Molecular evidence for transplacental transmission of Theileria equi from carrier mares to their apparently healthy foals

The intra-erythrocytic parasite Theileria equi is one of two tick-transmitted causative agents of equine piroplasmosis. Piroplasms of T. equi can be transmitted across the equine placenta and once a horse is infected, it appears to remain a lifelong carrier, since anti-theilerial drugs suppress but do not eliminate the parasite. Carrier mares may transmit the organism to their offspring and this may result in abortion or neonatal piroplasmosis, but observations by some researchers suggest that foals may be born as carriers yet remain apparently healthy. Using a T. equi-specific oligonucleotide probe, we have determined that transplacental transmission occurs early in equine foetal development and that carrier mares may give birth to healthy carrier foals. Investigation of parasite levels and the effect of maternal colostrum on the newborn suggests that colostral T. equi antibody may act to suppress parasitaemia in the newborn, reducing the incidence of clinical neonatal piroplasmosis.     

Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction

We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.     

Antimicrobial Activity of Tulathromycin and 14 Other Antimicrobials Against Virulent Rhodococcus equi In Vitro

This study determined the antimicrobial activity of tulathromycin against Rhodococcus equi in vitro. Ninety-eight virulent isolates of R. equi from equine clinical cases were examined, of which 20 isolates were macrolide resistant. A custom 96-well antimicrobial susceptibility testing plate was used, allowing 14 additional antimicrobials to be tested against R. equi. Isolates were cultured with various concentrations of antimicrobials, and minimal inhibitory concentration (MIC) values were determined. Tulathromycin was found to have poor activity in vitro against R. equi isolates susceptible or resistant to macrolides, with MIC50 and MIC90 values >64 ug/mL for all isolates. MIC values for other macrolides tested were similar to previously published data.     

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.     

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.     

Determination of the activity of ponazuril against Sarcocystis neurona in cell cultures

The present study examined the efficacy of ponazuril in inhibiting merozoite production of Sarcocystis neurona in cell cultures. Ponazuril inhibited merozoite production by more that 90% in cultures of S. neurona treated with 1.0 microg/ml ponazuril and greater than 95% inhibition of merozoite production was observed when infected cultures were treated with 5.0 microg/ml ponazuril. Ponazuril may have promise as a therapeutic agent in the treatment of S. neurona induced equine protozoal myeloencephalitis (EPM) in horses.     

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。     

In-vivo therapeutic efficacy trial with artemisinin derivative, buparvaquone and imidocarb dipropionate against Babesia equi infection in donkeys

The therapeutic efficacy of imidocarb, artesunate, arteether, buparvaquone and arteether+buparvaquone combination was evaluated against Babesia equi of Indian origin in splenectomised donkeys with experimentally induced acute infection. Efficacies of these drugs were tested by administering each drug or drug combination to groups of donkeys (having three donkeys each group). One group of donkey was kept as untreated control for comparing the results. Parasitaemia, haematology (WBC, RBC, PCV, granulocytes and haemoglobin), biochemical parameters (SAST, SALT, alkaline phosphatase, albumin/globulin ratio) were monitored at regular intervals. Individually, arteether and buparvaquone were found to have no parasite clearing efficacy and the treated animals died within 5-6 days after showing high parasitaemia and clinical symptoms of the disease. However, artesunate treated animals were able to restrict the parasite multiplication but only during the treatment period. Animals treated with imidocarb and arteether+buparvaquone combination were able to clear the parasite from the blood circulation after 2-5 days post-treatment (PT). After 55-58 days PT, recrudescence of B. equi parasite was observed in both these groups and a mean survival period of 66 days and 69 days, respectively, was recorded in these groups. Results of haemato-biochemical parameters had shown that imidocarb had deleterious effect on the liver function while on the other hand arteether+buparvaquone combination was found to be safe. This limited study indicates that arteether+buparvaquone combination could be a better choice than imidocarb for treating B. equi infection, but further trials are required in detail.     

Hematologic and immunophenotypic factors associated with development of Rhodococcus equi pneumonia of foals at equine breeding farms with endemic infection

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals and in immunocompromised people. In mice, both CD4+ and CD8+ T lymphocytes contribute to host defense against R. equi, but CD4+ T lymphocytes are required for pulmonary clearance of the bacteria. In this prospective study of 208 foals at two equine breeding farms with endemic R. equi infections, we collected peripheral blood samples at 2 and 4 weeks of age and at the time of diagnosis of R. equi pneumonia. Samples were analyzed for concentrations of total and differential leukocytes, EqCD4+ and EqCD8+ T lymphocytes, and B lymphocytes. Thirty (14.4%) foals developed R. equi pneumonia. At the 2nd week of life, affected foals had significantly lower concentrations of white blood cells (WBC) and segmented neutrophils, significantly lower proportions of EqCD4+ T lymphocytes, and significantly higher proportions of EqCD8+ T lymphocytes. The EqCD4:EqCD8 ratio was significantly lower for affected foals. At the 4th week of life, affected foals had significantly lower concentrations of segmented neutrophils and EqCD4+ T lymphocytes than did unaffected foals. The ratio of EqCD4:EqCD8 was significantly lower for affected foals. Two- and 4-week-old foals with ratios of EqCD4:EqCD8<3 were significantly more likely to develop R. equi pneumonia. There was a significant farm effect which diluted our statistical power to detect differences; however; after adjusting for the farm effect, 2-week-old foals with ratios of EqCD4:EqCD8<3 remained significantly more likely to develop R. equi pneumonia. There were no significant differences in immunophenotypic variables between affected foals (at the time of diagnosis) and age-matched control foals. These data suggest that there are hematologic and immunophenotypic differences between affected and unaffected foals during the first 2-4 weeks of life, prior to onset of clinical signs of R. equi pneumonia. These differences may represent important immunologic mechanisms associated with increased susceptibility of individual foals to infection with R. equi. Because there was considerable overlap between values for affected and unaffected foals, we cannot yet recommend immunophenotyping of foals at endemically-infected farms as a clinically useful screening tool to identify foals at increased risk of developing R. equi pneumonia.     

马泰勒虫病PCR检测方法的建立和应用

为寻求一种快速、有效的马泰勒虫 (Theileria equi,T.equi) PCR检测方法。基于马泰勒虫18S rRNA基因序列,在其V4高变区设计特异性引物Te-18F、Te-18R,通过PCR技术获得了531 bp的核酸片段。用该引物对马泰勒虫、马泰勒虫和驽巴贝斯虫混合样本、驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫基因组模板进行特异性试验。同时对马泰勒虫基因组模板进行不同浓度稀释后扩增,以便于确定试验的敏感性。用本试验建立的方法与常规显微镜镜检方法对45份马属动物血样进行检测。特异性试验显示,在被检测的9个样本中,只有马泰勒虫及马泰勒虫和驽巴贝斯虫混合模板中扩增出了符合大小的特异核酸片段。驽巴贝斯虫、尤氏泰勒虫、中华泰勒虫、环形泰勒虫、绵羊泰勒虫、吕氏泰勒虫和瑟氏泰勒虫的扩增结果均为阴性。灵敏度试验结果表明,PCR对马泰勒虫的扩增效率可达到10^-13。对本试验建立的PCR检测马泰勒虫方法评估结果显示,PCR对马泰勒虫的检出率为17.78%(8/45),显微镜镜检结果只有8.89%(4/45)两者的符合率为100%。本试验建立的马泰勒虫PCR检测方法不失,为一种好的检测方法。     

In vitro quantitative analysis of (3)H-uracil incorporation by Sarcocytis neurona to determine efficacy of anti-protozoal agents

Parasite-specific incorporation of (3)H-uracil was used to assess the replication of Sarcocystis neurona, a protozoal parasite associated with equine protozoal myeloencephalitis (EPM). Anti-protozoal drugs, pyrimethamine (0.01, 0.1 and 1.0microg/ml PYR), sulfadiazine (5microg/ml; SDZ), sulfamethoxazole (5microg/ml; SMZ), diclazuril (100ng/ml; DCZ), atovaquone (0.04ng/ml; ATQ), tetracycline (5microg/ml; TET) and the herbicide glyphosate (1.5 and 4.5mM; GLY) were studied with varying S. neurona parasite densities (2x10(1)-1.2x10(6)merozoites/well). A microtiter plate format was used to test these compounds, and incorporation of (3)H-uracil was determined using a semi-automated plate harvester and liquid scintillation counter. When PYR, DCZ, ATQ, SMZ, SDZ, and TET were tested, the assay was most reliable when parasite densities were greater than 9.0x10(4) individual merozoites per well. When the herbicide GLY was tested, as few as 900 individual merozoites were sufficient to demonstrate reduction in parasite proliferation. Of the anti-protozoal drugs commonly used to treat EPM, PYR was the most potent anti-S. neurona agent tested. The herbicide GLY appears to be more potent than all of the other compounds tested in vitro; however information regarding in vivo use of GLY is not available, and central nervous system penetration by this compound is unlikely. Incorporation of (3)H-uracil by replicating S. neurona is quantitative and can be used in a semi-automated assay. This in vitro assay is capable of high throughput screening of candidate drugs that may have applications in a clinical setting. Further studies using a wider range of drug concentrations with optimal numbers of merozoites are necessary to determine true potency of these agents.     

Species-specific serodiagnosis of equine piroplasma infections by means of complement fixation test (CFT), immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA)

The increasing horse trade requires a reliable immunodiagnosis of equine piroplasma infections due to import restrictions imposed by various countries, including the United States of America. It was the aim of our investigations to establish the suitability of serological tests for the detection of parasite carriers and, eventually, to differentiate between Babesia caballi and B. equi infections. The investigations were carried out on 11 ponies with experimentally-induced B. caballi and/or B. equi infection. The infections were confirmed by the demonstration of parasites in blood smears 2-13 days post infection (PI). The complement fixation test (CFT), the indirect immunofluorescence (IIF) and the enzyme-linked immunosorbent assay (ELISA) were employed for the demonstration of antibodies, and different antigen preparations were tested for their suitability. Antibodies could be demonstrated by all three tests. Complement-fixing antibodies disappear after 2-3 months PI in B. caballi-infected horses, while the IIF and ELISA gave positive results during latent infection. A reliable serodiagnosis thus requires the use of the CFT and IIF, since parasite carriers may appear seronegative by the CFT. Serological differentiation between B. caballi and B. equi was possible by CFT and, to a certain extent, by IIF during early infection, but not by ELISA. The successful treatment of B. caballi infections with Berenil could only be confirmed serologically by IIF.     

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.     

Surveillance programme for important equine infectious respiratory pathogens in the USA

The prevalence and epidemiology of important viral (equine influenza virus [EIV], equine herpesvirus type 1 [EHV-1] and EHV-4) and bacterial (Streptococcus equi subspecies equi) respiratory pathogens shed by horses presented to equine veterinarians with upper respiratory tract signs and/or acute febrile neurological disease were studied. Veterinarians from throughout the USA were enrolled in a surveillance programme and were asked to collect blood and nasal secretions from equine cases with acute infectious upper respiratory tract disease and/or acute onset of neurological disease. A questionnaire was used to collect information pertaining to each case and its clinical signs. Samples were tested by real-time PCR for the presence of EHV-1, EHV-4, EIV and S equi subspecies equi. A total of 761 horses, mules and donkeys were enrolled in the surveillance programme over a 24-month study period. In total, 201 (26.4 per cent) index cases tested PCR-positive for one or more of the four pathogens. The highest detection rate was for EHV-4 (82 cases), followed by EIV (60 cases), S equi subspecies equi (49 cases) and EHV-1 (23 cases). There were 15 horses with double infections and one horse with a triple infection. The detection rate by PCR for the different pathogens varied with season and with the age, breed, sex and use of the animal.     

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.     

In vitro erythrophagocytosis by cultured macrophages stimulated with extraneous substances and those isolated from the blood, spleen and bone marrow of Boran and N'Dama cattle infected with Trypanosoma congolense and Trypanosoma vivax

A standard radioactive chromium (51Cr) release assay was used to assess the in vitro phagocytosis and lysis of bovine erythrocytes by cultured splenic, bone marrow and peripheral blood monocyte-derived (PBM) macrophages isolated from healthy and Trypanosoma congolense and T. vivax-infected cattle of the Boran and N'Dama breeds. Recombinant cytokines (rHuTNF-alpha and rBolFN-gamma) and non-acid-dialysed peripheral blood mononuclear cell (PBMNC) culture supernatants stimulated these PBM for enhanced activities. The stimulants caused increases in the rate of erythrocyte phagocytosis and lysis by cultured PBM in a concentration-dependent manner. But very high stimulant concentrations caused deceased in vitro erythrophagocytosis. However, bacterial lipopolysaccharide (LPS) and acid-dialysed PBMNC culture supernatants did not cause any increase in cultured PBM erythrophagocytosis. In vitro erythrocyte phagocytosis and lysis by splenic, bone marrow and peripheral blood monocyte (PBM)-derived macrophages of Boran breed of cattle infected with Trypanosoma congolense increased from 14 days post-infection (DPI) onwards and thereafter maintained at various levels above pre-infection. Cultured splenic macrophages showed the greatest erythrocyte destruction capability while PBM-derived macrophages was the least. The rates of in vitro erythrocyte phagocytosis and lysis were higher with the cultured PBM of the Boran than those of the N'Dama cattle during T. congolense infection. The rate of in vitro erythrocyte destruction was however, similar in both groups of cattle during T. vivax infection. These results correlated positively with the dynamics and degree of anaemia developed by these groups of animals during both T. congolense and T. vivax infections. Cattle infected with T. congolense and T. vivax developed varying degrees of normocytic normochromic anaemia during infection. Boran cattle developed a more severe anaemia, and had to be treated with diminazine aceturate, than N'Dama cattle during T. congolense infection. Both breeds of cattle developed a milder but similar degree of anaemia during T. vivax infection. None of the animals were treated. The results of this study indicated a role of in vivo macrophage stimulatory factors, notably cytokines such as TNF-alpha and IFN-gamma in host's serum, as well as parasite antigens, which may act singly or in concert, in the process of enhanced erythrocyte destruction, hence anaemia by the mononuclear phagocytic system (MPS) during bovine trypanosomosis.     

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.     

Actions of non-steroidal anti-inflammatory drugs on equine leucocyte movement in vitro

The direct effects of four non-steroidal anti-inflammatory drugs (NSAIDs) on equine polymorphonuclear (PMN) and mononuclear (MN) leucocyte movement were investigated using two in vitro assay systems. The Boyden chamber microfilter technique measures both chemokinetic and chemotactic locomotion, and the agarose microdroplet assay measures solely chemokinesis. Zymosan-activated plasma (ZAP) and the synthetic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) were used as standard chemoattractants for PMN and MN leucocytes, respectively. The actions of six concentrations of each NSAID, indomethacin (50 microM-10 mM), phenylbutazone (10 microM-1 mM), oxyphenbutazone (2.5 microM-500 microM) and flunixin (0.1 microM-50 microM), in suppressing cell movement induced by ZAP and FMLP were investigated. All four drugs exerted inhibitory effects on induced movement of both cell types in the Boyden chamber assay, usually in a concentration-dependent manner, although oxyphenbutazone action on PMN cells occurred only at the highest concentration tested. Significant inhibition of PMN and MN cell locomotion was produced by indomethacin, flunixin and oxyphenbutazone, and inhibition of PMN movement by phenylbutazone occurred in the agarose microdroplet assay. Flunixin was the most potent of the four drugs investigated in both assay systems. The findings may be of importance to the use of phenylbutazone and flunixin as NSAIDs in equine medicine, since the concentrations used were similar to concentrations of both drugs and the phenylbutazone metabolite oxyphenbutazone previously reported to occur in equine plasma and inflammatory exudate.     

Review of triazine antiprotozoal drugs used in veterinary medicine

Triazines are relatively new antiprotozoal drugs that have successfully controlled coccidiosis and equine protozoal myeloencephalitis. These drugs have favorably treated other protozoal diseases such as neosporosis and toxoplasmosis. In this article, we discuss the pharmacological characteristics of five triazines, toltrazuril, ponazuril, clazuril, diclazuril, and nitromezuril which are used in veterinary medicine to control protozoal diseases which include coccidiosis, equine protozoal myeloencephalitis, neosporosis, and toxoplasmosis.     

Individual heterogeneity in erythrocyte susceptibility to Babesia divergens is a critical factor for the outcome of experimental spleen-intact sheep infections

Susceptibility of sheep erythrocytes to Babesia divergens was investigated in vitro and a high inter-individual variability in their ability to support parasite population development was demonstrated, with some individuals having refractory red blood cells (RBC). As neither changes in growth conditions nor the use of different B. divergens strains influenced the level of susceptibility, the main factor postulated for this variability is the erythrocyte itself. Sheep therefore represent an excellent in vitro model to study the parasite-erythrocyte interaction. In addition, the existence of refractory RBC should help in the identification of the erythrocyte components required for B. divergens development. Experimental infections were carried out on spleen-intact sheep characterized by refractory or fully susceptible erythrocyte types. These differences translated into the successful infection of only those animals with susceptible erythrocytes: infected animals showed no clinical signs, but maintained an asymptomatic persistent infection, as usually observed in the natural bovine host. Sheep therefore represent model organisms that can allow us to study interactions between B. divergens and its vertebrate host at different levels of biological organisation, from the target cell to the intact animal, and represent an experimental infection model of concomitant immunity. Only a low percentage (13%) of the sheep population tested possessed susceptible erythrocytes and the potential role of sheep as a natural host or reservoir of B. divergens is discussed.