Biomarkers of gastrointestinal nematodes in beef cattle raised in a tropical area.

Biomarkers are specific molecular, histological, or physiological characteristics of normal or pathogenic biological processes and are promising in the diagnosis of gastrointestinal nematodes (GINs). Although some biomarkers have been validated for infection by Ostertagia sp. in cattle raised in temperate regions, there is a lack of information for tropical regions. The aim of this project was to assess potential biomarkers and validate the most promising. In the first study, 36 bovines (Nelore breed) naturally infected by GINs were distributed into two groups: infected (not treated with anthelmintic) and treated (treated with fenbendazole on days 0, 7, 14, 21, 28, 42, and 56). The variables of interest were live weight, fecal egg count, hemogram, serum biochemical markers, phosphorus, gastrin, and pepsinogen. In the second step, pepsinogen was assessed in cattle of the Nelore breed distributed among three groups: infected (not treated with anthelmintic), MOX (treated with moxidectin), and IVM + BZD (treated with ivermectin + albendazole). In the first study, no difference between groups was found for weight, albumin, hematocrit (corpuscular volume [CV]), erythrocytes, or hemoglobin. Negative correlations were found between pepsinogen and both CV and albumin, and albumin was negatively correlated with the percentage of Haemonchus sp. in the fecal culture. Among the biomarkers, only pepsinogen differentiated treated and infected (beginning with the 28th day of the study). In the second study, a reduction in pepsinogen was found after anthelmintic treatment. Therefore, pepsinogen is a promising biomarker of worms in cattle naturally infected by the genera Haemonchus and Cooperia in tropical areas.

Efficiency of fluralaner pour-on in different strategic control protocols against Rhipicephalus microplus on Brangus cattle in a tropical area.

BACKGROUND: The occurrence of higher winter temperatures in Brazilian areas with tropical and highland climates may result in a fifth peak of tick populations during winter in addition to the four generations previously described. Therefore, a strategic control protocol was developed with treatments in two seasons with the objective of controlling the generations of ticks that occur in spring/summer and those that occur in autumn/winter. METHODS: The study was conducted in Mato Grosso do Sul, Brazil, from the beginning of the rainy season, November 2020, to October 2021. In a randomized block design, 36 calves were distributed into three groups: (i) negative control; (ii) traditional strategic control in one season (SC1S), at the beginning of the rainy season; and (iii) strategic control in two seasons (SC2S), at the beginning and end of the rainy season. The SC1S strategic control group was treated on day 0, November 2020, and twice more with intervals of 42 days. The SC2S group received three more treatments beginning on day 182, May 2021, with intervals of 42 days. All treatments consisted of 5% fluralaner (Exzolt® 5%) delivered via a pour-on dose of 1 mL/20 kg body weight. Counts of semi-engorged female ticks were performed on day 3 and every 14 days thereafter, and the animals were weighed at the same time. RESULTS: Fluralaner showed a mean efficacy of more than 95% up to day 294. The two treated groups showed a decrease (P < 0.05) in the average number of ticks on day 3. In the SC2S group, the means were close or equal to zero throughout the study, while in the SC1S group, the means did not differ (P > 0.05) from those of the control group from day 231 onward. The final mean weight gain of each group was 76.40 kg, 98.63 kg, and 115.38 kg for the control, SC1S, and SC2S groups, respectively, differing (P < 0.05) from each other. CONCLUSIONS: Therefore, three applications of fluralaner, with one application every 42 days from the beginning of the rainy season in the middle spring, resulted in effective tick control for 224 days. When three additional treatments were given in autumn/winter with intervals of 42 days between applications, tick counts were reduced throughout the year. This strategic control approach may be indicated in years with climatic conditions that allow that population peaks are expected to occur in the autumn/winter period.

Synergism of macrocyclic lactones against Haemonchus contortus.

A possible synergistic effect of macrocyclic lactones' (MLs) combination has been previously described against resistant gastrointestinal nematodes of cattle. In addition to synergism, drug-drug interactions between MLs can also result in additive or antagonistic effect, considering the different MLs pharmacokinetics, pharmacodynamics, and interactions with molecular mechanisms of resistance. Therefore, the aim of the current work was evaluated the effect of different MLs combinations against Haemonchus contortus. Infecting larvae of two isolates (one susceptible and one resistant to ivermectin) were used in the larval migration inhibition test. After estimating the half maximal effective concentration of abamectin (ABA), eprinomectin, (EPR), ivermectin (IVM), and moxidectin (MOX) for both isolates, combinations were delineated by a simplex-centroid mixture experiment, and the mixture regression analysis was applied to the special cubic model. A synergistic effect was found for the EPR + MOX against the susceptible isolate as well as the EPR + MOX, IVM + MOX, and ABA + EPR + IVM against the resistant isolate. An antagonistic effect of ABA + IVM + MOX was found against the susceptible isolate. For the susceptible isolate, a higher inhibition was found with greater proportions of EPR and lower proportions of the other drugs compared to the reference mixture. For the resistant isolate, inhibition greater than that of the reference mixture was found with higher proportions of IVM as well as lower proportions of the other drugs. The synergistic and antagonistic effects were dependent on the following: (a) parasite drug resistance profile, (b) the composition of the combination, and (c) the proportions used, with EPR and IVM exerting a greater impact on these effects.

Combination of quercetin and ivermectin: In vitro and in vivo effects against Haemonchus contortus.

The aim of the present study was to evaluate the in vitro effect of quercetin combined with ivermectin (IVM) on Haemonchus contortus larvae and adults with different resistance profiles and demonstrate the in vivo anthelmintic action of this combination when used in sheep naturally infected. The effect of combination was evaluated based on the analysis of the mean effective concentration (EC50) obtained for larvae using the larval migration inhibition test and for adults using the motility test on females. The tests with larvae and adults were conducted using isolates with different degrees of susceptibility to IVM (sensitive, intermediate and highly resistant). The in vivo effect was evaluated based on the reduction in the egg count (FEC) and reduction in the count of adult helminths recovered after parasitological necropsy. Using the combination of quercetin with IVM, it was observed that in larvae, quercetin did not significantly reduce the EC50 for IVM in the sensitive and highly resistant isolates, but led to a significant reduction in the EC50 for IVM in the intermediate isolate. In adults, quercetin did not significantly reduce the EC50 for IVM in any of the isolates. No significant effect of the combination was found regarding the reduction in FEC or total count of parasites. The results of the in vitro and in vivo tests performed in the present study on quercetin activity underscore the importance of evaluating resistance-reversing agents among different stages of parasite development as well as among isolates with different resistance profiles. The action of quercetin combined with IVM on the motility of H. contortus larvae and adults was influenced by the degree of resistance and development stage of the parasite. The combination was effective only on intermediate resistant larvae. No action of the combination against adults was found. Moreover, this combination, when administered through the intra-abomasal route, was not effective at reducing the FEC and parasite load of naturally infected sheep.

Discovery of potential ovicidal natural products using metabolomics.

Plant extracts are a potential source of new compounds for nematode control and may be an excellent alternative for the control gastrointestinal nematodes that are resistant to conventional anthelmintics. However, research involving natural products is a complex process. The main challenge is the identification of bioactive compounds. Online analytical techniques with universal detectors, such as high-performance liquid chromatography-mass spectrometry (HPLC-MS), together with metabolomics could enable the fast, accurate evaluation of a massive amount of data, constituting a viable option for the identification of active compounds in plant extracts. This study focused on the evaluation of the ovicidal activity of ethanol extracts from 17 plants collected from the Pantanal wetland in the state of Mato Grosso do Sul, Brazil, against eggs of Haemonchus placei using the egg hatchability test. The ethanol extracts were obtained using accelerated solvent extraction. The data on ovicidal activity, mass spectrometry and metabolomics were evaluated using HPLC-DAD-MS, partial least squares regression analysis (PLS-DA) and a correlation map (univariate correlation analyses) to detect compounds that have a positive correlation with biological activity. Among the ten metabolites with the best correlation coefficients, six were phenylpropanoids, two were triterpene saponins, one was a brevipolide, and one was a flavonoid. Combinations of metabolites with high ovicidal action were also identified, such as phenylpropanoids combined with the triterpene saponins and the flavonoid, flavonoids combined with iridoid and phenylpropanoids, and saponins combined with phenylpropanoid. The positive correlation between classes of compounds in plants belonging to different genera and biological activity (as previously identified in the literature) reinforces the robustness of the statistical data and demonstrates the efficacy of this method for the selection of bioactive compounds without the need for isolation and reevaluation. The proposed method also enables the determination of synergism among the classes, which would be impracticable using traditional methods. The present investigation demonstrates that the metabolomic technique was efficient at detecting secondary metabolites with ovicidal activity against H. placei. Thus, the use of metabolomics can be a tool to accelerate and simplify bioprospecting research with plant extracts in veterinary parasitology.

Biomarkers and renal arterial resistive index in dogs naturally infected with Leishmania infantum.

Canine visceral leishmaniasis frequently causes glomerulonephritis and tubulointerstitial nephritis, nephropathies for which diagnosis has been limited by the low sensitivity of traditional tests. The aim of this study was to evaluate serum cystatin C and urinary gamma-glutamyltransferase (uGGT) levels and the urinary GGT/urinary creatinine ratio (uGGT/uCR) and to measure the renal arterial resistive index (RARI) in dogs with leishmaniasis with varying degrees of renal injury based on the urine protein: creatinine ratio (UP/C) and serum creatinine (SCr) level. We tested 59 untreated adult dogs of both sexes and undefined breeds naturally infected with Leishmania infantum. The dogs were grouped into four groups based on UP/C and SCr level: group 1 (n = 15), dogs with SCr levels < 1.4 mg/dL and UP/C < 0.5; group 2 (n = 13), dogs with SCr levels < 1.4 mg/dL and UP/C of 0.5-1.0; group 3 (n = 16), dogs with SCr levels < 1.4 and UP/C > 1.0; and group 4 (n = 15), dogs with SCr levels > 1.4. A fifth group of healthy dogs (n = 10) was the control. uGGT concentrations and uGGT/uCR were higher in dogs with proteinuria and SCr < 1.4 mg/dL, whereas the serum cystatin C concentrations and RARI were higher only in dogs with SCr levels > 1.4. In conclusion, uGGT and uGGT/uCR may be useful tools for early detection and assessment of renal lesions associated with leishmaniasis; however, cystatin C is useful for monitoring the progression of kidney disease when measured sequentially.

Comparison of McMaster and Mini-FLOTAC fecal egg counting techniques in cattle and horses.

The aim of this study was to compare two fecal egg count (FEC) techniques; McMaster (McM) and Mini-FLOTAC (mF), for the detection of cattle and horse gastrointestinal nematode eggs, in different locations. Experiment 1: feces were collected from 16 cattle and FEC was performed individually, using mF with the sensitivity of 5 eggs per gram of feces (EPG) and McM with a sensitivity of 50 EPG at Empresa de Pesquisa Agropecuária de Minas Gerais - EPAMIG and the Laboratory of Parasitic Diseases of the University of Parana - LDP/UFPR. Experiment 2: Fecal samples from 30 horses were analyzed with mF (sensitivity of 5 EPG) and McM (sensitivity of 25 EPG) at the University of Mato Grosso do Sul - UFMS and LPD/UFPR. Experiment 3: feces were collected from 14 foals and FEC was performed using mF (sensitivity of 5 EPG); and McM (sensitivity of 25 EPG) only at the LPD/UFPR. For cattle, the average FEC of mF was 962 at LPD; and 1248 at EPAMIG; for McM it was 1393 at LPD and 1563 at EPAMIG. For horses, the FEC average using the mF was 650 at LPD and 469 at UFMS; and for McM it was 677 at LPD and 554 at UFMS. For foals, the average FEC for mF was 404 and 436 for McM. In all experiments, the standard deviation and the coefficient of variation values were significantly lower for mF. Therefore, it is recommended the use of the Mini-FLOTAC technique, which is a method with less variability and higher accuracy, particularly for animals with low FEC.