Prevalence of flukes (Fasciola hepatica and paramphistomids) in cattle in south-eastern Mexico.

The objective of this study was to determine the risk factors and prevalence of trematodes in south-eastern Mexico. The prevalence of trematodes was determined in 1010 bovines. The study was carried out from October 2018 (n=291) to December 2019 (n=719). Only in 2019 rumen and liver fluke eggs were differentiated. Faecal samples (n=311) were obtained from farms in southeast Mexico located in Tabasco, Chiapas and Campeche. In addition, the presence of flukes in liver and rumen from slaughtered cattle in abattoirs was recorded with a total of 408 samples. A logistic procedure was used to obtain the prevalence and the effect of main risk factors such as land physiography (flooded areas and hills), year, sex, animals' age and type of sample obtained (eggs in faeces and flukes). The general prevalence of flukes in cattle was 32.3 % in 2018 and 41.7 % in 2019. Prevalence of F. hepatica (liver fluke) was 18.6 % (134/719) and that of paramphistomids (rumen fluke) was 33.4 % (240/719). The infected cattle from the slaughterhouse indicated a lower prevalence of F. hepatica (1 %) and rumen fluke (26.7 %) than in farms detected by egg in faeces (41.8 % and 42.1 %, respectively). The physiographic zone was decisive in the presence of F. hepatica and rumen fluke, while sex did not represent a risk factor (P > 0.05). The environmental conditions of the Mexican southeast favour the presence of both liver and rumen fluke.

Prevalence of Neospora caninum, Toxoplasma gondii and Brucella abortus in water buffalo (Bubalus bubalis): Climatic and environmental risk factors in eastern and southeast Mexico.

A total of 600 serum samples from five farms were collected and the geographic coordinates of each farm were georeferenced to establish two Köppen climate classes (wet tropical and tropical rainforest). Serum samples were examined by commercially available enzyme-linked immunosorbent assay (ELISA) kits to detect antibodies against N. caninum and T. gondii, while anti-B. abortus antibodies were analysed using the Rose Bengal test and ELISA kit. The overall estimated prevalence of N. caninum, T. gondii and B. abortus was 41.2%, 30.7% and 0.5%, respectively. The highest percentages of positive buffaloes with neosporosis and toxoplasmosis were found in the states of Tabasco (46.7%, wet tropical climate) and Veracruz (32.7%, tropical rainforest climate), respectively. Multivariate logistic regression showed that N. caninum seropositivity in buffalos was associated with the normalized difference vegetation index (NDVI) (OR: 1.068; 95% CI: 1.026-1.112; P = 0.001) and tropical climate regions (OR: 1.737; 95% CI: 1.112-2.712; P = 0.015). In addition, statistical analysis revealed that sex (OR: 1.737, 95% CI: 1.685-9.737; P = 0.002), NDVI (OR: 1.382; 95% CI: 1.095-1.746; P = 0.007) and rainfall (OR: 1.478; 95% CI: 1.110-1.967; P = 0.007) were significant risk factors for T. gondii infections. These results indicate that the potential impact of the risk factors was different for each disease in buffaloes, which may be useful for developing regionally adapted control strategies and preventive measures.

Prevalence and seasonal variation of Fasciola hepatica in slaughtered cattle: the role of climate and environmental factors in Mexico.

The objective of this study is to determine the prevalence of Fasciola hepatica infection in cattle slaughterhouses, as well as its association with climatic/environmental factors (derived from satellite data), seasonality and climate regions in two states in Mexico. Condemned livers from slaughtered animals were obtained from three abattoirs in the states of Puebla and Veracruz. The overall prevalence of the parasite in cattle between January and December of 2017 was 20.6% (1407 out of 6834); the highest rate of condemnation was observed in Veracruz (26.3%; tropical climate), and the lowest rate was found in Puebla (15.5%; temperate climate). The seasonal prevalence of fluke infection was 18.6%, 14.8% and 28.4% during the wet season, and 17.1%, 12.4% and 22.8% during the dry season in the three abattoir sites, located in the districts of Zacatlán, Teziutlán and Ciudad Alemán, respectively. Liver condemnations due to bovine fasciolosis were prevalent in the Zacatlán, Teziutlán and Ciudad Alemán districts during summer, autumn and summer, respectively. Using generalized estimating equations analysis, we determined six variables - rainfall (wet/dry), land surface temperature day, land surface temperature night, normalized difference vegetation index, seasonality and climate regions (temperate/tropical) - to be significantly associated with the prevalence of condemned livers. Climate region was the variable most strongly associated with F. hepatica infection (odds ratio (OR) 266.59; 95% confidence interval (CI): 241.90-353.34), followed by wet and dry seasons (OR 25.56; 95% CI: 20.56-55.67).

Prevalence, economic assessment, and risk factors of gastrointestinal nematodes infecting herds in tropical, dry and temperate climate regions in Mexico.

This study investigated exposure to gastrointestinal (GI) nematodes on dairy cattle farms by antibody level determination in bulk tank milk (BTM) samples and its influence on production to detect the risk factors for infection in different climate regions in three states of Mexico. From January to April 2017, BTM samples were collected from 1058 dairy cattle herds and used to establish three Köppen climate classes (tropical, dry and temperate) and states of Mexico. A questionnaire on farm management was applied. The overall herd prevalence of parasites was 67.20%. The highest percentage of positive herds was detected in Veracruz state (78.45%). In addition, the highest prevalence among the climate regions was found in the tropical climate (78.59%). In general, production losses were approximately 1.37-1.78 kg of milk/cow per day. The annual costs of milk production losses per farm were estimated for three different climate regions, ranging between $5541.49 and 6982.50 US$, and those in the three states varied between 5974.10 and 8660.06 US$. The costs for anthelmintic treatments for young stock and adult cows ranged between 57.51 and 192.75 US$, respectively, among the three climate regions and between 46.02 and 189.49 US$, respectively, among the three states. The overall annual costs of milk yield loss per cow were estimated to be 150.74 US$ for the climate regions and 190.54 US$ for the three states of Mexico, followed by the treatment costs for young stock (4.02 US$) and adult cows (3.99 US$). The results suggest that the economic losses due to GI nematodes in Mexican dairy herds are approximately 248 million US$ per annum. Four final models were built based on multivariate logistic regression for potential statistical association from the ELISA results using climatic/environmental and management factors so that each model used different risk factors that were significantly associated with helminth infections in dairy herds.

Bulk tank milk ELISA to detect IgG1 prevalence and clustering to determine spatial distribution and risk factors of Fasciola hepatica-infected herds in Mexico.

Fasciola hepatica is a helminth parasite that causes huge economic losses to the livestock industry worldwide. Fasciolosis is an emerging foodborne zoonotic disease that affects both humans and grazing animals. This study investigated the associations between climatic/environmental factors (derived from satellite data) and management factors affecting the spatial distribution of this liver fluke in cattle herds across different climate zones in three Mexican states. A bulk-tank milk (BTM) IgG1 enzyme-linked immunosorbent assay (ELISA) test was used to detect F. hepatica infection levels of 717 cattle herds between January and April 2015. Management data were collected from the farms by questionnaire. The parasite's overall herd prevalence and mean optical density ratio (ODR) were 62.76% and 0.67, respectively. The presence of clustered F. hepatica infections was studied using the spatial scan statistic. Three marked clusters in the spatial distribution of the parasite were observed. Logistic regression was used to test three models of potential statistical association from the ELISA results using climatic, environmental and management variables. The final model based on climatic/environmental and management variables included the following factors: rainfall, elevation, proportion of grazed grass in the diet, contact with other herds, herd size, parasite control use and education level as significant predictors. Geostatistical kriging was applied to generate a risk map for the presence of parasites in dairy herds in Mexico. In conclusion, the spatial distribution of F. hepatica in Mexican cattle herds is influenced by multifactorial effects and should be considered in developing regionally adapted control measures.

Motility of Fasciola hepatica miracidia assessed with a computer-assisted sperm analyser.

The motility parameters of Fasciola hepatica miracidia were assessed at different temperatures and times post-hatching using computer-assisted sperm analysis. Eggs were incubated at 22 °C or 25 °C for 14 days. Five motion parameters were evaluated at different incubation temperatures up to 10 h post-hatching. No differences were observed in the percentage that hatched after incubation at the two different temperatures. However, the straight-line velocity of miracidia following incubation at 22 °C was significantly different from that observed at 25 °C (P< 0.01). All miracidium motion parameters at different post-hatching temperatures showed an overall decrease at the end of the experiment. Those miracidia hatching from eggs incubated at 25 °C had a higher velocity of 1673.3 µm/s compared with 1553.3 µm/s at 22 °C. Velocity parameters increased as the post-hatching temperature increased from 22 °C to 37 °C.

Proteolytic activity in Fasciola hepatica is reduced by the administration of cathepsin L mimotopes.

The objective of this study was to assess the proteolytic activity of Fasciola hepatica cathepsins in liver sections from mice vaccinated with phage clones of cathepsin L mimotopes, using the film in situ zymography technique. Female BALB/c mice were immunized three times with 2.5 x 10¹¹ phage particles without adjuvant. Animals vaccinated with phage clones produced high titres of anti-mimotope antibodies and a significant reduction in fluke burden was observed following challenge with metacercariae of F. hepatica. The proteolytic activity in hepatic tissue was reduced after the immunization with phage clones.

Moderate protection is induced by a chimeric protein composed of leucine aminopeptidase and cathepsin L1 against Fasciola hepatica challenge in sheep.

Leucine aminopeptidase (FhLAP) and cathepsin L1 (FhCL1) of Fasciola hepatica play a critical role in parasite feeding, migration through host tissue, and immune evasion. These antigens have been tested for immune protection as single components with variable degrees of success. The chimeric-protein approach could improve protection levels against fasciolosis. Previously, we reported the design and construction of a chimeric protein composed of antigenic sequences of FhLAP and FhCL1 of F. hepatica. The goal of the present study was to express and evaluate the immune-protective capacity of this chimeric protein (rFhLAP-CL1) in sheep. Animals were randomly allocated into five groups with five animals in each group. Groups 1, 2 and 3 were immunized twice with 100 µg, 200 µg and 400 µg of rFhLAP-CL1 emulsified with Quil A adjuvant, whereas groups 4 and 5 were the adjuvant control and infection control groups, respectively. The animals were then challenged with 200 metacercariae two weeks after the rFhLAP-CL1 booster. The fluke burden was reduced by 25.5%, 30.7% (p < 0.05) and 46.5% (p < 0.01) in sheep immunized with 100 µg, 200 µg and 400 µg of chimeric protein, respectively, in comparison to the infection control group. There was a reduction of 22.7% (p < 0.05) and 24.4% (p < 0.01) in fecal egg count in groups 2 and 3, respectively, compared to the infection control group. Sheep immunized with chimeric protein produced F. hepatica excretion-secretion product-specific total IgG antibody, which were increased after challenge. Moreover, the levels of rFhLAP-CL1-specific IgG1 and IgG2 isotypes in immunized sheep increased rapidly two weeks after the first immunization and were significantly more elevated than those of the control groups, indicating a mixed Th1/Th2 response. This is a preliminary evaluation of the chimeric protein rFhLAP-CL1 as a possible immunogen against F. hepatica infection in sheep.

Induction of immunity in sheep to Fasciola hepatica with mimotopes of cathepsin L selected from a phage display library.

An M13 phage random 12-mers peptide library was used to screen cathepsin L mimotopes of Fasciola hepatica and to evaluate their immunogenicity in sheep. Seven clones showed positive reactivity to a rabbit anti-cathepsin L1/L2 antiserum in ELISA, and their amino acid sequences deduced by DNA sequencing were tentatively mapped on the protein. Twenty sheep were randomly allocated into 4 groups of 5 animals each, for immunization with 1x10(14) phage particles of clones 1, 20, a mixture of 7 clones and PBS, without adjuvant at the beginning, and 4 weeks later. All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. The mean worm burdens after challenge were reduced by 47.61% and 33.91% in sheep vaccinated with clones 1 and 20, respectively; no effect was observed in animals inoculated with the clone mixture. Also, a significant reduction in worm size and burden was observed for those sheep immunized with clone 1. Animals receiving clone 20, showed a significant reduction in egg output. Immunization induced a reduction of egg viability ranging from 58.92 to 82.11%. Furthermore, vaccinated animals produced clone-specific antibodies which were boosted after challenge with metacercariae of F. hepatica.

In Vitro Anthelmintic Activity of Methanolic Extract from Caesalpinia coriaria J. Willd Fruits against Haemonchus contortus Eggs and Infective Larvae.

The aim of this study was to evaluate the in vitro lethal effect of a methanolic extract (ME) from Caesalpinia coriaria fruits against Haemonchus contortus eggs and infective larvae. The anthelmintic activity was assessed using the egg hatching inhibition assay (EHI) and the mortality test. The ME was assessed using five concentrations as follows: 6.15, 3.12, 1.56, and 0.78 mg/mL to eggs and 150, 100, 75, and 50 mg/mL to larvae, respectively. Ivermectin (5 mg/mL) was used as positive control and 4% methanol and distilled water were used as negative controls. The data of ovicidal and larvicidal effect were analyzed with a completely randomized design through ANOVA analysis using the general linear model (GLM) and lethal concentrations (LC50 and LC90) were estimated through a Probit analysis using the SAS program. A clear ME increased concentration dependence effect was observed in the EHI and mortality tests. The highest activity of the methanolic extract was observed at the highest concentration (P < 0.05) to obtain a similar effect to the positive control (ivermectin), with LC50 = 78.38 and 0.00064 mg/mL and LC90 =235.63 and 0.024 mg/mL, respectively, for larvae and eggs. The results indicate that the C. coriaria fruit ME possesses in vitro ovicidal and larvicidal properties (gallotannins: methyl gallate) against H. contortus that needs to be investigated more in vivo for the control of gastroenteric nematodes in ruminants.