Evaluation of frequency of antibodies against Neospora caninum, Toxoplasma gondii and Brucella melitensis, risk factors and spatial distribution of infection in goat and sheep flocks from Argentina.

Neospora caninum, Toxoplasma gondii and Brucella melitensis are pathogens that cause abortion in small ruminants. Besides, B. melitensis and T. gondii are zoonotic pathogens. The aim of this study was to describe the frequency of antibodies against N. caninum, T. gondii and B. melitensis in sheep and goats from three provinces of the center region of Argentina. In addition, the spatial distribution of the infected flocks/herds and risk factors were evaluated. A cross-sectional study was conducted from 2015 through 2016. Serum samples from 4783 goats and 1524 sheep from 186 goat, 51 sheep and 38 mixed flocks/herds were analyzed. Competitive inhibition enzyme-linked immunosorbent assay (ciELISA) and indirect fluorescent antibody test (IFAT) were performed for detection of antibodies against N. caninum and IFAT for T. gondii. The buffered plate antigen test and complement fixation test were performed for detection of antibodies against B. melitensis. The frequency of anti-T. gondii antibodies was 41.2% and 29.7% for sheep and goats, respectively. The frequency of anti-N. caninum antibodies was 17.2% and 14% for sheep and goats, respectively. About 97.1% of the sheep flocks, 79.4% of the goat herds and the 91.3% of the mixed flocks had seropositive animals to T. gondii. About 61.8% of the sheep flocks, 58% of the goat herds and the 82.6% of the mixed flocks had seropositive animals to N. caninum. All the analyzed animals were negative to anti-B. melitensis antibodies. For T. gondii, a significant cluster of high risk of seropositive flocks/herds was detected in the littoral of the Parana River. The province of origin of the flock/herd was the only variable associated to T. gondii positivity (p = 0.003). Animals from Santiago del Estero and Santa Fe Provinces had 3.48 and 1.77 times more risk to be positive to T. gondii than animals from Entre Ríos Province, respectively. For N. caninum, a cluster of high risk of seropositive flocks/herds was detected in the north of Santa Fe Province. The only explanatory variable associated to N. caninum positivity was animal species (p = 0.003). Sheep had 1.73 times more risk to be positive to N. caninum than goats. The absence of antibodies against B. melitensis in all the analyzed animals is an important finding for the public health of the region. Since bordering provinces have infected flocks/herds, brucellosis in small ruminants should be under epidemiologic surveillance in the region.

Development, validation and field evaluation of an indirect ELISA for the detection of antibodies against Brucella abortus in bulk and individual milk samples in dairy cattle.

Brucellosis is an abortigenic and zoonotic disease. In cattle, it is mainly caused by Brucella abortus. The disease is endemic in low- and middle-income countries, being considered a neglected zoonotic disease. In these countries, it is of high importance to develop and validate sensitive, specific and low-cost diagnostic assays for brucellosis. The aim of the present study was the development of an indirect enzyme-linked immune assay (iELISA) to detect anti-B. abortus antibodies in milk samples. We purified the lipopolysaccharide antigen from B. abortus and produced an anti-bovine IgG monoclonal antibody to develop an iELISA (iELISAINTA). The iELISAINTA was validated using 1730 bulk milk samples and 1734 individual milk samples. The sampled dairy herds had at least 3 years of consistency at their positive or negative official brucellosis status. Individual milk samples were taken in parallel with serum samples from the cows. The status of the cows was defined by the result of the complement fixation test (CFT) performed with their serum sample. The reproducibility of the assay was evaluated in two laboratories. In addition, we evaluated the performance of the assay in the field, using 4385 bulk milk samples and 968 individual milk samples. The results of the iELISAINTA were compared with those obtained using the officially accepted brucellosis techniques: iELISA from Canada (iELISACFIA) in milk samples, and the buffered plate antigen (BPA) and the CFT in serum samples. At validation, the sensitivity (Se) of the iELISAINTA in bulk milk samples was 98.61 %, and the specificity (Sp) 98.79 % with a ≥ 10 % of positivity (PP) cutoff. In individual milk samples, the Se was 98.04 %, and the Sp 98.56 % with a ≥ 16 PP cutoff. The chance-corrected agreement kappa value (κ) between the results obtained in the different laboratories was κ = 0.87. In the field evaluation, in bulk milk samples the κ value between the iELISAINTA and the iELISACFIA was κ = 0.86. On individual milk samples, the κ values were: between the iELISAINTA and the iELISACFIA κ = 0.79, between the iELISAINTA and BPA was κ = 0.85, and between the iELISAINTA and CFT κ = 0.82. The developed iELISAINTA showed a very good performance and it could be used as a screening assay for anti-B. abortus antibodies detection in individual milk samples and for epidemiologic surveillance in bulk milk samples.

Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle.

A nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) assay based on the amplification of the Anaplasma spp. highly conserved msp5 gene and posterior digestion with HindIII endonuclease was developed and evaluated in field samples. Results were compared using an nPCR specific for Anaplasma marginale (nPCR-Am) based on the msp1ß gene and an nPCR specific for A. centrale (nPCR-Ac) based on the msp2 operon (msp2-o) gene. Amplicons dilutions of msp1ß and msp5 of A. marginale and msp2-o and msp5 of A. centrale and dilutions of parasited erythrocytes (PE) with A. marginale and A. centrale were used to determine the detection limits. The results were 20 DNA copies/reaction and 30 PE for A. marginale and A. centrale by nPCR-RLFP and nPCR-Am/Ac. A mix of msp5-Am and msp5-Ac was used to evaluate the interference of msp5 from one species for the detection of the other. Co-amplification of the DNA from both species was observed up to a 1:7 ratio of one species to the other. Field samples positive for Anaplasma spp. antibodies (n = 260) from 32 herds were evaluated. Strength of agreement between results by nPCR-RFLP and nPCR-Am or nPCR-Ac was 78% (κ = 0.44) and 94% (κ = 0.85), respectively. Thirty-four samples were positive for A. marginale by nPCR-RFLP but negative by nPCR-Am. msp1ß amplicons of 10 samples from 5 herds with discrepancies between nPCR-Am and nPCR-RFLP results were cloned and sequenced. The analysis of the msp1ß sequence showed several mutations in the target region of the internal forward primer that would explain the failure in the amplification. Only 10 of the 20 coinfections identified by nPCR-Ac/nPCR-Am were detected by nPCR-RFLP. nPCR-RFLP is a sensitive, low-cost and accessible molecular method for low-complexity laboratories. More studies are needed to establish in which circumstances coinfections can be underestimated.

Neospora caninum truncated recombinant proteins formulated with liposomes and CpG-ODNs triggered a humoral immune response in cattle after immunisation and challenge.

Abortions caused by Neospora caninum are a serious problem in cattle production and require effective immunoprophylaxis. The objective of this work was to assess the humoral immune response to four recombinant (r) N. caninum antigens in cattle after immunisation and challenge. MIC1 and MIC3 proteins from the micronemes, SRS2 from the surface of tachyzoites, and GRA7 from the dense granules were expressed as truncated recombinant proteins in Escherichia coli. Cationic liposomes (Lip) and CpG oligodeoxynucleotides (CpG-ODNs) were used as adjuvant. Steers were assigned to three groups of six steers each and were inoculated twice subcutaneously, 21 days apart. The rP + Lip + CpG-ODN group received the truncated recombinant proteins rMIC1, rMIC3, rSRS2 and rGRA7 formulated with the adjuvant; the Lip + CpG-ODN group received the adjuvant alone; and the PBS group received sterile phosphate-buffered saline. All steers were subcutaneously challenged with the NC-1 strain of N. caninum 35 days after the second dose of immunisation. Steers from the rP + Lip + CpG-ODN group developed specific IgG, IgG1 and IgG2 against the four recombinant proteins after immunisation. After challenge, IgG against rMIC1 and rMIC3 was detected in rP + Lip + CpG-ODN group and against rSRS2 and rGRA7 in all groups. IgG1 and IgG2 against the four recombinant proteins remained high after challenge in the rP + Lip + CpG-ODN group. Indirect ELISA detected anti-N. caninum antibodies after challenge in all groups, with the highest level of antibodies being detected in the rP + Lip + CpG-ODN group. The recombinant vaccine formulated with rMIC1, rMIC3, rSRS2 and rGRA7 using Lip + CpG-ODN as adjuvant was immunogenic in cattle and the humoral immune response after challenge was enhanced in vaccinated cattle.

Validation and field evaluation of a competitive inhibition ELISA based on the recombinant protein tSAG1 to detect anti-Neospora caninum antibodies in sheep and goats.

Neospora caninum is a protozoan parasite that causes abortion and reproductive failure in small ruminants. We validated and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1) from N. caninum for the detection of anti-N. caninum antibodies in sheep and goat flocks. The assay was validated using 80 positive and 142 negative serum samples from sheep and goats analyzed by IFAT and immunoblot (IB). ciELISAtSAG1 was then used to evaluate the prevalence of anti-N. caninum antibodies in 1449 goats from 143 flocks and 385 sheep from 40 flocks and compared to IFAT. The prevalence of anti-Toxoplasma gondii antibodies was evaluated by IFAT. The ciELISAtSAG1 cut-off was ≥ 36 percent inhibition, with a diagnostic sensitivity of 100.0 % (95 % CI = 95.4-100.0 %) and a diagnostic specificity of 98.6 % (95 % CI = 95.0-99.8 %) relative to the agreement between IFAT and IB. The field evaluation revealed a concordance between ciELISAtSAG1 and IFAT of 97.4 %, with an agreement (κ) of 0.90 for sheep sera, and a concordance of 96.5 % with κ = 0.85 for goat sera. The overall prevalence of anti-N. caninum antibodies in sheep was 14.3 % by IFAT and 15.8 % by ciELISAtSAG1. In goats, prevalence was 12.9 % by IFAT and 14.6 % by ciELISAtSAG1. The overall prevalence of anti-T. gondii antibodies was 28.8 % in goats and 43.8 % in sheep. The ciELISAtSAG1 could be useful for large-scale detection of anti-N. caninum antibodies in sheep and goats, and for seroepidemiological investigations due to its appropriate sensitivity and specificity, and the simplicity of production.

Evaluation of a competitive inhibition ELISA based on the recombinant protein tSAG1 to detect anti-Neospora caninum antibodies in cattle.

Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. There are no treatments or vaccines available; disease control is based on diagnosis and herd management strategies. We developed, validated, and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1), expressed in Escherichia coli, and the RafNeo5 monoclonal antibody (ciELISAtSAG1). A criterion based on the 3-y sequential serologic analysis of 230 dairy cows by IFAT was used as the gold standard. The assay was validated using 860 serum samples from cows that were consistently positive or negative by IFAT throughout the study period. ciELISAtSAG1 was then used to evaluate the prevalence of neosporosis in 16 beef cow herds (22 samples per herd, 352 total samples). The results were compared with those from IFAT and a commercial cELISA (cELISAVMRD). The ciELISAtSAG1 cutoff was ≥ 29%I, with a diagnostic sensitivity of 98.7% (95% CI = 96.8-99.7%) and a diagnostic specificity of 97.9% (95% CI = 96.4-99.0%). Concordance among IFAT, cELISAVMRD, and ciELISAtSAG1 was 90.3%. The agreement (κ) between ciELISAtSAG1 and the other 2 tests was ≥ 0.81. The overall prevalence of neosporosis in the 16 beef herds was 30% (range: 5-60%). The ciELISAtSAG1 could be useful for large-scale detection of anti-N. caninum antibodies in cattle and seroepidemiologic investigations, given its appropriate sensitivity and specificity, and the simplicity of production.

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.

Immunisation of cattle against Babesia bovis combining a multi-epitope modified vaccinia Ankara virus and a recombinant protein induce strong Th1 cell responses but fails to trigger neutralising antibodies required for protection.

Protection against the intraerythrocytic protozoan parasite Babesia bovis depends on both strong innate and adaptive immune response, this latter involving the presentation of parasite antigens to CD4+ T-lymphocytes by professional antigen-presenting cells. Secretion of Th1 cytokines by CD4+ T cell is also very important for isotype switching to IgG2, the best opsonising antibody isotype in cattle, to target extracellular parasites and parasite antigens displayed at the erythrocyte surface. In the field of vaccinology, heterologous prime-boost schemes combining protein-adjuvant formulations with a modified vaccinia Ankara vector expressing the same antigen have demonstrated the induction of both humoral and cellular immune responses. It has been previously demonstrated that MVA-infected dendritic cells can present antigens in the context of MHC II and activate CD4+ T cell. These results support the use of the MVA viral vector for a pathogen like Babesia bovis, which only resides within erythrocytes. In this study, 13-15-months-old Holstein-Friesian steers were immunised with a subunit vaccine as a prime and a modified vaccinia Ankara vector as a boost, both expressing a chimeric multi-antigen (rMABbo - rMVA). This antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: merozoite surface antigen - 2c (MSA - 2c), rhoptry associated protein 1 (RAP - 1) and heat shock protein 20 (HSP20). Responses were compared with the Babesia bovis live attenuated vaccine used in Argentina (R1A). Eleven weeks after the first immunisation, all bovines were challenged by the inoculation of a virulent B. bovis strain. All groups were monitored daily for hyperthermia and reduction of packed cell volume. Both the rMABbo - rMVA and R1A vaccinated animals developed high titters of total IgG antibodies and an antigen-specific Th1 cellular response before and after challenge. However, all rMABbo - rMVA steers showed clinical signs of disease upon challenge. Only the R1A live vaccine group developed an immune response associated with in vitro neutralising antibodies at a level that significantly inhibited the parasite invasion. The lack of protection observed with this recombinant formulation indicates the need to perform further basic and clinical studies in the bovine model in order to achieve the desired effectiveness. This is the first report in which a novel vaccine candidate against Babesia bovis was constructed based on a recombinant and rationally designed viral vector and evaluated in the biological model of the disease.

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.

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-Neospora caninum antibodies in cattle.

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero-American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1-7) and three enzyme-linked immunosorbent assays (ELISAs 1-3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the "Pre-test information," which uses previous epidemiological and serological data; (2) the "Majority of tests," which classifies a serum as positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.

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.