Use of a nonlinear model to estimate milk losses due to subclinical mastitis in Holstein-Zebu cows.

The objective of this observational study was to estimate milk loss from subclinical mastitis (SCM) using a nonlinear one-phase exponential decay function fitted to the relationship between the sum of the California mastitis test scores of productive quarters (x1, 0 to 16) and daily milk production per cow (y, kg/day). The function was y = (a - b)e-c1*x1 + b, where a is the predicted y when x is zero, b is the y when x1 tends to infinity, and c1 is a rate constant. The fitted function was y = (10.02 - 5.76)e-0.06137*x1 + 5.76, with an adjusted coefficient of determination (R2adj) of 0.0692, a standard deviation of the residual (Sy.x) equal to 13.49, and an Akaike's information criterion corrected (AICC) of 3902.8. To this function, we added the multiplicative factors test day (x2, 1 to 12), the number of productive quarters (x3, 2, 3 or 4), months of age of cow (x4, 25 to 209), and days in milk (x5, 2 to 196), to adjust the decay function for their effects. The fitted model was y = [(9.65 - 3.95)e-0.03415*x1 + 3.95] (x2-0.1081) (x30.1049) (x40.1589) (x5-0.1972) with R2 of 0.3166, Sy.x = 11.38, and AICC = 3821.9. We used the adjusted decay function parameters to compute a conversion factor (CF, 0 to 1) each x1 integer value from 0 to 16 to project its actual y to that of an SCM-free udder (x = 0). Then, the CF for a x1 of 0 equals to 9.65/9.65 = 1, and for a x1 of 8 equals 8.28, so its CF would be 8.29/9.65 = 0.8588. A cow with a 5.5 kg/day milk production at an x1 score of 8 would have an SCM-free production of 5.5/0.8588 = 6.4 kg/day, and its daily milk loss would be 6.4-5.5 = 0.9 kg/day. The difference, multiplied by MX$ 5.50 (US$ 0.275), would result in an economic loss of 4.95 Mexican pesos per day. This procedure makes possible the timely on-site estimation of economic losses due to subclinical mastitis.

Effect of entomopathogenic native fungi from paddock soils against Rhipicephalus microplus larvae with different toxicological behaviors to acaricides.

The objectives of this study are to: (1) evaluate the in vitro acaricidal effect of 54 Metarhizium anisopliae strains, six Beauveria bassiana strains and one Purpureocilium lilacinum strain, against the larvae of two populations of Rhipicephalus microplus (multi-resistant and susceptible to chemical acaricides); and (2) determine the lethal concentrations required to eliminate the 50% (LC50) and 99% (LC99) of larvae through the use of entomopathogenic fungi (EF) with high acaricidal effects. The mortality percentage was evaluated by larval immersion tests at a dose of 1 × 108 conidia/mL for each fungal strain. For calculating LC50 and LC99, four doses (1 × 108, 1 × 107, 1 × 106 and 1 × 105) were used. Nine strains of M. anisopliae and the P. lilacinum strain showed a high mortality percentage in the R. microplus larvae of both populations. The best strains that showed the lowest values of LC50 and LC99 for tick elimination were MaV50 and PlV01. In conclusion, several strains of entomopathogenic fungi showed a high acaricidal effect against the R. microplus larvae of both populations, suggesting that these fungi might be a promissory adjuvant in the control of R. microplus, including those who are resistant. Finally, the discovery of a P. lilacinum strain with a high acaricidal effect is also reported.

Anthelmintic effect of 2H-chromen-2-one isolated from Gliricidia sepium against Cooperia punctata.

Gliricidia sepium is a tropical legume with known anthelmintic-like properties. The aim of this study was to: (1) perform a bio-guided fractionation of an acetonic extract of G. sepium leaves using the egg hatch assay (EHA); (2) elucidate the anthelmintic (AH)-like phytochemical using nuclear magnetic resonance (NMR); and (3) assess the ultrastructural damage of the Cooperia punctata treated eggs. The anthelmintic activity of G. sepium was traced from an acetonic extract using the EHA. Phytochemicals were isolated through silica gel columns and elucidated through spectroscopic measurements (1H and 13C). Final fraction was evaluated with EHA at decreasing concentrations of: 1.100; 0.500, 0.250, 0.125, 0.060, 0.001 and 0.00001 mg mL-1. Egg hatching inhibition was calculated using the formula: 100*(1-HT/HC). The maximal half of effective concentration (EC50) was calculated with GraphPad. Bio-guided isolation procedures lead to the elucidation of 2H-chromen-2-one, which inhibited both hatching and embryo development of C. punctata (EC50 of 0.024 ± 0.082 mg mL-1) (P < 0.05). Scanning and Transmission Electron Microscopy (SEM and TEM) revealed electrodensity alterations and fractures in the eggshell layers. After toxicity evaluations and in vivo assessment, 2H-chromen-2-one can be suggested as a novel AH-phytochemical for reducing larval density in pastures and worm burdens inside the host.

In vitro antihelmintic effect of fifteen tropical plant extracts on excysted flukes of Fasciola hepatica.

BACKGROUND: Fasciolosis due to Fasciola hepatica is the most important hepatic disease in veterinary medicine. Its relevance is important because of the major economical losses to the cattle industry such as: reduction in milk, meat and wool production; miscarriages, anemia, liver condemnation and occasionally deaths, are estimated in billons of dollars. The emergence of fluke resistance due to over or under dosing of fasciolides as well as environmental damage produced by the chemicals eliminated in field have stimulated the need for alternative methods to control Fasciola hepatica. The aim of this study was to evaluate the in vitro anthelmintic effect of fifteen tropical plant extracts used in tradicional Mexican medicine, on newly excysted flukes of Fasciola hepatica. RESULTS: The flukes were exposed in triplicate at 500, 250 and 125 mg/L to each extract. The efficacy was assessed as the mortality rate based on the number of live and dead flukes after 24, 48 and 72 h post-exposure. The plants with anthelmintic effect were evaluated once again with a concentration of 375 mg/L in order to confirm the results and to calculate lethal concentrations at 50%, 90% and 99% (LC(50), LC(90), and LC(99)). Plant extracts of Lantana camara, Bocconia frutescens, Piper auritum, Artemisia mexicana and Cajanus cajan had an in vitro anthelmintic effect (P <0.05). The LC(50), LC(90) and LC(99) to A. mexicana, C. cajan and B. frutescens were 92.85, 210.44 and 410.04 mg/L, 382.73, 570.09 and 788.9 mg/L and 369.96, 529.94 and 710.34 mg/L, respectively. CONCLUSION: It is concluded that five tropical plant extracts had promising anthelmintic effects against F. hepatica. Further studies on toxicity and in vivo biological evaluation in ruminant models might help to determine the anthelmintic potential of these plant extracts.

Prevalence of cutaneous pathological conditions and factors associated with the presence of skin wounds in working equids in tropical regions of Veracruz, Mexico.

A wide spectrum of welfare issues is encountered on working equids. The objectives of this study were: (i) to determine the prevalence of cutaneous lesions in working equids in tropical regions of the North-Central of Veracruz State, Mexico, (ii) to identify the most prevalent cutaneous pathological conditions in this population of working equids, and (iii) to identify risk factors associated to the presence of work-related skin wounds. A number of 467 working equids presented for treatment at Donkey Sanctuary Mobile Clinic Program - National Autonomous University of Mexico from seven different villages in the North-Central region of Veracruz State were evaluated. Animals with signs of cutaneous pathology underwent a physical examination and samples were obtained of exudates, hair, and tissue for cytology examination, fungal cultures, and dermo-histopathology examination. The overall prevalence of cutaneous pathological conditions in working equids was 20.6 %. The prevalence per species was 22.6 % for horses, 18.2 % for donkeys, and 14.3 % for mules. The most common cause of skin lesions was trauma (abrasions, lacerations, and abscesses) followed in frequency by parasitic dermatitis and sarcoids. No strong associations among species were observed (P > 0.20). Multivariate analysis showed that there was greater association between BCS and age with the presence of skin wounds (P < 0.05). Equids who's BCS was rated as "moderate" as well as adult animals showed significantly more skin wounds compared with the young and the elderly (P < 0.05). These findings can help to develop a better management practices to improve the welfare of equids in tropical regions of Veracruz, Mexico.

First Record of Flavocillium subprimulinum (Cordycipitaceae, Hypocreales) in Mexico: Morphological and Molecular Characterisation, Nematocidal Activity of Its Liquid Culture Filtrates against Haemonchus contortus and Protease Activity.

This is the first record of the fungus Flavocillium subprimulinum in Mexico. The isolate was taxonomically characterised and cultured in potato dextrose broth (PDB), Czapek-Dox broth (CzDoxB), and sweet potato dextrose broth (SPDB) to obtain its filtrates (FLCF). The nematocidal activity (NA) of three FLCF concentrations was assessed against Haemonchus contortus L3. Protease activity (PA) was assessed with SDS-PAGE, followed by a zymogram. The NA of the FLCF reached 94.43% in PDB and 95.82% in CzDoxB, respectively, at 100 mg/mL. Lower mortality (64%) was found in SPDB at 100 mg/mL. SDS-PAGE showed bands (in PBS) of ~25, ~40, and ~55 kDa. The zymogram showed protein bands (PBs) with PA in the media, including PBs of ~14, ~40, and ~55 kDa. This study establishes the basis for exploring the potential use of this fungus against H. contortus, which is considered the most pathogenic parasite affecting lambs.

Gastrointestinal nematode burden in working equids from humid tropical areas of central Veracruz, Mexico, and its relationship with body condition and haematological values.

The east coast of Veracruz, Mexico, has an important equine population used for working in rural production systems. The objectives of this study were (1) to calculate the prevalence of tropical working equids (donkeys, mules and horses) infected with gastrointestinal nematodes (GINs) and the GINs involved, and (2) to measure the body condition score (BCS) and haematological values for each working equid and its relationship with faecal worm egg count (EPG). One hundred and forty working equids were randomly selected from five different villages along the central coast of the state of Veracruz and faecal and blood samples were obtained from each animal. Gastrointestinal parasite burdens were determined using the McMaster technique. Packed cell volume, total plasma proteins, red blood cell count and white blood cell count were measured from each blood sample. Prevalence of infected equids was higher than 90 %. Mules had the highest median faecal worm egg counts (875 EPG), followed by horses and donkeys with 400 EPG. There was no correlation between EPG and BCS or haematological values (p > 0.05). Results suggest that despite the high prevalence and parasite burdens, equids involved in this trial are not being seriously affected. This study provides information which might help in designing future strategies to control nematode infections in working equids in the Mexican tropics; more emphasis should be placed on other inputs (nutrition perhaps), with individual anthelminthic treatment to those animals with the highest EPG or when signs present themselves.

Testing Efficacy of a Conserved Polypeptide from the Bm86 Protein against Rhipicephalus microplus in the Mexican Tropics.

Rhipicephalus microplus economically impacts cattle production in tropical and subtropical countries. Application of acaricides constitutes the major control method; however, inadequate use has increased resistant tick populations, resulting in environmental and cattle product contamination. Anti-tick vaccines based on the Bm86 antigen are an environmentally friendly, safe, and economically sustainable alternative for controlling R. microplus infestations. Nevertheless, variable efficacy has been experienced against different geographic tick strains. Herein, we evaluated the efficacy of a conserved polypeptide Bm86 derived from a Mexican R. microplus strain previously characterized. Twelve cows were assigned to three experimental groups and immunized with three doses of the polypeptide Bm86 (pBm86), adjuvant/saline alone, and Bm86 antigen (control +), respectively. Specific IgG antibody levels were measured by ELISA and confirmed by Western blot. In addition, the reproductive performance of naturally infested R. microplus was also determined. The more affected parameter was the adult female tick number, with a reduction of 44% by the pBm86 compared to the controls (p < 0.05), showing a vaccine efficacy of 58%. Anti-pBm86 IgG antibodies were immunogenic and capable of recognizing the native Bm86 protein in the eggs, larvae, and guts of R. microplus. The negative correlation between antibody levels and the reduction of naturally tick-infested cattle suggested that the effect of the polypeptide Bm86 was attributed to the antibody response in immunized cattle. In conclusion, the polypeptide Bm86 showed a specific immune response in cattle and conferred protection against R. microplus in a Mexican tropical region. These findings support further experiments with this antigen to demonstrate its effectiveness as a regional vaccine.

Assembly and Analysis of Haemonchus contortus Transcriptome as a Tool for the Knowledge of Ivermectin Resistance Mechanisms.

Haemonchus contortus (Hc) is an important parasitic nematode of small ruminants. In this study we assembled the transcriptome of Hc as a model to contribute to the knowledge about the profile of the differential gene expression between two Mexican Hc strains under different anthelmintic resistance statuses, one susceptible and the other resistant to ivermectin (IVMs and IVMr, respectively), in order to improve and/or to have new strategies of control and diagnosis. The transcript sequence reads were assembled and annotated. Overall, ~127 Mbp were assembled and distributed into 77,422 transcript sequences, and 4394 transcripts of the de novo transcriptome were matched base on at least one of the following criteria: (1) Phylum Nemathelminthes and Platyhelminthes, important for animal health care, and (2) ≥55% of sequence identity with other organisms. The gene ontology (GO) enrichment analysis (GOEA) was performed to study the level of gene regulation to IVMr and IVMs strains using Log Fold Change (LFC) filtering values ≥ 1 and ≥ 2. The upregulated-displayed genes obtained via GOEA were: 1993 (for LFC ≥ 1) and 1241 (for LFC ≥ 2) in IVMr and 1929 (for LFC ≥ 1) and 835 (for LFC ≥ 2) in IVMs. The enriched GO terms upregulated per category identified the intracellular structure, intracellular membrane-bounded organelle and integral component of the cell membrane as some principal cellular components. Meanwhile, efflux transmembrane transporter activity, ABC-type xenobiotic transporter activity and ATPase-coupled transmembrane transporter activity were associated with molecular function. Responses to nematicide activity, pharyngeal pumping and positive regulation of synaptic assembly were classified as biological processes that might be involved in events related to the anthelmintic resistance (AR) and nematode biology. The filtering analysis of both LFC values showed similar genes related to AR. This study deepens our knowledge about the mechanisms behind the processes of H. contortus in order to help in tool production and to facilitate the reduction of AR and promote the development of other control strategies, such as anthelmintic drug targets and vaccines.

Rotational Grazing Modifies Rhipicephalus microplus Infestation in Cattle in the Humid Tropics.

Rotational grazing has been mentioned as a potential tool to reduce losses caused by high tick loads. This study aimed: (1) to evaluate the effect of three grazing modalities (rotational grazing with 30- and 45-day pasture rest and continuous grazing) on Rhipicephalus microplus infestation in cattle, (2) to determine population dynamics of R. microplus in cattle under the three grazing modalities mentioned in the humid tropics. The experiment was carried out from April 2021 to March 2022 and consisted of 3 treatments of grazing with pastures of African Stargrass of 2 ha each. T1 was continuous grazing (CG00), and T2 and T3 were rotational grazing with 30 (RG30) and 45 d of recovery (RG45), respectively. Thirty calves of 8-12 months of age were distributed to each treatment (n = 10). Every 14 days, ticks larger than 4.5 mm were counted on the animals. Concomitantly, temperature (°C), relative humidity (RH), and rainfall (RNFL) were recorded. Animals in the RG45 group had the lowest count of R. microplus compared to the RG30 and CG00 groups; these results suggest that RG45 days of rest could be a potential tool to control R. microplus in cattle. Yet, we also observed the highest population of ticks on the animals under rotational grazing with a 30-day pasture rest. A low tick infestation characterized rotational grazing at 45 days of rest throughout the experiment. The association between the degree of tick infestation by R. microplus and the climatic variables was nil (p > 0.05).

In vitro anthelmintic activity of five tropical legumes on the exsheathment and motility of Haemonchus contortus infective larvae.

This study investigated the in vitro anthelmintic (AH) activity of five tropical legume plants [Arachis pintoi CIAT 22160 (A.p. 22160), Gliricidia sepium, Cratylia argentea (C.a. Yacapani), C. argentea CIAT 22386 (C.a. 22386), C. argentea Veranera (C.a. Veranera)] against Haemonchus contortus infective larvae and the role of tannins/polyphenolic compounds in the AH effect. Lyophilized leaf extracts of each plant were evaluated using the Larval Exsheathment Inhibition Assay (LEIA) and the larval migration inhibition assay (LMIA). The role of tannins/polyphenolic compounds in the AH effect was evaluated in both assays using polyethylene glycol (PEG) to remove tannins from the solutions. At the highest concentration (1200µg of extract/ml), A. pintoi 22160, C.a. Yacapani, C.a. Veranera and C.a. 22386 completely inhibited the exsheathment process of H. contortus (P<0.01). At the same concentration (1200µg of extract/ml), the inhibition of larval migration for C.a. 22386, C.a. Veranera and G. sepium was 66.0%, 35.9% and 39.2% (relative to the PBS control), respectively. In both bioassays (LEIA and LMIA), the AH effect shown by each plant was blocked after the addition of polyethylene glycol (PEG), corroborating the role of tannins/polyphenolic compounds.

Prevalence and risk factors of coccidiosis in calves from Veracruz, México.

The objectives of the present study were: (1) to determine the prevalence of Eimeria spp. sporulated oocysts in calves from 26 Municipalities in the Central Zone of the State of Veracruz, Mexico, (2) to identify the Eimeria spp. infecting calves, and (3) to identify the risk factors associated with the presence of Eimeria spp. in tropical cattle. A total of 930 individual fecal samples were analyzed by using the McMaster technique; then, oocysts were maintained in 2.5% potassium dichromate to allow sporulation. The general prevalence of calves with Eimeria spp. oocysts was 39.7% (370/930). Of a total of 10 identified species, Eimeria canadensis was the most observed, followed by Eimeria bovis and Eimeria zuernii. The statistical analysis showed an association between the age of the calves (5 to 9 months), the presence of other animals, the physiographic regions and the restricted type of husbandry with the presence of Eimeria spp. in calves (P<0.05). Protective risk factors, such as: routine coprological analysis was also associated with a decrease in infection. These data demonstrate the presence of coccidia in cattle from the State of Veracruz, additionally to the necessary measures that must be taken to control this parasitosis.

Arthrobotrys musiformis (Orbiliales) Kills Haemonchus contortus Infective Larvae (Trichostronylidae) through Its Predatory Activity and Its Fungal Culture Filtrates.

Haemonchus contortus (Hc) is a parasite affecting small ruminants worldwide. Arthrobotrys musiformis (Am) is a nematode-trapping fungi that captures, destroys and feeds on nematodes. This study assessed the predatory activity (PA) and nematocidal activity (NA) of liquid culture filtrates (LCF) of Am against Hc infective larvae (L3), and additionally, the mycochemical profile (MP) was performed. Fungal identification was achieved by traditional and molecular procedures. The PA of Am against HcL3 was performed in water agar plates. Means of non-predated larvae were recorded and compared with a control group without fungi. LCF/HcL3 interaction was performed using micro-tittering plates. Two media, Czapek−Dox broth (CDB) and sweet potato dextrose broth (SPDB) and three concentrations, were assessed. Lectures were performed after 48 h interaction. The means of alive and dead larvae were recorded and compared with proper negative controls. The PA assessment revealed 71.54% larval reduction (p < 0.01). The highest NA of LCF was found in CDB: 93.42, 73.02 and 51.61%, at 100, 50 and 25 mg/mL, respectively (p < 0.05). Alkaloids and saponins were identified in both media; meanwhile, coumarins were only identified in CDB. The NA was only found in CDB, but not in SPDB. Coumarins could be responsible for the NA.

Entomopathogenic Fungi for Tick Control in Cattle Livestock From Mexico.

Ticks are one of the main economic threats to the cattle industry worldwide affecting productivity, health and welfare. The need for alternative methods to control tick populations is prompted by the high prevalence of multiresistant tick strains to the main chemical acaricides and their ecological consequences. Biological control using entomopathogenic fungi (EPF) is one of the most promising alternative options. The objective of this paper is to review the use of EPF as an alternative control method against cattle ticks in Mexico. Metarhizium anisopliae sensu lato (s.l.) and Beauveria bassiana s.l. are the most studied EPF for the biological control of ticks in the laboratory and in the field, mainly against Rhipicephalus microplus; however, evaluations against other important cattle ticks such as Amblyomma mixtum and R. annulatus, are needed. A transdisciplinary approach is required to incorporate different types of tools, such as genomics, transcriptomics and proteomics in order to better understand the pathogenicity/virulence mechanism in EPF against ticks. Laboratory tests have demonstrated the EPF efficacy to control susceptible and resistant/multiresistant tick populations; whereas, field tests have shown satisfactory control efficiency of M. anisopliae s.l. against different stages of R. microplus when applied both on pasture and on cattle. Epidemiological aspects of ticks and environmental factors are considered as components that influence the acaricidal behavior of the EPF. Finally, considering all these aspects, some recommendations are proposed for the use of EPF in integrated control schemes for livestock ticks.

Protective efficacy of the peptide Subolesin antigen against the cattle tick Rhipicephalus microplus under natural infestation.

The cattle tick Rhipicephalus microplus affect animal health, welfare, and cattle production in tropical and subtropical zones of the world. Anti-tick vaccines have been an effective alternative for cattle tick control instead of traditional chemical products. To date, Subolesin antigen has shown efficacy for the control of tick infestation in cattle, and previous studies showed that one peptide derived from this protein has demonstrated to elicit a strong and specific humoral immune response. Based on these findings, herein we characterized the efficacy of the peptide Subolesin for the control of cattle tick, R. microplus infestation under field conditions. Twenty-four female calves were assigned to four experimental groups and immunized with three subcutaneous doses of the peptide Subolesin, Bm86, both antigens (dual vaccine) and adjuvant/saline alone, respectively. Serum antibody levels (IgG) were assessed by ELISA and confirmed by Western blot; also, reproductive performance of naturally infested R. microplus was determined. The results showed that immunizations with the experimental antigens reduced tick infestations with vaccine's efficacy of 67 % (peptide Subolesin), 56 % (Bm86), and 49 % (dual vaccine) based on adult tick numbers, oviposition, and egg fertility between vaccinated and control animals. Peptide Subolesin-immunized calves developed a strong humoral immune response expressed by high anti-pSubolesin IgG levels, and the Western blot analysis confirmed that it is immunogenic. Cattle receiving Bm86 and dual vaccine showed less protection, although Bm86 was within the range reported previously. The negative correlation between antibody levels and reduction of naturally infested R. microplus strongly suggested that the effect of the vaccine was the result of the antibody response in immunized cattle. In conclusion, it was demonstrated that the peptide Subolesin induced a specific immune response in cattle under field conditions, resulting in reduced R. microplus populations in subsequent generations. Finally, integrated tick control must consider anti-tick vaccines as a cost-effective, sustainable, and successful tool for controlling cattle tick infestations.

The immune gene expression induced by Cooperia punctata in naturally infected calves with resistance and susceptible phenotype traits.

The analysis of the immune gene expression was performed in Zebu × Holstein calves with resistant and susceptible phenotypes naturally infected with Cooperia punctata. Fourteen calves of 4 months old were grazed for 11 weeks under a tropical climate. The parasitic infection showed an average epg value of 1055 ± 1155 and an IgG optical density of 0.814 ± 0.0.037 with statistic differences among the different weeks (p < 0.05), and a pcv value of 24 ± 2.0 % (p > 0.05). High variation in epg value was observed, between 7 ± 7.14 and 4657 ± 1886, and, based on these differences; the infected hosts were classified as five resistant calves with epg ≤ 200 and nine susceptible calves with epg ≥ 300. Moreover, IgG levels displayed statistical differences between resistance and susceptible calves to C. punctata infection. The immune gene expression was analysed in three resistant and susceptible calves, respectively. Nine cytokine genes and the FCεR1A receptor were analysed at the 3rd and 11th weeks post-infection. In the first period upregulation was found, from 2.19- to 9.45-fold, (p < 0.05) for IL-2, -5, - 6, -10, TGF-ß and FCεR1A in the resistant group; the expression was decreased at the 11th week with low level of IgG. In contrast, downregulation for susceptible calves was found for nine immune genes and upregulation for INF-γ in both periods together with increased IgG levels. In conclusion, immune gene expression was regulated at the begging infection of C. punctata in resistant grazing calves. In contrast, suppression of important genes was involved in calves susceptible to C. punctata.

Effect of an Arthrobotrys musiformis (Fungi: Orbiliales) culture filtrate on the population of gastrointestinal parasitic nematode eggs in faeces of grazing lambs.

This study assessed the anthelmintic activity of the oral administration of a free-spore culture filtrate of the nematophagous fungus (NF) Arthrobotrys musiformis (M-10) on gastrointestinal parasitic nematodes (GIN) in naturally infected lambs. The fungus was grown on potato-dextrose agar plates (PDA) and transferred to a fermented rice medium (FRM). After 40-day incubation the total amount of FRM with the growing fungi was transferred to a flask shaker with distilled water for a 24 h period. The fungus was centrifuged and filtered. Three groups of six naturally-infected lambs (>1000 epg) each were treated once as follows: Group 1) 63.8 mg/kg A. musiformis culture filtrate (CF) (per os); Group 2) Levamisole 7.5 mg/ml (intramuscularly), Group 3) 15 ml of distilled water (per os). Faecal samples were individually collected on days -2, 0, 2, 4, 6, 8 and 10 after treatment. For each experimental group, mean egg shedding was calculated and transformed (log 10 [epg + 1]). Means between the fungal filtrate group and the negative control were analysed using a T-Student Test. The faecal egg count reduction test (FECRT) was performed in groups treated with CF and Levamisole in relation to the control group (water) were 36.8-57.4% and 89-95.4%, respectively., although due to the difference between groups, no statistical significance was found (p > 0.05). The use of A. musiformis CF appears to be a good alternative treatment, although, more studies should be performed to establish the use of these fungal products as potential tools for GIN control.

Molecular survey of pyrethroid resistance mechanisms in Mexican field populations of Rhipicephalus (Boophilus) microplus.

Susceptibility to synthetic pyrethroids (SP s) and the role of two major resistance mechanisms were evaluated in Mexican Rhipicephalus microplus tick populations. Larval packet test (LPT), knock-down (kdr) PCR allele-specific assay (PASA) and esterase activity assays were conducted in tick populations for cypermethrin, flumethrin and deltamethrin. Esterase activity did not have a significant correlation with SP s resistance. However a significant correlation (p < 0.01) was found between the presence of the sodium channel mutation, and resistance to SP s as measured by PASA and LPT respectively. Just over half the populations (16/28) were cross-resistant to flumethrin, deltamethrin and cypermethrine, 21.4% of the samples (6/28) were susceptible to all of the three pyrethroids 10.7 of the samples (3/28) were resistant to flumethrin, 3.4 of the samples (1/28) were resistant to deltamethrin only and 7.1% (2/28) were resistant to flumethrin and deltamethrin. The presence of the kdr mutation correlates with resistance to the SP s as a class. Target site insensitivity is the major mechanism of resistance to SP s in Mexican R. microplus field strains, involving the presence of a sodium channel mutation, however, esterase-based, other mutations or combination of mechanisms can also occur.

Phylogenetic Relationships and Acaricidal Effects of Beauveria bassiana Obtained from Cattle Farm Soils Against Rhipicephalus microplus.

The objectives of the present study were to isolate Beauveria bassiana strains from cattle farm soils, analyze the phylogenetic relationships among the fungal strains isolated from these soils, and determine the acaricidal effect of B. bassiana isolates on engorged Rhipicephalus microplus tick strains resistant or susceptible to chemical acaricides. Six strains of B. bassiana were obtained and isolated from cattle farm soils in the Mexican tropics using the Galleria bait method, and their acaricidal effect was assessed against 2 populations of R. microplus ("Media Joya" chemical acaricide-resistant strain or "CLAR" chemical acaricide-susceptible strain) using the adult immersion test. The BbV03 strain produced 86.7% and 60% mortality in resistant and susceptible ticks on day 20, respectively, whereas the BbV04 strain produced 66.7% and 53.5% mortality in resistant and susceptible ticks on day 20, respectively. The BbV03 and BbV04 strains reduced egg laying on both R. microplus populations. There was no statistical difference in the acaricidal effect of B. bassiana strains among chemical acaricide-susceptible or -resistant R. microplus populations ( P > 0.05). The BbV03 strain was the most virulent against R. microplus with an LC50 of 2 × 107 and LC99 of 7 × 108 conidia/ml. We found that the 6 B. bassiana isolated clustered in the same clade with other previously reported B. bassiana strains (from GenBank) but were separated into 3 different sub-clades. This study shows that some B. bassiana strains are a promising coadjuvant alternative for biological tick control, including tick populations that are resistant to chemical acaricides. Beauveria bassiana is present in the pastures of tropic cattle farms, and there are genetic variations between B. bassiana strains living in this ecosystem that might play an important role in the natural control of R. microplus in cattle farm paddocks.

Effect of Gliricidia sepium leaves intake on larval establishment of Cooperia punctata in calves and bio-guided fractionation of bioactive molecules.

The aims of this study were: 1) to assess the anthelmintic effect of Gliricidia sepium on the establishment of C. punctata third-stage larvae (L3) in calves, and 2) to isolate and to elucidate an anti-exsheathment phytochemical from the plant offered during the trial. Twelve ¾ Holstein × Zebu calves were divided in two experimental groups: control (T1) and treatment (T2) (n = 6). After adaptation, each calf was infected with an oral dose of 400 C. punctata L3/Kg LW. Basal diet consisted of Digitaria decumbens hay (6.27% CP) and commercial concentrate (12% CP). In addition, during the experimental period T2 received fresh G. sepium leaves (26.88% CP) ad libitum. On day 9 post-infection, three calves per treatment were randomly selected for slaughter, and worm counts were performed. Larval establishment rates obtained were 13.44 ±â€¯0.13% and 3.1 ±â€¯1.42% for T1 and T2, respectively (P < .05). The reduction of larval establishment was 76.9%. The total length of worms recovered from the animals was also affected by the intake of G. sepium (P < .05). Phytochemicals present in G. sepium leaves offered to calves were isolated through silica gel columns and elucidated through Magnetic Nuclear Resonance (1H and 13C). Bio-guided isolation procedures lead to the elucidation of Oxytroside (Kaempferol 3-O-rhamnopyranosyl-(1 → 6)-ß-D-glucopyranoside-7-O-rhamnopyranoside), which fully inhibited the C. punctata exsheathment process (2400 µg mL-1). Gliricidia sepium represents an alternative to prevent severe C. punctata infections by reducing larval establishment in cattle.