Risks of cattle babesiosis (Babesia bovis) outbreaks in a semi-arid region of Argentina.

The epidemiology of Babesia bovis was studied in terms of enzootic stability/instability and husbandry and abiotic factors influencing B. bovis transmission rate in northeastern Santiago del Estero province, Argentina. The area is of limited suitability for its only vector in Argentina, the tick Rhipicephalus microplus. The proportion of calf herds in a state of enzootic stability/instability to B. bovis was determined and husbandry practices and abiotic factors associated with variations in B. bovis transmission rates were explored using a cross-sectional observational study design. Daily probability of infection (inoculation rate, h) with B. bovis was calculated from age-specific seroprevalence via ELISAi in 58 herds of 4.5-8.5-month-old calves. Herds were considered to be in enzootic instability (EI) when h < 0.005, and therefore inferred to be at risk of babesiosis outbreaks. Husbandry practices associated with differences in B. bovis transmission were analyzed using generalized linear models. Sixty-two percent of herds were found to be in an EI situation for B. bovis. Calves raised exclusively on permanent pastures -where higher cattle density is achieved- were exposed to higher B. bovis inoculation rates (h = 0.0063, 95% CI 0.0032-0.0123) than those reared under forage combinations (h = 0.0024, 95% CI 0.0011-0.0051) (P =  0.05). In addition, calves from herds located in the area of intermediate suitability for R. microplus development were more likely to become infected with B. bovis (h = 0.0067, 95% CI 0.0037-0.0121) than those reared in the ecologically unfavorable area for the vector (h = 0.0023, 95% CI 0.0010-0.0049) (P =  0.02). Neither the frequency of treatment with acaricides nor the use of long-acting acaricides to control R. microplus influenced the inoculation rate (P =  0.99 and P =  0.26, respectively). This result indicates that current R. microplus control schemes are not effective in reducing B. bovis transmission. Enzootic instability still prevails in the study area despite the drastic changes occurred in cattle production system. However, 38% of herds did reach enzootic stability; therefore, a specific epidemiological status cannot be assumed at a regional level. Yearly determination of the immunological status of each calf cohort is considered a proper approach to decision-making in vaccination against B. bovis.

Pheno- and genotyping of Brucella abortus biovar 5 isolated from a water buffalo (Bubalus bubalis) fetus: First case reported in the Americas.

An isolate of Brucella spp. from an aborted water buffalo (Bubalus bubalis) fetus was characterized based on its pheno- and genotype. The phenotype was defined by carbon dioxide requirement, hydrogen sulfide production, sensitivity to thionin and basic fuchsin and agglutination with Brucella A and M monospecific antisera. The genotype was based on the amplification of the following genes: bcsp31, omp2ab, and eri and the species-specific localization of the insertion sequence IS711 in the Brucella chromosome via B. abortus-B. melitensis-B. ovis-B. suis (AMOS)-PCR. Unexpectedly, the isolate showed a phenotype different from B. abortus bv 1, the most prevalent strain in cattle in Argentina, and from vaccine strain 19, currently used in bovines and water buffaloes. Genotyping supported the phenotypic results, as the analysis of the omp2ab gene sequence showed an identical pattern to either B. abortus bv 5 or B. melitensis. Finally, the AMOS PCR generated a 1700-bp fragment from the isolate, different than those amplified from B. abortus bv 1 (498bp) and B. melitensis (731bp), confirming the presence of B. abortus bv 5. The OIE/FAO Reference Laboratory for Brucellosis confirmed this typing. This is the first report of B. abortus bv 5 from a water buffalo in the Americas.

Validation and field evaluation of a competitive enzyme-linked immunosorbent assay for diagnosis of Babesia bovis infections in Argentina.

Infections by Babesia bovis limit cattle production and cause important economic losses in tropical and subtropical areas around the world. Monitoring of calf sera can be used to detect unprotected cattle herds and to decide on strategic control measures, as well as for epidemiological studies. Merozoite surface antigen 2c (MSA-2c) is an immunodominant surface protein expressed in B. bovis merozoites and sporozoites and contains B-cell epitopes that are conserved among geographic isolates. A monoclonal antibody against recombinant MSA-2c (rMSA-2c) was previously shown to inhibit the binding of anti-B. bovis antibodies to a parasite B-cell epitope in a competitive enzyme-linked immunosorbent assay (cELISA) format. In the work at hand, the parameters of this cELISA were reevaluated and adjusted when necessary, and a cutoff value was determined by receiver operator characteristic (ROC) curve analysis of a total of 357 bovine sera of known reactivity, as assessed by indirect immunofluorescence assay (IFAT). The established rMSA-2c cELISA demonstrated a specificity of 98% and a sensitivity of 96.2%. An additional set of 303 field bovine sera from regions where ticks are endemic and tick-free regions of Argentina was tested by both rMSA-2c cELISA and IFAT, and the results were shown to be in very good agreement (kappa index, 0.8325). The performance shown by rMSA-2c cELISA in the detection of B. bovis-specific antibodies and its suitability for standardization and large-scale production, as well as the possibility of its application in most veterinary diagnostic laboratories, make the assay a powerful tool for the surveillance of herd immunity as a strategic measure for the control of bovine babesiosis.

Improved molecular tools for detection of Babesia bigemina.

Molecular detection of Babesia bigemina involves a nested PCR protocol and reverse line blot hybridization (RLBH) assay based on the 18S gene. In this study, we report the development of molecular tools for improving B. bigemina detection in bovine blood-a one-step PCR assay based on the amplification of rap-1a paralogous and a new RLBH Babesia spp. 18S probe. The one-step PCR assay is highly specific, with an estimated analytical sensitivity corresponding to 0.00002% parasitemia. The RLBH assay, with a new B. bigemina probe, allows the detection of all tested B. bigemina isolates showing no cross-hybridization with B. bovis 18S gene. By developing this highly specific and sensitive one-step PCR and upgrading the RLBH assay for B. bigemina, we have improved molecular assays which, together with serologic methods, provide valuable tools for epidemiologic studies of bovine babesiosis.

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences.

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (> or =80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick-pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick-pathogen interactions.

Development of an immunosensor for the diagnosis of bovine anaplasmosis.

An optical immunosensor based in major surface protein 5 (MSP5) of Anaplasma marginale was developed towards detection of anti-Anaplasma sp. antibodies in acute infection as well as in vaccinated cattle. This study was performed using recombinant MSP5 covalently immobilised in controlled pore glass (CPG) beads to detect anti-MSP5 antibodies in serum samples. The quantification is based on the measurement of the Cy5 fluorescence of the detection antibody, anti bovine IgG, after reaction with serum. Sera were collected in enzootic and tick-free regions of Argentina. The immunosensor showed a detection range of 1.2 g/ml to 48 g/ml of antibody in sera, with a sensitivity of 93% and a specificity of 70%. The optical immunosensor developed is suitable for quantification of antibodies in sera of naturally or experimentally infected animals.

Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA.

An indirect enzyme linked immunosorbent assay (iELISA) for diagnosis of anaplasmosis using undiluted individual milk samples from dairy cows was developed. The recombinant 19 kDa major surface protein 5 (rMSP5) of Anaplasma marginale was used as antigen. A monoclonal antibody against bovine IgG1 conjugated with peroxidase and the chromogen 3,5,3',5'-tetramethylbenzidine were used in the test. Strong and weak, positive and negative milk samples were set up as reference controls. Results were expressed as percentage of positivity (PP) contrasting with the strongest positive control. The test was evaluated in two groups (G1 and G2) of lactating dairy cows from herds located in A. marginale non-endemic areas of Argentina. The infection status of both groups, G1 (n=128) sampled after anaplasmosis outbreak, and G2 (n=216) free of anaplasmosis was established by polymerase chain reaction (PCR). Serum samples of cows from G1 and G2 were analyzed by card agglutination test (CAT) and competitive ELISA (cELISA), while the novel iELISA was evaluated in their corresponding milk samples. At a cutoff of 42 PP, the ELISA has 98% sensitivity and 95% specificity. A significant difference (P<0.0001) was found between the mean PP value of negative samples from G1 (17.4+/-14.9), and G2 (8.6+/-7.1). The agreement and kappa (kappa) value between iELISA and PCR was 96%, kappa=0.919; between iELISA and CAT was 97%, kappa=0.880; and between iELISA and cELISA was 97%, kappa=0.899. These results strongly support the usefulness of iELISA to detect A. marginale antibodies in milk. Additional studies are necessary to define the ability of the milk iELISA to detect not only acutely infected, but also carrier cattle.