A technique to establish a laboratory colony of Boophilus microplus infected with Babesia bigemina

A technique was evolved for the establishment and maintenance of a colony Boophilus microplus free of infection with Anaplasma marginale and Babesia spp., and for their subsequent infection with a pure isolate of Babesia bigemina. Confirmation was obtained that the ticks are infected normally during the last 24 h of attachment on the host. The life cycle of Boophilus microplus was described for a single situation on the Atlantic Coast of Colombia.     

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).     

Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis and B bigemina transmitted by Boophilus microplus

OBJECTIVE: To assess the effect of breed of cattle on the transmission rates of and innate resistance to Babesia bovis and B bigemina parasites transmitted by Boophilus microplus ticks. DESIGN: Groups of 56 purebred B indicus and 52 B indicus cross B taurus (50%, F1 generation) steers were placed in a paddock seeded with and also naturally infested with B microplus which were the progeny of females ticks fed on B taurus cattle specifically infected with a virulent isolate of B bovis. The cattle were placed in the infested paddock 50 days after seeding had started. PROCEDURE: Cattle were inspected from horseback daily for 50 days. Clinically ill cattle were brought to yards and assessed by monitoring fever, depression of packed-cell volume, parasitaemia and severity of clinical signs. Any animals that met preset criteria were treated for babesiosis. Blood samples were collected from all cattle on day 28, 35 and 42 after exposure and antibodies to Babesia spp and packed cell volume measured. RESULTS: All steers, except for one crossbred, seroconverted to B bovis and B bigemina by day 35 and 75% of the crossbred steers showed a maximum depression in packed cell volume of more than 15% due to infection with Babesia spp compared with only 36% of the B indicus group. Ten of the 52 crossbreds and 1 of the 56 B indicus steers showed severe clinical signs. Two of the crossbreds required treatment of which one died 2 weeks after initial treatment. CONCLUSIONS: Pure-bred B indicus cattle have a high degree of resistance to babesiosis, but crossbred cattle are sufficiently susceptible to warrant the use of preventive measures such as vaccination. Transmission rates of B bovis and B bigemina to B indicus and crossbred cattle previously unexposed to B microplus were the same.     

Factors affecting the detachment rhythm of engorged Boophilus microplus female ticks (Acari: Ixodidae) from Charolais steers in New Caledonia

As in most parts of the world where the cattle tick Boophilus microplus is established, resistance of ticks to acaricides occurs in New Caledonia. In order to implement laboratory resistance tests on larvae, engorged females collected in suspected farms are necessary. Investigations on the detachment schedule of the engorged females were conducted to explain certain field situations such as the lack or scarcity of engorged females on highly infested cattle driven from the pasture to the pen in the morning. Three experiments on Charolais steers naturally infested on pastures showed that: (1) engorged female burdens at sunrise are similar whether the steers spend the night in pasture or in a pen; (2) compared with steers maintained in a pen, morning detachment of females increases when the steers stay on the pasture or move from the pasture to the pen; (3) detachment rhythm of engorged females on steers staying the morning in a pen, is not influenced by feeding activity or exposition of steers to sun; (4) detachment occurs earlier for females attached on anatomical sites exposed to sun, and earlier from these sites for the steers in pasture or walking than for steers in a pen.     

Effect of different methods of maintenance on the pathogenicity and infectivity of Babesia bigemina for the vector Boophilus microplus

Observations were made on the effects of five different methods of laboratory maintenance on the infectivity and virulence of Babesia bigemina for the tick Boophilus microplus. The original isolate was highly infective and virulent, causing premature death of engorged female ticks and reduced egg production. Maintenance of the strain by syringe passage in unsplenectomised calves at six to 10 week intervals reduced both its infectivity and virulence for ticks. When slow passages were preceded by a series of rapid passages in splenectomised calves, the changes to the strain were less pronounced. The other three procedures, rapid syringe passage in splenectomised calves and tick passage in either splenectomised or intact calves, had no statistically significant effect on the characteristics measured.     

Detection of Babesia bigemina in cattle of different genetic groups and in Rhipicephalus (Boophilus) microplus tick

Babesia bigemina infections were investigated in four genetic groups of beef cattle and in Rhipicephalus (Boophilus) microplus engorged female ticks. Blood samples and engorged female ticks were collected from 15 cows and 15 calves from each of the following genetic groups: Nelore, Angus x Nelore, Canchim x Nelore, and Simmental x Nelore. Microscopic examination of blood smears and tick hemolymph revealed that merozoites of B. bigemina (6/60) as well as kinetes of Babesia spp. (9/549) were only detected in samples (blood and ticks, respectively) originated from calves. PCR-based methods using primers for specific detection of B. bigemina revealed 100% infection in both calves and cows, regardless the genetic group. Tick infection was detected by nested-PCR amplifications showing that the frequency of B. bigemina was higher (P<0.01) in female ticks collected from calves (134/549) than in those collected from cows (52/553). The frequency of B. bigemina was similar in ticks collected from animals, either cows or calves, of the four genetic groups (P>0.05).     

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.     

The inability of a South African Babesia bovis vaccine strain to infect Boophilus microplus

A strain of Babesia bovis that had been attenuated by rapid syringe passage through a series of 23 splenectomized calves was unable to infect its vector Boophilus microplus. An attempt to transmit the attenuated Australian Babesia bigemina G strain with a South African strain of B. microplus was likewise unsuccessful. The epidemiological implication of these observations in terms of babesiosis control is discussed.     

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.     

Detection of Babesia bigemina infection in strains of Rhipicephalus (Boophilus) microplus collected from outbreaks in south Texas

The sudden death of several cattle infested experimentally with Rhipicephalus (Boophilus) microplus led to a clinical investigation into the reasons for the unexpected mortality. Microscopic evidence for Babesia bigemina infection was found in blood smears from the affected animals and a PCR assay was designed to detect the presence of B. bigemina and Babesia bovis in all R. microplus strains received and propagated at the laboratory. The assay utilizes a nested PCR approach with the first PCR amplifying a well-conserved segment from the Babesia 18S ribosomal RNA gene followed by a nested PCR with Babesia species-specific primers and annealing temperatures enabling amplification of the 18S ribosomal RNA gene fragment specific to either B. bigemina or B. bovis. DNA from groups of 50 larvae was extracted using a rapid DNA preparation protocol, which consisted of grinding the frozen tick larvae in PCR buffer and boiling the mixture for 5min. The assay sensitivity allowed for the detection of the equivalent of a single infected tick larva. R. microplus eggs were also analyzed, but yolk protein viscosity created inconsistent results with the crush and boil DNA isolation protocol, necessitating the use of a more extensive proteinase K digestion-based DNA purification method. We detected the presence of B. bigemina in all strains of R. microplus currently reared at the laboratory and 4 of 26 strains collected from infestation outbreaks in Texas by the U.S. Cattle Fever Tick Eradication Program.     

Proteomic profiling of Rhipicephalus (Boophilus) microplus midgut responses to infection with Babesia bovis

Differences in protein expression in midgut tissue of uninfected and Babesia bovis-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus, were investigated in an effort to establish a proteome database containing proteins involved in successful pathogen transmission. The electrophoretic separation of midgut membrane proteins was greatly improved by using liquid-phase isoelectric focusing combined with one-dimensional or two-dimensional (2-D) gel electrophoresis. A selection of differentially expressed proteins were subjected to analysis by capillary-HPLC-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Among the identified Babesia-affected tick midgut proteins were six proteins that are implicated in signaling processes, including three Ca(2+)-binding proteins, a guanine nucleotide-binding protein, a protein with signal peptide activity and a translocon-associated receptor protein. Up-regulation of five metabolic enzymes indicated parasite-induced changes in electron and proton transport, protein processing and retinoic acid metabolism. Among the down-regulated proteins were a molecular chaperone, a cytoskeletal protein and a multifunctional protein of the prohibitin family. Identification of these proteins may provide new insights into the molecular interactions between B. bovis and its tick vector, and could lead to identification of anti-tick and transmission-blocking vaccine candidates.     

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.     

Bovine babesiosis: severity and reproducibility of Babesia bovis infections induced by Boophilus microplus under laboratory conditions

A total of 61 intact Holstein-Friesian calves were exposed to Babesia bovis (= B argentina) by the injection of infected blood or the application of infected Boophilus microplus larvae. Tick-induced infections were uniformly severe, even when induced by relatively small numbers of infected ticks. In contrast, calves infected with carrier blood experienced mild, subclinical reactions despite detectable parasitaemia. The greater severity of tick-induced reactions appeared to be due to the large number of infective doses injected by each infected tick rather than greater virulence of tick versus blood-origin babesiae. The severity of tick-induced babesiosis was related to the age of the experimental calves, with more severe reactions and high mortality occurring among older animals. The mean daily temperature rise increased with age and was highest in 15 animals dying of babesiosis. No relationship could be found between peripheral blood parasitaemia, packed cell volume reduction and mortality in tick-induced infections. One thousand larvae from infected colonies induced babesiosis in all of 29 animals exposed. Although larval transmission of B bovis is difficult to quantitate, there would seem to be no objection to using a controlled tick-borne challenge in the laboratory for assessment of susceptibility.     

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.     

Light and electron microscopic observations on the development of Babesia bigemina in larvae, nymphae and non-replete females of Boophilus decoloratus

In Boophilus decoloratus infected by transovarian passage with B. bigemina, primary schizogony occurred as a continuous repetitive process in all 3 stages of the tick's life cycle spent on the host. The primary schizonts and the large merozoites (= vermicules) produced by them were observed in the gut epithelium, haemocytes, muscles, ad peritracheal cells. Secondary schizogony which led to the formation of small merozoites (= infective forms) occurred mainly in the salivary glands, but was also observed in the cortex of the synganglion. Mature small merozoites were observed in nymphal and adult ticks only. An infective stabilate was prepared from nymphae collected on Day 14 and Day 15 post larval infestation. The infections resulting from intravenous injection of the stabilate had a prepatent period of 8 days.     

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.     

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.     

Detection of Babesia bigemina DNA in ticks by DNA hybridization using a nonradioactive probe generated by arbitrary PCR

The Dig-labeled probe specific to Babesia bigemina generated from monomorphic RAPD fragment of approximately 873 bp size amplified by a 10 mer CGGTGGCGAA, detected up to 100 ng of template DNA. This nonradioactive probe also detected B. bigemina in preparations of larval tick DNA from two of the five samples on dot-blot hybridization.