Experimental concurrent infection of sheep with Oestrus ovis and Trichostrongylus colubriformis: effects of antiparasitic treatments on interactions between parasite populations and blood eosinophilic responses

The purpose of this experiment was to determine if an earlier infection with Oestrus ovis would down regulate an infection with Trichostrongylus colubriformis when the larvae of O. ovis were expelled from the nasal cavities of sheep by a specific treatment. Three groups of five lambs were used: group 1 was artificially infected with O. ovis larvae and later with T. colubriformis, group 2 received O. ovis larvae and later was treated with ivermectin 14 days before being infected with T. colubriformis. Group 3 was infected with T. colubriformis only. The criteria examined were: the effects on nematode egg excretion, worm fecundity, nematode burdens and the kinetics of blood eosinophils. Significant decreases of nematode egg excretion, worm fecundity, nematode burdens were observed in group 1 compared to group 3. However, no changes were observed in either group 2 or 3. In group 2 it was noted that antiparasitic treatment induced a rapid decrease in blood eosinophils to a range close to the non-infected control group and this was associated with the removal of the down regulation effects of nematode burdens. This experiment showed that there is no cross immunity between O. ovis and T. colubriformis and that eosinophils may act against any parasite without specific priming.     

Oestrus ovis in sheep: relative third-instar populations, risks of infection and parasitic control.

Oestrus ovis (L.) (Diptera: Oestridae), the nasal bot fly, has a relatively short free-living life cycle outside of the host, and therefore it is necessary to know when the parasitic period occurs in order to prevent the clinical signs and economic losses caused by this parasite. The length of this parasitic portion of the life cycle is quite variable: a few weeks to several months depending on the season and climatic conditions. Surveys of Oestrus ovis larval populations in sheep show different results on the number of generations according to the local climate. Mean monthly larval profiles of L1 and L3 burdens of sheep from West African Sahelian countries, Mediterranean countries (Morocco, Tunisia and Sicily) and Southwest France were compared. Valuable information on the suspected extension of the fly season is obtained showing the period of infection in each area. This knowledge will be a valuable tool to help in choosing the right treatment at the right period.     

Experimental concurrent infection of sheep with Oestrus ovis and Trichostrongylus colubriformis: negative interactions between parasite populations and related changes in the cellular responses of nasal and digestive mucosae

Concurrent infections of sheep with Oestrus ovis and trichostrongyles of the digestive tract are common in the field. In order to examine the possible occurrence of interactions between these two parasites and the consequences on parasite biology and the associated pathophysiological changes, an experimental study was conducted on four groups of na?ve sheep. Group O was infected repeatedly with O. ovis larvae from D0 to D42 of experiment; group T received a single infection of Trichostrongylus colubriformis on D49; sheep from group OT received both infections, and animals from group C remained uninfected as controls. Parasitological and pathophysiological parameters were measured regularly. At necropsy (D94), the mucosal cellular responses in the nasal cavities and digestive tract from all animals were analysed from histological sections. Infection of the digestive tract with nematodes did not modify the biology of Oestrus populations. In contrast, the presence of O. ovis was related to significant reductions in nematode egg excretion, worm fecundity and worm burdens. These changes were associated with significant modifications in tissular populations of mast cells, globule leucocytes and eosinophils in the respiratory and digestive tracts. These results indicate that parasitic infection in one particular anatomical site induces "at distance" inflammatory reactions of the whole mucosal system. The relationships between the cellular response and the changes in worm biology are discussed.     

Parasitism by Oestrus ovis: influence of sheep breed and nematode infections

Previous studies showed that Santa Ines (SI) hair sheep were more resistant to gastrointestinal nematode infections (GIN) than Ile de France (IF) sheep. The present experiment aimed to evaluate if that reported resistance difference against GIN also occurred against Oestrus ovis infestation and also to evaluate the influence of O. ovis infestation on the gastrointestinal nematodes (GIN) infections. SI (n=12) and IF (n=12) young male lambs were weaned at 2 months of age and moved to a paddock (0.3 ha) with Brachiaria decumbens grass, where they also received concentrate ration. The animals were kept together during the experimental period (September to early December 2009). Fecal and blood samples were taken from all animals every 2 weeks and body weight and nasal discharge score (oestrosis clinic signs) were recorded on the same occasion. In early December 2009, all lambs were sacrificed and O. ovis larvae and GIN were recovered, counted and identified according to the larval stage. All animals were infested by different larval instars of O. ovis without any statistical difference between breeds (P>0.05). The SI lambs had an average of 24.8 larvae, and the intensity of infection ranged between 14 and 39 larvae, while the IF lambs showed an average of 23.5 larvae with the minimum and maximum from 11 to 36 larvae, respectively. SI lambs presented the lowest nematode fecal egg counts (FECs) and the lowest mean numbers of Haemonchus contortus, Trichostrongylus colubriformis and Strongyloides papillosus, however, there was no significant differences between group means (P>0.05). Inverse relationship between numbers of O. ovis larvae and gastrointestinal nematodes was observed in both breeds. SI sheep showed a significant increase in blood eosinophils and total IgE serum levels and these variables were negatively correlated with nematode FEC. A negative correlation was observed between total IgE serum level and H. contortus burden in both breeds. In conclusion, there was no breed difference regarding O. ovis infestation and in each breed, animals with more nasal bot fly larvae tended to display smaller worm burden.     

Genetic structure of Oestrus ovis populations in sheep and goats.

A genetic analysis using RAPD markers was performed on 12 natural populations of Oestrus ovis (Linné, 1761). Three-hundred and six O. ovis larvae (first, second and third instars) were randomly recovered in nasal cavities of sheep and goats naturally infected in Algeria, Ethiopia, France, Mauritania, Rumania and Tunisia and were analysed by 56 RAPD fragments. The results showed a high diversity within all samples. A significant genetic divergence was showed by discriminant analyses among the 12 populations sampled (p<0.0001). Moreover, discriminant analyses showed significant differentiation (p<0.0001) between O. ovis larva populations of sheep and goats and also among samples collected in the same region.     

Seroprevalence of Oestrus ovis infection in sheep in southwestern Germany

The aim of the survey was to determine the seroprevalence of Oestrus ovis infection in flocks in southwestern Germany. Serum samples collected from 1497 sheep (>6 months of age) of 110 flocks in 1997 and 1998 were examined for antibodies to crude somatic antigens of O. ovis second-stage larvae using an ELISA test. Data on the farm management were obtained by a questionnaire. Overall, 76% of the flocks had at least one seropositive animal, and the seroprevalence of anti-Oestrus antibodies was 50% in sheep. Flock size was the only risk factor significantly associated with the detection of antibodies. Larger flocks (>50 ewes) were more likely to be seropositive than smaller ones. These results show that Oestrus infections are widespread in sheep in southwestern Germany. Further investigations are required to estimate the economic importance of oestrosis and the efficiency of control measures.     

Regulation of Teladorsagia circumcincta and Trichostrongylus colubriformis worm populations by grazing sheep with differing resistance status

In an experiment lasting 4 years, changes in the Teladorsagia circumcincta and Trichostrongylus colubriformis populations were compared in lambs and adult sheep with differing resistance statuses. Two flocks of 30 rams (resistant R and susceptible S) grazed separate pastures and 8 rams were slaughtered in the middle and at the end of each grazing season. Five groups of tracer lambs were added each year to estimate the pasture infectivity and were killed for worm counts. The availability of animals with differing resistance statuses (rams and tracer lambs) and differing levels of infection made it possible to investigate the number, size and fecundity of worms of these two species. The inflammatory response was measured in the rams by counting the globule leukocytes, mast cells and eosinophils in the fundic, pyloric and intestinal mucosa. In the tracer lambs, the daily egg production by the female worms of both species was negatively correlated with the worm burden. Worm length accounted for 60 and 70% of the variation in the number of eggs in utero for T. circumcincta and T. colubriformis respectively. Worm length was closely associated with the resistance status of the host; there were greater differences between lambs, and S and R rams for T. colubriformis. T. circumcincta worm lengths were not affected by the worm number. Globule leukocyte counts were related to the worm burdens, and mast cell counts to worm length in the R and S rams. The number, size and fecundity of the worms may well be regulated by similar mechanisms in both species, but T. colubriformis seemed to be more intensively regulated than T. circumcincta. This finding could be useful in devising more effective methods of parasite control.     

Epidemiology of Oestrus ovis infection of sheep in the highveld of Zimbabwe

During a period of 13 months, 507 heads of sheep, obtained from an abattoir near Harare, were examined for infection with Oestrus ovis larvae. The prevalence of infection varied from 6 to 52%, the highest being in November and the lowest in April. The mean annual larval burden was 1.12. The maximum number of larvae recovered from a single head was 57 in the month of November. Two larval peaks were observed, the first and highest in November and the second in August/September. Some flies are present throughout the year, except in May. There are at least 3 generations of flies per year. The wet summer period from January to May seems to be unfavourable, as very few flies are present. There is no overwintering of first instar larvae in the heads of sheep.     

Evaluation of a direct ELISA for the serodiagnosis of Oestrus ovis infections in sheep.

Oestrosis is a parasitic disease of sheep and goats caused by the nasal bot fly Oestrus ovis. In the United Kingdom the economic losses as a result of infestation can be considered negligible, but the differentiation of O ovis cases from more serious diseases such as listeriosis, gid and sheep scab is of considerable importance. Currently, diagnosis of oestrosis relies on the subjective observation of clinical signs or the demonstration of larvae postmortem. This paper assesses the effectiveness of a direct enzyme-linked immunosorbent assay (ELISA) using a crude somatic antigen from first-stage larvae (L1) in the serodiagnosis of oestrosis. The system has been validated with sera from both endemic and non-endemic areas and the results correlated with the clinical data found postmortem. The sensitivity and specificity of the assay were 97.4 per cent and 97.6 per cent, respectively, using a cut-off point based on 35 per cent binding of a reference positive control serum.     

Regulation of Trichostrongylus vitrinus and T colubriformis populations in naturally infected sheep in the Netherlands

During a study on the epidemiology of gastrointestinal helminth infections in sheep at the University of Utrecht (1974 to 1977) information was obtained on the population regulation of small intestinal Trichostrongylus species. Comparison of the cumulative burdens in tracer lambs with those in flock lambs showed an accumulation of Trichostrongylus species in the latter until autumn followed by an expulsion of worms. The dominating species, T vitrinus, was expelled earlier than T colubriformis. The proportion of inhibited early third stage Trichostrongylus species larvae in tracer lambs was low, but it tended to increase in autumn. In most flock lambs the levels of inhibition were also low. In non-lactating ewes, populations consisted predominantly of inhibited early third stage larvae but when adults were present they tended to be mainly T colubriformis. Similar populations were seen in some lactating ewes. In other lactating ewes Trichostrongylus species populations with low proportions of inhibited early third stage larvae which were dominated by T vitrinus, indicated a relaxation of resistance around parturition.     

Towards a lower prevalence of Oestrus ovis infections in sheep in a temperate climate (south west France)

Oestrus ovis larvae are obligatory parasites of the nasal and sinus cavities of sheep and goats. In the temperate climate of western Europe, fly attacks occur between May and October and the first stage larvae arrest their development within the host between October and February. Oestrosis clinical signs such as nasal discharge and sneezing are well known by sheep breeders in southwest France. According to veterinarian recommendations, most of them treat their animals with long lasting fasciolicides once a year at least, mainly during the fly activity period and at the beginning of the hypobiotic period (when the parasitic population is only constituted of larvae). The consequences of these therapeutic programs were analysed in a local slaughterhouse by larval counts. Both prevalence and intensities of O. ovis infections decreased between 1989-1991 (before the use of systematic treatments) and 1996-1998 (after the spread of these treatments). The use of systematic treatments during the fly activity period and the beginning of the hypobiotic period seems to be very efficient in O. ovis control and could theoretically lead to a possible 'eradication' program as with cattle hypodermosis. Nevertheless the presence of parasites in apparently healthy goats, the possibility for a fly generation to develop before the first treatment in July-August and the succession of several fly generations all around the year in southern Mediterranean and tropical countries will maintain O. ovis infections. Furthermore, there are increased concerns about drug residues on consumer health and environment and this is the basis for the prospect of alternative strategies in O. ovis control.     

Comparison of infection rates of Oestrus ovis between sheep and goats kept in mixed flocks

Oestrosis is a nasal myiasis of sheep and goats caused by larvae of the fly Oestrus ovis and can lead to severe clinical signs, which together with the disturbance caused by the adult fly may result into serious economic losses. Infection rates and larval burdens are always higher in sheep than in goats after either natural or artificial infestation. The aim of this study was to compare the host preference of the adult fly O. ovis between sheep and goats in mixed flocks, where they are kept together under the same husbandry conditions and hence, are very similarly exposed to the fly preference. Blood sera samples were collected from a total of 397 sheep and 335 goats, from 43 mixed flocks located at different regions of Greece. Antibodies specific to O. ovis IgG were measured by ELISA. A flock was considered positive when at least one individual was positive, i.e. showed a seropositivity of >or=20% in relation to positive control sera. A total of 193 (48.6%) sheep and 58 (17.9%) goats were found to be seropositive against O. ovis. Thirty-eight (88.4%) out of 43 flocks had at least one seropositive animal. The mean seroconversion against O. ovis in animals from the different flocks was 38.6% and 13.6% for sheep and goats, respectively, whereas the variance of infection within each flock was 0-100%. The mean seropositivity between sheep that were found to be positive or negative was 60.6% and 5.4%, respectively, whereas the corresponding values between goats were 35.2% and 5.2%, respectively. No significant difference in the seroconversion values was noted between flocks from the different areas (P=0.817), whereas a very significant difference was observed between animal species (P=0.001). However, there was no significant difference when seroconversion comparisons were made within samples of the same animals species, sheep or goats from different flocks of all the regions included in the study (P=0.695). The results of this study clearly demonstrate that O. ovis has a widespread distribution in Greece, and the seroprevalence is significantly higher in sheep than goats (P=0.001).     

Evidence for the presence of nematode-derived acetylcholinesterase in sheep infected with Trichostrongylus colubriformis

The distribution of acetylcholinesterase isoenzymes in the ovine intestinal nematode Trichostrongylus colubriformis was compared with that in chronically infected and worm-free lambs. Total acetylcholinesterase activity in homogenates of adult T colubriformis was resolved into five isoenzyme peaks following gel electrophoresis and specific esterase staining. Two isoenzymes with electrophoretic mobilities similar to those present in adult worm homogenates were detected in mucosal homogenates and plasma extracts from all of six sheep chronically infected with T colubriformis, but not in similar preparations from two uninfected animals.     

Effects of molybdenum intake on primary infection and subsequent challenge by the nematode parasite Trichostrongylus colubriformis in weaned Merino lambs.

In a study designed to test the effect of molybdenum (Mo) on resistance to trichostrongylosis, the Mo content of the diet during primary infection of 8-month-old Merino lambs with Trichostrongylus colubriformis was found to affect the number of worm eggs found in the faeces during challenge and the total number of worms in the jejunum after six weeks of challenge infection. The optimal intake of Mo in this experiment was in the range of 4-8 mg sheep(-1)day(-1), approximately equivalent to feeding a diet containing 6-10 mg Mo kg(-1)dry matter (DM) and to 0.15-0.30 mg Mo kg(-1)liveweight. Lambs fed Mo at this rate showed a 90 per cent reduction in faecal egg count and total worm count six weeks after challenge compared with lambs fed quantities of Mo outside this range. The mechanism by which Mo exerted these effects was not defined, although the interactions of molybdenum, worm establishment and faecal egg count suggested that this trace element may be acting via an effect on the host's acquired immune response. This hypothesis is supported by the observed enhancement of immune responses (intestinal antibody and granulocyte numbers and in vitro worm-specific proliferation of lymphocytes) associated with Mo intake of 0.15-0.3 mg kg(-1)Lwt.     

Total and nematode-specific IgE responses in intestinal lymph of genetically resistant and susceptible sheep during infection with Trichostrongylus colubriformis

Total and antigen-specific IgE responses in afferent (AIL) and efferent (EIL) intestinal lymph of sheep with a nematode resistant (R) or susceptible (S) genotype during challenge infection with the intestinal nematode parasite Trichostrongylus colubriformis were examined. Within each sheep line, lambs with a nematode naive or nematode field-primed pre-challenge status were used. Total IgE level in AIL and EIL was dependent on nematode infection and was further influenced by genotype or the immune phenotype (nematode immune mean FEC+/-SDM=77+/-179 or non-immune mean FEC+/-SDM=4016+/-4318) of the animal. During T. colubriformis challenge immune animals had higher levels of total IgE in lymph than non-immune sheep, R line sheep had higher concentrations of total IgE than S line sheep, and field-primed animals had higher total IgE levels than nematode naive animals. Concentrations of total IgE were consistently higher in AIL than EIL or serum and were higher in lymph draining the proximal than the distal jejunum demonstrating that polyclonal IgE in AIL was largely derived from the intestinal mucosa of the anatomical compartment where the nematodes reside. The consistently higher concentration of total IgE in AIL was dependent on phenotype or genotype and in S genotype sheep also on the pre-challenge status. Concentrations of nematode specific IgE were significantly higher in EIL than AIL indicating a preference for the production of IgE reacting with excretory secretory products of the infective T. colubriformis larvae in the regional lymph node.     

Cytokine and antibody subclass responses in the intestinal lymph of sheep during repeated experimental infections with the nematode parasite Trichostrongylus colubriformis

The expression of interleukin (IL)-4, IL-5, IL-10, IL-13, TNF-alpha and IFN-gamma genes, and parasite-specific IgM, IgG1, IgG2, IgA and total IgE levels, were monitored daily in intestinal lymph of sheep infected repeatedly with the nematode parasite Trichostrongylus colubriformis. Host genotype had a significant influence on IL-13 gene activity, with resistant-line (R) sheep consistently expressing higher levels of mRNA than susceptible-line (S) sheep. Mean gene expression of IL-13, IL-4 and IFN-gamma did not differ significantly between the first and second nematode challenge. Field-primed R and S as well as field-primed R and na?ve S sheep had lower mean gene expression of IL-5 and IL-10, respectively, during the second when compared to primary challenge. Genes for IL-13 and IL-5 were transiently and strongly up-regulated after nematode infection, particularly in animals with previous exposure to nematodes. Genes for TNF-alpha and IFN-gamma were also transiently up-regulated, but to a lesser extent and more typically after primary challenge. Na?ve sheep of both genotypes produced relatively little antibody response after primary challenge. A second nematode challenge resulted in large increases in the lymphatic levels of all antibody sub-classes which were significant for adult antigen-specific IgA and larval antigen-specific IgG1. In na?ve S line sheep, the larval-specific IgA and IgG2 response appeared delayed when compared to the R line animals. Field-primed R and S line sheep had relatively high lymphatic IgG1 levels prior to experimental infection and these did not change significantly afterwards. These results demonstrate that during nematode infections, the intestinal micro-environment of sheep is transiently skewed towards Th2 cytokine dominance, although IFN-gamma gene expression continues. This response is accompanied by increases of nematode-specific IgG1, IgA, IgG2 and IgM, as well as of total IgE in lymph plasma.