be confirmed using molecular methods if available.