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.     

Seroepidemiologic studies on Babesia equi and Babesia caballi infections in horses in Jilin province of China

The prevalence of equine piroplasmosis caused by Babesia equi and Babesia caballi in northeast China has remained unknown, although the People's Republic of China is recognized as an endemic country for the diseases. In the present study, we investigated the prevalence of equine piroplasmosis in Jilin province, a part of northeast China. A total of 111 serum samples were taken from horses in eastern Jilin, and examined for diagnosis of B. equi and B. caballi infections by the enzyme-linked immunosorbent assays with recombinant antigens, equi merozoite antigen-1 and P48, respectively. Of the 111 samples, 38 (34%) and 36 (32%) samples were sero-positive for B. equi infection and B. caballi infection, respectively. In addition, 14 (12%) samples were sero-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 northeast China.     

A serological study of Babesia caballi and Theileria equi in Thoroughbreds in Trinidad

Ninety-three (93) horses were investigated for serum antibodies to Theileria equi (T. equi) and Babesia caballi (B. caballi) using the immunofluorescent antibody test (IFAT). Seventy-seven (82.8%) horses were seropositive; 31 (33.3%) were positive to T. equi compared to 64 (68.8%) to B. caballi while 18 (19.4%) horses were seropositive to both parasites. No significant differences in antibody frequencies among females and males for either T. equi or B. caballi were noted. Differences in seropositivity to B. caballi among age groups were not significant. Antibodies to T. equi were more frequent than to B.caballi in the age group 5 years and over than in the 1-2 and 2-4 years age groups (p<0.05). Unlike T. equi antibodies, B. caballi antibodies in horses in the county of Caroni were significantly less frequent when compared to other counties (p<0.05). Of 18 (19.4%) clinically ill horses, seven (42.9%) had clinicopathological evidence of anemia. Only one-third (6 of 18) horses were positive for the parasite on Wright-Giemsa stained blood smears and anemia was present in only 2. We report here that B. caballi and not T. equi may be the more common agent of piroplasmosis in Trinidad.     

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.     

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.     

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.     

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。     

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.     

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.     

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.     

Repeated high dose imidocarb dipropionate treatment did not eliminate Babesia caballi from naturally infected horses as determined by PCR-reverse line blot hybridization

Imidocarb treatment of horses infected with Babesia caballi is supposed to eliminate the infection, but data on the efficacy of this treatment is scarce. The study presented here concerns four Paso Fino horses, which were imported into the island of Curacao on the basis of a piroplasmosis negative complement fixation test (CFT). Upon re-testing with an indirect fluorescent antibody test immediately after arrival in Curacao, two horses appeared to have antibodies to B. caballi and all horses had antibodies to Theileria equi. Subsequent testing with polymerase chain reaction combined with a reverse line blot yielded positive results for both agents in all four horses. Treatment with five consecutive doses of imidocarb dipropionate (4.7 mg/kg BW im q 72 h), temporarily resulted in negative results, but B. caballi and T. equi were detected again in the samples taken at 6 and 18 weeks after completion of the treatment. These results confirm that the CFT is not a suitable test for pre-import testing and that even high dose treatment with imidocarb may not be capable of eliminating B. caballi and T. equi infections from healthy carriers.     

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.     

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.     

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.     

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.     

Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood

With the aim of developing more simple diagnostic alternatives, a differential single-round and multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of Babesia caballi and Babesia equi, by targeting 18S ribosomal RNA genes. The multiplex PCR amplified DNA fragments of 540 and 392 bp from B. caballi and B. equi, respectively, in one reaction. The PCR method evaluated on 39 blood samples collected from domestic horses in Mongolia yielded similar results to those obtained from confirmative PCR methods that had been established earlier. Thus, the single-round and multiplex PCR method offers a simple tool for the differential diagnosis of B. caballi and B. equi infections in routine diagnostic laboratory settings as well as in epidemiological studies.     

A survey for piroplasmids in horses and Bactrian camels in North-Eastern Mongolia

Equine piroplasmosis caused by Babesia caballi and Theileria equi is widespread in Asia. The presence of these haemozoans in Mongolia was previously confirmed in domestic as well as in reintroduced Przewalski horses in which they cause significant pathology. The data on occurrence of piroplasms from Bactrian camels in Asia is lacking. A total of 192 horses, 70 Bactrian camels, and additional 16 shepherd dogs from the Hentiy province were included in our study. No clinical signs typical for piroplasmid infection were observed during the field survey. Microscopic examination revealed the presence of T. equi in blood smears from 67% of examined horses, with camels and dogs being negative. A two step PCR approach was used to detect piroplasms in peripheral blood. In the first "catch all" PCR reaction, amplification of the 496 bp-long fragment of the SSU rRNA gene enabled the detection of Babesia and Theileria spp. Second round multiplex PCR reaction used for species discrimination allowed the amplification of T. equi- and B. caballi-specific 340 bp and 650 bp-long regions of the SSU rRNA, respectively. This assay detected T. equi in 92.7% of horses, while the infections with B. caballi and dual infections were rare. In both PCR setups, camels and dogs were negative indicating that in the studied region, these hosts do not share piroplasms with horses.     

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.     

Serodiagnosis of experimental and natural Babesia equi and B. caballi infections

The sensitivity and specificity of the complement fixation (CF) test for the diagnosis of Babesia infections in equines was assessed, using the indirect fluorescent antibody (IFA) test as a reference. Antibodies were first detected between 11 and 20 days post infection (dpi) in the CF test and between 7 and 14 dpi in the IFA test in ponies infected experimentally with B. equi (USDA strain). The CF test became negative in four of five ponies 63-174 dpi although B. equi was demonstrated microscopically in two of these four ponies up to 364 and 455 dpi. The IFA test remained positive up to 476 dpi (end of the examination period). Ponies infected experimentally with B. caballi (USDA strain) showed positive reactions in the CF test at first between 13 and 15 dpi and in the IFA test 10 or 11 dpi. The CF test became negative in two of three ponies 80 and 140 dpi, whereas the IFA test remained positive up to 190 dpi (end of the examination period). Cross-reactions of sera with heterologous antigens occurred at dilutions of 1/5 in the CF test and up to 1/20 in the IFA test. A total of 3944 CF tests was performed on 3765 horses from various European countries during 1980-1984. Sera that gave positive or trace CF reactions were retested in the IFA test. All 123 CF-positive sera were also IFA-positive and 26 of 31 sera (B. equi) and 11 of 32 sera (B. caballi) showing CF trace reactions were positive in the IFA test. Sera of two CF-negative horses were positive in the IFA test (B. equi); one of these horses was also positive upon microscopic examination. In seven of 21 horses repeatedly examined over longer periods the IFA titers (B. equi) persisted for up to 454 days longer than the CF titers. Sera of horses from highly endemic areas gave the following reactions: Sudan, 62 of 91 sera CF- and 86 of 91 IFA-positive; Zaire, 58 of 75 sera CF- and 72 of 75 IFA-positive; Columbia, 51 of 56 sera CF- and 56 of 56 IFA-positive; Brazil, 17 of 25 sera CF- and 21 of 25 IFA-positive. Only B. equi infections were demonstrated in Zaire. The combined use of the CF and IFA tests is recommended for safe identification of equine Babesia infections.     

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.