Development and evaluation of a TaqMan® real-time PCR assay for species-specific detection of Ehrlichia canis.

The aim of this work was to develop a real-time PCR assay with a TaqMan® probe that detects a species-specific part of the 16S rDNA gene of Ehrlichia canis. Canine blood samples (n = 207), collected and tested by a conventional PCR assay within a study conducted by De Salvo et al., were simultaneously analyzed with the novel designed real-time PCR, and the results of both assays were compared. The agreement between the two methods was 97.6 % with a kappa value of 0.92186. Hereby, the standard error was 0.034416 and the 95 % confidence interval from 0.8544 to 0.98931. While the conventional PCR assay showed false negative results (2.42 %; 5/207), the real-time PCR assays showed a specificity of 100 %. The results of the current study showed that the developed assay presents sensitivity and specificity for the detection of E. canis in blood samples, adding a new tool for the diagnosis of this pathogen.

Evaluation of the impact of successive acaricide treatments on resistance evolution in Rhipicephalus microplus populations: Monodrugs versus drug combinations.

The aim of this work was to comparatively evaluate the evolution of resistance in Rhipicephalus microplus tick populations exposed to successive treatments with monodrug-based formulations and combinations of them in the same commercial formulation. Thirty-six heifers, naturally infested with R. microplus, were divided into three groups (G) and subjected to three successive treatments, on days 0 (Nov-2021), 43 (Jan-2022) and 78 (Feb-2022), with the following formulations: I) ivermectin 3.15% (Ivomec Gold®) (GI), II) fipronil 1% (Ectoline®) (GII) and III) a combination of fipronil 2% and ivermectin 1% (Vaquero®) (GIII). From Nov-2021 to Dec-2022, counts of R. microplus were periodically performed to evaluate the tick infestation. Engorged females were collected at the beginning and end of the trial to determine the evolution of tick resistance to ivermectin and fipronil by in vitro bioassays. At the end of trial, GII and GIII had higher tick counts (39.18 ± 11.88 and 38.33 ± 14.31, respectively) than group I (5.11 ± 6.24) (P<0.05). The in vitro results shows that the resistance ratio (RR50) values after the treatments were higher for fipronil (5.584 and 5.649 for GII and GIII, respectively) than for ivermectin (1.165 and 1.088 for GI and GIII, respectively). In the group treated with the combination (GIII), the RR50 increased for both drugs simultaneously. These results suggest that the successive use of drug combinations could exacerbate the problem of multi-resistance of R. microplus to chemical acaricides.

Analysis of sequence variations in the GABA gated chloride gene associated with resistance to fipronil in Rhipicephalus (Boophilus) microplus ticks from Argentina.

This study aimed to evaluate the sequence variations of the GABA-Cl gene of Rhipicephalus (Boophilus) microplus ticks from Argentina with different resistance levels to fipronil. Genomic DNA was obtained from engorged females (n = 50) of fipronil-susceptible and resistant tick field populations from five localities in northern Argentina. Tick populations were tested for fipronil resistance by in vitro larval packet test. DNA encoding amino acid residues 264-360 of the GABA-Cl was amplified by PCR. PCR products were sequenced and the translated amino acid sequences were aligned and compared among each other and with sequences of the GABA-Cl belonging to the Brazilian and Uruguayan reference strains fipronil-susceptible (Mozo) and resistant (RFSan and Juarez). All the GABA-Cl amino acid sequences obtained from the R. microplus specimens from Argentina were identical, regardless of the fipronil resistance-susceptibility status of each population. Mutations present in GABA-Cl sequence of RFSan and Juarez strains were not present in any of the populations from Argentina analyzed. These results indicate the failure of this gene as a molecular marker of resistance to fipronil, at least in the R. microplus populations from northern Argentina.

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to fluazuron in Argentina.

The aim of this work is to report the presence of resistance to fluazuron in a population of Rhipicephalus microplus in Argentina. The evidence was obtained from field and in vitro trials. In the field trial, cattle infested with ticks was treated with two commercial formulations of fluazuron. The in vitro trial (adult immersion test, AIT) was performed by using technical grade fluazuron. In the field trial, there were no significant differences between the treated and control groups between days 2 and 34 post-treatment. The only exceptions (treated group I in day 14 post-treatment, treated group II in days 23 and 29 post-treatment) had a significantly lower tick load than the untreated group, but the efficacy was not higher than 70%. Viable engorged females were collected on both groups of treated bovines in all counts, and the production of viable larvae was not precluded with the application of the two commercial formulations of fluazuron evaluated in this study. The results obtained with the in vitro assay (AIT) also indicate that the R. microplus population tested in this work has a higher level of resistance to fluazuron than another susceptible field strain. The integrated analysis of the field and in vitro trials clearly reveals the emergence of resistance to fluazuron in a R. microplus population from Argentina. This diagnosis of resistance does not imply that the fluazuron has lost its functionality at a regional scale, but it highlights the need to establish control strategies that minimize the use of this drug in order to preserve its functionality as an acaricide.

Successive treatments with ivermectin (3.15%) to control the tick Rhipicephalus (Boophilus) microplus in cattle: Pharmacokinetic and efficacy assessment.

This study aimed to evaluate the pharmacokinetics, the potential accumulation in the body of treated animals and the efficacy of ivermectin long-acting formulation (3.15%) against the cattle tick Rhipicephalus (Boophilus) microplus in a scheme of three successive treatments. Fifteen 12-month-old heifers, naturally infested with R. microplus, were divided into two groups (G). Cattle from GI (n = 10) were subjected to three treatments with ivermectin 3.15% (IVOMEC GOLD®, Merial Argentina S.A.) at a rate of 1 mL/50 kg on days 0, 35, and 70. Cattle from GII (n = 5) were not treated. From day 1 to 202 post-treatment blood samples were taken to measure ivermectin concentrations by HPLC and female ticks (4.5-8 mm) were counted to evaluate the efficacy of the treatment. The level of tick resistance to ivermectin was evaluated before and after finishing the scheme of successive treatments by larval immersion test (LIT) bioassay from engorged females collected from GI. The area under the concentration vs. time curves (AUC0-35d) obtained post-second treatment was 1.51 ± 0.39-fold higher than those observed post-first treatment (P<0.05). The mean plasma concentrations of ivermectin 3.15% at 20 days after the first, second and third treatment were 17.0, 27.5 and 37.8 ng/mL, respectively (P<0.01). The elimination half-life of ivermectin post-third treatment was significantly longer than that was previously reported after a single dose (P<0.01). Values of therapeutic efficacy percentage reached 75.6% post-first treatment and between 95.9 and 100% after the second treatment. Ticks evaluated by LIT showed a significant increase in lethal concentrations after treatments. Although the efficacy level was high, the successive treatments with long-acting ivermectin formulation generate a significant accumulation of drug in plasma and could increase the levels of resistance to this drug in the tick population.

Sensitivity of Anaplasma marginale genotypes to oxytetracycline assessed by analyzing the msp1α gene in experimentally infected cattle.

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/αßßßßГ 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.

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.

Efficacy of long-acting oxytetracycline and imidocarb dipropionate for the chemosterilization of Anaplasma marginale in experimentally infected carrier cattle in Argentina.

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.

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.

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to ivermectin in Argentina.

Resistance to ivermectin in populations of the cattle tick Rhipicephalus microplus in Argentina was diagnosed in this work. The in vitro larval immersion test (LIT) was used to determine quantitatively the levels of resistance to ivermectin in different populations of R. microplus. Additionally, field trials to control natural infestations of R. microplus on cattle with a commercial formulation of ivermectin 3.15% were carried and jointly analyzed with the in vitro assays. The phenotypic response of the populations analyzed was not uniform. Five of them were classified as susceptible, four populations as resistant, and one in the category "incipient resistance". Regarding the field trials, the therapeutic efficacy in a population classified with LIT as susceptible achieved values higher than 94% two weeks after treatment, and no reproductively viable females were observed after the second day post-treatment. Conversely, the values of efficacy percentage in a population (named as "San Martín") classified with LIT in the category "incipient resistance" never exceeded the 70.8%, and engorged females were collected in practically all counts. The population "San Martín" was classified in the category "incipient resistant" with LIT analysis, but the field trial unambiguously shows that this tick population is resistant. The comparison of the results obtained with LIT in vitro assays and through field trials shows that biased estimations of resistance levels may occur when resistance ratios (RR) values are ≤2, and additional field efficacy trials could be needed to know with precision the status of the tick populations evaluated.

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.

A vaccine using Anaplasma marginale subdominant type IV secretion system recombinant proteins was not protective against a virulent challenge.

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.

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.

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.