Investigating the benefits of targeted selective treatment according to average daily weight gain against gastrointestinal nematodes in Morada Nova lambs.

The objective of this study was to evaluate a method of targeted selective treatment (TST) of Morada Nova lambs, based on the average daily weight gain (DWG). For this, 114 lambs in the rainy and 102 in the dry season were randomized into three treatments: control (CT), routine (RT, treated every 42 days), and targeted selective (TST, treated according to DWG). Packed cell volume (PCV) and eggs per gram of feces (EPG) tests were performed. The anthelmintic resistance of parasites was assessed by the EPG count reduction test (FECRT), RESISTA-Test©, and molecular test. For CT, RT, and TST groups, the following results were obtained: mean EPG values were 4665.1, 3063.5, and 3462.1 in the rainy season and 4475.1, 1341.7, and 2863.4 in the dry season, respectively; mean PCV values were 32.1, 33.4, and 32.3% in the rainy season and 33.9, 36.0, and 35.1% in the dry season; mean DWG readings were 0.087, 0.101, and 0.094 kg in the rainy season and 0.102, 0.113, and 0.112 kg in the dry season; efficacies of levamisole in FECRT were 66.4, 24.1, and 76.4% in the rainy and 90.7, 12.4, and 64.8% in the dry season, respectively; in the RESISTA-Test©, the LC50 values were 0.482, 1.926, and 0.117 µg.mL-1 in the rainy and 0.437, 0.851, and 0.045 µg.mL-1 in the dry season, respectively; the frequencies of the homozygous-resistant genotype were 57.1, 71.4, and 40.0% in the rainy and 47.8, 55.9, and 41.9% in the dry season. In conclusion, TST reduced the development of resistance through refugia maintenance, without productive losses, contributing to the sustainability of sheep breeding.

Local and systemic immune mediators of Morada Nova lambs with divergent Haemonchus contortus resistance phenotypes.

AIMS: Local and systemic immune mediators of Morada Nova lambs with divergent Haemonchus contortus resistance phenotypes were evaluated. METHODS AND RESULTS: Lambs were ranked through faecal egg counts (FEC) after two parasitic challenges with 4,000 H.contortus L3 . After the second challenge, the lambs underwent a third artificial infection and were euthanized 7 days later. Immune-related genes were quantified locally in abomasal mucosa and lymph nodes (CD4, IFNγ, IL4, IL5, IL13, IL2RA and MS4A2) and systemically in the whole blood (IL4 and IL13). Anti-H. contortus IgG and IgA antibodies and eosinophils and mast cells counts were also investigated. Resistant animals presented higher systemic IgG and IgA titres, both negatively correlated with FEC. Susceptible animals had higher blood levels of IL4 transcripts. At the local level, resistant lambs had higher eosinophils counts and superior MS4A2 levels in abomasal fundic mucosa, besides higher IgA levels in abomasal mucus, while susceptible lamb had superior IL4 expression in abomasal lymph nodes. CONCLUSION: These data indicate that resistant lambs had an immune response mediated by antibody-mediated cytotoxicity. Also, the systemic humoral profile, particularly IgA isotype, seems to be a good resistance marker for Morada Nova sheep, as we found differences between groups even when FEC did not differ.

Innate Immune Responses Associated with Resistance against Haemonchus contortus in Morada Nova Sheep.

The immune response against Haemonchus contortus infections is primarily associated with the Th2 profile. However, the exact mechanisms associated with increased sheep resistance against this parasite remains poorly elucidated. The present study is aimed at evaluating mediators from the innate immune response in lambs of the Morada Nova Brazilian breed with contrasting H. contortus resistance phenotypes. Briefly, 287 lambs were characterized through fecal egg counts (FEC) and packed cell volume (PCV) after two independent experimental parasitic challenges with 4,000 H. contortus L3. 20 extreme resistance phenotypes (10 most resistant and 10 most susceptible) were selected, subjected to a third artificial infection with 4,000 L3, and euthanized 7 days later. Tissue samples were collected from abomasal fundic and pyloric mucosa and abomasal lymph nodes. Blood samples were collected at days 0 and 7 of the third parasitic challenge. RNA was extracted from tissue and blood samples for relative quantification of innate immune-related genes by RT-qPCR. For the abomasal fundic mucosa, increased TNFα and IL1ß expression levels (P < 0.05) were found in the susceptible animals, while resistant animals had IL33 superiorly expressed (P < 0.05). Higher levels (P < 0.05) of TLR2 and CFI were found in the abomasal pyloric mucosa of resistant animals. TNFα was at higher levels (P < 0.05) in the blood of susceptible lambs, at day 0 of the third artificial infection. The exacerbated proinflammatory response observed in susceptible animals, at both local and systemic levels, may be a consequence of high H. contortus parasitism. This hypothesis is corroborated by the higher blood levels of TNFα before the onset of infection, which probably remained elevated from the previous parasitic challenges. On the other hand, resistant lambs had an enhanced response mediated by TLR recognition and complement activation. Nevertheless, this is the first study to directly associate sheep parasitic resistance with IL33, an innate trigger of the Th2-polarized response.

Viability of Haemonchus placei parasitism in experimentally infected young goats.

The present study aimed to evaluate the viability of Haemonchus placei parasitism in experimentally infected goats. For that, 14 75 days old kids male Saanen kids were placed in one of the four experimental groups: GI - infected with 5000 H. placei L3 (n = 4); GII - infected with 5000 H. contortus L3 (n = 4); GIII - infected with 2500 H. contortus L3 + 2500 H. placei L3 (n = 4), and GIV - control, inoculated with distilled water (n = 2). Each kid received, orally, the infective dose in a single inoculum. Based on daily fecal egg counts, the average pre-patent period was determined as 24 days for H. contortus, and 31 days for H. placei. Regarding the Haemonchus spp. recovered at necropsy, the experimental groups GI, GII, and GIII had, respectively, an average of 25.5, 619.5, and 724.75 (120 H. placei, and 604.75 H. contortus) adult specimens, and no immature forms. Under the conditions of this study, the viability of goat infection by H. placei was confirmed, although, with low susceptibility. Nevertheless, the parasitism of this helminth species was more intense when associated with H. contortus. This fact indicates that in common grazing between cattle and young goats, when the latter end up ingesting both Haemonhcus species, especially in a mixed infection, H. placei may also parasitize them.

Evaluation of Tagetes patula (Asteraceae) as an ecological alternative in the search for natural control of the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The cattle tick Rhipicephalus microplus is a great sanitary problem and causes huge losses to livestock, being a vector of important diseases. The aim of this work was to verify the action of plant-derived material obtained from Tagetes patula on the life cycle of R. microplus, as well as to analyze the action of these extracts on ovary cells of engorged females. In the Adult Immersion Test, the crude ethanolic extract (TpEtOH) showed an efficacy of 99.2% (LC50 = 18.60 mg mL-1). From the repellency test, it was found that both the TpEtOH and essential oil were 100% efficient on the larval stage. In addition, in the Larval Package Test, a mortality of 98.37% was obtained with TpEtOH (LC50 = 3.798 mg mL-1). From the microscopic analysis of ovary cells, morphological changes in the chorion and cytoplasm of oocytes were observed, with vacuolization around the germinal vesicle and disorganization of the pedicular cells, suggesting an interference in the normal embryogenic formation of the larvae. These results demonstrate that T. patula extracts interfere at all stages of development of the ixodid, from eggs to adults forms, and have a pronounced repellent effect. In addition, the results of the cytotoxicity assays performed on keratinocytes, as well as previous information on oral and dermal acute toxicity (LD50 > 4000 mg kg-1), attest that T. patula can be a safe alternative for ectoparasitic control, representing an alternative for development of a formulation used to help control populations of ticks in the field.

Comparative study of hatching estimation methods of Rhipicephalus (Boophilus) microplus eggs.

Reproductive parameters of Rhipicephalus (Boophilus) microplus are often evaluated. They are good indicators of resistance to commercial acaricides and of plant extracts' efficacy. The objective of this study was to compare the techniques: visual estimation and quantification by sampling used in the Adult Immersion Test (AIT) to calculate the hatching rate of eggs. Engorged females collected from cattle were subjected to the AIT with plant extracts and kept in an incubator for oviposition. The egg hatching was evaluated in 210 syringes by visual estimation (%). Then, eggs and larvae were counted into samples of 100 individuals, in three repetitions by stereo microscope. Significant differences were found between the two tests (p≤ 0.05). The egg hatching average of visual estimation was higher than the quantification by sampling, 56.8-48.0, respectively (correlation = 0.85). We found that the visual assessment leads to a higher estimate of larvae in relation to eggs, because the infertile eggs can be concealed in the center of the syringe. In quantification by sampling, no statistical differences (p = 0.99) were observed in the pairwise counts between the three samples (48.1 ± 26.6%, 47.8 ± 26.9%, 48.1 ± 26.5%) (correlation of repetitions = 0.96). This suggests that counting one sample is sufficient and the result should not differ much, regardless of the evaluator. Regarding the cutoff point of tick resistance status (95%), both methods are reliable. This study contributes to improvement of the AIT and can stimulate researchers to choose more accurate techniques for the assessment of egg hatching.

Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus.

Due to the great economic impact of Haemonchus contortus on sheep farming, there is an increasing number of studies addressing host resistance against this nematode, including identification of directly related immune mechanisms. In this context, relative gene expression by RT-qPCR have been largely used, due to its rapidity, high sensitivity, specificity, and reproducibility. Although, appropriate reference gene selection is crucial for accurate interpretation of results. In this study, five reference genes (GAPDH, G6PDH, YWHAZ, ACTB, and B2M) were tested for expression stability in abomasum (fundic and pyloric regions) and abomasal lymph nodes of Morada Nova sheep classified as resistant (n = 5) or susceptible (n = 5) to H. contortus infection in a flock of 151 animals. GAPDH combined with YWHAZ were selected as reference genes for abomasal fundic region and abomasal lymph nodes, whereas YWHAZ was the most stable gene for abomasal pyloric region. These genes presented the lowest intra- and inter-group variations and, consequently, highest stability. In contrast, expression of G6PDH was the least stable in all tissues. The impact of reference gene selection was demonstrated by relative quantification of a pro-inflammatory cytokine (TNFα) in abomasal fundic region. Significant differences in TNFα expression levels between resistant and susceptible groups were only observed when the most stable genes (GAPDH combined with YWHAZ) or GAPDH were used as reference genes, whereas no significant differences were observed when other tested reference genes were used. It was demonstrated that normalization of expression data using inappropriate reference genes may significantly influence interpretation results.

Neither quantification by qPCR nor quantitative Elisa can be used to discriminate Angus cattle for resistance/susceptibility to Babesia bovis.

With the aim of finding quantitative phenotypic traits that can be used to discriminate the levels of resistance/susceptibility to Babesia bovis, we estimated the repeatability and correlation between the level of infection, determined by the number of copies of a fragment of the gene that encodes cytochrome B (NC mt-cyB) of B. bovis, and the levels of the anti-B. bovis antibodies, in blood samples collected from 51 Angus cattle on two different occasions. Samples with the anticoagulant EDTA were used for DNA extraction and without anticoagulant for separation of the blood serum. The quantification of the NC mt-cyB of B. bovis was carried out by the quantitative PCR technique (qPCR), while the anti-B. bovis IgG antibody titers (S/P) were quantified by the ELISA method. The NC and S/P data were log10-transformed to improve the approximation to the normal distribution and were analyzed using mixed models. The correlations between NC mt-cyB and S/P were estimated, as well as the repeatability values for each trait. The results obtained showed the high sensitivity of the techniques, with 100% of the animals being positive for B. bovis, detected by both the serological and molecular tests. The correlations estimated between NC and S/P were low, 0.10 and 0.12, in the first and second collection, respectively. The repeatability estimated for NC was 0.06, whereas for the S/P it was 0.42. The low correlations between S/P and NC in the two collections demonstrated that the variation in the NC value is independent of the level of antibodies. This results indicated that animals with a higher levels of antibodies against B. bovis in the first collection continued to have a higher levels in the second one. However, the very low values for the repeatability value of NC, and for the correlations between S/P and NC, demonstrates that neither NC or S/P could be used to discriminate animals for resistance/susceptibility to B. bovis.

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.

In vitro efficacy of plant extracts and synthesized substances on Rhipicephalus (Boophilus) Microplus (Acari: Ixodidae).

Herbal drugs have been widely evaluated as an alternative method of parasite control, aiming to slow development of resistance and obtain low-cost biodegradable parasiticides. This study evaluated the in vitro efficacy on Rhipicephalus (Boophilus) microplus of extracts from Carapa guianensis seed oil, Cymbopogon martinii and Cymbopogon schoenanthus leaf essential oil, and Piper tuberculatum leaf crude extract and similar synthesized substances. In the immersion test, engorged females were evaluated in five dilutions ranging from 10% to 0.030625% concentration. In the larval test on impregnated filter paper, the concentration ranged from 10% to 0.02%. The treatments and controls were done in three replicates. Chemical analysis of the oils was performed by gas chromatography. The main compounds were oleic acid (46.8%) for C. guianensis and geraniol for C. martinii (81.4%), and C. schoenanthus (62.5%). The isolated and synthesized substances showed no significant effect on larvae and adult. C. martinii and P. tuberculatum showed the best efficacy on the engorged females. The LC(50) and LC(90) were 2.93% and 6.66% and 3.76% and 25.03%, respectively. In the larval test, the LC(50) and LC(90) obtained for C. martinii, P. tuberculatum, and C. schoenanthus were 0.47% and 0.63%, 0.41% and 0.79%, 0.57% and 0.96%, respectively. The fact that geraniol is present in greater quantities in C. martinii explains its higher activity in relation to C. shoenanthus. It is necessary to validate the in vivo use of safe and effective phytoparasiticidal substances. Efforts should be focused on developing formulations that enhance the efficacy in vivo and lengthen the residual period.

Anthelmintic action of eprinomectin in lactating Anglo-Nubian goats in Brazil.

Eprinomectin is the only avermectin approved for use to control gastrointestinal nematodes in lactating cows. Some studies in the USA and Europe have also demonstrated that this drug is highly effective in small ruminants. The aim of the present work was to evaluate the anthelmintic efficacy of pour-on eprinomectin in Anglo-Nubian goats at the end of lactation. Twenty-four goats were used, naturally infected with gastrointestinal nematodes, and divided into two groups: control and treated with eprinomectin (Eprinex, Merial, pour-on 0.5%) at a dose of 850 microg/kg. Counts were made of the eggs per gram (EPG) of feces, along with coprocultures, on days -7, 0, 4, 8, 11, 15, 18, 22, 25 and 29. The milk production of each group was recorded throughout the experiment. The coprocultures detected 98% Haemonchus contortus and 2% Oesophagostomum. There was no statistically significant difference (P > 0.05) in daily milk output between the two groups. Eprinomectin at the tested dosage was not effective (P > 0.05) in reducing the EPG. Positive results would serve as basis for use of an avermectin without residues in dairy goats, providing a scientific basis for greater food safety.