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.     

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).     

Survival of larvae of Boophilus annulatus, Boophilus microplus, and Boophilus hybrids (Acari: Ixodidae) in different temperature and humidity regimes in the laboratory.

The survival period for larvae of Boophilus annulatus (Say), Boophilus microplus (Canestrini) and hybridized Boophilus ticks was determined by exposure to various combinations of temperature (20, 25, 30 and 35°C) and relative humidity (32, 63, 75, 84 and 97% RH) in the laboratory. Results indicated that within a given temperature and RH regime, there was no difference (P> 0.05) in larval survival among the three species tested, indicating that these ticks respond similarly over a wide range of temperature and RH combinations. Larval survival in all three species was longest (P < 0.05) at 20°C and either 84 or 97% RH. With each increase in temperature at the 84 and 97% RH treatment levels, there was a corresponding significant (P < 0.05) decrease in larval survival. When the temperature reached 35°C at all humidities or when the RH was 63% or less at all temperatures, the mean larval survival period was 43 days or less in all cases and little difference (P> 0.05) was observed among the treatment regimes included. Results suggest that at a RH of 75% and more, the temperature is the determining factor in larval survival, whereas at a RH of 63% and less the RH is the determining factor in larval survival, regardless of temperature.     

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.     

Effect of infection by Babesia spp. on the development and survival of free-living stages of Boophilus annulatus

Oviposition, egg hatching and survival of newly-hatched larvae of Boophilus annulatus were studied in relation to infection by Babesia species and different temperature regimens. Infection of female ticks by Babesia bigemina or B. bovis had no effect on the time elapsed between engorgement and oviposition. The duration of oviposition was shorter in infected females incubated at 25 degrees C or 35 degrees C and infected females laid fewer eggs than the controls. No larvae hatched at 16 degrees C. B. bigemina-infected eggs hatched more quickly than uninfected eggs at 35 degrees C. The hatching percentage of B. bigemina-infected eggs was reduced by 50% at an incubation temperature of 25 degrees C and by 75% at 35 degrees C. At 16 degrees C there was no difference in the duration of survival of infected and non-infected larvae but at 25 degrees C and 35 degrees C the mean survival period of infected larvae was significantly lower than those of controls.     

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.     

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.     

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.     

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.     

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 note on the transmission of Babesia bovis (syn B argentina) by the one-host tick, Boophilus microplus.

Boophilus microplus infected with Babesia bovis were transferred artificially from one splenectomised calf to another during each moult in the parasitic life cycle of the tick. Eggs from the engorged female ticks recovered at the end of the cycle were incubated and the resulting larvae used to infest more splenectomised calves. Babesia bovis was transmitted only by the original larvae used at the commencement of the experiment and it was concluded that the protozoan parasite did not persist in an infective form in the ticks beyond the larval stage.     

Eclosion blocking effect of ethanolic extract of Leucas aspera (Lamiaceae) on Rhipicephalus (Boophilus) annulatus

The crude ethanolic extract of aerial parts of Leucas aspera was tested for its acaricidal properties against Rhipicephalus (Boophilus) annulatus. The per cent adult mortality, inhibition of fecundity and hatching of laid ova were studied at concentrations of 1.56, 3.13, 6.25, 12.5, 25, 50 and 100mg/ml. Adult tick mortality was significant at the highest concentration tested. Inhibition of fecundity of treated groups differed significantly from control and was concentration dependent. L. aspera extract also produced complete failure of eclosion of eggs from the treated ticks even at lower dilutions of the extract.     

Experimental transmission of field Anaplasma marginale and the A. centrale vaccine strain by Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus ticks

The cattle rickettsia Anaplasma marginale is distributed worldwide and is transmitted by about 20 tick species, but only Rhipicephalus simus, a strictly African tick species, has been shown to transmit the vaccine strain of A. centrale. The aim of the present study was to examine transmission of field strains of A. marginale and of the vaccine strain of A. centrale by three tick species -Hyalomma excavatum, Rhipicephalus sanguineus and Rhipicephalus (Boophilus) annulatus - to susceptible calves. Two genetically distinct Israeli field strains of A. marginale, tailed and non-tailed (AmIsT and AmIsNT, respectively), were efficiently transmitted by R. sanguineus, whereas H. excavatum transmitted only the tailed isolate, and R. (Boophilus) annulatus did not transmit A. marginale. None of the three tick species transmitted A. centrale. By means of msp1a primers in PCR assays, amplicons of similar sizes were obtained from either A. marginale-infected calves that were used for acquisition feeding, from R. sanguineus fed on the infected calves, or from calves to which anaplasmosis had been successfully transmitted by these ticks. Although an A. centrale-specific fragment was amplified from salivary glands of R. sanguineus, no transmission to susceptible cattle occurred during 3 months of observation, and anaplasmosis was not induced in splenectomized calves that were subinoculated with blood from calves on which R. sanguineus had fed.     

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.     

Survival of larvae of Boophilus annulatus, Boophilus microplus, and Boophilus hybrids (acari: ixodidae) in different temperature and humidity regimes in the laboratory

The survival period for larvae of Boophilus annulatus (Say), Boophilus microplus (Canestrini) and hybridized Boophilus ticks was determined by exposure to various combinations of temperature (20, 25, 30 and 35°C) and relative humidity (32, 63, 75, 84 and 97% RH) in the laboratory. Results indicated that within a given temperature and RH regime, there was no difference (P > 0.05) in larval survival among the three species tested, indicating that these ticks respond similarly over a wide range of temperature and RH combinations. Larval survival in all three species was longest (P < 0.05) at 20°C and either 84 or 97% RH. With each increase in temperature at the 84 and 97% RH treatment levels, there was a corresponding significant (P < 0.05) decrease in larval survival. When the temperature reached 35°C at all humidities or when the RH was 63% or less at all temperatures, the mean larval survival period was 43 days or less in all cases and little difference (P > 0.05) was observed among the treatment regimes included. Results suggest that at a RH of 75% and more, the temperature is the determining factor in larval survival, whereas at a RH of 63% and less the RH is the determining factor in larval survival, regardless of temperature.     

Prevalence and molecular detection of Anaplasma marginale, Babesia bovis and Babesia bigemina in cattle from Puntarenas Province, Costa Rica

A study was conducted in 2008 to determine the prevalence of Anaplasma and Babesia infections in cattle in the Puntarenas Province of Costa Rica. Blood samples were taken from a total of 449 cattle during the month of March at 30 farms in the region of Espiritu Santu, Costa Rica. Commercially available enzyme-linked immunosorbent assays (ELISA) were used to determine presence of antibodies to Babesia bigemina and Anaplasma marginale, and real-time PCR was used to determine the presence of DNA from the disease-causing organisms. The ELISA results indicated that 87.5% of the cattle sampled were positive for antibodies to A. marginale, while 59.1% were positive for antibodies to B. bigemina. The real-time PCR results showed that 235 cattle were carrying A. marginale DNA (56.9%), 6 with B. bigemina DNA (1.34%), and 2 with B. bovis DNA (0.45%).