Sheep as a new experimental host for Babesia divergens

Babesia divergens was cultivated in sheep erythrocytes in RPMI 1640 supplemented with 10% Fetal Calf Serum (FCS) or sheep serum. In vitro cultures in sheep red blood cells were initiated with human erythrocytes infected in vitro with B. divergens Rouen 1987 or with gerbil blood infected with several isolates from bovine origin. After the first subcultures on sheep erythrocytes, a ten-fold multiplication of the parasites was obtained within 48 h. Erythrocytes from three splenectomized sheep were infected in vitro with B. divergens; when parasitaemia reached 10%, the animals were inoculated with homologous parasitized erythrocytes. All sheep expressed hyperthermia with a peak between the 6th and the 9th day post-infection (p-i) and a transitory parasitaemia 10 days p-i. In vitro primary cultures were performed on two of these sheep, demonstrating the parasite persistence at very low parasitaemia in the infected animals. Splenectomized sheep can be used as a new model for B. divergens chronic infection.     

Heterologous strain immunity in bovine babesiosis using a culture-derived soluble Babesia bovis immunogen

The cross-protective capacity of culture-derived soluble immunogens against heterologous Babesia bovis strains from different geographical locations of Latin America was examined. Susceptible yearling cattle were either immunized with immunogens derived from Venezuelan or Mexican strains, or were administered a multi-component immunogen containing antigens of the Australian, Mexican and Venezuelan strains. Cattle were challenged with virulent B. bovis organisms of the Argentinian, Colombian, Ecuadorean, Mexican and Venezuelan strains. The major parameters used to evaluate cross-protection were the following: presence, level and duration of parasitemia; maximal PCV reduction; level and duration of fever; determination of fibrinogen and cryofibrinogen; homologous and heterologous antibody levels; and net gains in body weight. Results showed good protection with a Venezuelan B. bovis immunogen after homologous and heterologous challenge exposures. A low degree of cross-immunity was observed when cattle vaccinated with the Mexican immunogen were challenged with each of the heterologous strains.     

Development of a Babesia rodhaini mouse assay for quality control of the serum component of a bovine babesiosis vaccine

The rodent babesia, Babesia rodhaini, survived equally well in basal medium containing either 10 per cent rat serum or 10 per cent bovine serum. As a result, a B rodhaini mouse assay is now performed routinely to determine the suitability of batches of bovine serum for use in a commercial Babesia bovis vaccine issued in Australia. Heat inactivation of bovine serum at 56 degrees C for two hours did not affect the survival of either B bovis or B rodhaini.     

An ELISA for the diagnosis of Babesia ovis infection utilizing a synthetic, Babesia bovis-derived antigen

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of Babesia ovis antibodies is described. In an initial study, a crude Babesia bovis antigen and a synthetic B. bovis-derived antigen (designated 11C5) were used to screen 46 B. ovis-positive and 55 negative sheep sera. A 95% correlation between the two antigenic preparations was found with the positive sera; no negative sera gave positive reactions. The synthetic antigen was then used in the screening of 1466 sera collected from sheep from 18 regions of Turkey. A high incidence of B. ovis-positive reactions was found from all regions (60-80%) in sheep over 1 year old, while from two smaller samples the incidence in young sheep was much less (28 and 52%). This test is superior to existing ones because the synthetic antigen can be produced in a highly reproducible state, is specific and is stable over extended periods of time.     

Inhibition of Babesia divergens in cattle by oxytetracycline

The effects of continuous oxytetracycline administration on the development of parasitaemia of Babesia divergens during both natural and artificial infections were studied. During natural exposure on grazing heavily infested with Ixodes ricinus, seven out of 42 cattle with no previous exposure to tick-borne diseases were injected every four days with a long acting preparation of oxytetracycline at a dose rate of 20 mg/kg. During the six week grazing period 21 untreated cattle developed a patent parasitaemia of B divergens and all became seropositive by the fluorescent antibody test. In contrast, no parasites were observed in treated cattle and antibody titres remained low. Artificial infections were studied with different dose levels of oxytetracycline and their effects on antibody stimulation noted. First, four groups of cows were infected with 10(8) erythrocytes infected with B divergens, three groups being injected every four days with the long acting oxytetracycline formulation at dose levels of 20, 10 and 5 mg/kg, respectively. The highest level completely inhibited parasite replication and antibody formation; the same was observed in one animal dosed at 10 mg/kg but the remainder, plus those treated at 5 mg/kg, developed both low parasitaemia and high antibody titres. The untreated cows developed severe babesiosis. A further untreated control group was added and three weeks after cessation of oxytetracycline treatment all were infected with 10(9) erythrocytes infected with a homologous isolate of B divergens. The controls, plus those in which the previous infection had been completely inhibited, developed severe clinical babesiosis but the remainder were refractory to parasite development.     

The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay

ABSTRACT: Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.     

Utilization of culture-derived soluble antigen in the latex agglutination test for bovine babesiosis and anaplasmosis

A simple lated agglutination test (LAT) for the diagnosis of Babesia bovis and Anaplasma marginale infection in cattle was developed using cell culture-derived soluble antigens to sensitize latex particles. The conditions to perform the test were established as follows: a 2% suspension of polystyrene latex particles (0.8 μm diameter) in 0.15 M glycine buffer pH 8.3 containing 0.2% disodium-EDTA was used to sensitize an equal vlume of antigen at a final antigen concentration between 0.625×–1.25× of the original antigen concentration of the supernatant culture medium. The latex particles were sensitized for 15 min at 56°C, The test, which uses heat-inactivated sera, was performed at room temperature by mixing one drop of each antigen and serum on a glass slide. T he LAT showed a high degree of specificity and sensitivity when compared with the babesiosis indirect fluorescent antibody (IFA) and anaplasmosis capillary tube-agglutination (CA) tests. The LAT possesses appropriate stability and simplicity suitable for field purposes.     

Chemotherapy of Babesia divergens in the gerbil, Meriones unguiculatus

It was found that surprisingly low doses of four babesicides were effective against Babesia divergens in gerbils and it was concluded that this was due to the involvement of host resistance, which may be of a non-specific nature. The efficacy of the drugs relative to each other was the same in gerbils as in cattle and this host-parasite system is evidently more suitable for the screening of babesicides than are other rodent babesia systems. The prophylactic dose of imidocarb dipropionate required to provide a similar degree of protection in gerbils as in cattle was found to be much higher and was very close to toxic levels. Challenge infections resulted in sterile immunity. Acute babesiosis in gerbils could be cured with all four drugs if parasitaemias were below approximately 45 per cent and packed cell volumes above 18 per cent at treatment.     

A novel 57-kDa merozoite protein of Babesia gibsoni is a prospective antigen for diagnosis and serosurvey of canine babesiosis by enzyme-linked immunosorbent assay

We isolated a novel single copy gene encoding a 57-kDa merozoite protein of Babesia gibsoni (BgP57). The nucleotide sequence of the cDNA was 2387 bp with an open reading frame (ORF) of 1644 bp encoding a 57-kDa predicted polypeptide having 547 amino acid residues. The recombinant BgP57 (rBgP57) without a predicted signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein. Western blotting showed that the corresponding native protein was 57-kDa, consistent with molecular weight of predicted mature polypeptide. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP57 detected specific antibodies in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with antibodies to B. canis sub-species and closely related apicomplexan parasites indicating that the rBgP57 was a specific antigen for B. gibsoni antibodies. The diagnostic performance of ELISA based on rBgP57 using 107 sera from B. gibsoni-naturally infected dogs was the same as the previously identified rBgP32 but performed better than the previously studied rBgP50. Although, seminested PCR detected higher proportions (82%) of positive samples than the ELISAs, the Mcnemar's chi-square test showed that there was no significant difference in relative effectiveness of rBgP57-ELISA and seminested PCR (chi(2)=2.70; P=0.1003) in identifying positive samples. The rBgP57-ELISA when used in combination with rBgP32-ELISA and rBgP50-ELISA appeared to improve sensitivity of the rBgP57-ELISA for detection of B. gibsoni antibodies. Overall, the rBgP57-ELISA and seminested PCR when used in combination, could improve epidemiological surveys and clinical diagnosis of B. gibsoni infection.     

Use of cerebellar brain smears in the diagnosis of babesiosis (Babesia bovis) in cattle

Summary Cerebral and cerebellar smears were made from 4 animals acutely reacting toBabesia bovis and 94 animals free from clinical babesiosis. The brain smears were stained by the Giemsa method and examined for the presence ofB. bovis parasites. In animals showing clinical babesiosis capillaries congested with parasitised erythrocytes were abundant in cerebral and cerebellar smears. Results obtained from both types of brain smears in animals free from clinical babesiosis agreed closely (83% conformity) as to the presence or absence of parasites. A third group of 39 animals from which cerebral and cerebellar smears were taken was also examined serologically by the indirect immunofluorescent antibody test (IFAT); about 69% of the IFAT positive and doubtful animals showed parasites in the cerebellar brain smears. The existence of false negatives in the IFAT test has been shown and discussed. It has been concluded that cerebellar samples obtained through the foramen occipitale can be used for the microscopic detection ofB. bovis parasites in latently infected bovines. This method can also be used in field cases suspected of cerebral babesiosis permitting brain sampling without resorting to the opening of the skull. Such an approach might prove particularly useful in areas where rabies occurs and the animal's head has to be sent to a diagnostic centre.

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El Empleo De Frotis De Cerebelo En El Diagnostico De Babesiosis (Babesia Bovis) En Bovinos

Resumen Se hicieron frotis de cerebro y cerebelo de 4 animales con síntomas agudos de babesiosis porBabesia bovis y de 94 animales clínicamente sanos. Los frotis se tiñeron con Giemsa y se examinaron por la presencia deB. bovis. En animales enfermos, los capilares del cerebro y cerebelo se encontraron congestionados y repletos de eritrocitos parasitados. Los resultados obtenidos en ambos tipos de frotis en animales libres de la enfermedad clínica, estuvieron de acuerdo (83% de conformidad) en cuanto a la presencia o ausencia de parásitos. Un tercer grupo de 39 animales de los cuales se tomaron frotis de cerebro y cerebelo, se examinaron también serológicamente mediante la técnica indirecta de anticuerpos fluorescentes (TIAF); cerca del 69% de animales positivos y dudosos por immunofluorescencia revelaron parásitos en los frotis de cerebelo. La existencia de falsos positivos se demostró mediante TIAF. Se concluye que los frotis de cerebelo tomados a través del agujero occipital pueden usarse para detectarB. bovis microscópicamente en animales con infecciones latentes. Este método se puede utilizar en el campo para no tener que abrir el craneo, sobre todo en áreas en donde existe rabia y las cabezas se envían al laboratorio.

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Examen De Frottis De Matière Cervelette Pour Le Diagnostic De La Babésiose (babesia bovis) Du Bétail

Résumé Des frottis de cerveau et de cervelet ont été effectués à partir de 4 animaux atteints de babésiose aiguë et de 94 autres n'ayant présenté aucun symptôme de la maladie. Les frottis ont été colorés au Giemsa et examinés pour mettre en évidence la présence deB. bovis. Chez les animaux atteints de babésiose clinique, les capillaires étaient congestionnés avec présence abondante d'érythrocytes parasités. Il existe une excellente concordance (83 p.100) entre les résultats des examens des deux types de frottis, qu'il s'agisse d'absence ou de présence des parasites. Un troisième groupe de 39 animaux dont les frottis de cerveau et de cervelet ont été faits ont également fait l'objet d'une étude sérologique pour déceler des anticorps par le test de l'immunofluorescence indirecte (I.F.A.T.); environ 69 p.100 des animaux reconnus positifs et douteux par l'I.F.A.T. ont montré des parasites dans les frottis du cervelet. L'existence de cas négatifs faux dans le test IFAT a été montrée et discutée. Il en a été conclu que les échantillons de cervelet obtenus à travers le foramen occipital peuvent être utilisés pour la détection microscopique deB. bovis chez des animaux en état d'infection latente. Cette méthode peut être également utilisée en brousse lors de suspicion de babésiose cérébrale car elle permet d'obtenir les prélèvements de tissus cérébraux nécessaires sans ouverture de la boite cranienne. Une telle approche peut être particulièrement utile dans les régions où sévit la rage et où il est nécessaire d'expédier la tête des animaux suspects à un centre de diagnostic.

     

Molecular cloning and genetic polymorphism of Babesia capreoli gene Bcp37/41, an ortholog of Babesia divergens merozoite surface antigen Bd37

Babesia divergens and Babesia capreoli are closely related species with distinct host ranges, a zoonotic feature being described only for B. divergens. The two species are 99.8% similar in the 18S rDNA gene sequence and indistinguishable by morphological or serological means, leading to confusion as to their species status. The phylogenetic relatedness between the two species, and the frequent involvement of surface components in serological cross-reactions led us to postulate that an ortholog of Bd37, the merozoite surface antigen described for B. divergens, could also exist in B. capreoli. We were able to amplify a single partial PCR product from B. capreoli genomic DNA using primers specific for the B. divergens merozoite surface protein coding gene Bd37, and sequencing confirmed the presence of a Bd37 ortholog in B. capreoli, named Bcp37/41. The full sequences of the Bcp37/41 genes and their intron-exon structures were obtained for two cloned lines of B. capreoli. They suggest functional homologies between Bd37 and Bcp37/41 such as their surface localization, their role in immune escape mechanism and in the initial non-specific attachment to the erythrocyte. Restriction fragment length polymorphism (RFLP) analysis of the amplicons and partial sequencing revealed an extreme polymorphism within B. capreoli, greater than the one observed for its ortholog Bd37. Such a marker could thus be useful in epidemiological as well as phylogenetic studies.     

Babesia divergens: the immunisation of splenectomised calves during irradiated piroplasms.

Groups of three splenectomised calves were inoculated with 1 . 2 x 10(10) Babesia divergens-infected erythrocytes irradiated at 24, 28, 32, 36 and 40 kilorads. Control calves were inoculated with 1 . 2 x 10(7) or 1 . 2 x 10(4) non-irradiated parasites. While control animals all experienced severe reactions, animals receiving blood irradiated at 24, 28 and 32 kilorads had mild reactions and were solidly immune to an homologous challenge of 1 . 1 x 10(4) Babesia-infected erythrocytes. Animals receiving parasites irradiated at 36 and 40 kilorads had limited ability to resist the challenge.     

Babesia divergens: cloning of a Ran binding protein 1 homologue

Babesia divergens is an Apicomplexa transmitted to bovines by its acarian vector, the tick I. ricinus. Babesia divergens merozoites have an intraerythrocytic development in the blood of infected mammals. The nucleocytoplasmic transport system in this parasite is not yet characterized and no protein involvement in such transport has been described. In this report, we describe the cloning of a protein that shares important homologies with Ran binding protein 1. This protein in Eukaryote belongs to the nucleocytoplasmic transport system.     

Effects of rapid passage in the gerbil (Meriones unguiculatus) on the course of infection of the bovine piroplasm Babesia divergens in splenectomised calves

The antigenicity and virulence of the cattle piroplasm Babesia divergens was compared in splenectomised calves before and after adaptation to the gerbil. It was apparent that adaptation of B divergens to the gerbil did not result in attenuation of the parasite in cattle. The gerbil-adapted parasite had a shorter prepatent period, produced higher parasitaemias, and more severe haematological and biochemical changes than the initial bovine strain. The antigenicity of the parasite was unchanged by gerbil passage. The changes in the virulence of the gerbil-adapted parasite were attributed to selection of a rapidly replicating parasite.     

A strain of Babesia divergens, attenuated after long term culture

The Weybridge strain of Babesia divergens became less virulent after 18 months in culture and was believed to be attenuated. Inoculations with the attenuated line using as many as 5 x 10(8) infected erythrocytes failed to raise a normal infection in Mongolian gerbils (Meriones unguiculatus) whereas, with the line that had been passaged from culture through gerbils at bimonthly intervals, inoculations of 10(7) infected erythrocytes gave rise to fatal infections. Gerbils were immunised with the attenuated line using doses ranging from 5 x 10(5) to 5 x 10(7) infected erythrocytes. Parasitaemias in the infected gerbils did not exceed 0.7 per cent after immunisation. All animals were protected when challenged with the virulent line four weeks later. Control animals died four days after challenge infection.     

Canine babesiosis in Slovenia: molecular evidence of Babesia canis canis and Babesia canis vogeli

Canine babesiosis, caused by intraerythrocytic Babesia spp., is a tick-borne disease of worldwide importance. No information on canine babesiosis has been documented in Slovenia. Therefore, 238 dogs admitted to the Small animal clinic in Ljubljana from the years 2000 to 2002 were tested for the presence of babesial parasites in the blood. Based on clinical, microscopic and molecular investigations, 14 dogs (5.9%) were determined as being infected with babesiae. Clinical signs relating to acute haemolysis, fever, anorexia, depression and haematological abnormalities such as anaemia and thrombocytopenia were noticed in most of the 14 infected dogs. The morphology of the parasites was indicative of Babesia canis infection. Two subspecies were detected, namely B. canis canis (11 dogs, 4.6%) and B. canis vogeli (3 dogs, 1.3%) using PCR and subsequent sequence analysis of portions of nns rRNA gene. In addition, based on nucleotide sequence analysis, the 11 isolates of B. c. canis could be subdivided into three groups, whereas the three B. c. vogeli isolates were genetically identical. The results of this study demonstrate the presence of canine babesiosis due to B. c. canis and B. c. vogeli in Slovenia.     

Annual evolution of cattle immunity against Babesia divergens in Northern Switzerland

A seroepidemiological survey of Babesia divergens babesiosis was conducted on 9 farms in a focus of northern Switzerland (Clos-du-Doubs, Jura). During 1981, a total of > 300 cattle were examined for B. divergens antibodies with an indirect immunofluorescence test. Annual evolution of immunity was estimated 3 times during the year (April, July and December). Cattle were progressively sensitized against B. divergens during the grazing period. Considering all cattle, 33.9% possessed anti-B. divergens antibodies in spring, before the grazing period. This percentage increased in the summer (59.1%), reaching 90.6% in December.In the studied area, the most important calving period occurs in autumn and young calves do not leave the farm before they are 2 months old. Calves generally lacked maternal antibodies when they were moved to pastures for the first time. Although infested with B. divergens-infected ticks, they never presented acute symptoms of babesiosis. They appeared to be naturally resistant to the disease. Cattle from 9 months to 3 years old were more susceptible. In April, 73.4% did not show antibodies. During our study, 12 of the 15 cattle of that age which had babesiosis did not possess antibodies. Cattle > 3 years old were more protected. In April, only 51% lacked antibodies and 1 case of babesiosis was observed in this group.