Ignacio Ferre

Helminths of Wild Boar in Spain

Forty-seven wild boars (Sus scrofa) collected from two areas in the province of Valencia (eastern Spain) were examined for digestive and respiratory helminth parasites. Helminth species and their prevalence were Taenia hydatigena cysticercus (19%), Ascarops strongylina (87%), Physocephalus sexalatus (6%), Ascaris suum (2%), Metastrongylus spp. (85%), Capillaria sp. (2%) and Macracanthorhynchus hirudinaceus (21%). Trichinella spp. were not found. Physocephalus sexalatus, A. suum, Capillaria sp. and M. hirudinaceus were found only in wild boars collected from one area (National Hunting Reserve of Cortes de Pallás). Macracanthorhynchus hirudinaceus did not occur in hogs < 1-yr-old, whereas the intensity of Metastrongylus spp. infection was the greatest in this age group.

Plasma aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transpeptidase (GGT) activities in water buffaloes with experimental subclinical fasciolosis

The effect of chronic Fasciola hepatica infection on the activity of plasma aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transpeptidase (GGT) was investigated in water buffaloes dosed daily with 60 F. hepatica metacercariae over 20 days. Experimental fluke infection caused no clinical signs but provoked an increase in plasma level of IgG directed against F. hepatica from 4 weeks after infection. There was a significant increase in plasma AST from 6 weeks post-infection. Maximal values were reached at 14 weeks and remained significantly elevated by 23 weeks. Plasma GLDH was significantly elevated from 6 to 21 weeks post-infection. Significant increases in plasma GGT occurred from 8 to 26 weeks post-infection, reaching maximal values at 15 weeks. This study shows that plasma enzyme activities may be useful in studies of fluke-induced liver damage in water buffaloes.

Appetite depression in sheep experimentally infected with Fasciola hepatica L.

The purpose of this study was to determine the effects of experimental fasciolosis at various stages of development on the daily food intake of sheep. Five male Churra sheep, 4 months of age, were infected orally with 300 Fasciola hepatica metacercariae over a 30 day period. There was a significant increase in serum glutamate dehydrogenase (GLDH) activity from 40 days post-infection and in aspartate aminotransferase (AST) activity from 60 days post-infection. Both enzyme activities reached maximum levels in the serum of infected animals at 80 days and then progressively decreased. Serum gamma-glutamyltransferase (GGT) activity was significantly increased from 80 to 120 days post-infection. Glycaemia was significantly decreased from 60 days post-infection. The average daily food intake was shown to steadily decrease until approximately 100 days. The coincidence of decreased food intake with the period of significant increase, both in AST and GLDH activities, indicated that damage caused around the time of migration of immature flukes through the liver parenchyma may be involved in appetite depression.

Seroprevalence of Fasciola hepatica infection in sheep in northwestern Spain

To estimate the prevalence ofFasciola hepatica infection in sheep in the León province (northwestern Spain), we conducted a survey between October 1992 and May 1993. A total of 767 samples of feces and serum were collected from sheep over 1 year of age belonging to 152 flocks randomly selected from the 4 natural regions of León province. Samples were analyzed by a standard coprological sedimentation method and an indirect enzyme-linked immunosorbent assay (ELISA) using excretory-secretory products fromF. hepatica as the antigen. The results showed the feasibility of using the indirect ELISA to facilitate the serodiagnosis of ovine fasciolosis in seroepidemiology studies (95% sensitivity and >99% specificity). No serological cross-reaction with infection by the trematodeDicrocoelium dendriticum was found. Furthermore, a statistically significant association was demonstrated between the mean flock prevalence results as determined by ELISA (77.6%) and by coprological examination (23.7%;P<0.001). Differences in the results obtained by the two diagnostic methods could have been due to fluctuations in the numbers of fluke eggs detected in feces and to the persistence of specific antibodies in serum after and efficacious fasciolicide treatment. The median number ofF. hepatica eggs detected per gram of feces was 10 (range, 5–450 eggs/g feces). The geographical distribution ofF. hepatica infection in León province was similar in all natural regions, probably due to the observation that meteorological conditions are not limiting for the maintenance of the parasite life cycle in any area of the province and to the abundance of irrigated areas together with the lack of planned control strategies. No significant association between trematode infection and sheep breed, flock size, or number of treatments was found, but the results showed a significant association between infection and untreated sheep (P<0.05).To estimate the prevalence ofFasciola hepatica infection in sheep in the Leon province (northwestern Spain), we conducted a survey between October 1992 and May 1993. A total of 767 samples of feces and serum were collected from sheep over 1 year of age belonging to 152 flocks randomly selected from the 4 natural regions of Leon province. Samples were analyzed by a standard coprological sedimentation method and an indirect enzyme-linked immunosorbent assay (ELISA) using excretory-secretory products fromF. hepatica as the antigen. The results showed the feasibility of using the indirect ELISA to facilitate the serodiagnosis of ovine fasciolosis in seroepidemiology studies (95% sensitivity and >99% specificity). No serological cross-reaction with infection by the trematodeDicrocoelium dendriticum was found. Furthermore, a statistically significant association was demonstrated between the mean flock prevalence results as determined by ELISA (77.6%) and by coprological examination (23.7%;P<0.001). Differences in the results obtained by the two diagnostic methods could have been due to fluctuations in the numbers of fluke eggs detected in feces and to the persistence of specific antibodies in serum after and efficacious fasciolicide treatment. The median number ofF. hepatica eggs detected per gram of feces was 10 (range, 5–450 eggs/g feces). The geographical distribution ofF. hepatica infection in Leon province was similar in all natural regions, probably due to the observation that meteorological conditions are not limiting for the maintenance of the parasite life cycle in any area of the province and to the abundance of irrigated areas together with the lack of planned control strategies. No significant association between trematode infection and sheep breed, flock size, or number of treatments was found, but the results showed a significant association between infection and untreated sheep (P<0.05).

Serum and bile antibody responses (IgG and IgA) during subclinical Fasciola hepatica infection in sheep

The kinetics of serum and bile immunoglobulins (IgG and IgA) directed against Fasciola hepatica in the course of subclinical infection induced experimentally was investigated in sheep. Serum activities of glutamate dehydrogenase and gamma-glutamyltransferase were used as markers of the different fluke stages during infection and associated liver damage. Specific serum and bile immunoglobulins followed a similar kinetic pattern, increasing progressively from infection throughout the prepatent period and tended to decrease when adult flukes became established in the bile duct. IgA titres were lower than those of IgG. Specific IgG and IgA bile titres reached maximum values at 14 weeks postinfection that were considerably lower than the serum titres during the whole experimental period. The major bile immunoglobulins are probably derived directly from plasma. The immunoglobulin kinetic pattern could be related to changes in serum liver enzyme activities.

The effects of subclinical fasciolosis on hepatic secretory function in sheep

The effects of subclinical fasciolosis at various stages of its development on bile flow and bile acid secretion and on the hepatobiliary transport of bilirubin were investigated in experimentally infected sheep. Bile flow was significantly reduced by weeks 6–14 postinfection. This was accompanied by a decrease in bile acid secretion by weeks 6–8. Serum AST and GLDH activities and serum bile acid concentration were significantly elevated from weeks 6 to 14. Total serum bilirubin was maximally increased at 6 weeks postinfection and remained elevated at weeks 8 and 14. Increases corresponded to both unconjugated and conjugated fractions, although the conjugated/total bilirubin ratio was enhanced in all infected animals. Biliary bilirubin secretion declined from weeks 6 to 14. No alteration was detected in either uridine diphosphate (UDP)-glucuronosyltransferase activity, cytochrome P-450 concentration, or hematological markers of hemolysis. This study shows that the migration of immature flukes in the course of ovine fasciolosis causes a cholestatic phenomenon responsible for changes in serum and biliary bilirubin levels.

Is the anthelmintic effect of heather supplementation to grazing goats always accompanied by anti-nutritional effects?

To test the hypothesis that the beneficial anthelmintic effect of consuming moderate amounts of tannins may not always be accompanied by anti-nutritional effects in goats, two experiments were conducted. In the first, 48 Cashmere goats were randomly assigned to two treatments: supplementation with tannin-containing heather (6.4% total tannins) and non-supplementation. All goats grazed continuously from May to September under farm conditions in a mountainous area of northern Spain. The mean percentage of heather incorporated into the diet of the supplemented animals was 29.1%. Supplementation reduced the mean number of nematode eggs in faeces (P < 0.001) and the goat mortality rate (P < 0.05). The rumen ammonia concentration was markedly reduced in the goats receiving the heather supplement (160 v. 209 mg/l; P < 0.01), while volatile fatty acid (VFA) concentrations were significantly greater (63.0 v. 53.6 mmol total VFA/l; P < 0.05). The heather-supplemented goats also showed a lower loss of live weight (P < 0.01) and body condition score (P < 0.001). In the second experiment, batch cultures of rumen microorganisms with rumen fluid from nine goats whose diet included 29% heather - or not, were used to incubate three substrates (pasture, pasture + heather and pasture + heather + polyethylene glycol) to investigate in vitro ruminal fermentation. Differences (P < 0.01) among substrates were observed in terms of dry matter disappearance (DMD), in vitro true substrate digestibility (ivTSD), gas production and ammonia concentration, the greatest values always associated with the pasture substrate. Cultures involving rumen inoculum derived from goats receiving the heather-containing diet showed slightly lower DMD (46.9 v. 48.5 g/100 g; P < 0.05), ivTSD (64.6 v. 65.9 g/100 g; P < 0.10) and gas production (105 v. 118 ml/g; P < 0.001) values, but much greater total VFA concentrations (48.5 v. 39.3 mmol/l; P < 0.05), and suggest that the efficiency of ruminal fermentation in these animals was probably improved. Together, the results support the absence of a clear nutritional cost counteracting the beneficial anthelmintic effect of supplementing the diet of grazing goats with tannin-containing heather.

Experimental ovine fasciolosis: antipyrine clearance as indicator of liver damage

Antipyrine clearance was used to assess microsomal oxidative function in eight female Churra breed sheep at 20, 30, 40, 60, 80 and 100 days after infection by an oral administration of 150 metacercariae of Fasciola hepatica. Experimental infection was ascertained by an ELISA test and by faecal analysis. A significant increase in plasma glutamate dehydrogenase (GLDH) activity from 20 days post-infection and in gamma-glutamyltransferase (GGT) activity from 40 days post-infection was found. Both enzyme activities reached maximum levels in plasma of infected sheep at 80 days post-infection, progressively decreasing thereafter. A significant reduction in the total plasma clearance of antipyrine occurred from 60 to 100 days post-infection and a significant increase in mean residence time occurred by 80 days post-infection. The decrease of antipyrine metabolism coincided with the entrance of parasites in bile ducts and the highest liver damage caused by migrating juvenile flukes.

Influence of the gestational stage on the clinical course, lesional development and parasite distribution in experimental ovine neosporosis

Neospora caninum is considered one of the main causes of abortion in cattle, yet recent studies have also emphasised its relevance as an abortifacient in small ruminants. In order to gain deeper insight into the pathogenesis of ovine neosporosis, pregnant ewes were intravenously inoculated with 106 tachyzoites of the Nc-Spain7 isolate at days 40, 90 or 120 of gestation. Infection during the first term resulted in the death of all foetuses between days 19 and 21 post-infection, showing mainly necrotic lesions in foetal liver and the highest parasite DNA detection and burden in both placenta and foetal viscera. After infection at day 90, foetal death was also detected in all ewes, although later (34–48 days post-infection). In this group, lesions were mainly inflammatory. Foetal livers showed the lowest frequency of lesions, as well as the lowest parasite detection and burden. All ewes infected at day 120 delivered viable lambs, although 3 out of 9 showed weakness and recumbency. Neospora DNA was detected in all lambs but one, and parasite burden was similar to that observed in day 90 group. Lesions in this group showed more conspicuous infiltration of inflammatory cells and higher frequency in foetal brain and muscle when compared to both previous groups. These results highlight the crucial role that the stage of gestation plays on the course of ovine neosporosis, similar to that reported in bovine neosporosis, and open the doors to consider sheep as a valid model for exogenous transplacental transmission for ruminant neosporosis.

Experimental ruminant models for bovine neosporosis: what is known and what is needed

At present, bovine neosporosis is an important worldwide concern because of its wide geographic distribution and economic impact. Abortion is the main clinical sign of bovine neosporosis in both dairy and beef cattle. Ruminant challenge models are critical to evaluate potential vaccine candidates to help tackle bovine neosporosis and to study pathogenesis and host responses to infection. Several research groups have developed ruminant models of Neospora caninum infection independently of others, resulting in a high degree of variability due to the use of different species of animals, breeds, strains/isolates of N. caninum, doses, routes and times of inoculation. Standardization is greatly needed to advance research in a more collaborative, timely and efficient manner. In the absence of widely accepted international guidelines, this manuscript serves to summarize and discuss the different models and parameters currently in use. Parameters essential for the development of non-pregnant and pregnant ruminant models are outlined and the main knowledge gaps are identified. This information could act as the basis to develop a consensus for international standard guidelines for ruminant models of neosporosis that would be helpful for researchers in this field worldwide.