M. C. S. Oliveira

Evaluation of the Efficacy of Acaricides Used to Control the Cattle Tick, Rhipicephalus microplus, in Dairy Herds Raised in the Brazilian Southwestern Amazon.

The adult immersion test (AIT) was used to evaluate the efficacy of acaricide molecules used for control of Rhipicephalus microplus on 106 populations collected in five municipalities in the state of Rondônia in the Brazilian South Occidental Amazon region. The analysis of the data showed that the acaricide formulations had different efficacies on the tick populations surveyed. The synthetic pyrethroids (SPs) acaricides were the least effective (48.35–76.84%), followed by SP + organophosphate (OP) associations (68.91–81.47%) and amidine (51.35–100%). For the macrocyclic lactones (MLs), the milbemycin (94.84–100%) was the most effective, followed by spinosad (93.21–100%) and the avermectins (81.34–100%). The phenylpyrazole (PZ) group had similar efficacy (99.90%) to the MLs. Therefore, SP acaricides, including associations with OP, and formulations based on amidine presented low in vitro efficacy to control the R. microplus populations surveyed.

Haemonchus contortus: A multiple-resistant Brazilian isolate and the costs for its characterization and maintenance for research use

The aim of this work was to determine the resistance level of Haemonchus contortus isolated from the Santa Inês flock of the Embrapa (Brazilian governments Agricultural Research Company), Southeast Livestock Unit (CPPSE), as well as to determine costs of characterizing and maintaining this isolate in host donors. Forty-two male Santa Inês lambs were experimentally infected with 4000 H. contortus infective larvae of the field isolate of CPPSE, called Embrapa2010, and divided into six treatment groups, which received triclorfon, albendazol plus cobalt sulfate, ivermectin, moxidectin, closantel and levamisole phosphate, as well as a negative control group (water). Egg per gram (EPG) counts were performed at 0, 3, 7, 10 and 14 days post treatment when the animals were slaughtered for parasite count. The data were analyzed using the RESO statistical program, considering anthelmintic resistance under 95% of efficacy. EPG and worm count presented a linear and significant relation with 94% determination coefficient. The susceptibility results obtained by RESO through both criteria (EPG and worm count) were equal, except for closantel, showing that the isolate Embrapa2010 is resistant to benzimidazoles, macrocyclic lactones and imidazothiazoles. The need of a control group did not appear to be essential since the result for susceptibility in the analyses with or without this group was the same. Suppression in egg production after treatment did not occur in the ivermectin and moxidectin groups. In the control group, the establishment percentage was just 12.5 because of the low number of third-stage larvae, resistance (innate and infection immunity) of the animals studied plus good nutrition. Drug classes presented similar efficacy between adults and immature stages. The costs for isolate characterization were calculated for 42 animals during 60 days. The total cost based on local market rates was approximately US

Detection of Babesia bigemina in cattle of different genetic groups and in Rhipicephalus (Boophilus) microplus tick.

8000. The precise identification of Brazilian isolates and their establishment in host donors would be useful for laboratorial anthelmintic resistance diagnoses through in vitro tests, which has an annual cost of approximately US

Gastrointestinal nematode infection in beef cattle of different genetic groups in Brazil.

2500 for maintenance in host donors.

In vitro acaricidal activity of neem (Azadirachta indica) seed extracts with known azadirachtin concentrations against Rhipicephalus microplus

Babesia bigemina infections were investigated in four genetic groups of beef cattle and in Rhipicephalus (Boophilus) microplus engorged female ticks. Blood samples and engorged female ticks were collected from 15 cows and 15 calves from each of the following genetic groups: Nelore, Angus x Nelore, Canchim x Nelore, and Simmental x Nelore. Microscopic examination of blood smears and tick hemolymph revealed that merozoites of B. bigemina (6/60) as well as kinetes of Babesia spp. (9/549) were only detected in samples (blood and ticks, respectively) originated from calves. PCR-based methods using primers for specific detection of B. bigemina revealed 100% infection in both calves and cows, regardless the genetic group. Tick infection was detected by nested-PCR amplifications showing that the frequency of B. bigemina was higher (P<0.01) in female ticks collected from calves (134/549) than in those collected from cows (52/553). The frequency of B. bigemina was similar in ticks collected from animals, either cows or calves, of the four genetic groups (P>0.05).

Resistance of beef cattle of two genetic groups to ectoparasites and gastrointestinal nematodes in the state of São Paulo, Brazil

Resistance to natural infection by gastrointestinal nematodes was compared in 67 female calves of the following genetic groups: Nelore (NX); 1/2 Senepol+1/2 Nelore (SN); and 1/2 Aberdeen Angus+1/2 Nelore (AN). The NX (n=26), SN (n=23) and AN (n=18) animals were monitored for 14 months, during which they remained without treatment, allowed to graze in a tropical environment. Eggs per gram of feces (EPG), coprocultures and packed cell volume (PCV) were carried out monthly. No significant effects of the interaction between the genetic groups and month/year of collection and the genetic group on the EPG were found, but there was a significant influence of the month of collection (P<0.01). The monthly PCV measurements did not differ for the animals of the three genetic groups and there was no association found between the EPG and PCV. The animals of the SN and NX groups showed similar numbers of EPG with results zero, while for the AN group these numbers were significantly lower (P<0.05). Although the NX group had a large number of EPG with results zero, it also contained many animals with high counts, meaning this group had higher averages during the entire study period. The following nematode genera were found in the coprocultures: Haemonchus, Cooperia, Oesophagostomum and Trichostrongylus, the latter in smallest proportion. There was no significant difference between the genetic groups for averages of all parasites identified, except Cooperia, which were present in higher numbers in the animals of the NX group (P<0.05). The results obtained in this experiment suggest that the use of Bos taurus x Bos indicus crossbreeds can be a good strategy to reduce the use of chemical control in Brazil.

mRNA profile of Nellore calves after primary infection with Haemonchus placei.

The effect of four extracts from neem seeds (Azadirachta indica) containing 2000, 5000, 9000 and 10,000 ppm of azadirachtin A (AZA), quantified by high-performance liquid chromatography (HPLC) and diluted to 1.25%; 2.5%; 5.0%; 10.0% and 12.8% was verified by in vitro tests with engorged females and larvae of the cattle tick Rhipicephalus microplus. The results from the bioassays with the engorged females showed that the main toxic effect of the extracts was reduction of the reproductive parameters, with a sharp drop in the number of eggs laid and the hatching rate, mainly when the extracts were diluted to 10.0% and 12.8%. The product effectiveness (PE) calculations for all the solutions tested showed that the AZA solution at 10,000 ppm (N10) was the most effective. However, statistical analysis of the PE data obtained for the proportional AZA concentrations in the different diluted extracts showed significance (P<0.05) of the effects included in the model (extract dilution, principle effect (classificatory) of the assay (extract) and the interaction between the two), indicating significant variations due to the dilution, the test and the interaction between the two factors in the tests with engorged females. For solutions N2, N5, and N9, it was not possible to estimate LC(90) values in the dilution range tested. The lowest LC(50) was observed for extract N5, and although extract N10 was the only extract for which the LC(90) could be estimated within the range tested, the LC(50) was higher than for N5 and N9. These results suggest that substances other than AZA present in the extracts influenced the efficacy, especially up to a certain LC range. In the tests with larvae, no mortality was observed, indicating zero effectiveness of all the extracts tested. The results of the tests with engorged females showed that the neem extracts had acaricide activity, inhibiting egg laying and the larval hatching rate. Complementary studies are necessary to develop new methods to isolate and/or identify other substances besides AZA contained in this plant, to enable using products made from it as acaricides.

Quantitative study of Babesia bovis infection in beef cattle from Sao Paulo state, Brazil

The resistance to infestations by ectoparasites and infections by gastrointestinal nematodes was studied in 45 animals (males and females) of two genetic groups: purebred Nelore (NI, n=28) and Three-Cross (1/2 Angus+1/4 Canchim+1/4 Nelore - TC, n=17). The animals were monitored for 24 months, during which they were left to graze in tropical pastures without receiving treatment for parasites. Each month the animals were examined for infestations by external parasites, to count the numbers of cattle ticks Rhipicephalus microplus with diameter greater than 4.5mm present on the left side, horn flies (Haematobia irritans) present in the lumbar region and botfly larvae (Dermatobia hominis) present on the entire body. The H. irritans counts were performed with the aid of digital photographs. At the time of examination, fecal samples were collected to count the eggs per gram (EPG) and to perform coprocultures, and peripheral blood samples were drawn to determine the packed cell volume (PCV) and to count the eosinophils. For statistical analysis, the count data were transformed into log₁₀ (n+1), where n is the number of parasites. For PCV, significant effects (P<0.05) were found for collection month (CO), genetic group (GG) and gender (SX), with means and respective standard errors of 41.5 ± 0.65% for the NI animals, 39.3 ± 0.83% for the TC, 41.5 ± 0.72% for the females and 39.3 ± 0.77% for the males. Regarding the eosinophil counts, only the effect of sex was significant (P<0.01), with means and respective standard errors of 926.0 ± 46.2/μL, for males and 1088.0 ± 43.8/μL of blood, for females. The NI animals presented lower mean counts for all the external parasites compared to the TC animals (P<0.01). For ticks, the transformed means followed by standard errors for the NI and TC animals were 0.06 ± 0.01 and 0.34 ± 0.02, while for horn flies these were 0.92 ± 0.05 and 1.36 ± 0.06 and for botfly larvae they were 0.05 ± 0.03 and 0.45 ± 0.05, respectively. The average EPG values were only influenced by CO (P<0.01). The coprocultures revealed the presence of the following endoparasites: Haemonchus spp., Cooperia spp., Oesophagostomum spp. and Trichostrongylus spp., the last in smaller proportion. There were no significant differences between the genetic groups for the endoparasite loads, except for Cooperia spp., which were present in greater number (P<0.05) in the NI group. The results obtained in this experiment confirm previous findings of greater susceptibility of the Nelore breed to Cooperia spp. and high resistance to ectoparasites.

Babesia bovis and Babesia bigemina infection levels estimated by qPCR in Angus cattle from an endemic area of São Paulo state, Brazil

Haemonchus parasites are responsible for many losses in animal production. However, few studies are available, especially of zebu cattle. In this study, we investigated mRNA differences of immune response genes in naïve Nellore calves infected with Haemonchus placei, relating these differences to patterns of cellular infiltrate. Calves were infected with 15,000 H. placei L3 larvae and after 7 days lymph node and abomasum tissues were collected. IL-2, IL-4, IL-8, IL-12, IL-13, IFN-γ, MCP-1, lysozyme, pepsinogen and TNF-α genes were evaluated by qPCR. Mast cells, eosinophils and globular leukocytes were counted by abomasum histology. In the infected group, IL-4, IL-13 and TNF-α were up-regulated in the abomasal lymph node. In the abomasum, IL-13 increased and TNF-α was down-regulated (p<0.05). No differences were detected for mast cells and eosinophil counts in abomasal tissue (p>0.05). We conclude that for this infection time, there was Th2 polarization but that cellular infiltrate in abomasal tissue takes longer to develop.

Pyrethroid and organophosphate pesticide resistance in field populations of horn fly in Brazil: Pesticide resistance in the horn fly

The qPCR technique with SYBR Green was used to estimate the prevalence and level of Babesia bovis infection in beef cattle raised in areas endemic for babesiosis in Brazil, where the animals were continuously exposed to ticks (Rhipicephalus microplus). This is the first report in which qPCR was used to quantify and compare B. bovis DNA in blood of different cattle breeds. Blood samples were collected from 150 animals (75 cows and 75 calves) of the Angus and Nelore breeds and the first generation of an Angus and Nelore cross (AxN). Blood samples from the jugular vein were used for DNA extraction and determination of packed cell volume (PCV), while samples from peripheral veins were used for microscopic parasite detection. Although no piroplasms of B. bovis were found in blood smears, DNA amplification using qPCR revealed that all of the 150 animals, except two calves and one cow, were positive. The number of copies of B. bovis DNA was higher (p<0.05) in the Angus than in the Nelore and AxN animals, for both calves and cows, but no significant difference was found between the Nelore and AxN groups. These results suggest that a heterotic effect was present, since the results from the crossbred animals significantly deviated from the mean of the two parental groups, while closely approaching that of the Nelore group. In the Nelore and AxN groups, calves showed higher infection levels than cows (p<0.05), while for the Angus group the difference was found to be non-significant. Within each animal age group, the breed groups with higher infection levels were those with lower PCV values. However, within each breed group, no significant correlations were found between the number of DNA copies and PCV according to animal age. The qPCR method applied here allowed the observation that although there are no differences in the prevalence of infection among breed groups, Nelore and AxN cattle are able to maintain infection by B. bovis at lower levels than the Angus cattle.