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%).     

Comparison of blood smear and indirect fluorescent antibody techniques in detection of haemoparasite infections in trade cattle in Nigeria

The incidence of blood parasites in trade cattle was surveyed with emphasis on tick-borne parasites, using blood smears and immunofluorescent antibody (IFA) techniques. With the blood smear method, about 9 and 8.9% of cattle examined were found positive for Babesia bigemina and Anaplasma marginale, respectively. Percentage infections with other parasites were 3.33, 1.92, 0.75, 0.75 and 0.58, respectively, for Babesia bovis, Trypanosoma brucei, Anaplasma centrale, Eperythrozoon and Theileria species as well as Trypanosoma congolense. The incidence of A. marginale infection was at its peak during the rainy season while B. bigemina was most prevalent during the dry season. There were mixed infections of Anaplasma and Babesia (1.42%); Babesia and trypanosomes (1.00%); Babesia and Eperythrozoon (0.75%) and Babesia and Theileria (0.75%). Using the indirect fluorescent antibody test, 93, 55 and 68% of cattle sera examined were found to be positive for B. bigemina, B. bovis and A. marginale, respectively. Forty-nine percent of the positive sera of B. bigemina had highest titres. The importance of using serological means for determining the endemic levels of tick-borne diseases in cattle in Nigeria is discussed.     

Prevalence of Anaplasma marginale, Babesia bigemina and B. bovis in Nigerian cattle using serological methods

Serum samples collected randomly from 500 cattle from the 10 northern states of Nigeria were tested for antibodies against Anaplasma marginale by indirect fluorescent antibody (IFA), card agglutination (CT) and capillary tube-agglutination (CA) tests. The serum samples were also examined for antibodies to Babesia bigemina and B. bovis by the IFA test only. Of the serum samples tested, 79.4% had antibodies against A. marginale by the IFA test, 40 and 25% in the CT and CA tests, respectively. The IFA test results for B. bigemina and B. bovis were 29.4 and 14.1%, respectively.     

The impact of 2 dipping systems on endemic stability to bovine babesiosis and anaplasmosis in cattle in 4 communally grazed areas in Limpopo Province, South Africa

A 12-month study was conducted in 4 communal grazing areas in the Bushbuckridge region, Limpopo Province, South Africa. The main objective was to investigate the impact of reduced acaricide application on endemic stability to bovine babesiosis (Babesia bigemina and Babesia bovis) and anaplasmosis (Anaplasma marginale) in the local cattle population. To this end 60 cattle in each communal grazing area were bled at the beginning and the conclusion of the experimental period and their sera were assayed for B. bovis, B. bigemina and Anaplasma antibodies. Cattle in the intensively dipped group were dipped 26 times and maintained on a 14-day dipping interval throughout the study, whereas cattle in the strategically dipped group were dipped only 13 times. Three cattle, from which adult ticks were collected, were selected from each village, while immature ticks were collected by drag-sampling the surrounding vegetation. During the dipping process, a questionnaire aimed at assessing the prevalence of clinical cases of tick-borne disease, abscesses and mortalities was completed by an Animal Health Technician at each diptank. An increase in seroprevalence to B. bovis and B. bigemina and a decrease in seroprevalence to Anaplasma was detected in the strategically dipped group while in the intensively dipped group the converse was true. Amblyomma hebraeum was the most numerous tick species on the cattle, and Rhipicephalus (Boophilus) microplus was more plentiful than Rhipicephalus (Boophilus) decoloratus. Drag samples yielded more immature stages of A. hebraeum than of Rhipicephalus (Boophilus) spp. The incidence of clinical cases of tick-borne disease and of abscesses increased in the strategically dipped group at the start of the survey.     

Epidemiology of tick-borne diseases of cattle in Botshabelo and Thaba Nchu in the Free State Province

A seroepidemiological study was conducted on 151 cattle from the Botshabelo and Thaba Nchu areas in the central Free State Province of South Africa, two areas where small scale, peri-urban cattle farming is practised. An indirect fluorescent antibody test was used to test for Babesia bigemina and B. bovis antibodies. To test for Anaplasma marginale antibodies a competitive inhibition enzyme-linked immunosorbent assay method was used. There were no significant differences in serological test results between the cattle from Botshabelo and those from Thaba Nchu. The herd (two areas combined) had an average seroprevalence of 62.42% to B. bigemina, 19.47% to B. bovis and 98.60% to A. marginale. Based on the percentage of cattle that were seropositive to B. bigemina the immune status of cattle in the Botshabelo-Thaba Nchu area is approaching a situation of endemic stability. With reference to A. marginale, the high seroprevalence is indicative of a situation of endemic stability. The occurrence of B. bovis antibodies in the cattle is difficult to explain as Boophilus microplus ticks do not occur in the area in which the study was conducted.     

Productivity and health effects of anaplasmosis and babesiosis on Bos indicus cattle and their crosses, and the effects of differing intensity of tick control in Australia

Tick fever is an important disease of cattle where Rhipicephalus (Boophilus) microplus acts as a vector for the three causal organisms Babesia bovis, Babesia bigemina and Anaplasma marginale. Bos indicus cattle and their crosses are more resistant to the clinical effects of infection with B. bovis and B. bigemina than are Bos taurus cattle. Resistance is not complete, however, and herds of B. indicus-cross cattle are still at risk of babesiosis in environments where exposure to B. bovis is light in most years but occasionally high. The susceptibility of B. indicus cattle and their crosses to infection with A. marginale is similar to that of B. taurus cattle. In herds of B. indicus cattle and their crosses the infection rate of Babesia spp. and A. marginale is lowered because fewer ticks are likely to attach per day due to reduced numbers of ticks in the field (long-term effect on population, arising from high host resistance) and because a smaller proportion of ticks that do develop to feed on infected cattle will in turn be infected (due to lower parasitaemia). As a consequence, herds of B. indicus cattle are less likely than herds of B. taurus cattle to have high levels of population immunity to babesiosis or anaplasmosis. The effects of acaricide application on the probability of clinical disease due to anaplasmosis and babesiosis are unpredictable and dependent on the prevalence of infection in ticks and in cattle at the time of application. Attempting to manipulate population immunity through the toleration of specific threshold numbers of ticks with the aim of controlling tick fever is not reliable and the justification for acaricide application should be for the control of ticks rather than for tick fever. Vaccination of B. indicus cattle and their crosses is advisable in all areas where ticks exist, although vaccination against B. bigemina is probably not essential in pure B. indicus animals.     

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.     

Preliminary observations on ticks and tick-borne diseases in the north west province of Cameroon. I. Babesiosis and anaplasmosis.

During a survey on ticks and tick-borne diseases in the North-Western Cameroon at the Bamenda cattle market, the ticks identified were Boophilus annulatus (20%) and B. decoloratus (80%). More than 50% of the ticks were collected during the dry season. Of 524 blood smears 47.3% were positive for Babesia bovis, 31.1% for B. bigemina and 2.2% for Anaplasma marginale. 19.4% were negative.     

Epidemiology of bovine tick-borne diseases in southern Italy

This investigation was carried out in an area covering part of three southern Italian regions: Campania, Basilicata and Apulia. Eighty-one farms were involved using the formula suggested by Thrusfield; they were equally distributed over the area which was subdivided into 81 geo-referenced sub-areas. In May and June 1999 from a total of 506 cattle, older than 18 months, blood-samples were taken and ticks were collected and identified. Serum samples were tested for antibodies of Bahesia bigemina, Babesia bovis and Anaplasma marginale with an ELISA technique. Eight farms (9.8%) out of the 81 examined were positive for B. bigemina only, 3 (3.7%) for A. marginale only, and 70 (86.4%) for both. None of the animals of any farm was found to be positive for B. bovis. Out of the 506 sera tested, 117 (23.1 %) were positive for B. bigemina only, 58 (11.5%) forA. marginale only and 250 (49.4%) for both species; 81 (16.0%) were negative for all of them. Ticks were collected on animals on 62 (76.5%) out of the 81 farms. Adult ticks (1 410) were collected and identified; the highest number belonged to the Rhipicephalus bursa species (65.5%), followed by Rhipicephalus turanicus (8.6) and Haemaphysalis punctata (8.4). The results showed that B. bigemina, A. marginale and their potential vectors are common in the area examined and indicated that there is a risk for animals imported from tick-borne disease-free areas.     

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.     

Susceptibility of Bos indicus and Bos taurus to Anaplasma marginale and Babesia bigemina infections

The reaction of Bos taurus and pure-bred Bos indicus heifers to infection with the intraerythrocytic parasites Anaplasma marginale and Babesia bigemina was studied. B. bigemina infection at 18 months and A. marginale infection at 13 or 24 months resulted in slightly less severe reactions in pure-bred Bos indicus cattle than in Bos taurus. In both breeds, the reaction to A. marginale infection was more severe in older cattle. The severity of B. bigemina infection was not affected by a previous infection with A. marginale.     

Tick-borne diseases of cattle in Paraguay. II. Immunisation against anaplasmosis and babesiosis

A total of 102 susceptible adult Holstein Friesian cattle imported into an area of Paraguay where anaplasmosis and babesiosis are endemic were immunised by infection with Anaplasma centrale and attenuated forms of Babesia bigemina and Babesia bovis obtained from Uruguay. The results indicated that the attenuated forms of both Babesia species protected cattle against heterologous field challenge whereas A. centrale did not invariably confer sufficient protection against a field challenge of A. marginale.     

Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

Immunity following use of Australian tick fever vaccine: a review of the evidence

OBJECTIVE: To review the evidence available on the degree and duration of immunity provided by Australian tick fever vaccines against Babesia bovis, B. bigemina and Anaplasma marginale infections in Australia and overseas. BACKGROUND: Vaccines containing attenuated strains of B bovis and B bigemina as well as A. centrale grown in splenectomised calves have been used in Australia since 1964 to immunise cattle against tick fever. About 800,000 doses of vaccine are supplied annually and much of the evidence for protection is field evidence rather than conventional immunological measures or pen trials. CONCLUSIONS: Immunity to Babesia bovis and B. bigemina--A single inoculation generally provides sound, long-lasting protection both in Australia and overseas. No evidence was found of a loss of immunity with time. Vaccine failures to B. bovis do occur, but are uncommon and evidently caused by a number of factors, including immune responsiveness of the vaccinated animals, and immunogenicity of the vaccine strain. Immunity to Anaplasma marginale--The vaccine containing A. centrale provides partial, variable protection against A. marginale. Protection against challenge in Australia is adequate in most cases to prevent disease and use of the vaccine in this country appears to be justified. Protection against antigenically diverse, highly virulent stocks of A. marginale in other countries is, at times, clearly inadequate and better vaccines are required in situations where the challenge is severe.     

Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.     

Current status of bovine haemoparasitic diseases in Martinique (French West Indies)]

A serological survey using indirect immunofluorescence (IFAT) for bovine babesiosis (Babesia bovis and B. bigemina) and card test for anaplasmosis, indicates that these haemoparasites are widespread in Martinique. The high prevalences (62% for B. bovis, 52% for B. bigemina and 43% for Anaplasma marginale) lead to the hypothesis of an unstable epizootic situation for these three haemoparasitic diseases. However, the number of smears examined was too low to evaluate their clinical incidence. Both the American and Cuban card tests gave similar results in the detection of antibodies to A. marginale. Theileria mutans is described for the first time in Martinique. Trypanosomosis (Trypanosoma vivax) has disappeared from Martinique, on clinical and serological evidence.     

Molecular identification of tick-borne pathogens in Nigerian ticks

A molecular epidemiology investigation was undertaken in two Nigerian states (Plateau and Nassarawa) to determine the prevalence of pathogens of veterinary and public health importance associated with ticks collected from cattle and dogs using PCR, cloning and sequencing or reverse line blot techniques. A total of 218 tick samples, Amblyomma variegatum (N=153), Rhipicephalus (Boophilus) decoloratus (N=45), and Rhipicephalus sanguineus (N=20) were sampled. Pathogens identified in ticks included piroplasmids (Babesia spp., Babesia bigemina and Babesia divergens), Anaplasma marginale and Rickettsia africae. Piroplasmids were identified in A. variegatum, A. marginale was found in R. decoloratus, while R. africae was detected in all tick species examined. Ehrlichia spp. and Theileria spp. were not identified in any of the ticks examined. Of the 218 ticks examined, 33 (15.1%) contained pathogen DNA, with the presence of B. divergens and R. africae that are zoonotic pathogens of public health and veterinary importance. The variety of tick-borne pathogens identified in this study suggests a risk for the emergence of tick-borne diseases in domestic animals and humans, especially amongst the Fulani pastoralists in Plateau and Nassarawa states of Nigeria.     

Ticks and tick-borne disease in Guatemalan cattle and horses

Blood samples and ticks were collected from 48 cattle and 74 horses from seven sites in the Peten region of Guatemala. Data on body condition, mucous membrane capillary refill time and tick infestation levels were recorded for each animal in the study. Horses had significantly higher levels of tick infestation than cattle, as well as poorer body condition scores. Seroprevalence of Babesia spp. was 95.8% for B. bovis in cattle, 89.6% for B. bigemina in cattle, and 92.7% for B. equi in horses. Seroprevalence of Anaplasma marginale in cattle was 87.5%, similar to reports in animals from other regions of Central America. This is the first time that A. phagocytophilum has been reported in animals from this region, with overall PCR-prevalence of 27.6% in cattle and horses, and seroprevalence of 28.4% (52% in cattle and 13% in horses). An agent was identified with serological cross-reactivity and close genetic relatedness to Ehrlichia ruminantium, but further testing confirmed that the agent in Guatemalan cows was not the agent of heartwater. Ticks were identified to species with the predominant species identified on cattle as Boophilus microplus and Amblyomma cajennense, while Anocentor nitens and A. cajennense were most commonly found on horses. Prevalence of infection, tick infestation levels, host factors and environmental data were analyzed for association; A. nitens was significantly associated with A. phagocytophilum prevalence by village.     

Molecular and serological prevalence of Babesia bovis and Babesia bigemina in cattle from central region of Syria

A total of 207 bovine blood samples were collected from clinically healthy cattle bred in central region of Syria and examined by Giemsa-stained blood smears, nested PCR, ELISA, and IFAT to determine the molecular and serological prevalence of Babesia bovis and B. bigemina. All samples were negative to Babesia spp. by microscopic examination of blood smears. On the other hand, the overall prevalence of B. bovis and B. bigemina was 9.18% and 15.46% by nPCR, 15.46% and 18.84% by ELISA, and 18.36% and 21.74% by IFAT, respectively. Mixed infections were detected in a total of 5 samples (2.4%) by nPCR, 16 (7.73%) by ELISA and 27 (13.04%) by IFAT. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location. These data provide valuable information regarding the occurrence and epidemiology of B. bovis and B. bigemina infections in Syrian cattle, which can be employed in developing rational strategies for disease control and management.