Eimeria spp. in dairy calves in Uruguay. Identification, dynamics of oocyst excretion and association with the age of calves.

Eimeria bovis, Eimeria zuernii, Eimeria ellipsoidalis, Eimeria auburnensis, Eimeria canadensis and Eimeria alabamensis were identified on different dairy farms in Uruguay. The most prevalent species were E. bovis and E. zuernii, which were mainly found in the feces of calves with diarrhea. The dynamics of oocyst excretion were evaluated via the weekly determination of oocysts per gram (OPG) values in fecal samples from 97 calves over seven months. Three groups of calves were formed according to their age in days: Group 1 (1-20 days old), Group 2 (21-40 days old) and Group 3 (41-65 days old). In Group 1, the median OPG was zero, and the maximum OPG was 1,680. In Group 2, the median OPG was between zero and 8,240, and the maximum OPG was 428,800. In Group 3, the median OPG was between zero and 220, and the maximum OPG was 16,000. For the evaluation of the relationship between OPG and age group, a proportional odds model was built. Two samples from 60 bovines evaluated in Group 2 and in Group 3 were selected. OPG was categorized as negative, moderate (lower than or equal to 4,000) or high (greater than 4,000). Calves of Group 2 (21-40 days old) were significantly (p < 0.001) more affected by eimeriosis than calves of Group 3 (41-65 days old). Considering that diarrhea in calves is a multifactorial disease, eimeriosis should be considered when evaluating the control measures for diarrhea syndrome, particularly in calves of 21-40 days of age.

Mutations in Rhipicephalus microplus GABA gated chloride channel gene associated with fipronil resistance.

The tropical cattle tick, Rhipicephalus microplus, is one of the most damaging parasites that affects cattle in tropical and subtropical regions in the world. Tick resistance to acaricides is dispersed worldwide and a number of associated mutations in target site genes have been described. Phenylpyrazole (e.g. fipronil) and cyclodiene (e.g. lindane, dieldrin) insecticides both have the same mode of action blocking the GABA-gated chloride channel encoded by the GABA-Cl gene. A conserved mutation, rdl (resistance to dieldrin) is found across a number of arthropods resistant to cyclodienes and phenylpyrazoles. In ticks, the mutation T290 L, was identified in the second transmembrane (TM2) domain of the GABA-gated chloride channel of Australian cattle tick populations that are resistant to dieldrin. Recently, cross-resistance between fipronil and lindane was reported in R. microplus populations obtained from Uruguay and Brazil. The objective of the present study was to identify mutations in the GABA-Cl gene associated with fipronil resistance. Genomic DNA was obtained from engorged females from fipronil-susceptible and resistant populations sampled from Uruguay and Brazil (n = 166). Initially, it was searched the T290 L mutation described in Australia; however, this mutation was not detected in individuals from resistant populations from either country. The sequencing of a fragment of the GABA-Cl gene revealed nucleotide polymorphisms in fipronil- and lindane-resistant ticks in two populations from Uruguay and five from Brazil. Five amino acid substitutions were present in the resistant strains. Two different substitutions were found in an alanine residue (A286S and A286 L) that is homolog to rdl mutations in fipronil-resistant individuals of other arthropod species. Four other amino acid substitutions (S281 T, V317I, T328 A and A329S) were present in some resistant strains, always with the mutation A286S. This is the first documentation of mutations in the GABA-Cl gene associated with fipronil-resistant in R. microplus.

Cross-resistance between fipronil and lindane in Rhipicephalus (Boophilus) microplus.

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), is one of the most damaging parasites of cattle in tropical and subtropical regions. Several chemical groups have been used for its control, including cyclodienes (lindane and dieldrin). In Uruguay and Brazil these products were used at the beginning of the 1960s and during a few years. Fipronil and lindane act on the same target site. In both countries, southern cattle tick resistance to fipronil has sometimes developed quickly after only a few acaricide treatments (three to seven). The objective of the present study was to determine cross-resistance between fipronil and lindane in southern cattle ticks from Uruguay and Brazil. Initially, the FAO's (Food and Agricultural Organization) larval packet test with lindane was applied to a fipronil-resistant strain and to susceptible field populations. Mozo and POA strains were used as the susceptible controls. A larval immersion test was used to assess fipronil toxicity. Of fifteen fipronil-resistant field populations that were tested with lindane, eleven were lindane-resistant and three were susceptible. The last three populations had incipient resistance to fipronil. Finally, cross-resistance between fipronil and lindane in the southern cattle tick is reported in this study for the first time.

Applicability of in vitro bioassays for the diagnosis of ivermectin resistance in Rhipicephalus microplus (Acari: Ixodidae).

The applicability of laboratory bioassays to diagnose ivermectin (IVM) resistance in Rhipicephalus microplus was evaluated. Adult immersion tests (AITs), larval immersion tests (LITs) and larval packet tests (LPTs) were performed to characterise the effects of ivermectin toxicity on adults and larvae of a susceptible reference strain. The AIT was determined to be a reasonable assay but requires a large number of individuals to attain interpretable results. The LIT and LPT were validated with an IVM resistant strain, revealing resistance ratios (RRs) of 6.73 and 1.49, respectively. In a field survey, nine different populations of cattle tick from the states of São Paulo and Mato Grosso do Sul, Brazil, were analysed with the LIT. Populations without previous exposure to ivermectin exhibited RRs between 0.87 and 1.01. Populations previously exposed to IVM showed RRs between 1.83 and 4.62. The LIT was more effective at discriminating between resistant and susceptible populations than the LPT. The use of the LIT is recommended for the diagnosis of ivermectin resistance in R. microplus.

Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay.

Rhipicephalus (Boophilus) microplus is an important cattle pest in Uruguay, and the law regulates its control. It is resistant to organophosphates, synthetic pyrethroids and, as recently discovered, to fipronil. Resistance to macrocyclic lactones (MLs) and amitraz have not been documented; however, veterinarians and farmers have reported treatment failures. The objective of the present work was to study the susceptibility of cattle tick strains from different Uruguayan counties to ivermectin (IVM) and fipronil by using the Larval Immersion Test (LIT). The Mozo strain was used as the susceptible reference strain. From 2007 to 2009, twenty-eight tick populations were collected from different cattle farms with and without history of IVM or fipronil use. A probit analysis estimated dose-mortality regressions, lethal concentrations (LC), and confidence intervals. The resistance ratio (RR) was determined at the LC(50) and LC(90) estimates. To classify a tick population in relation to resistance, three categories based on a statistical analysis of LC and RR between field populations and Mozo strains were defined: susceptible (no differences), incipient resistance (differences and RR(50)<2) and resistant (differences and RR(50)≥2). Eighteen field populations were tested with IVM and five of them presented a RR(50) range between 1.35 and 1.98 and the LC(50/90), which is statistically different from the Mozo strain (incipient resistance). However, the RR(90) increases ≥2 in four of the populations, confirming that tick resistance to IVM is emergent. The low RR values obtained could be a result of a low frequency of treatments with IVM. Twenty-seven tick populations were tested with fipronil and six were diagnosed as resistant according to the LIT. Cross-resistance was not observed between fipronil and IVM on these tick populations. The current study presents different R. (B.) microplus populations with an incipient resistance to IVM, and indicates that the fipronil tick resistance is restricted to certain areas in Uruguay.

Diagnoses of fipronil resistance in Brazilian cattle ticks (Rhipicephalus (Boophilus) microplus) using in vitro larval bioassays.

Fipronil is a phenylpyrazolic insecticide that is widely used in agriculture and has been recently used to control the cattle tick, Rhipicephalus (Boophilus) microplus. Because of the serious problems associated with resistance to the available acaricides, this product has been used as an important alternative to control acaricide-resistant ticks. The objective of this work was to analyse the fipronil sensitivity of ticks that were collected from farms with a history of fipronil use by larval bioassays. A total of 11 Brazilian tick populations were studied: one population from Rio Grande do Sul, one population from Mato Grosso do Sul and nine populations from São Paulo. To validate the assays, susceptible reference strains, POA (Porto Alegre, Brazil) and Mozo (Dilave, Uruguay), and ticks from six different farms that never used fipronil were tested. The resistance of various tick populations to technical grade fipronil (95.3%) was primarily evaluated using the larvae immersion test (LIT) and the larval packet test (LPT), when a sufficient number of larvae was collected. Using the LIT, the resistance ratios (RR(50)) of the tick populations from Rio Grande do Sul and Mato Grosso do Sul were 14.9 and 2.6, respectively, and the populations derived from São Paulo had RR(50)s ranging from 2.5 to 6.9. Four populations were evaluated with the LPT, and two populations displayed lower RR(50), while other populations displayed higher RR(50) than those determined by the LIT. This article reports the first cases of fipronil resistance in Brazil and highlights the LIT as a more sensitive technique for the evaluation of fipronil resistance in R. (B) microplus ticks. We suggest the use of the LIT as an evaluation tool for monitoring fipronil resistance in the control programmes of R. (B) microplus.

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) resistance to fipronil in Uruguay evaluated by in vitro bioassays.

Rhipicephalus (Boophilus) microplus obtained from four local populations in Uruguay (2007-2008) were subjected to various bioassay techniques to determine the presence of fipronil resistance within the country. Resistance ratios (RRs) obtained by larval immersion test varied between 3.3 and 3635 for tick populations subjected to treatment with fipronil for the last 3-7 years. The highest RR was observed in the population which received fewer treatments. Using discriminating concentration (8ppm) for larval immersion test, all field strains were correctly diagnosed as fipronil-resistant. This study presents the first diagnoses of cattle tick resistance to fipronil in Uruguay's field populations. It also highlights the importance of the possible conflict between programs to control agricultural pests and cattle ticks. The findings provide valuable information for selection and adoption of new control alternatives to manage drug resistance exhibited by cattle ticks.

In vitro tests to establish LC50 and discriminating concentrations for fipronil against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and their standardization.

Laboratory test was carried out on larvae and adults of the cattle tick, Rhipicephalus (Boophilus) microplus, to determine fipronil toxicity. Adult immersion test (AIT, N=26), larval immersion test (LIT, N=71) and larval packet test (LPT, N=41) were standardized using susceptible strain (Mozo). Dose-response curves were compared with a fipronil resistant strain. Four variables were analyzed from AIT results: mortality, weight of eggs on day 7 and on day 14, index of fertility, and index of fecundity. For larval test, dose mortality curves were analyzed. In spite of the high LC(50) variability, all variables determined for AIT were appropriate to discriminate both strains. AIT and LIT had more sensitivity than LPT, with larger resistance factors. It was used two times LC(99.9) as discriminating doses (DCs) following FAO suggestion. For mortality by AIT, LIT and LPT the DCs were estimated: 4.98ppm, 7.64ppm and 2365.8ppm, respectively, for Mozo strain. DCs mortality values estimated for resistant strain by AIT, LIT and LPT were: 6.96x10(5)ppm, 343.26ppm and 5.7x10(3)ppm, respectively and their respective resistant factors were: 202.4, 5.36 and 1.52. Protocols for AIT, LIT and LPT have been presented in this paper.