Biological parameters for Rhipicephalus microplus in the field and laboratory and estimation of its annual number of generations in a tropical region.

The present study aimed to evaluate biological parameters of Rhipicephalus microplus in the non-parasitic phase in both field and laboratory conditions. It also aimed to assess correlations between duration (in days) of these parameters and climatic variables (humidity, rainfall, and soil temperature) and to estimate the annual number of generations of R. microplus in a tropical region. The non-parasitic phase of R. microplus in field and laboratory conditions was evaluated throughout the course of two years. A pasture was infested with engorged female of R. microplus, and biological parameters, including female pre-oviposition, female oviposition, egg mass incubation, larval pre-hatching phase, larval maturation, and larval longevity, were evaluated concomitantly with the collection of data on climatic conditions. The same parameters were also evaluated in a climatized chamber in the laboratory. The total duration of the non-parasitic phase in the field was longer in the dry season (1st and 4th life-cycle repetitions) than in the rainy season (2nd, 3rd, and 5th repetitions). Tick biological parameters for the non-parasitic phase in the laboratory were similar to those obtained in the field during the rainy season. The evaluated biological parameters were influenced mainly by environmental and ground-level temperatures, which modified egg mass incubation, larval pre-hatching, and larval longevity periods and, consequently, the total duration of the non-parasitic phase of the tick. The annual number of generations for the tick was estimated at five per year, which is alarming because it represents an increase, and so new studies into strategic control are needed.

Avermectin toxicity in bovines less than thirty days old.

The present study was designed to evaluate possible adverse effects of different dosages of avermectins (abamectin and a combination of ivermectin + abamectin) administered subcutaneously in calves less than one month of age. Clinical as well as biochemical parameters of blood and cerebrospinal fluid (CSF) were evaluated for animals subjected to various treatments. Thirty-five calves were divided into seven groups of five animals each: T01 (control), T02 (abamectin 200 µg/kg), T03 (abamectin 400 µg/kg), T04 (abamectin 600 µg/kg), T05 (ivermectin 450 µg/kg + abamectin 250 µg/kg), T06 (ivermectin 900 µg/kg + abamectin 500 µg/kg) and T07 (ivermectin 1350 µg/kg + abamectin 750 µg/kg). Cerebrospinal fluid and blood samples were collected on day zero (treatment day) and on days 1, 7 and 14 following treatment. The biochemical parameters measured in blood samples were total protein and the AST, GGT and alkaline phosphatase enzymes. In CSF samples, color, aspects, pH, density, cellularity, total proteins, glucose and the CK, ALT and LDH enzymes were evaluated. No neurological toxicity or biochemical changes in the CSF or blood that could be correlated to administration of tested products were observed in this study. Though it is well known that abamectin may lead to intoxication in bovines less than four months of age, based on data from the present study, no adverse clinical effects to the CSF or blood were observed in calves <30 days old that had received up to 600 µg/kg abamectin formulations and up to 1350 µg/kg ivermectin +750 µg/kg abamectin associations. It is possible that the level and activity of the multiple drug resistance gene (MDR1 - ABCB1) present in the calves may have influenced the results obtained in this study. Future studies should be carried out.

Experimental infection of calves with Haemonchus placei or Haemonchus contortus: Assessment of clinical, hematological and biochemical parameters and histopathological characteristics of abomasums.

The present study had the primary objective of evaluating clinical, hematological and biochemical parameters, as well as observing anatomical and histopathological characteristics of abomasums, from calves prime-infected with Haemonchus contortus or H. placei. Ten male Holstein newborns were subdivided in three groups (GI placebo; GII infected with H. contortus; GIII inoculated with H. placei). Eye mucosa staining was evaluated. Hematological and biochemical tests were performed on animals. The euthanasia of all ten experimental calves was performed on the 42nd day post-inoculation. Fragments were collected from each of all 10 abomasums for histopathological analysis. Discrete submandibular edema was diagnosed in animals from both infected groups (H. contortus or H. placei). However, there were no significant changes (P > 0.05) in the color of the ocular mucosa of calves from all three experimental groups across the entire experimental period. Hematological and biochemical changes diagnosed on animals could not be linked to infections by species of Haemonchus spp. Regarding histopathological exams, it was possible to diagnose hypertrophy, hyperplasia, binucleated cells, inflammatory infiltrate, multifocal hemorrhage and edema in abomasums from calves of both groups infected with H. placei and H. contortus. It can, thus, be concluded that not only are calves susceptible to infections by both Haemonchus species, but they can also present clinical changes and similar anatomic histopathological lesions independent of being infected by Haemonchus placei or Haemonchus contortus. These results reflect a negative effect on helminth control by mixed grazing between sheep and cattle, especially when using calves.

Ten years later: Evaluation of the effectiveness of 12.5% amitraz against a field population of Rhipicephalus (Boophilus) microplus using field studies, artificial infestation (Stall tests) and adult immersion tests.

Using field trials, artificial infestations (Stall tests) and in vitro adult immersion tests, the present study evaluated the acaricidal efficacy of 12.5% amitraz administered via whole body spraying against a Rhipicephalus (Boophilus) microplus population that did not have any contact with chemical products belonging to this acaricide family for 10 years (approximately 40 generations). Two natural infestation trials, two artificial infestation trials (Stall tests) and two adult immersion tests were performed in two different stages in 2005 and 2015. Between 2002 and 2015, the bovine herd of this property was formed by approximately 450 animals from the Simmental breed that were divided into nine paddocks formed by Cynodon dactylon (L.) Pers. For the natural infestation experiments in 2005 and 2015, we selected nearly 70 animals naturally infested with ticks from the same herd that belonged to the "São Paulo" farm located in São José do Rio Pardo, São Paulo, Brazil. Field studies were performed in the same paddock (9). To evaluate anti-R. (B.) microplus activity in the artificially infested cattle (Stall tests) and adult immersion tests, two experiments of each methodology were performed at CPPAR (the Center of Research in Animal Health located on the FCAV/UNESP campus in Jaboticabal, São Paulo, Brazil) in 2005 and 2015. R. (B.) microplus used in the artificial infestation, and adult immersion test experiments were obtained from paddocks 1-9 in 2005 and 2015 from the commercial farm where the field studies were performed. Based on the obtained results, it was possible to conclude that amitraz use in rotation with pyrethroids every 28 days for three consecutive years (2002-2004) previous to the beginning of the first trial (2005) was sufficient to generate a R. (B.) microplus strain resistant to amitraz. Moreover, using field trials, artificial infestations (Stall tests) and adult immersion tests, we verified that 40 generations of the tick species with no contact to the aforementioned compound (amitraz) were not sufficient to revert or modify the efficacy/resistance of amitraz for this analyzed R. (B.) microplus strain. The reversion of amitraz efficacy values in R. (B.) microplus may only occur when resistance of the field strain is incipient. Alternatively, the differences in the results may be due to differences in the Rhipicephalus spp. species between current study locations. Therefore, future studies must be performed to prove this hypothesis.

Susceptibility of helminth species from horses against different chemical compounds in Brazil.

By means of parasitological necropsies, the present study aimed to evaluate, in six experiments, the degree of susceptibility or resistance of different helminth species which naturally infect horses to ivermectin 0.2 mg/kg, abamectin 0.2 mg/kg, moxidectin 0.4 mg/kg, trichlorfon 35 mg/kg, ivermectin 0.2 mg/kg+praziquantel 2.5 mg/kg, abamectin 0.2 mg/kg+praziquantel 2.5 mg/kg and ivermectin 0.2 mg/kg+6.6 mg/kg pyrantel. At experimental day zero, the horses were allocated to treatment groups based on average counts of strongylid eggs per gram of feces (EPG) obtained on days -3, -2 and -1. Oxyuris sp. infections were confirmed as positive or negative. All the animals in the six experiments were naturally infected by this helminth species. Each group (control or treated) consisted of six animals. All the assessed Habronema muscae populations analyzed were susceptible to ivermectin, abamectin and moxidectin. Of the six Trichostrongylus axei populations, four were susceptible to ivermectin, abamectin, moxidectin, trichlorfon and ivermectin+praziquantel, and two were resistant to abamectin+praziquantel and ivermectin+pyrantel. Both Strongyloides westeri populations analyzed were susceptible to ivermectin, abamectin, moxidectin and abamectin+praziquantel. For O. equi, resistance was found in four different populations treated with ivermectin, abamectin, moxidectin, trichlorfon and ivermectin+praziquantel. Only combinations of abamectin+praziquantel and ivermectin+pyrantel were effective against this parasite species. All the large strongyles diagnosed in the present study (Strongyus edentatus, Strongyus vulgaris and Triodontophorus serratus) were susceptible to all the chemicals tested, with the exception of trichlorfon. Of the Cyathostominae populations, one was diagnosed as resistant to ivermectin and another to trichlorfon. The remaining populations from this nematode group were considered to be sensitive to ivermectin, abamectin, moxidectin, ivermectin+praziquantel, abamectin+praziquantel and ivermectin+pyrantel. New studies should be performed in different regions to evaluate the efficacy of trichlorfon in others field populations of helminthes.

Susceptibility of Rhipicephalus (Boophilus) microplus to ivermectin (200, 500 and 630 µg/kg) in field studies in Brazil.

The present study aimed to determine the susceptibility of 17 Rhipicephalus (Boophilus) microplus populations, originating in the Southeast and Southern regions of Brazil, to different ivermectin concentrations (200, 500 and 630 µg/kg), administered through subcutaneous or topical (pour-on) routes. R. (B.) microplus populations from the states of Minas Gerais (seven populations), São Paulo (seven populations) and Paraná (three populations) were chosen for the tests. The selected cattle were allocated to treatment groups on day 0, and block formation was based on the arithmetic mean of female ticks (4.5-8.0 mm long) counted on three consecutive days (-3, -2 and -1). To evaluate the therapeutic and residual efficacies of these formulations, tick counts (females ranging from 4.5 to 8.0 mm long) were performed on days 3, 7 and 14 post-treatment, and continued on a weekly basis thereafter until the end of each experiment. The results obtained throughout this study, utilizing field efficacy studies, allowed us to conclude that the resistance of R. (B.) microplus against 200 and 500 µg/kg ivermectin is widely disseminated because all tick populations that had contact with these specific concentrations were diagnosed as resistant. However, it is possible to infer that R. (B.) microplus resistance against 630 µg/kg ivermectin was also widespread, diagnosed at six of ten analyzed properties. Resistance of these ectoparasites to 630 µg/kg ivermectin is most likely emerging in three other populations of R. (B.) microplus. Strategies of resistance management need to be quickly determined to keep the selection pressure at a minimum level in Brazil.

Nematode resistance to ivermectin (630 and 700µg/kg) in cattle from the Southeast and South of Brazil.

Two high doses of ivermectin (630µg/kg and 700µg/kg) that are sold commercially in Brazil were evaluated in dose-and-slaughter trials with 144 naturally nematode-infected cattle from eight regions within the states of Minas Gerias, São Paulo and Rio Grande do Sul in Brazil. Treatment groups were based on fecal egg counts 1, 2, and 3days before treatment; all animals studied had a minimum egg count of at least 500 eggs per gram of feces (EPG). Post-mortem analyses were conducted on day 14. The highest levels of resistance to ivermectin were found for Haemonchus placei, Cooperia punctata and Oesophagostomum radiatum; all populations of H. placei were resistant to the 630µg/kg dose, and 67% were resistant to 700µg/kg; 86% of C. punctata were resistant to the 630µg/kg dose, and 33% were resistant to 700µg/kg. A combined analysis revealed that 57% of O. radiatum were resistant to the lower dose of ivermectin. H. placei, C. punctata and O. radiatum, in order, were the nematode populations with the highest indices of resistance, whereas Trichostrongylus axei was the most susceptible to 630 and 700µg/kg dosages of ivermectin. The results of helminthic resistance to ivermectin for different populations of H. placei and C. punctata described in the present study support previous literature data, in which a small decrease in the average parasitic burden of C. punctata and a consequent increase of H. placei were observed in cattle from the Southeast, South and Center-West regions of Brazil.

Effects of fluazuron (2.5 mg/kg) and a combination of fluazuron (3.0 mg/kg) + abamectin (0.5 mg/kg) on the reproductive parameters of a field population of Rhipicephalus (Boophilus) microplus on experimentally infested cattle.

The current study assessed the deleterious effects of two formulations of fluazuron (2.5 mg/kg fluazuron and a combination of 3.0 mg/kg fluazuron + 0.5 mg/kg abamectin) on the reproductive parameters of engorged Rhipicephalus (Boophilus) microplus females that naturally detached from experimentally infested cattle in two experiments. Based on the obtained results, it can be concluded that fluazuron (2.5 mg/kg) showed deleterious effects only on the hatchability percentage in the period of 2-7 post-treatment days of this ectoparasite's engorged females. On the other hand, the fluazuron (3.0 mg/kg) + abamectin (0.5 mg/kg) combination presented harmful effects over reproductive parameters of this tick species. Further studies should be performed with a known tick population (reference susceptible strain) to determine the effect of these formulations on the reproductive parameters and to elucidate if this field population is fluazuron resistant.

Efficacy of fipronil (1.0 mg/kg) against Rhipicephalus (Boophilus) microplus strains resistant to ivermectin (0.63 mg/kg).

The present study aimed to evaluate the efficacy of fipronil (1 mg/kg) against three strains of ivermectin-resistant Rhipicephalus (Boophilus) microplus (R. (B.) microplus), naturally infesting cattle from different states of Brazil. Three rural properties with a history of macrocyclic lactones ineffectiveness against the cattle tick, and low frequency use of fipronil in the herd, were selected for the study. The animals were randomized according to the mean tick counts, performed on days -3, -2 and -1, into three groups with 10 animals each: T01, control (saline solution); T02, subcutaneous ivermectin (0.63 mg/kg) and T03, topical fipronil (1 mg/kg). Treatment was performed on day 0. Counts of partially engorged female ticks were performed on days 3, 7 and 14 post-treatment (DPT), and then every 7 days until the 49th DPT. In all three experiments, it was possible to diagnose resistance of R. (B.) microplus to ivermectin (0.63 mg/kg). The maximum efficacy (arithmetic mean) obtained for ivermectin was 64% in experiment II. On the other hand, the formulation containing fipronil (1 mg/kg) reached high efficacy values (≥97%) in all three experiments. The results from all experiments in this study demonstrate the high efficacy of 1mg/kg fipronil, administered pour-on in naturally infested cattle, against strains of R. (B.) microplus that are resistant to 630 mcg/kg ivermectin.

Evaluation of clinical safety and anthelmintic efficacy of aurixazole administed orally at 24 mg/kg in cattle.

The current study evaluated, in vivo, the clinical safety and the anthelmintic efficacy of 24% aurixazole (24 mg/kg), administered orally, in bovines. Two experiments were conducted: the first one evaluating the clinical safety of 24% aurixazole (24 mg/kg) in cattle, and a second one evaluating the anthelmintic efficacy of aurixazole (24 mg/kg) against gastrointestinal nematodes on naturally infected cattle. Based on the results of clinical safety, no alterations on clinical and haematological signs and on the biochemical values obtained in animals treated orally with aurixazole 24 mg/kg were observed. Regarding the results of reduction or efficacy, obtained by eggs per gram of faeces (EPG) counts, the formulation of aurixazole reached values superior to 99% (arithmetic means) in all post-treatment dates. In two occasions, this formulation reached maximum efficacy (100%). Comparing these results with the reduction percentages obtained by EPG counts, it is possible to verify that the values obtained by all three formulations were compatible with the efficacy results. Aurixazole reached maximum efficacy (100%) against Haemonchus placei, Cooperia spatulata and Oesophagostomum radiatum. Against Cooperia punctata, this formulation reached an efficacy index of 99.99%. Regarding aurixazole, no specific trials were conducted on the field in order to evaluate the behaviour of this molecule against helminths that are resistant to other molecules, specially isolated levamisole and disophenolat. Due to this fact, future studies will be necessary to assess the effectiveness of aurixazole against strains of nematodes that are resistant to levamisole and disophenolat, but the results of clinical safety and efficacy described in this study allow us to conclude that the aurixazole molecule, concomitantly with other measures and orally administered formulations, can be another important tool in the control of nematodes parasitizing bovines.

Effects of macrocyclic lactones on the reproductive parameters of engorged Rhipicephalus (Boophilus) microplus females detached from experimentally infested cattle.

The present study therefore assessed the deleterious effects of MLs (ivermectin, abamectin, doramectin and moxidectin) on the reproductive parameters of engorged Rhipicephalus (Boophilus) microplus females that naturally detached from experimentally infested cattle in two experiments. The following reproductive parameters of engorged female ticks were analyzed: female weight, egg mass weight, percentage of hatchability, percentage of reduction in oviposition, percentage of reduction in hatchability, reproductive efficiency and percent control/efficacy of formulations with respect to reproductive parameters. In the experiment I, statistical analysis of the data grouped into 5-days intervals revealed that pour-on application of abamectin (500 mcg/kg) had significantly (p ≤ 0.05) reduced engorged female weight, egg mass weight and percent hatchability on days 6-15, 6-20 and 11-20 post-treatment (p. t.) compared to the respective data for detached and pre-selected engorged females in the control group. The abamectin, demonstrated 33.41% of reduction in oviposition, 6.77% in hatchability and abamectin efficacy was of 13.99%. In the experiment II, statistically significant reductions (p ≥ 0.05) were observed in animals treated subcutaneous with ivermectin (630 mcg/kg), doramectin (700 mcg/kg) and moxidectin (1000 mcg/kg) relative to the control for days 6-40, 6-48 and 6-40 p. t., respectively. Ivermectin reduced hatchability only on days 16-20 p. t., whereas doramectin significantly reduced (p ≤ 0.05) hatchability on days 6-10 and 16-35 p. t. For moxidectin, deleterious effects on hatchability were observed on days 16-35 p. t. The percent reductions in oviposition of engorged female ticks were 46.31%, 62.17% and 61.02% with ivermectin, doramectin and moxidectin treatments, respectively. The percent efficacy of the formulations on the reproductive parameters of engorged female ticks was 21.22% for ivermectin, 36.03% for doramectin and 35.45% for moxidectin. Among the MLs assessed, doramectin and moxidectin had the highest acaricidal efficacies and the most deleterious effects on the reproductive parameters of engorged R. (B.) microplus females. However, future studies will be necessary to assess the extent to which these effects, along with acaricidal activity, can be used to control the ectoparasite in cattle.

In vitro and in vivo evaluation of the activity of pineapple (Ananas comosus) on Haemonchus contortus in Santa Inês sheep.

The development of resistance to anthelmintics has prompted research into alternative methods of controlling intestinal nematodes in ruminants. This study aimed to assess the activity of Ananas comosus on Haemonchus contortus in Santa Inês sheep. The aqueous extract of pineapple skin (AEPS), bromelain from pineapple stems (B4882) and residue from pineapple processing was evaluated in in vitro and in vivo tests. The enzymatic activity of substances was analyzed by the azocasein method. The egg hatch test (EHT) and larval development test (LDT) were performed using the Embrapa2010 isolate of H. contortus. In the in vivo test, 36 sheep artificially infected with H. contortus were divided into six groups: G1: 2 g/kg BW of the aqueous extract administered for three days; G2: 2 g/kg BW of the industrial pineapple residue for 60 days; G3: 180 mg/animal of bromelain in a single dose; G4: negative control I; G5: positive control (levamisole phosphate); and G6: negative control II. The eggs per gram (EPG) in the feces were counted till 28 days after treatment. LC50 and LC90 were obtained by the probit procedure, while the in vivo test results were analyzed by GLM. The aqueous extract in the in vitro and in vivo test, the bromelain and industrial residue presented 0.102, 0.157, 1.864 and 0.048 enzyme units/mL, respectively. In the egg hatch test, the LC50 and LC90 were respectively 31 and 81 mg/mL for the aqueous extract and 0.50 and 2 mg/mL for bromelain. In the larval development test, the LC50 and LC90 were respectively 1.7 and 7.3 mg/mL for the aqueous extract and 0.019 and 0.086 mg/mL for bromelain. In the in vivo test, the general efficacies of the treatments in relation to the negative control were 22.6%, 42.2%, 3.65% and 89% for the aqueous extract, industrial pineapple residue, bromelain and positive control respectively. The transformed EPG values were 3.19 ± 0.59, 3.32 ± 0.25, 2.85 ± 0.66, 3.44 ± 0.50, 2.28 ± 0.93 and 2.75 ± 0.94 for the aqueous extract, industrial residue, bromelain, negative control I, positive control and negative control II respectively. The results for all the treated groups differed significantly (p<0.05) from the positive control, and although the residue presented efficacy of 42.2%, there was no statistical difference (p>0.05) in relation to the negative control. Therefore, both the aqueous extract and bromelain were effective in vitro, but showed reduced anthelmintic efficacy in vivo. For the pineapple residue, the 42.2% in vivo efficacy in reducing the EPG and the possibility of reducing environmental contamination through reuse of industrial residue indicate it can also be useful for control of this parasite.

Molecular and serological detection of tick-borne pathogens in dogs from an area endemic for Leishmania infantum in Mato Grosso do Sul, Brazil.

Tick-borne pathogens affect a wide range of vertebrate hosts. To identify tick-borne pathogens among dogs from Campo Grande, MS, Brazil testing seropositive for Leishmania infantum (syn. L. chagasi), a serological and molecular study was conducted to detect Ehrlichia canis, Anaplasma platys and Babesia vogeli in 60 serum and spleen samples. A confirmatory diagnosis of L. infantum based on serological and molecular assays was also performed, as was sequence alignment and phylogenetic analysis to assess the identity of the parasite species infecting these animals. IgG antibodies to Ehrlichia spp., B. vogeli and L. infantum were found, respectively, in 39 (65%), 49 (81.6%) and 60 (100%) of the sampled dogs. Twenty-seven (45%), fifty-four (90%), fifty-three (88.3%), two (3.3%) and one (1.6%) dog were positive, respectively, for E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. and Anaplasma sp. in PCR assays. After sequencing, the amplicons showed 99% of identity with E. canis, B. vogeli, A. platys and Leishmania chagasi isolates. The findings of this study indicate that L. infantum-seropositive dogs from Campo Grande are exposed to multiple tick-borne pathogens, which should therefore be included in the differential diagnosis of dogs with clinical suspicion of leishmaniasis.

Detection of Toxoplasma gondii in the reproductive system of male goats.

Male goats of mating age serologically negative for Toxoplasma gondii were divided into three groups: GI--controls (placebo) (n = 2); GII--infected with 1 x 106 tachyzoites (RH strains) (n = 2); and GIII--infected with 2 x 105 oocysts (P strains) (n = 2). Clinical, hematology, parasite and serology tests and studies of parasites in the semen through bioassay and polymerase chain reaction (PCR), and in reproductive organs (bioassay) were performed to assess toxoplasma infection. Serological titers peaked at 4096 in two animal groups infected with the protozoan. The bioassays allowed an early detection of protozoa in semen samples of tachyzoite-inoculated animals. T. gondii DNA was identified through PCR in the semen in five (Days 5, 7, 28, 49, and 70) and two (both at day 56) different days post-inoculation in GII and GIII animals, respectively. It was also possible to detect T. gondii DNA in reproductive organs (prostate pool, testicles, seminal vesicle and epididymis) of goats inoculated with either tachyzoites or oocysts. The present study suggests the possibility of venereal transmission of T. gondii among goats and it should be further assessed.