Development and field evaluation of an ELISA to differentiate Anaplasma marginale-infected from A. centrale-vaccinated cattle.

Immunization of calves with Anaplasma centrale is used to prevent acute anaplasmosis caused by A. marginale. Natural and vaccine-acquired immunity is detected through serologic tests based primarily on A. marginale recombinant major surface protein 5 (MSP5m) because it has 91% identity with MSP5 from A. centrale (MSP5c). We developed a displacement, double-antigen, sandwich ELISA (ddasELISA) to detect antibodies against A. marginale or A. centrale. For ddasELISA validation, we analyzed serum samples positive for antibodies against Anaplasma spp. from cattle naturally infected with A. marginale (n = 300) or vaccinated with A. centrale (n = 255). Species-specific nested PCR (nPCR) assays were used to confirm infection. The optical density (OD) values obtained from antibodies directed at unique epitopes of A. marginale (ODAm) or A. centrale (ODAc) were used in the formula ODAm/ODAc. If the derived ratio was >0.38, the serum sample was considered positive for antibodies against A. marginale, with 98.9% sensitivity and 98.0% specificity. In a field evaluation, we analyzed 702 Anaplasma spp. antibody-positive serum samples from 34 herds by ddasELISA and nPCR; 571 were classified by ddasELISA as A. marginale-infected or A. centrale-vaccinated, with 84% agreement (κ = 0.70) between ddasELISA and nPCR. Our results indicate that ddasELISA could be used as a cost-effective alternative to molecular techniques to confirm infection with A. marginale in countries in which prevention is based on vaccination with A. centrale.

Molecular detection and phylogenetic characterization of Theileria equi in horses (Equus caballus) from a peri-urban area of Argentina.

To investigate the presence of Theileria equi in an endemic area of equine piroplasmosis 42 horses (Equus caballus) from Corrientes City, Argentina were sampled. Eighty-one percent (34 blood samples) of the analyzed horses were tested positive to the presence of piroplasmid 18S rDNA. All these samples could be identified as T. equi by amplifying the specific EMA-1 (merozoite antigen 1) gene of this species. Phylogenetic analysis of an obtained 18S rDNA complete sequence from one strain resulted in the identification of this sample as T. equi sensu stricto (genotype A). This study presents the first molecular detection and characterization of T. equi by the complete 18S rDNA sequence in Argentina. Based on these results further studies should be carried out to investigate the distribution and heterogeneity of presented genotypes of T. equi in Argentina, which is essential for the diagnosis, prevention and treatment of equine piroplasmosis.

Ixodes silvanus n. sp. (Acari: Ixodidae), a new member of the subgenus Trichotoixodes Reznik, 1961 from northwestern Argentina.

Females, nymphs, and larvae of Ixodes silvanus n. sp. collected from birds and from the vegetation in northwestern Argentina (Yungas Phytogeographic Province) are described herein. The new species belongs to the subgenus Trichotoixodes (Acari: Ixodidae). The female is diagnosed by a combination of the following characters: scutum with setae moderately long and more numerous in central field, fewer and moderately long setae on lateral fields, and inconspicuous setae in anterior field; basis capituli subtriangular dorsally; porose areas large and irregular in shape, lacking distinct margins; auriculae with straight edges diverging posterolaterally and ending with small blunt processes; hypostome narrow and pointed with dental formula 4/4 in the anterior third, then 3/3 and 2/2 near the base; coxae I with two spurs, sub-equal in size, internal slightly slimmer than external. The nymph is diagnosed by notum with numerous and long setae, ventral surface covered by numerous whitish setae, scutum with short scapulae and few and shallow punctations, setae on scutum few, short and irregularly distributed, basis capituli sub-triangular dorsally with posterior margin straight, cornua large and directed postero-laterally, auriculae large and projected laterally, lateral margin of basis capituli above auriculae with a lateral and triangular projection, hypostome pointed with dental formula 3/3 in the anterior third and then 2/2, and coxa I with two short, sub-equal, triangular spurs. The diagnostic characters of the larva are: basis capituli dorsally sub-triangular with lateral angles acute and posterior margin straight, auriculae as large triangular lateral projections, hypostome with apex bluntly pointed and dental formula 3/3 in the anterior third and then 2/2, coxa I with two short, sub-equal, triangular spurs, and pattern of dorsal and ventral body setae. This new species is phylogenetically related to Ixodes brunneus, Ixodes turdus and Ixodes frontalis, and the principal hosts for all its parasitic stages are birds.

A Trypanosoma species detected in Rhipicephalus (Boophilus) microplus ticks from Argentina.

Specimens of a Trypanosoma sp. were found in a haemolymph sample of Rhipicephalus microplus from Argentina. Polymerase chain reaction (PCR) was done targeting the SSU rRNA gene of Trypanosoma spp. and a fragment of 2300 base pairs (bp) was amplified, subsequently a phylogenetic analysis was conducted, based on an alignment of 905 bp, containing the sequence of the Argentina isolate and sequences of different Trypanosoma species retrieved from GenBank. Phylogenetic analysis revealed that this trypanosome is not related to Trypanosoma theileri as was previously thought, instead the strain of Trypanosoma detected in this study can be provisionally determined as belonging to the recently described organism Trypanosoma rhipicephalis. Furthermore, phylogenetic analysis performed in this work revealed that T. rhipicephalis belongs to a novel clade of tick-related trypanosomes, most with limited genetic data, for which essential aspects of both the vertebrate and invertebrate life cycles are lacking. The lack of basic information restricts the inferences that can be done from the present finding and, in addition, points out a clear knowledge gap in the biology of this group of trypanosomes.

Development and evaluation of a double-antigen sandwich ELISA to identify Anaplasma marginale-infected and A. centrale-vaccinated cattle.

Bovine anaplasmosis is a worldwide infectious disease caused by the intraerythrocytic bacterium Anaplasma marginale, which is transmitted by ticks and fomites. A. centrale is a less virulent subspecies used as a live vaccine in cohorts of 8- to 10-mo-old calves that did not naturally reach enzootic stability. We developed 3 variants of a double-antigen sandwich ELISA (dasELISA) using a recombinant major surface protein 5 (MSP5) from A. marginale (dasELISAm) or from A. centrale (dasELISAc) or using MSP5 from both organisms (dasELISAmc). Each dasELISA was tested for the detection of antibodies against A. marginale and A. centrale. The tests were validated using serum samples from cattle not infected with Anaplasma spp. (n = 388), infected with A. marginale (n = 436), and vaccinated with A. centrale (n = 358), confirmed by nested PCR. A total of 462 samples were compared with a commercial competitive ELISA (cELISA). For dasELISAm, dasELISAc, and dasELISAmc, specificities were 98.7%, 98.7%, and 97.4%, and overall sensitivities were 92.6%, 85.7%, and 97.4%, respectively. For A. marginale-infected and A. centrale-vaccinated cattle, sensitivities were 97.7% and 86.3% for dasELISAm, and 77.7% and 95.5% for dasELISAc, respectively. Sensitivity of dasELISAmc was similar for both groups (>96%). The agreement rate between dasELISAmc and cELISA was 96.3% (κ = 0.92); the former test allowed earlier detection of seroconversion of vaccinated cattle than did cELISA. Based on these results, the test could be used to 1) determine the enzootic stability or instability of anaplasmosis in calves, 2) conduct epidemiologic studies, and 3) evaluate the immunogenicity of A. centrale live vaccine.

The Ixodes ricinus complex (Acari: Ixodidae) in the Southern Cone of America: Ixodes pararicinus, Ixodes aragaoi, and Ixodes sp. cf. I. affinis.

The goal of this study was to clarify the taxonomic status of the Ixodes ricinus complex in the Southern Cone of America, by using morphological characters and molecular markers (mitochondrial 16SrDNA and cox1 genes). The morphological analysis indicates that three different taxa of the I. ricinus complex occur in this region: Ixodes pararicinus, Ixodes aragaoi, and Ixodes sp. cf. I. affinis. The most prominent diagnostic character among them is the size of scutal punctations in both male and female ticks. In the males of Ixodes sp. cf. I. affinis, the punctations on the central field and along the median marginal groove of the scutum are clearly larger than in the males of I. aragaoi and I. pararicinus, while the punctations of I. aragaoi are larger but less numerous than in I. pararicinus. The punctations in Ixodes sp. cf. I. affinis females are larger and deeper than in females of I. aragaoi and I. pararicinus, and those of I. aragaoi are slightly larger than in I. pararicinus. The length of the lateral posterior denticles of the male hypostome is comparatively longer in I. aragaoi than in the other two species, and longer in Ixodes sp. cf. I. affinis than in I. pararicinus. In the 16S analysis, I. pararicinus and I. aragaoi are monophyletic (99% and 98% bootstrap support, respectively), while Ixodes cf. I. affinis does not represent a single lineage. In the cox1 analysis, both I. pararicinus and I. aragaoi are well-defined taxa, but the bootstrap support for Ixodes sp. cf. I. affinis is low (67%). In general, there are considerable 16SrRNA differences among lineages of Ixodes sp. cf. I. affinis from different geographical areas. These results may be indicative of the existence of different species. The populations morphologically compatible with I. affinis from Argentina, Colombia, Panama, Belize, and USA should be provisionally named as Ixodes sp. cf. I. affinis until an integrative taxonomic work with further evidence redefines whether or not this taxon actually represents a species complex.

The presence of Borrelia theileri in Argentina.

The presence of Borrelia theileri in Argentina is confirmed after recording the spirochete from a bovine in northern Argentina. The analysis of sequences of the flagellin gene (fla) and length of Borrelia spp. specimens on thick blood films shows that the local isolate clusters within a well-supported clade with B. theileri isolates from different geographical origins, confirming the presence of B. theileri in Argentina. The mean length of 30 specimens of B. theileri was 12.89 µm (standard deviation 2.88 µm, range 9.35-20.16 µm). The only known vector of Borrelia theileri in northern Argentina is the cattle tick Rhipicephalus microplus, therefore Borrelia infection should be regarded as a potential complication of other cattle tick-borne diseases such as babesiosis, especially on cattle introduced from areas free of R. microplus. The possibility of serologic cross-reaction with B. theileri must not be minimized in studies of other spirochaetes in the R. microplus infested region of Argentina.

Development of a novel fusion protein with Anaplasma marginale and A. centrale MSP5 improved performance of Anaplasma antibody detection by cELISA in infected and vaccinated cattle.

Detection of antibodies to Anaplasma spp. using commercial competitive enzyme-linked immunosorbent assay (ccELISA) is based on the recombinant major surface protein 5 fused to maltose binding protein (MBP-MSP5) or glutathione S-transferase (GST-MSP5). To avoid false positive reactions due to the presence of antibodies against E. coli MBP in cattle, previous sera absorption is required. This study evaluated the replacement of MBP-MSP5 or GST-MSP5 antigens by the truncate MSP5 (residues 28-210) of A. marginale (tMSP5m), A. centrale (tMSP5c) and fusion protein MSP5 (tMSP5cm), expressed without N-terminus transmembrane helix in the ccELISA test. Immunoreactivity was evaluated by western blot using monoclonal antibodies against the tMSP5 and by in-house cELISA (hcELISA) with purified tMSP5m, tMSP5c or tMSP5cm using sera from cattle infected with A. marginale (n = 226) or vaccinated with A. centrale (n = 173) and uninfected cattle (n = 216). Results of hcELISA were compared with those of ccELISA. Recombinant protein was expressed highly soluble (> 95%) in E. coli without a molecular chaperone. Specificity of the hcELISA-tMSP5m, -MSP5c or -tMSP5cm was identical to (99.5%) and greater than that in ccELISA (96.3%). Sensitivity of hcELISA-tMSP5m and ccELISA was identical (95.5%), but lower than that of hcELISA-tMSP5cm (96.2%) and -tMSP5c (97.2%). The analysis of vaccinated cattle by hcELISA-tMSP5c showed sensitivity of 99.4%. In summary, the generation of fusion MSP5 A. marginale-A. centrale protein without transmembrane helix was a very effective method to express the recombinant protein highly soluble in the bacterial cytoplasm and contributed to an increased test performance for detecting antibodies in cattle naturally infected with A. marginale or vaccinated with A. centrale.

Molecular evidence of Babesia species in Procyon cancrivorus (Carnivora, Procyonidae) in Uruguay.

The crab-eating raccoon (Procyon cancrivorus) is a carnivore widely distributed from southern Central America to all South American countries except Chile. In the Southern cone of America, P. cancrivorus has been found parasitized by several Amblyomma spp. Particularly, in Uruguay, A. aureolatum is the only tick found in this wild carnivore. Piroplasmid hemoparasites were found in Procyon lotor from North America and Japan. In this work, molecular evidence Babesia sp. DNA was found in blood and tissues from road-killed P. cancrivorus from different locations in Uruguay. PCRs targeting 18S rRNA gene were carried out. Subsequently, the obtained amplicons were sequenced and full-length sequences was assembled. A phylogenetic tree was constructed and revealed that the Babesia sp. found in this work clustered with other 18sRNA sequences of Babesia spp. obtained from P. lotor from Japan and USA, along with Babesia spp. of maned wolf and I. ovatus. This is the first report of molecular evidence of Babesia sp. parasitizing P. cancrivorus.

Biochemical characterization of thioredoxin reductase from Babesia bovis.

This paper addresses the identification, cloning, expression, purification and functional characterization of thioredoxin reductase from Babesia bovis, the etiological agent of babesiosis. The work deals with in vitro steady state kinetic studies and other complementary analyses of the thioredoxin reductase found in the pathogenic protist. Thioredoxin reductase from B. bovis was characterized as a homodimeric flavoprotein that catalyzes the NADPH-dependent reduction of Trx with a high catalytic efficiency. Moreover, the enzyme exhibited a disulfide reductase activity using DTNB as substrate, being this activity highly sensitive to inhibition by Eosin B. The thioredoxin reductase/thioredoxin system can reduce oxidized glutathione and S-nitrosoglutathione. Our in vitro data suggest that antioxidant defense in B. bovis could be supported by this enzyme. We have performed an enzymatic characterization, searching for targets for rational design of inhibitors. This work contributes to the better understanding of the redox biochemistry occurring in the parasite.

Efficiency of a recombinant MSA-2c-based ELISA to establish the persistence of antibodies in cattle vaccinated with Babesia bovis.

Bovine babesiosis is caused by Babesia bovis and B. bigemina in Argentina. These protozoans are prevalent north of parallel 30 degrees S, where their natural vector Rhipicephalus (Boophilus) microplus is widespread. To prevent babesiosis outbreaks in endemic areas, an increasing population of 4-10-month-old calves are vaccinated with low virulence B. bovis R1A (BboR1A) and B. bigemina S1A (BbiS1A) strains. In non-endemic areas, an additional calf population is also vaccinated and boostered as adults, before they are relocated to R. microplus-endemic areas of the country. Serological tests are currently utilized not only to determine the status of natural Babesia spp. infections, but also to confirm the infection caused by vaccine strains. For this purpose, an indirect enzyme immunoassay (ELISA) based on the recombinant major surface antigen-2c (rMSA-2c) of B. bovis expressed in Escherichia coli, was standardized using sera from Babesia spp. experimentally infected cattle. ELISA(rMSA-2c) was validated using sera obtained weekly during 336 days from steers primed and boostered with BboR1A and/or BbiS1A on days 0 and 154, then compared with the immunofluorescent-antibody test (IFAT). Western blot (WB) protein analysis was used to confirm the specificity of the immune response to rMSA-2c. The sensitivity and specificity for ELISA(rMSA-2c) were 92 and 96% after the Babesia spp. priming and 88 and 73% after the boostering immunization, respectively. The sensitivity and specificity for IFAT were 99 and 90% after priming and 92 and 98% after boostering, respectively. Unlike IFAT, ELISA(rMSA-2c) detected a remarkable delayed booster response and a significant drop in specificity between 35 and 84 days after the booster immunization. Simultaneously, 87.5% of cattle boostered with B. bigemina showed cross-reactions in the ELISA(rMSA-2c), particularly between 63 and 77 days after the inoculation. A reaction against E. coli was observed, since bands of approximately 40 and/or 42kDa were detected using sera from cattle before and after Babesia spp. inoculations. ELISA(rMSA-2c) showed to be useful between 42 and 98 days after priming with Babesia spp. live vaccine to evaluate the success of infecting cattle. However, after boostering the test showed low specificity.