Babesia spp. infection in Boophilus microplus engorged females and eggs in Sao Paulo State, Brazil

Babesia spp. infections were investigated in Bos taurus x Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01).     

PCR-based detection of the transovarial transmission of Uruguayan Babesia bovis and Babesia bigemina vaccine strains

Bovine babesiosis is responsible for serious economic losses in Uruguay. Haemovaccines play an important role in disease prevention, but concern has been raised about their use. It is feared that the attenuated Babesia bovis and Babesia bigemina vaccine strains may be transmitted by the local tick vector Boophilus microplus, and that reversion to virulence could occur. We therefore investigated the possibility that these strains could be transmitted via the transovarial route in ticks using a Babesia species-specific polymerase chain reaction (PCR) assay. DNA was extracted from the developmental stages of the tick vector that had fed on calves immunized with the haemovaccine. It was possible to detect Babesia DNA not only in adult ticks, but also in their eggs and larvae. In addition, it was shown that calves infested with larvae derived from eggs laid by ticks fed on acutely infected calves, were positive for Babesia using PCR. Caution should therefore be shown with the distribution of the haemovaccine in marginal areas. It is still advisable that suitable tick control measures be used to prevent transovarial transmission and the potential risk of attenuated Babesia reverting to virulence.     

Babesia bovis and B. bigemina DNA detected in cattle and ticks from Zimbabwe by polymerase chain reaction

From blood collected from 94 cattle at 12 locations in the eastern and northeastern areas of Zimbabwe, DNA was extracted and analysed by polymerase chain reaction with primers previously reported to be specific for Babesia bigemina and Babesia borvis. Overall, DNA of Babesia bigemina was detected in the blood of 33/94 (35%) cattle and DNA from B. bovis was detected in 27/58 (47%) of cattle. The prevalence of DNA of B. bigemina was significantly higher in young animals (<2 years) (23/46) than in animals over 2 years of age (10/48; chi2= 8.77; P <0.01%). Although tick sampling was not thorough, Boophilus decoloratus could be collected at 7/9 sites sampled and Boophilus microplus at 4/9 sites. Of the 20 B. decoloratus allowed to oviposit before PCR analysis, 1 (5%) contained DNA that could be amplified with primers for B. bigemina while 12 (60%) were positive with primers for B. bovis. Of the B. microplus allowed to oviposit, 11/16 (69%) were positive for B. bovis DNA by PCR and 2/16 (12%) were positive for B. bigemina.     

一种牛源扇头蜱及其携带梨形虫种类鉴定

为了鉴定从汉中市牛体表采集到的蜱种类及其携带病原梨形虫(Piroplasma)的种类,在形态学初步观察的基础上,用PCR技术基于线粒体16SrDNA对蜱种类进行了分子鉴定,并基于梨形虫18S rRNA基因分别检测蜱体内携带巴贝斯虫属(Babesia)、泰勒虫属(Theileria)等病原情况.结果显示,所采集到的67只...     

Molecular detection of Babesia bovis and Babesia bigemina in white-tailed deer (Odocoileus virginianus) from Tom Green County in central Texas

Serologic and molecular evidence suggest that white-tailed deer in South Texas and North Mexico carry the agents of bovine babesiosis, Babesia bovis and Babesia bigemina. To determine if white-tailed deer in central Texas, which is outside the known occurrence of the vector tick at this time, harbor these parasites, blood samples from free-ranging and captive white-tailed deer (Odocoileus virginianus) in Tom Green County were tested by polymerase chain reaction (PCR) assays for B. bovis and B. bigemina 18S rDNA. Of the 25 samples tested, three (12%) were positive by nested PCR for B. bovis. This identity was confirmed by sequence analysis of the cloned 18S rDNA PCR product. Further confirmation was made by sequence analysis of the rRNA internal transcribed spacer (ITS) 1, 5.8S rRNA gene, and ITS 2 genomic region in two (representing samples from two different ranches) of the B. bovis positive samples. Three samples were positive by B. bigemina nested PCR, but sequencing of the cloned products confirmed only one animal positive for B. bigemina; Theileria spp. DNA was amplified from the other two animal samples. In addition to Theileria spp., two genotypically unique Babesia species sequences were identified among the cloned sequences produced by the B. bigemina primers in one sample. Phylogenetic analysis showed no separation of the deer B. bovis or B. bigemina 18S rDNA, or deer B. bovis ITS region sequences from those of bovine origin. Clarification of the possible role of white-tailed deer as reservoir hosts in maintaining these important pathogens of cattle is critical to understanding whether or not deer contribute to the epidemiology of bovine babesiosis.     

Transmission of Babesia spp by the cattle tick (Boophilus microplus) to cattle treated with injectable or pour-on formulations of ivermectin and moxidectin

OBJECTIVE: To assess the efficacy of ivermectin and moxidectin to prevent transmission of Babesia bovis and Babesia bigemina by Boophilus microplus to cattle under conditions of relatively intense experimental challenge. DESIGN: Naive Bos taurus calves were treated with either pour-on or injectable formulations of either ivermectin or moxidectin and then exposed to larvae of B microplus infected with B bovis or larvae or adults of B microplus infected with B bigemina. One calf was used for each combination of haemoparasite, B microplus life stage, drug and application route. PROCEDURE: Groups of calves were treated with the test drugs in either pour-on or injectable formulation and then infested with B microplus larvae infected with B bovis or B bigemina. B bigemina infected adult male ticks grown on an untreated calf were later transferred to a fourth group of animals. Infections were monitored via peripheral blood smears to determine haemoparasite transmission. RESULTS: Cattle treated with either pour-on or injectable formulations of ivermectin and moxidectin became infected with B bovis after infestation with infected larvae. Similarly, larvae infected with B bigemina survived to the nymphal stage to transmit the haemoparasite to animals treated with each drug preparation. Cattle treated with pour-on formulations of ivermectin and moxidectin then infested with adult male ticks infected with B bigemina did not become infected with B bigemina whereas those treated with the injectable formulations of ivermectin and moxidectin did show a parasitaemia. CONCLUSIONS: Injectable or pour-on formulations of ivermectin and moxidectin do not prevent transmission of Babesia to cattle by B microplus. Use of these drugs can therefore not be recommended as a primary means of protecting susceptible cattle from the risk of Babesia infection.     

Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction

The potential role of Boophilus microplus as a natural tick vector of Babesia equi and Babesia caballi in Brazilian horses was assessed using nested polymerase chain reaction (PCR)-based marker assay. B. equi merozoite-specific 218bp gene fragment was detected in almost 96% of horse blood samples, and 45.3-62.5% of females, eggs, larvae, and nymphs of B. microplus collected from 47 horses at Campo Grande in the State of Matto Grosso, Brazil. Except for the partially-fed female ticks, the B. caballi-specific 430bp gene fragment was amplified from horse blood samples, and all developmental stages. Parasite DNA from both species was detected in horse blood samples and B. microplus, with the preponderance of B. equi DNA. No DNA samples were positive solely for B. caballi parasite. Only 32% of the Giemsa-stained thin blood smears were positive for Babesia parasites, as against detection of B. equi parasite DNA in 95.7% of the blood samples by nested PCR. We have obtained molecular evidence that strengthens earlier experimental and ultrastructural studies in Brazil incriminating B. microplus as a natural vector of B. equi, and possibly of B. caballi. The detection of B. equi and B. caballi DNA in eggs and larvae of B. microplus is likewise suggestive of the possibility of both transovarial and transstadial parasite transmission in this tick vector.     

Seroprevalence of Babesia bovis and Babesia bigemina in cattle in the Soutpansberg region, Limpopo Province, South Africa, associated with changes in vector-tick populations

A survey was conducted at 30 communal dip tanks and on 5 commercial farms in Limpopo Province, South Africa, during 1999 and 2000 to determine the seroprevalence of antibodies to Babesia bovis and Babesia bigemina. Cattle seropositive for B. bovis were found in 97% of the herds on communal land; the overall seroprevalence changed little between 1999 (63.3%) and 2000 (62.4%). All herds surveyed were infected with B. bigemina, and overall seroprevalence decreased significantly from 56.1% in 1999 to 49.3% in 2000. In herds on communal land in Sour Lowveld Bushveld, overall seroprevalence of B. bovis increased from 70% in 1999 to 80% in 2000, while seroprevalence of B. bigemina decreased from 70% in 1999 to 30% in 2000. This was possibly due to an influx of Rhipicephalus (Boophilus) microplus that occurred at the time. In commercially farmed herds the seroprevalence to B. bovis increased significantly from 19% in 1999 to 57.5% in 2000. All commercial herds in the survey tested positive to B. bigemina, with a seroprevalence of 48.3% in 1999 and 47.5% in 2000. During 1999, cattle in 60% of the dip tank/farm herds with only R. (B.) microplus present were approaching endemic stability to both B. bovis and B. bigemina. In 2000, 60% of the herds with only R. (B.) microplus present were approaching endemic stability for B. bovis, while only 45% were approaching endemic stability for B. bigemina. Those dip tanks/farms where only R. (B.) microplus was recorded had a significantly higher seroprevalence of B. bovis than those where both tick species were present.     

Comparison of duplex PCR and microscopic techniques for the identification of Babesia bigemina and Babesia bovis in engorged female ticks of Boophilus microplus

A single-step duplex polymerase chain reaction (PCR) technique and traditional microscopic examination of haemolymph smears were used to detect Babesia bigemina and/or Babesia bovis infection in engorged female ticks of Boophilus microplus recovered from calves raised in an endemic area of the State of Minas Gerais, Brazil. In the PCR amplification of tick-derived DNA, pairs of oligonucleotide primers specific for a 278-bp sequence from B. bigemina and for a 350-bp sequence from B. bovis were used conjointly. The microscopic examination of haemolymph revealed that 16.7% of the engorged ticks were infected with Babesia spp., although no significant differences (rho > 0.05) were found in the infection rate of ticks collected from calves of different age groups. PCR analysis showed that 77.8% of the engorged ticks whose haemolymph contained sporokinetes were infected with B. bigemina, 7.8% with B. bovis and 14.4% with both protozoan species. However, the PCR assay further revealed that, amongst the engorged female ticks whose haemolymph was apparently negative for the presence of sporokinetes, 15.6% were infected with B. bigemina, 2.2% with B. bovis and 10.0% with both species. The duplex PCR method is thus more efficient and sensitive than the microscopic assay and also permits facile identification of the protozoa species present in engorged female ticks.     

A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina

The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. B. bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.     

Cross-reaction of tick salivary antigens in the Boophilus microplus-cattle system

Calves were immunized with Boophilus microplus saliva, filtered through Millipore membranes, in Freund's complete adjuvant. Serum samples were tested by passive hemagglutination against Babesia bigemina, Anaplasma marginale, B. microplus larvae extract, Stomoxys calcitrans extract and B. microplus saliva. After immunization, titers to saliva, larval tick-extract and to S. calcitrans were increased. The challenge with live tick larvae enhanced the formation of antibodies against larva extract, fly extract and tick saliva, which supports the idea that under natural controlled conditions this cross-reactivity could occur.     

Detection of bovine babesiosis in Mozambique by a novel seminested hot-start PCR method

Babesiosis is a tick borne disease (TBD) caused by parasites of the genus Babesia, with considerable worldwide economic, medical, and veterinary impact. Bovine babesiosis and other TBDs were considered responsible for 50% of the deaths of cattle that occurred in Mozambique in the first year after importation from neighbouring countries. Here, we present the detection of Babesia bigemina and Babesia bovis in cattle from Mozambique using two distinct PCR methods. For this study, blood samples were collected in one farm located near Maputo city. The DNA samples were analyzed using a previously described nested PCR and a novel hot-start PCR method. Primers were selected for the hot-start PCR based on the putative gene of an undescribed aspartic protease named babesipsin, present in both B. bovis and B. bigemina. The combination of hot-start polymerase and long primers (29-31 bp) were in this study determinant for the successful amplification and detection in only one PCR. With a seminested approach the sensitivity was further increased. The babesipsin seminested hot-start PCR was in this study more sensitive than the nested PCR. A total of 117 field samples were tested by seminested hot-start PCR, and 104 were positive for B. bigemina (90%), 97 were positive for B. bovis (82%), 86 were mixed infections (52%) and only 2 were negative for both Babesia species (1.7%). The results confirm that this area of Mozambique is endemic for babesiosis, and that this TBD should be regarded as a threat for imported cattle.     

A specific DNA probe which identifies Babesia bovis in whole blood.

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.     

Comparison of diagnostic methods to detect piroplasms in asymptomatic cattle

This study was carried out to compare different diagnostic techniques to reveal the presence of piroplasms in asymptomatic cattle kept at pasture. Nineteen blood samples were collected from animals of two different areas of Emilia Romagna Region of Italy and processed for microscopic observation, PCR, serological test (IFAT) for Babesia bovis and Babesia bigemina antibodies and in vitro cultivation. The cultures were performed on both bovine and ovine erythrocytes. Seventeen blood smears (89%) were positive for piroplasms, while PCR was positive on 18 samples (95%). DNA sequencing of 18S rRNA identified the piroplasms as Theileria spp. In vitro cultures were successful for 6 samples (32%) cultured on bovine blood and subsequent identified these as Babesia major by PCR. On IFAT analyses of 16 samples, 36.8% resulted positive for B. bovis and 31.6% positive for B. bigemina. These results show, in the same animals, the co-infection with Babesia spp. and Theileria spp.; the detection of B. major was possible only using the in vitro cultures.     

Comparison of different direct diagnostic methods to identify Babesia bovis and Babesia bigemina in animals vaccinated with live attenuated parasites

Blood smear examination, flow cytometry, duplex Polymerase Chain Reaction (PCR), and duplex nested PCR (nPCR) were evaluated for detection of Babesia bigemina and Babesia bovis infections in cattle vaccinated with live attenuated strains. Two groups of four cattle were immunized with either B. bigemina (Bi) or B. bovis (Bo). On day 23 post inoculation (PI), Bi cattle were vaccinated with B. bovis (BiBo) and Bo cattle were vaccinated with B. bigemina (BoBi). Babesia bigemina was first detected by blood smear examination 7.5+/-3.5 days PI in the Bi group and 32.2+/-1.7 days PI in the BoBi group. The first occurrence of B. bovis in blood smears was 8.0 days PI in the Bo group and 36.0+/-2.6 days PI in the BiBo group. Flow cytometry detected parasitized erythrocytes on day 1.7+/-1.5 and 2.2+/-1.5 PI in the Bi and Bo groups, respectively, but did not discriminate between the two Babesia spp. Duplex PCR detected B. bigemina on day 4.0+/-0.8 and 26.0+/-0.8 PI in the Bi and BoBi groups, respectively, and B. bovis on day 4.0 and 25.3+/-0.5 PI in the Bo and BiBo groups, respectively. The duplex nPCR detected B. bigemina on 3.0+/-0.8 and 25.0+/-0.0 days PI in the Bi and BoBi groups, respectively, and 4.7+/-1.7 and 27.7+/-6.2 days PI in the Bo and BiBo groups, respectively. Duplex nPCR outperformed the other tests in terms of specificity and sensitivity, indicating that it is the most useful method for identifying Babesia spp. in cattle following vaccination.     

Seroepidemiological studies of bovine babesiosis on Pemba Island, Tanzania.

Serological evidence of infection with Babesia bovis and Babesia bigemina at a number of sites in Pemba was obtained using an enzyme-linked immunosorbent assay (ELISA) capable of detecting the appropriate parasite-specific antibody. Overall, 96% of animals were found to be positive for B. bovis, 88% were positive for B. bigemina and 88% were positive for both Babesia species. Antibody to B. bovis and B. bigemina was detected early in life in a number of calves born on Pemba, and was considered to be of maternal origin. The amount of maternal antibody in the serum of individual animals fell throughout the first 3 months of life. Later in life, antibody levels increased, probably in response to Babesia infection from natural tick challenge. These results suggest that infection with both Babesia parasites is widespread throughout Pemba and that both parasites probably exist in an enzootically stable situation.     

Detection of haemoparasites in cattle by reverse line blot hybridisation with a note on the distribution of ticks in Sicily

A reverse line blot hybridisation (RLB) of 21 oligonucleotides with polymerase chain reaction (PCR) amplified regions of 16S rRNA (Ehrlichia/Anaplasma group) or 18S rRNA (Babesia/Theileria group) genes of haemoparasites detected Theileria annulata, T. buffeli/orientalis, Babesia bovis, B. bigemina, B. divergens, Ehrlichia bovis, Anaplasma marginale, A. centrale and unknown species within the Rickettsia tribe.A very high prevalence of mixed infections was detected, which indicated that animals infected with Babesia spp. were also infected with Theileria spp. and/or Anaplasma spp.The tick distribution appeared to be seasonal with Hyalomma marginatum as the most frequently observed tick and Boophilus annulatus and Ixodes ricinus as the least frequently observed ticks. Other species identified in the 818 ticks collected during the five sampling periods between April 1998 and November 1999 included H. lusitanicum, Rhipicephalus sanguineus group, R. bursa, Dermacentor marginatus, Haemaphysalis punctata, B. annulatus and I. ricinus.     

Detection of Theileria and Babesia in brown brocket deer (Mazama gouazoubira) and marsh deer (Blastocerus dichotomus) in the State of Minas Gerais, Brazil

Intraerythrocytic protozoan species of the genera Theileria and Babesia are known to infect both wild and domestic animals, and both are transmitted by hard-ticks of the family Ixodidae. The prevalences of hemoprotozoa and ectoparasites in 15 free-living Mazama gouazoubira, two captive M. gouazoubira and four captive Blastocerus dichotomus from the State of Minas Gerais, Brazil, have been determined through the examination of blood smears and the use of nested polymerase chain reaction (nPCR). The cervid population was inspected for the presence of ticks and any specimens encountered were identified alive under the stereomicroscope. Blood samples were collected from all 21 animals, following which blood smears were prepared, subjected to quick Romanowsky staining and examined under the optical microscope. DNA was extracted with the aid of commercial kits from cervid blood samples and from tick salivary glands. The nPCR assay comprised two amplification reactions: the first was conducted using primers specific for a 1700 bp segment of the 18S rRNA gene of Babesia and Theileria species, whilst the second employed primers designed to amplify a common 420 bp Babesia 18S rRNA fragment identified by aligning sequences from Babesia spp. available at GenBank. The ticks Amblyomma cajennense, Rhipicephalus microplus and Dermacentor nitens were identified in various of the cervids examined. Of the animals investigated, 71.4% (15/21) were infected with hemoprotozoa, including Theileria cervi (47.6%), Theileria sp. (14.3%), Babesia bovis (4.8%) and Babesia bigemina (4.8%). However, only one of the infected wild cervids exhibited accentuated anaemia (PCV=17%). This is first report concerning the occurrence of Theileria spp. in Brazilian cervids.     

Detection of Babesia bigemina in cattle by a radioimmunoassay incorporating specifically depleted antigen

It was observed that mild acidification (pH less than 4.0) together with solvent extraction of the soluble sonicate of a crude preparation of Babesia bigemina infected cattle erythrocytes caused a quantitative loss of B. bigemina-specific antigen. Cross-reacting antigen activities with Babesia bovis remained intact. These properties were utilized in an assay system wherein antibody response to the specifically depleted antigen preparation was subtracted from the response to the initial crude preparation leaving the net B. bigemina response. The radioimmunoassay based on this antigen system was verified using sera from known negative cattle and from cattle previously infected with B. bigemina, B. bovis or Anaplasma marginale. The following discrimination values were obtained: B. bigemina-positive sera less than 2% false negatives; negative sera, 2% false positives; B. bovis-positive sera, 4% false positives; A. marginale-positive sera, 0% false positives. Levels of cross-reactivity in the false positive results were in the "suspect" rather than positive class and in the case of B. bovis-positive sera, may have been due to non-specific antibodies induced by blood inoculation. In animals naturally infected with B. bovis only, there were no false positive reactions. B. bigemina antibodies were readily detectable in field sera for at least 10 months post-infection following infection by the cattle tick Boophilus microplus. This assay overcomes the problems of currently used tests for B. bigemina infection as it is both sensitive and specific and is able to discriminate between both field and laboratory infections of B. bigemina and B. bovis.     

Effect of different methods of maintenance on the development and morphology of Babesia bigemina in the gut of Boophilus microplus

The development and morphology of Babesia bigemina in the gut of Boophilus microplus ticks were studied during laboratory maintenance of the babesia by four methods: tick transmission in unsplenectomised (intact) calves; tick transmission in splenectomised calves; syringe passage at intervals of five days or less in splenectomised calves; and syringe passage at intervals of six weeks or more in intact calves. The first method had no apparent effect on the development of B bigemina in ticks compared to that of the original isolate. The other three methods had obvious effects, the most pronounced being increased numbers of babesial forms with processes, particularly during early stages of development. The findings emphasise the importance of maintaining laboratory strains of parasites by natural means in life cycle studies.