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The aim of this longitudinal study was to characterize the parasitemia of Neospora caninum and the associated immunological parameters in naturally infected beef cows for 10 months. The following groups were established: Neospora caninum seropositive pregnant cows (+Preg, n = 7), seropositive non-pregnant cows (+Npreg, n = 7), seronegative pregnant cows (-Preg, n = 4), and seronegative non-pregnant cows (-Npreg, n = 4). Several samples were obtained for absolute and relative leukocyte counting, cytokines IL-10, IL-12, α-TNF, and γ-IFN quantification, specific IgG, IgG1, and IgG2 and avidity and N. caninum DNA molecular detection and quantification. The +Preg group had a higher frequency and concentration of N. caninum DNA in PBMC in the last third of pregnancy compared to +Npreg ( p <0.05), with 22 and 8% of detection, respectively. Parasitemia correlated positively with IgG titers and negatively with IgG1/IgG2 ratio ( p <0.05). On day 222 of the assay, the +Preg group had the lowest total leukocyte counting ( p <0.05). The +Preg group had a higher concentration of IgG and higher avidity in the last third of gestation compared to +Npreg ( p <0.05). Avidity correlated with total IgG and IgG2 ( p <0.05). All +Preg cows gave birth to clinically healthy but seropositive calves before colostrum intake, therefore, the congenital transmission was 100% efficient. Only a complete N. caninum genotype from a placenta and a partial genotype from cow #3 of the group +Preg were achieved by multilocus microsatellite analysis. Overall, N. caninum parasitemia is frequent in seropositive beef cows during the last third of gestation. This correlates with higher antibody levels and a decrease in total leukocyte counting. The precise timing of the parasitemia may be used for diagnosis purposes and/or for design strategies to avoid vertical transmission. Further studies are needed to identify the immune molecular mechanisms that favor parasitemia during gestation in chronically infected cattle., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gual, Campero, Hecker, Regidor-Cerrillo, Leunda, Odeón, Campero, Torioni de Echaide, Echaide, Estein, Ortega-Mora and Moore.)

The aim of this study was to evaluate the influence of the long-acting oxytetracycline (OTC) treatment on A. marginale genotypes of the isolate S1P, by analyzing the msp1α genotype based on a microsatellite (ms) and tandem repeat sequences (TRS) located at the 5´ end of the gene. DNA samples were obtained from a longitudinal study of chemosterilization; 10 2-year-old steers were experimentally infected with blood from a splenectomized calf inoculated with the A. marginale isolate S1P. All the steers had received a first dose of 20 mg kg -1  OTC to treat acute disease, and once recovered all steers received a sterilizing treatment based on three doses of 20 mg kg -1  OTC 7 days apart. Blood from two steers not sterilized by the treatment was inoculated into two splenectomized calves (receptors) 104 days after treatment. DNA samples (S) used for msp1α amplification were obtained from i) the donor calf (S0), ii) 10 steers during acute disease (S1), after the first antibiotic treatment (S2), and after the chemosterilization procedure (S3 and S4), and iii) two receptor calves (S5). Thirty clones from the donor calf and at least 5 clones from the other DNA samples were analyzed. The genotype E/αββββГ msp1α identified in the donor calf and steers, before OTC treatment, was not detected either in steers that continued infected after the sterilizing treatment or in the receptor calves, in which only genotype C/EϕFF msp1α was observed. These results highlight the existence of A. marginale genotypes with different sensitivity to OTC and the importance of other variables to successfully sterilize the carriers., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The expansion of anaplasmosis to non-endemic areas in Argentina has created the need for specific treatments to eliminate Anaplasma marginale from carriers. The most recent studies have failed to chemosterilize A. marginale infections. In this work, we compare the efficacy of long-acting oxytetracycline (OTC) and imidocarb dipropionate (IMD) to chemosterilize the A. marginale infection. For this purpose, twenty steers were randomly clustered into two groups of ten animals each 78 days after A. marginale experimental infection (day 0). Cattle from group 1 (G1) were treated with three doses of 20 mg kg -1 of OTC (Terramycin® LA, 200 mg/ml) 7 days apart by intramuscular injection. Cattle from G2 were treated with two doses of 5 mg kg -1 of IMD (Imizol®, 120 mg/ml) 14 days apart by intramuscular injection. The efficacy of sterilizing treatments was evaluated by detection of DNA by nested PCR, anti-MSP5 antibodies by ELISA and by inoculation of splenectomized calves with blood from the steers 104 days post-treatment (dpt). The results showed 50% efficacy of the OTC treatment to chemosterilize persistent A. marginale infections in cattle and the failure of the IMD treatment under the evaluated conditions. The persistence of specific antibody levels in the sterilized animals (56 dpt) was shorter than the period of DNA detection. The ELISA was the test of choice to confirm the sterilizing outcome after 60 dpt. In spite of its limitations, the sterilization of A. marginale carrier status using OTC, could be useful for high-value bovines in non-endemic areas., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 (ciELISA tSAG1 ). 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. ciELISA tSAG1 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 (cELISA VMRD ). The ciELISA tSAG1 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, cELISA VMRD , and ciELISA tSAG1 was 90.3%. The agreement (κ) between ciELISA tSAG1 and the other 2 tests was ≥ 0.81. The overall prevalence of neosporosis in the 16 beef herds was 30% (range: 5-60%). The ciELISA tSAG1 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.

Anaplasma marginale is the most prevalent tick-borne livestock pathogen with worldwide distribution. Bovine anaplasmosis is a significant threat to cattle industry. Anaplasmosis outbreaks in endemic areas are prevented via vaccination with live A. centrale produced in splenectomized calves. Since A. centrale live vaccine can carry other pathogens and cause disease in adult cattle, research efforts are directed to develop safe recombinant subunit vaccines. Previous work found that the subdominant proteins of A. marginale type IV secretion system (T4SS) and the subdominant elongation factor-Tu (Ef-Tu) were involved in the protective immunity against the experimental challenge in cattle immunized with the A. marginale outer membrane (OM). This study evaluated the immunogenicity and protection conferred by recombinant VirB9.1, VirB9.2, VirB10, VirB11, and Ef-Tu proteins cloned and expressed in E. coli. Twenty steers were randomly clustered into four groups (G) of five animals each. Cattle from G1 and G2 were immunized with a mixture of 50 μg of each recombinant protein with Quil A® or Montanide™ adjuvants, respectively. Cattle from G3 and G4 (controls) were immunized with Quil A and Montanide adjuvants, respectively. Cattle received four immunizations at three-week intervals and were challenged with 107 A. marginale-parasitized erythrocytes 42 days after the fourth immunization. After challenge, all cattle showed clinical signs, with a significant drop of packed cell volume and a significant increase of parasitized erythrocytes (p<0.05), requiring treatment with oxytetracycline to prevent death. The levels of IgG2 induced in the immunized groups did not correlate with the observed lack of protection. Additional strategies are required to evaluate the role of these proteins and their potential utility in the development of effective vaccines., Competing Interests: The authors have declared that no competing interests exist.

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 aim of the present study was to characterize the specific immune response in prepubertal female calves inoculated with Neospora caninum. Forty-eight N. caninum-seronegative 6-month-old Angus female calves were randomly allocated into two groups: group A calves were inoculated subcutaneously (sc) with 1 × 10 6 tachyzoites of the low virulence NC-Argentina LP1 isolate in sterile phosphate-buffered saline (PBS); group B calves were mock inoculated sc with sterile PBS. Calves from group A developed a specific immune response characterized by the production of IgG antibodies and the expression of IFN-γ and TNF-α cytokines. Animals did not present any febrile reaction or reactions at the site of inoculation. Although chronic N. caninum infection was developed in 50% of calves of group A after inoculation, according to the presence of antibodies against rNc-SAG4, antigen characteristic of bradyzoites, N. caninum antibodies dropped below the cut-off of ELISA from day 210 post-inoculation onwards. Future trials using the same group of inoculated animals will allow the characterization of the evolution of the immune response during pregnancy and to determine whether the immunization with the local isolate is able to prevent congenital transmission and to protect against heterologous challenges.

Background: Tritrichomonas foetus is the etiologic agent of the sexually transmitted disease Bovine Trichomonosis (BT). In Argentina, BT is endemic and represents a relevant health problem that causes reproductive inefficiency in cattle and large economic losses. Metronidazole is the drug of choice in the treatment of BT. Treatment has been associated with a temporary resolution of the clinical signs but is not able to control the disease. In recent years, the apparition of in vivo and in vitro aerobic and anaerobic resistance leading to ineffective treatments has been reported., Aims: Thus, the aim of the present study was to explore the susceptibility of six different isolates of T. foetus under aerobic (AC) and anaerobic (ANC) conditions., Results and Discussion: Six isolates of T. foetus were obtained from samples of preputial smegma of bovine origin. Values of minimum lethal concentration and minimum inhibitory concentration were higher than those observed in other works and represent current data in Argentina and provide information to establish new treatment protocols.

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.

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5' ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

Babesia bigemina infection of mature bovine erythrocytes results in new proteins specifically exposed on the parasitized cell surface. Monoclonal antibody (MAb) 64/32 binds a protein, designated p94, on B. bigemina-infected erythrocytes but not on either uninfected or B. bovis-parasitized erythrocytes. However, p94 was not encoded by B. bigemina and was not a parasite-modified erythrocyte membrane protein. In contrast, we showed that p94 could be eluted from the infected erythrocyte surface and was identified as specifically bound immunoglobulin M (IgM) heavy chain for the following reasons: (i) MAb 64/32 bound a reduced molecule of 94 kDa in both infected erythrocyte lysates and normal bovine serum; (ii) MAb 64/32 bound a 94-kDa molecule in reduced preparations of purified IgM; (iii) an anti-bovine mu heavy-chain MAb, BIg73, reacted specifically with the surface of infected erythrocytes and bound the 94-kDa molecule in lysates of infected erythrocytes, normal bovine serum, and purified IgM; and (iv) immunoprecipitation of infected erythrocyte lysates with MAb 64/32 depleted the 94-kDa antigen bound by anti-mu MAb BIg73 and vice versa. Binding of IgM to the infected erythrocyte surface was detected in vivo early in acute parasitemia and occurred during both the trophozoite and merozoite stages of intraerythrocytic parasitism. The common feature of IgM binding to the parasitized erythrocyte surface among otherwise genetically and antigenically distinct B. bigemina strains is suggestive of an advantageous role in parasite survival in vivo.

A live, frozen experimental vaccine containing Babesia bovis and Babesia bigemina multiplied in vitro was stored in liquid nitrogen after simultaneous cryopreservation using glycerol as cryoprotectant. The viability of the vaccine was tested by inoculating (subcutaneously) three groups of seven steers each, 2, 12 and 24 h after thawing at 40 degrees C and dilution to obtain a dose of 2 x 10(7) of each organism. All vaccinated cattle developed detectable parasitaemia in thin and/or thick blood smears. No statistically significant differences in the prepatent period were detected amongst the cattle groups (analysis of variance). This prepatent period was 12.3 days for B. bovis and 8.4 days for B. bigemina. Vaccinal organisms derived from in vitro culture systems may replace antigens obtained from in vivo culture to produce vaccine against cattle babesiosis.

Attenuated Babesia bigemina were multiplied in vivo and in vitro to vaccinate two groups (Groups 2 and 3) of Holstein Friesian heifers. Another group (Group 1) of heifers was vaccinated twice with purified soluble antigens obtained from the supernatant of in vitro culture combined with saponin. All these heifers plus controls (Group 4) were inoculated with heterologous pathogenic B. bigemina 5 months later. Heifers vaccinated with live organisms (Group 2 and 3) were able to stand the challenge without specific treatment whereas the opposite occurred in heifers vaccinated with soluble antigens (Group 1) and controls (Group 4). Antibody titres were higher in heifers inoculated with soluble antigens than in heifers inoculated with live B. bigemina multiplied in vivo, indicating that antibody titres may not be a proper indicator of protection. In vitro culture of this protozoan is probably a better source of live antigens for vaccine production than in vivo culture. These kind of immunogens can fill the gap until improved vaccines are available.