In vitro ovicidal and larvicidal activity of a hydroalcoholic extract and its fractions from Cyrtocarpa procera fruits on Haemonchus contortus.

This study describes the in vitro anthelmintic effect of a hydroalcoholic extract (HA-E) and its fractions from Cyrtocarpa procera fruits against Haemonchus contortus eggs and infective larvae. The HA-E was subjected to bipartition using ethyl acetate, which resulted in an aqueous fraction (Aq-F) and an organic fraction (EtOAc-F). The HA-E and both fractions were tested using the egg hatching inhibition assay (EHIA) and the larval mortality test (LMT). Fractionation of the EtOAc-F was achieved using different chromatographic processes, i.e., open glass column and HPLC analysis. Fractionation of the EtOAc-F gave 18 subfractions (C1R1-C1R18), and those that showed the highest yields (C1R15, C1R16, C1R17 and C1R18) were subjected to anthelmintic assays. The HA-E and the EtOAc-F displayed 100% egg hatching inhibition at 3 and 1 mg/mL, respectively, whereas Aq-F exhibited 92.57% EHI at 3 mg/mL. All subfractions tested showed ovicidal effect. Regarding the larval mortality test, HA-E and EtOAc-F exhibited a larvicidal effect higher than 50% at 50 and 30 mg/mL, respectively. The subfractions that showed the highest larval mortality against H. contortus were C1R15 and C1R17, with larval mortalities of 53.57% and 60.23% at 10 mg/mL, respectively. Chemical analysis of these bioactive subfractions (C1R15 and C1R17) revealed the presence of gallic acid, protocatechuic acid, and ellagic acid. This study shows evidence about the ovicidal and larvicidal properties of C. procera fruits that could make these plant products to be considered as a natural potential anthelmintic agents for controlling haemonchosis in goats and sheep.

Phenolic compounds in the fruits of Enterolobium cyclocarpum and the inhibition of Haemonchus contortus eggs.

The fabacean Enterolobium cyclocarpum is a tree with multiple uses in production systems and its fruits serve as food for livestock, in addition, they contain phenolic compounds. The ovicidal effect of the secondary compounds of the Enterolobium cyclocarpum fruits extracted with hydroalcoholic solvent (HA-E) and the aqueous (Aq-F) and organic (AcOEt-F) fractions was evaluated. Additionally, a phytochemical analysis of the extract and fractions was performed by high performance liquid chromatography (HPLC). The HA-E showed an ovicidal effect close to 100% with the concentration evaluated of the 100 mg/mL. The fractionation of the extract allowed to potentiate the activity in the Aq (94.05% at 12 mg/mL) and AcOEt (99.45% at 3 mg/mL) fractions. The secondary compounds extracted from the fruits of E. cyclocarpum in the HA-E and fractions were flavonols, coumaric and ferulic acids and other derivatives of hydroxycinnamic acid and were responsible for the ovicidal activity observed against H. contortus.

Phenolic Acids and Flavonoids from Pithecellobium dulce (Robx.) Benth Leaves Exhibit Ovicidal Activity against Haemonchus contortus.

Pithecellobium dulce (Robx.) Benth is an arboreal legume used in traditional medicine for the treatment of several ailments, including a number of intestinal disorders, and as a natural deworming. The objective of this study was to evaluate the ovicidal activity of a hydroalcoholic extract (HA-E) and its fractions (aqueous, Aq-F and organic, EtOAc-F) from P. dulce leaves, as well as subfractions (C1F1-C17) obtained from EtOAc-F against Haemonchus contortus eggs. The HA-E, Aq-F, and EtOAc-F were assessed at 0.03-5.00 mg/mL and subfractions (C1F1-C17) were assessed at 0.06-1.00 mg/mL. The HA-E and Aq-F showed an ovicidal activity close to 100% at 2.5 mg/mL, and EtOAc-F displayed the highest anthelmintic effect (100% at 0.25 mg/mL). Meanwhile, the sub-fractions with the highest ovicidal effect were C1F6, C1F9, C1F10, C1F11, and CIF13. The main compounds identified in the most active fractions (C1F9, C1F11, and C1F13) were kaempferol (1), quercetin (2), coumaric acid (3), ferulic acid (4), luteolin 7-O-rhamnoside (5), quercetin 3-O-rhamnoside (6), and a caffeoyl derivate (NI). The results indicate that P. dulce leaves exhibit a potent anthelmintic activity and contain bioactive compounds able to inhibit egg hatching in H. contortus. Therefore, this plant could be used for the control of gastrointestinal nematodes in small ruminants.

In vitro and in vivo anthelmintic properties of Caesalpinia coriaria fruits against Haemonchus contortus.

The purpose of the present study was to assay the in vitro and in vivo anthelmintic activity (AA) of Caesalpinia coriaria (Cc) mature fruits against the nematode Haemonchus contortus (Hc). The Hc infective larvae were used to assess the in vitro AA through larval mortality assay. The exposure of larvae to the different treatments was performed in 96-well microtitration plates. The treatments were as follows: hydroalcoholic extract (HA-E, at 25-100 mg/mL), aqueous fraction (Aq-F, at 12.5-50 mg/mL), organic fraction (EtOAc-F at 12.5-50 mg/mL), compounds (1, methyl gallate and 2, gallic acid at 1.25-10 mg/mL), positive control (ivermectin at 5 mg/mL) and two negative controls (distilled water and 4% methanol). After exposure, dead and live larvae were quantified and results were compared to their controls. The in vivo assay was carried out by a faecal egg count reduction test (FECRT); artificially infected goat kids (F1: Boer x Nubia) were treated with Cc ground dried fruits to assess the AA. The treatments were established as follows: G1-untreated goats (negative control), G2-goats dewormed with ivermectin (positive control), G3-goats fed with Cc mature fruits (10% of their diet). Results in both in vitro and in vivo assays were analysed using an ANOVA through random design, applying a general linear model and mixed models. The in vitro results showed an evident larvicidal effect of the HA-E, EtOAc-F from Cc, indicating that the compound responsible for the AA was gallic acid. The results of the in vivo study corroborated the anthelmintic properties of Cc, reaching 78.6% reduction in the elimination of Hc eggs per gram of faeces. This plant represents a potential natural anthelmintic for the control of haemonchosis in goats under grazing conditions. Future studies should standardise the Cc extract or dried fruits for use in the management of nematodiasis in goat herds.

Effect of soybean grain (Glycine max L.) supplementation on the production and fatty acid profile in milk of grazing cows in the dry tropics of Mexico.

Improving the lipid profile in milk of cows with the use of soybean grain (Glycine max L.) can be favored in the grazing systems in the dry tropics of Mexico. The objective was to evaluate the milk production, the chemical composition, and the fatty acids profile (FAs) of the milk of cows in continuous grazing and supplemented with and without ground roasted soybean in the dry tropics of Mexico. Ten cows randomly distributed in two equal groups were used. Daily during confinement for milking, the cows individually received the treatments on dry basis T0: supplement with 4.6 kg commercial concentrate® without soybean, T1: supplement with 3.7 kg commercial concentrate® with 380 g of soybean. During the 78 days of the experiment, milk production was measured in all cows, and samples were collected to determine the chemical composition and FAs profile. Milk production, protein, milk total fat, lactose, and non-fat solids did not vary with treatment (p >0.05). Linoleic acid content (C18: 2, cis, cis-∆9, ∆12) increased by 22% in milk fat of cows of the T1 (p ˂0.05). The sum of the mono- and polyunsaturated FAs 29.1%, the ratio of saturated-unsaturated FAs (1.65), and the atherogenicity index (1.71) also improved in the milk of cows supplemented with T1 (p ˂0.05). It was concluded that ground roasted soybean supplement in the diet of grazing dairy cows did not affect production and did improve the lipid profile in milk fat with favorable index to promote human health.

Vaccination with cathepsin L phage-exposed mimotopes, single or in combination, reduce size, fluke burden, egg production and viability in sheep experimentally infected with Fasciola hepatica.

Fascioliasis is a worldwide emergent zoonotic disease that significantly constrains the productivity of livestock. In this study, fluke burdens, liver fluke size and biomass, faecal eggs counts, serum levels of hepatic enzymes and immune response were assessed in sheep vaccinated with peptide mimotopes of cathepsin L and infected with metacercariae. A total of 25 sheep were allocated randomly into five groups of five animals each, and experimental groups were immunised with 1 × 1013 filamentous phage particles of cathepsin L1 (CL1) (TPWKDKQ), CL2 (YGSCFLR) and mixtures of CL1 + CL2 mimotopes, in combination with Quil A adjuvant, and wild-type M13KE phage in a two-vaccination scheme on weeks 0 and 4. The control group received phosphate-buffered saline. All groups were challenged with 300 metacercariae two weeks after the last immunisation and euthanised 16 weeks later. The CL1 vaccine was estimated to provide 57.58% protection compared with the control group; no effect was observed in animals immunised with CL2 and CL1 + CL2 (33.14% and 11.63%, respectively). However, animals receiving CL2 had a significant reduction in parasite egg output. Vaccinated animals showed a significant reduction in fluke length and width and wet weights. In the CL1 group, there was a significant reduction in the total biomass of parasites recovered. Egg development was divided into seven stages: dead, empty, unembryonated, cell division, eyespot, hatched and hatching. The highest percentage of developmental stages was detected for vaccinated sheep administered CL1 + CL2 with cell division, and the lowest percentage was observed in the hatching stage. Furthermore, a significant difference in all developmental stages was observed between vaccinated animals and the control group (P < 0.01). The levels of anti-phage total IgG in immune sera increased significantly at four weeks after immunisation and were always significantly higher for cathepsin L vaccine group than in the challenged control group. Total IgG was inversely and significantly correlated with worm burden in the CL1 group.

Vaccination with cathepsin L mimotopes of Fasciola hepatica in goats reduces worm burden, morphometric measurements, and reproductive structures.

Fasciolosis is a worldwide emergent zoonotic disease that significantly constrains the productivity of livestock. We conducted an experimental trial with four groups of five goats each, vaccinated with 1 × 1013 phage particles of cathepsin L1 (CLI: DPWWLKQ), CL1 (SGTFLFS), and CL2 (PPIRNGK) mimotopes combined with Quil A adjuvant. Animals received a booster four weeks later. The control group received phosphate-buffered saline. All animals were infected with 200 Fasciola hepatica metacercariae at week six and euthanised 16 weeks later. The percentage of significant worm reduction in CL1 (DPWWLKQ), CL1 (SGTFLFS), and CL2 (PPIRNGK) compared to the control group were 55.40%, 70.42% (P < 0.05), and 32.39%, respectively. Vaccinated animals showed a significant reduction in faecal egg production and egg viability. A significant reduction in the total biomass of parasites recovered was observed in the CL1 (DPWWLKQ) and CL1 (SGTFLFS) groups. In goats vaccinated with CL2 (PPIRNGK), fluke length and width were smaller than those in the control group. Furthermore, animals receiving CL mimotopes showed a significant reduction in the total area of reproductive structures. Goats immunised with phage-displayed mimotopes produced significantly high titres of specific IgG1 and IgG2 isotypes, indicating a mixed Th1/Th2 response. The liver fluke burdens in goats vaccinated with CL1 (DPWWLKQ) and CL1 (SGTFLFS) were significantly correlated with IgG and IgG1 levels.

Phage display-based vaccine with cathepsin L and excretory-secretory products mimotopes of Fasciola hepatica induces protective cellular and humoral immune responses in sheep.

Fasciolosis is a foodborne zoonotic disease that affects grazing animals and causes substantial economic losses worldwide. Excretory/secretory (E/S) products and cathepsin L mimotopes from Fasciola hepatica were used to immunise experimentally infected sheep against liver flukes. The level of protection was measured in terms of fluke burden, morphometric measurements and faecal egg counts, as well as the humoral and cellular immune responses elicited. Five groups of 5 sheep each were immunised with 1 × 1013 phage particles of cathepsin L1 (group 1: SGTFLFS), cathepsin L1 (group 2: WHVPRTWWVLPP) and immunodominant E/S product (group 3) mimotopes with Quil A adjuvant, and wild-type M13KE phage (group 4) at the beginning and as a booster two weeks later. The control group received phosphate-buff ;ered saline. All groups were challenged with 300 metacercariae at week four and slaughtered 18 weeks later. The mean fluke burdens after challenge were reduced by 52.39 % and 67.17 % in sheep vaccinated with E/S products (group 3) and cathepsin L1 (group 1: SGTFLFS), respectively; no eff ;ect was observed in animals inoculated with cathepsin L1 (group 2: WHVPRTWWVLPP). Animals vaccinated showed a significant reduction in fluke length and width, wet weights and egg output Sheep immunised with phage-displayed mimotopes induced the development of specific IgG1 and IgG2, indicating a mixed Th1/Th2 immune response. Measurement of cytokine levels revealed higher levels of IFN-γ as well as lower production of IL-4 in sheep vaccinated with the mimotope peptide of F. hepatica. Fluke-specific production of IFN-γ in immunised animals was significantly correlated with fluke burden (P < 0.01). As helminth infection progressed, increased levels of IL-4 were evident in the wild-type M13KE phage (group 4) and the control groups (group 5), accompanied by a downregulation of IFN-γ production. Vaccinated animals with cathepsin L1 (group 1: SGTFLFS) showed that amino acids located in the middle (64SG65) of the linear sequence and C-terminal end (314TFLFS318) were associated with significant protection.

Antibacterial activity of compounds isolated from Caesalpinia coriaria (Jacq) Willd against important bacteria in public health.

Antimicrobial resistance has been increasing in recent years and is most frequently found in pathogenic microorganisms resistant or multiresistant to drugs. The secondary metabolites of plants have been evaluated as alternatives for control and treatment of these microorganisms. The aim of this study was to isolate and identify the secondary metabolites with antibacterial activity from Caesalpinia coriaria (Jacq) Willd fruit. Hydroalcoholic extract (CCHA), was subjected to a bipartition with ethyl acetate giving two fractions an aqueous (Aq-F) and an organic (EtOAc-F). The isolation of bioactive fraction (EtOAc-F) allowed obtain two important compounds, methyl gallate (1) and gallic acid (2). These compounds were identified by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). The CCHA, both fractions and the isolated compounds were evaluated in vitro to determine their Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Listeria monocytogenes and Staphylococcus aureus. Gallic acid (2) showed the lowest MIC on S. typhi, (0.156 mg/mL), L. monocytogenes and S. aureus (1.25 mg/mL), while methyl gallate (1) had the best inhibitory effect against E. coli and P. aeruginosa (1.25 mg/mL). On the other hand, methyl gallate (1) showed the best MBC on P. aeruginosa (2.50 mg/mL), and gallic acid (2) had the lowest MBC on P. aeruginosa and L. monocytogenes. In conclusion, methyl gallate (1) and gallic acid (2) are the compounds responsible for the antibacterial activity of Caesalpinia coriaria fruit.

Susceptibility of ruminal bacteria isolated from large and small ruminant to multiple conventional antibiotics.

The pivotal aim of the present context was to isolate diversified group of bacteria from the ruminants and to evaluate their antibiogram pattern against 22 antibiotics of 14 different classes. The bacterial isolates from small and large ruminant (sheep, cattle and calves) were isolated from the rumen based on various colonies morphology, and subjected for preliminary antibiotics susceptibility assay using disc diffusion method. The most sensitive isolates (based on zone of inhibition) were selected for determining the minimum inhibitory concentration (MIC) of each antibiotic ranging from 1 to 256 µg/mL. Results revealed the concentration dependent growth inhibitory property of antibiotics a species-specific process. The maximum tolerable concentration (MTC) of each antibiotic was further determined using disc diffusion method, and results exhibited that the tolerance nature of ruminal isolates to antibiotics is a species-specific mechanism. Based on the MIC and MTC values of antibiotics, amikacin, ciprofloxacin, and amoxicilline were observed to be the most potent antibiotics in terms of inhibiting the growth of ruminal isolates. In brief, the findings of the current study showed that despite the overexploitation of antibiotics as additives in the animal's feed, most of the ruminal isolates are sensitive to multiple conventional antibiotics tested. The growth inhibitory trait of antibiotics proves these antimicrobials a propitious agent against the pathogenesis of ruminal isolates in livestock.

Bulk tank milk prevalence and production losses, spatial analysis, and predictive risk mapping of Ostertagia ostertagi infections in Mexican cattle herds.

This study investigated the prevalence, production losses, spatial clustering, and predictive risk mapping in different climate zones in five states of Mexico. The bulk tank milk samples obtained between January and April 2015 were analyzed for antibodies against Ostertagia ostertagi using the Svanovir ELISA. A total of 1204 farm owners or managers answered the questionnaire. The overall herd prevalence and mean optical density ratio (ODR) of parasite were 61.96% and 0.55, respectively. Overall, the production loss was approximately 0.542 kg of milk per parasited cow per day (mean ODR = 0.92, 142 farms, 11.79%). The spatial disease cluster analysis using SatScan software indicated that two high-risk clusters were observed. In the multivariable analysis, three models were tested for potential association with the ELISA results supported by climatic, environmental, and management factors. The final logistic regression model based on both climatic/environmental and management variables included the factors rainfall, elevation, land surface temperature (LST) day, and parasite control program that were significantly associated with an increased risk of infection. Geostatistical kriging was applied to generate a risk map for the presence of parasite in dairy cattle herds in Mexico. The results indicate that climatic and meteorological factors had a higher potential impact on the spatial distribution of O. ostertagi than the management factors.

Antibacterial activities of tannic acid against isolated ruminal bacteria from sheep.

This present study was conducted to evaluate the antimicrobial effects of tannic acid (TA) against isolated ruminal bacteria from adult sheep. Rumen samples were collected from two (2) adults sheep, and a total of nine (9) ruminal bacteria were isolated from the sample. The sensitivity of the ruminal bacteria isolates to 0.63, 1.25, 2.50, 5.00 and 10.00 mg TA/mL of growth medium was determined using clearance zone (CZ) of Kirby-Bauer disc diffusion susceptibility test. There was observable increase in the sensitivity of all bacterial isolates as the level of TA increases. Not all bacterial isolates have the capacity to tolerate more than 1.25 mg TA/mL. The result shows that only 20% of the bacterial isolates had the capacity to tolerate 0.63 and 1.25 mg of tannic acid per liter. This concentration of tannic acid would be equivalent to 2% tannin in the diet of ruminant. Our findings shows that increase in concentration of tannic acid completely inhibited the ruminal bacteria from the sheep rumen.

Comparative Diagnosis of Serum IgG1 and Coproantigen ELISA for Fasciolosis Detection of Goats in Mexico.

The objective of present study was to determine the prevalence of natural caprine fasciolosis in the Mixteca region of Mexico using coproantigen and serum IgG1 ELISA tests for comparative purposes. A total of 1070 serum and faecal samples were analyzed for IgG1 antibodies and coproantigens, using ELISA with E/S products as antigen and a monoclonal antibody-based sandwich ELISA. Prevalence of 73.46% was found using the serological ELISA and a percentage of 77.20 was found for coproantigen ELISA. The diagnostic sensitivity and specificity for serum ELISA were 86.7% and 96.4%, and for the coproantigen ELISA they were 93.1% and 97.8%, respectively. The seropositive samples were further categorized as low, medium, or high positivity. Results show a great proportion of low and medium positive goats when the serum ELISA test was used. Correlation coefficients between coproantigens and seropositivity were statistically significant (P < 0.01) for low seropositivity (r = 0.93) and medium seropositivity (r = 0.84). The accuracy of faecal antigen ELISA was higher compared to indirect ELISA serological test. Two ELISAs were shown to be useful for demonstrating the current status of F. hepatica infection in the endemic areas and can be employed in studies on epidemiology as well as anthelmintics treatment for preventing economic loss and the risk of transmission to humans.

Prevalence of bovine subclinical mastitis, its etiology and diagnosis of antibiotic resistance of dairy farms in four municipalities of a tropical region of Mexico.

A region-wide survey was conducted in the tropical area of Tierra Caliente, State of Guerrero, Mexico to estimate the prevalence of subclinical bovine mastitis (SCM), distribution of mastitis pathogens, and in vitro antimicrobial susceptibility of different mastitis pathogens in dairy farms. In total, 1036 quarter milk samples were obtained from 259 cows at 87 different dairy farms. Collected quarter milk samples were submitted for California Mastitis Test (CMT), bacteriological examination, and testing for antimicrobial susceptibility. Overall prevalence of SCM in the studied area was 20.5 %. Prevalence in the different regions was as follows: 28 % in Arcelia municipality, 21 % in Tlalchapa municipality, 19.4 % in Pungarabato municipality, and 14.3 % in Finch Cutzamala municipality. Of all positive isolates, 97.5 % were Gram-negative bacteria. Moreover, of all positive isolates, 37.5 % were Proteus vulgaris, 25 % Salmonella spp., 12.5 % Enterobacter aerogenes, and 10 % Escherichia coli. Klebsiella pneumonia and E. coli were sensitive for netilmicin antimicrobial. However, E. coli was sensitive for pefloxacin and gentamicin with a sensitivity for pefloxacin for E. aerogenes, while Staphylococci were sensitive for gentamicin and dicloxacillin. It could be concluded that practices such as the implementation of mastitis control programs, improved milking hygiene together with an intramammary treatment with netilmicin, pefloxacin, and gentamicin antimicrobials should be considered for mastitis prevention in the study area of Tierra Caliente, in the tropical area of Guerrero, Mexico.

Productive response of lambs fed Crescentia alata and Guazuma ulmifolia fruits in a tropical region of Mexico.

In vitro gas production with and without polyethylene glycol (PEG) of the fruits of Crescentia alata and Guazuma ulmifolia was evaluated, the degradation kinetics of lamb diets with added fruit of the tree was determined, and the ration intake and growth rate of lambs fed these diets were measured. Twenty-five entire male lambs of 23.5 ± 0.44 kg body weight were used and distributed in treatments: T0 (control without fruit); T1 and T2, 15 and 30 % of the fruit of C. alata; and T3 and T4, 15 and 30 % of the fruit of G. ulmifolia. Data variables chemical composition, fermentation kinetic, and digestibility in vitro were analyzed by a completely randomized design and data production response factorials design of five treatments by three evaluation periods. The total phenolic content (TP) (23.0 g/kg DM) was higher (P < 0.01) in the fruits of G. ulmifolia. The addition of PEG increased (P < 0.05) in vitro gas production (156.6 mL/g DM) in fruits of G. ulmifolia. In the fermentation kinetics, the total gas volume was higher (P < 0.01) at T0 (b = 293 mL/g DM), and the rate of degradation (c) but Lag time (t lag) was not different. In animal response, total dry matter intake was higher in lambs that received T4 (1.35 kg), and the daily weight gain and feed conversion did not differ (P > 0.05) among lambs receiving the treatments. Thirty percent G. ulmifolia fruit added in the diet increased dry matter intake and improved feed conversion but did not increase weight gain.

Use of three fodder trees in the feeding of goats in the subhumid tropics in Mexico.

Chemical composition, in vitro gas production with and without polyethyleneglycol (PEG-4000 MW), and in vitro digestibility of dry matter (IVDMD) and organic (IVOMD) foliage from Pithecellobium dulce, Gliricidia sepium and Haematoxylum brasiletto were determined. The preference test was run for 15 days: the first 10 days as adaption period and the 5 days served as evaluation period. It was conducted in ten developing female Creole goats of 6 months old, weighing 14 ± 2.0 kg in order to determine goat preference for any of the three foliages. Productive performance of 35 male creole kids of 6 months old (14 ± 3.0 kg) was also determined by ad libitum feeding of the foliage of the tree: the 30 and 15 % of each of the P. dulce (T1, T2), G. sepium (T3, T4), and H. brasiletto (T5, T6) foliages were added to the experiment diets, while T7 served as control diet that did not contain any foliage. The crude protein (CP), total phenols (TP), condensed tannins (CT), IVDMD, and IVOMD were different among the foliages. The PEG determined the biological activity of the TP and CT of H. brasiletto. Goats preferred to consume the foliage of P. dulce because of its higher content of CP and IVDMD and low content of TP and CT. In the productive response, dry matter intake (DMI) was higher in kids fed T1 diet and was stimulated by higher IVDMD and IVOMD, which resulted in the higher daily weight gain (DWG). The contribution with TP and CT of H. brasiletto to T5 and T6 and the rejection by the animals of G. sepium in T3 and T4 explain the negative effects on the DMI and the DWG. Findings of the study suggested higher kid performance for P. dulce foliage. Possible attributes may include its better CP, low TP and CT, and higher digestibility.