In vitro and in vivo effects of neem tree (Azadirachta indica A. Juss) products on larvae of the sheep nose bot fly (Oestrus ovis L. Díptera: Oestridae).

Two studies were carried out in order to test the effects of neem tree extracts (Azadirachta indica A. Juss) on sheep bot fly larvae (Oestrus ovis L. Diptera: Oestridae). First, aqueous extracts from neem seeds (ASNE) at 0, 5 y 10% (w/v) concentrations were tested on larval mortality in vitro. In a second study, the effect of oral administration with neem seed meal (0, 100 y 200mg/kg) and neem leaves (1% of diet) on number of larvae found at necropsy and larval development was evaluated in experimentally O. ovis-infected sheep. Results in Experiment 1 showed a significant (P<0.05) effect of ASNE on time to L1 mortality in a dosis-dependent manner. In Experiment 2, oral administration of seeds or leaves did not affect the number of larvae found at necropsy of the sheep, but interfered with larval development and there was a tendency to reduce larval weight at the end of the infection period (55d).

Sheep and goat immune responses to nose bot infestation: a review.

Oestrus ovis L. (Diptera: Oestridae) is a cosmopolitan agent of myiasis in sheep and goats. The parasitic phase begins after adult females deposit first-stage larvae (L1) into the nostrils of hosts; these larvae develop into L2 and L3 in the nasal and sinus horn cavities. Sneezing and nasal discharges are the major clinical signs in infected animals. The pathogenesis of O. ovis infection is caused by: (a) the trauma resulting from the mechanical action of spines and hooks during larval movement on mucosal membranes, and, more importantly, (b) an allergenic reaction provoked by molecules excreted/secreted by larvae, of which salivary antigens are those mainly recognized by the host's immune system. The recruitment of immune reactive cells increases gradually from the nasal to sinus cavities in infected hosts. Mast cells, eosinophils, macrophages and lymphocytes are always more numerous in infected than non-infected animals. Humoral (antibody) systemic response of immunoglobulin G (IgG) usually reaches seroconversion 2-4 weeks post-first infection and the highest levels are observed during the development of L2 and L3 larvae. Local antibody responses include specific IgG, which has been found to negatively correlate with larval survival and development. Hypersensitivity reaction, immunomodulation, immunization trials and mixed infections of O. ovis and helminths are discussed.

Practical aspects of equine parasite control: a review based upon a workshop discussion consensus.

Development of resistance of several important equine parasites to most of the available anthelmintic drug classes has led to a reconsideration of parasite control strategies in many equine establishments. Routine prophylactic treatments based on simple calendar-based schemes are no longer reliable and veterinary equine clinicians are increasingly seeking advice and guidance on more sustainable approaches to equine parasite control. Most techniques for the detection of equine helminth parasites are based on faecal analysis and very few tests have been developed as diagnostic tests for resistance. Recently, some molecular and in vitro based diagnostic assays have been developed and have shown promise, but none of these are currently available for veterinary practice. Presently, the only reliable method for the detection of anthelmintic resistance is a simple faecal egg count reduction test, and clinicians are urged to perform such tests on a regular basis. The key to managing anthelmintic resistance is maintaining parasite refugia and this concept is discussed in relation to treatment strategies, drug rotations and pasture management. It is concluded that treatment strategies need to change and more reliance should now be placed on surveillance of parasite burdens and regular drug efficacy tests are also recommended to ensure continuing drug efficacy. The present review is based upon discussions held at an equine parasite workshop arranged by the French Equine Veterinary Association (Association Vétérinaire Equine Française, AVEF) in Reims, France, in October 2008.

Eosinophils in Haemonchus contortus-infected resistant and susceptible breeds of sheep: abomasal tissue recruitment and in vitro functional state.

A comparative experimental study was performed between Barbados Black Belly (resistant) and INRA-401 (susceptible) breeds of sheep in which primary infection with Haemonchus contortus was terminated on day 16. Measurements of parasite burden, abomasal tissue eosinophilia, IL-4, IL-5 and IL-13 mRNA transcripts in abomasal mucosa, and in vitro larval killing abilities of blood eosinophils were performed. The results show that: (1) worm burden was significantly lower and blood eosinophilia higher in the Black Belly than in the INRA breed. (2) Abomasal cytokine expression was noticed but no difference existed between the two breeds. (3) Three out of four Black Belly sheep had higher tissue eosinophil numbers compared to the INRA sheep (more eosinophils observed in the pyloric than in the fundic region in both breeds). (4) No significant difference was observed in the in vitro larval immobilizing potential of eosinophils between the two breeds. Collectively, abomasal eosinophil number and larval killing abilities of blood eosinophils do not seem to explain the difference in worm burden between the two breeds.

Specific IgG antibody responses in Oestrus ovis L. (Diptera: Oestridae) infected sheep: associations with intensity of infection and larval development.

Larvae of Oestrus ovis (Diptera: Oestridae) are ubiquitous parasites of nasal and sinusal cavities of sheep and goats. According to the chronobiology of O. ovis infections in Sardinia and the seasonal pattern of the IgG response, the optimal period to investigate the relationships between O. ovis larval populations and intensity of local and systemic IgG antibody responses was mid-July in the summer season. Sarda x Lacaune ewes (n=186), divided into three ram-families were used in the study. Systemic and local IgG responses were measured by ELISA tests using second stage larval crude extracts (L2CE) and L2 (L2SGC) and L3 (L3SGC) salivary gland contents as coating antigens. The number of larval instars, larval length of L1, L2 and L3 larvae, and larval weight of L2 and L3 larvae were individually recorded after ewe necropsy. Negative correlations among larval establishment and/or larval development on the one hand and intensity of local or systemic IgG responses on the other hand were found in two out of three studied ram-families.

[Does 2003 summer heat wave modified prevalence of bovine helminthosis in southwestern France?]. / La prevalence des helminthoses bovines a-t-elle été modifite par la canicule de l'éit 2003 dans le sud-ouest de la France?

The heat wave of 2003 summer had serious consequences on helminths prevalence and epidemiology. Comparison of 1917 cattle epg counts in southwestern France for years before and after showed that prevalence and mean numbers of Fasciola hepatica, paramphistomids, Dicroccelium lanceolatum and gastrointestinal strongyles eggs were significantly reduced. Nevertheless, the next year the epg counts were quickly increasing. Several factors may have induced a more or less long modification: strains of helminths may become adapted, a new spreading of intermediate hosts may occur with a modified ecology and finally moving of hosts and reservoirs may be amongst causes of epidemiological changes. Finally, after a heat wave the anthelmintic pressure may be maintained, as parasites seem quickly become adapted to a novel and modified environment.

Proteolytic activity in salivary gland products of sheep bot fly (Oestrus ovis) larvae.

This study identified and characterized hydrolytic enzymes in salivary gland products of Oestrus ovis larvae. Third instars were collected from the heads of slaughtered goats. Salivary glands were extracted, their products obtained by centrifugation and the enzymatic profile determined. Optimum pH, temperature of maximum proteolytic activity, thermal stability, and resistance of salivary gland products were determined on collagen and subclasses of proteases were identified using protease inhibitors. Zymograms were used to determine the molecular weight of proteases. Antigenic protein bands were revealed by immunoblotting using sera obtained from experimentally infested goats. Seven positive enzymatic activities were detected in salivary gland products: acid phosphatase, naphthol-AS-BI-phosphohydrolase, esterase (C4), esterase lipase (C8), leucine arylamidase, alpha-glucosidase and N-acetyl-beta-glucosaminidase. Optimum pH for proteolytic activity was 8.0; proteolytic activity increased with temperature (10-50 degrees C) then drastically decreased at 60 degrees C. Proteases in O. ovis salivary gland products belong to the serine subclass. In Zymograms, bands of proteolytic activity were detected in the 20-63 kDa range; the immunoblot showed three antigenic bands, one of them related to a protease band (63 kDa). Serine proteases in O. ovis salivary gland products are most likely involved in larval nutrition and host immuno-modulation.

Immune response to Haemonchus contortus infection in susceptible (INRA 401) and resistant (Barbados Black Belly) breeds of lambs.

The immune responses to Haemonchus contortus were compared in studies in resistant Barbados Black Belly (BBB) and susceptible INRA 401 (INRA) breeds of lambs. The cytokine patterns indicated a Th2-biased response in both breeds. A more persistent and elevated Th2 cytokine mRNA transcription and blood eosinophilia were noted in the BBB lambs. However, at days 4 and 30 post-infection, abomasal recruitment of eosinophils and mast cells were similar between the two breeds. Following primary infections, the BBB demonstrated a substantially lower faecal egg count compared to the INRA lambs. Furthermore, worm counts at 4 and 30 days post-infection, and adult female worm size and in utero egg counts 30 days after the first infection were significantly lower in the BBB than in the INRA breed. In the INRA breed, re-infection caused a significant reduction in most parasitological parameters compared with those observed after the primary infection. A similar response was not observed in the BBB sheep. In conclusion, while the major driving force in the response to H. contortus infection is a Th2-biased immunity in which the BBB showed its maximal performance during the primary infection, the INRA breed performed better after re-infection compared to its response to first exposure.

Serological survey of caprine toxoplasmosis in Ethiopia: prevalence and risk factors.

The study was conducted to determine the prevalence and risk factors of toxoplasmosis in goats in Southern and central Ethiopia between October 2005 and May 2006. A total of 641 goats sera were tested using Modified Direct Agglutination Test (MAT), of which 480 (74.8% CI: 71.3, 78.2) were found to be positive. The highest prevalence was recorded in South Omo zone (82%) while the lowest was observed in East Shewa zone (62.2%). The study revealed that goats raised in southern Ethiopia are at a greater risk of acquiring T. gondii infection (OR = 2.55, CI: 1.726, 3.776; p = 0.000) than those which are raised in central Ethiopia. The prevalence of anti T. gondii antibody was significantly higher in older goats than in kids (OR = 2.33, CI: 1.490, 3.655; p < 0.0002) and in females than in males (p < 0.0007; OR = 0.68, CI: 0.542, 0.849). No significant difference was observed among goats kept under various husbandry practices. The high prevalence of toxoplasmosis in Ethiopian goats suggests a high risk of human infections. Further epidemiological investigation, isolation and genotyping of T. gondii are planned.

Trends and challenges in the effective and sustainable control of Haemonchus contortus infection in sheep. Review.

Haemonchosis, with its very wide distribution, has become a very important production constraint in sheep farms in tropical, subtropical and temperate regions worldwide. Various intrinsic and extrinsic factors determine the survival of Haemonchus contortus and hence the development of the disease in the animal. In general, control of gastrointestinal nematode infestation in sheep relies heavily on anthelmintic treatments. However, the indiscriminate use of these drugs has led to the widespread emergence of drug resistant strains of parasites, that has necessitated the development and use of various parasite control methods such as grazing management, biological agents and vaccines and the selection of resistant breeds of animals, with or without moderate use of anthelmintics. The ultimate goal of such control programs is to enhance productivity, while minimising risks regarding drug resistance and consumer and environmental concerns. This review attempts to highlight the different methods employed in the control of haemonchosis in sheep and the practical limitations associated with both control programs and the internal and external factors associated with the parasite and its microenvironment.

Effects of immunization of Pelibuey lambs with Oestrus ovis digestive tract protein extracts on larval establishment and development.

Larval midgut proteins of hematophagous parasites contain strong antigens that can be used for host immunization. This concept has been applied for immunization of Pelibuey sheep against Oestrus ovis L. (Diptera: Oestridae). The aim of this study was to examine the effect of immunization on larval establishment (LE) and development. Immunized lambs (I, n = 6) received two injections of crude gut membrane protein extracts (GMPE) from third instar larvae with Freund's incomplete adjuvant (FIA) on days 0 (Day of first immunization) and 21 (0.4 and 0.45 mg GMPE/lamb, respectively). The control group (C, n = 5) received physiological saline with FIA. Lambs were challenged with first instars on Day 29 (20 larvae) and Day 43 (25 larvae). Blood samples were collected biweekly and IgG titers were analyzed by ELISA. All lambs were slaughtered on Day 90 and number of larvae recovered, larval stage and larval weight were recorded at necropsy. No significant effect of immunization on LE (C = 28.9%; I = 31.0% P > 0.05) was observed. Antibody titers were higher in the immunized group on Day 28 (P < 0.05), but subsequently similar in both groups. Larval physiological age and weight were also significantly (P < 0.05) affected by immunization. Immunization of Pelibuey lambs with GMPE did not affect LE but did delay O. ovis larval development.

Efficacy of eprinomectin pour-on against gastrointestinal nematodes and the nasal bot fly (Oestrus ovis) in sheep.

The efficacy of the pour-on formulation of eprinomectin, at a dose rate of 0.5 mg/kg bodyweight, was assessed in sheep against three main species of gastrointestinal nematodes and against the nasal bot fly, Oestrus ovis, and some pharmacokinetic parameters were determined for 21 days after the treatment. By comparison with untreated control sheep, infected experimentally with Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis, eprinomectin was 100 per cent effective against the two abomasal species and 99.5 per cent effective against T. colubriformis. In ewes naturally infected with the nasal bot fly, the efficacy of the drug against O. ovis was 97.7 per cent. The mean (se) systemic area under the curve (AUC) was 56.0 (26.2) ng/day/ml and the mean residence time was 5.3 (1.0) days, but there were wide variations between individual sheep.

Host specificity of Haemonchus spp. for domestic ruminants in the savanna in northern Ivory Coast.

Three Haemonchus species (Haemonchus contortus, Haemonchus placei and Haemonchus similis) live in sympatry in the savanna of North Côte d'Ivoire. Four domestic ruminant hosts (zebu-cattle, taurine-cattle, sheep and goats) share the same pastures throughout the year. Our post-mortem data imply that a Haemonchus spp. circulation exists among the different hosts species in the savanna of North Côte d'Ivoire. Under field conditions, mixed congeneric infections were the rule except in sheep where mono-specific H. contortus infections were the most frequent. The associations H.contortus+H.placei and H.placei+H.similis were dominant in goats and in cattle, respectively. In the populations examined, triple infections occurred in 11.5% of zebu-cattle and 29% of the taurine-cattle. Considering the intensities of infections, H. contortus was the main Haemonchus species of sheep and goats whereas H. similis was very rare in these host species. About 10% of the worms recovered in goats belong to the H. placei species. In cattle, H. contortus was very rare. H. placei was the dominant species in zebu-cattle (58.7% of the identified worms) and in taurine-cattle (73.9%) nevertheless, the proportion of H. similis was higher in zebu-cattle (38.6%) than in taurine-cattle (21.3%). Only few hybrids between H. placei and H. similis were found. The importance of such Haemonchus spp. circulation is discussed.

Prevalence, abundance and site distribution of equine small strongyles in Normandy, France.

Forty-two horses from Normandy (France) were examined post-mortem for small strongyle infections from October to March. In the positive horses, total worm numbers ranged from 234 to 90,247 (mean 11,297). Encysted larvae represented the major part of the total cyathostome burdens with a high percentage (83%) being early third stage larvae. They were mostly recovered from the caecum (48%) and ventral colon (40%) and were less present in the dorsal colon (12%). Adult cyathostomes were mainly located in the ventral colon (64%) and less frequently in the dorsal colon (27%) and caecum (9%). Twenty species of Cyathostominae were identified. The 10 most prevalent species (in sequence of prevalence) were Cyathostomum coronatum, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, Poteriostomum imparidentatum, Cyathostomum labiatum, Cylicocyclus ultrajectinus, Cylicostephanus calicatus and Cylicostephanus minutus which comprised 84% of the total adult population. Twelve species showed a site preference in the ventral colon, five in the dorsal colon and only one in the caecum while two species were collected in nearly equal numbers from the ventral and dorsal colon. The number of species per horse ranged from 1 to 12 with a median of 5. Infections with singletons occurred in 12.5% of the positive horses while multiple infections were encountered in 87.5%. A positive correlation was found between the intensity of cyathostome infection and its diversity, measured either by the number of occurring species or Shanon indexes.

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.

Mast cell and eosinophil mucosal responses in the large intestine of horses naturally infected with cyathostomes.

From December 1998 to March 2000, caecum and ascendant colon of 42 horses naturally infected with cyathostomes were collected during routine necropsy or from a local slaughterhouse. Changes in the numbers of mucosal and submucosal mast cells (MMC and SMMC), intraepithelial, mucosal and submucosal eosinophils (IE, ME and SME) in the large intestine were investigated by histochemical techniques in relation to the worm burdens. The effect of age was examined in three subgroups: 6-24-month-old horses (group 1), 2-10-year-old horses (group 2) and horses more than 10 years of age (group 3). No globule leucocytes were detected in any sections. No significant variations with breed or sex were observed in cell counts. The main variations were higher eosinophil counts in groups 2 and 3 and a marked increase of the MMC counts in the oldest horses (group 3). For each cell type, the infiltration was homogeneous and generalised along the large intestine. In the whole horse sample, the IE numbers were the only parameters that correlated with the MMC and SMMC counts. Very few significant relationships were found between mast cells and eosinophils in groups 1 and 3, whereas numerous positive correlations were recorded in group 2. In the whole horse sample, several correlations were found between different cell counts and cyathostome burdens. The numbers of larvae, adult worms, and the total worm burdens were related to some of the tissular eosinophil counts while the percentage of early third stage larvae (EL3) was linked to mast cell densities. These relations between cells and worm populations showed variations with age. In group 1, most of the significant associations were found between eosinophil counts (IE and SME) and the total numbers of larvae and worms; in group 2, they were noticed between the three eosinophil types and the total cyathostome burdens. In group 3, a MMC hyperplasia was observed and correlations were mostly recorded between these MMC and the total numbers of adult worms or the percentage of EL3. Several associations were also detected between eosinophils (mainly ME and/or IE) and different cyathostome burdens. These variations in the relationship between inflammatory cells and cyathostomes seemed to be consistent with the cellular changes observed among the three age groups. These results suggest that eosinophil and mast cell infiltrations quantified in the large intestine wall might be associated with cyathostome infection.

Oestrus ovis (Diptera: Oestridae): sheep humoral immune response to purified excreted/secreted salivary gland 28 kDa antigen complex from second and third instar larvae.

The potential of Oestrus ovis larvae excretory-secretory products (ESP), crude extract, salivary gland contents (SGc) and digestive tube contents (DTc) proteins from O. ovis larvae as antigens in serological assay were analyzed and compared. This study demonstrated that excretory-secretory proteins were produced mainly by digestive tube and salivary gland. The main antigenic component of ESP was a 28kDa protein complex (pc28) synthesized by the salivary gland ring. After purification, its diagnostic value was estimated by an ELISA using sheep sera sampled in summer (n=30) and winter (n=69) in order to investigate the relative importance of wintering hypobiotic first instar larvae on animal responsiveness. ELISA test based on pc28 antigen was compared to a reference ELISA test based on second instar larvae crude extract. After comparison it appeared that the pc28 test was much more specific (87%) and more sensitive (90%) in summer. Anti-pc28 antibody titers were significantly higher in summer than in winter demonstrating the impact of summer developing larvae on the humoral systemic immune response. This data was correlated to the maturation of larvae in summer as the number of larvae at necropsy was significantly higher in winter, when the larval population is only represented by first instar larvae, than in summer. No correlation was observed between the mean antibody titers and the number of larvae.

Comparison of nematode infections of the gastrointestinal tract in Angora and dairy goats in a rangeland environment: relations with the feeding behaviour.

A study was conducted in order to examine the possible influence of feeding behaviour on differences in the diversity and the intensity of nematode infections of the gastrointestinal tract between two breeds of goats placed in the same rangeland environment. Within the same flock bred on rangeland conditions, 10 Saanen dairy goats and 14 Angora goats were surveyed for 5 months (from May to September) and parasitological and behavioural parameters were measured. The level of nematode infection was estimated by monthly measurements of faecal egg counts whereas the feeding behaviour was assessed every fortnight. During the whole survey, nematode egg excretion was repeatedly higher in the Angora goats compared to the Saanen ones and significant differences were observed in May and September. This difference in egg excretion was related to major difference in feeding behaviour between the two breeds. In contrast with the Saanen does, Angora goats did not show any browsing and resting activities at any date of observation. Hence, Angora goats can be considered mainly as grazers whereas Saanen goats exhibit feeding activities close to browsers. The relative influence of feeding behaviour and host immune response on the susceptibility to gastrointestinal nematode infection in goats is discussed.

Scanning electron microscopy of larval instars and imago of Oestrus caucasicus (Grunin, 1948) (Diptera: Oestridae).

Oestrus caucasicus (Grunin, 1948) is a larval parasite of the nasal cavities of Capra caucasica, Capra ibex and Capra pyrenaica. This study is the first description of the parasite using scanning electron microscopy. The first larval instar shows minor differences with Oestrus ovis. The second larval instar shows important synapomorphic features common to Oestrus ovis but, also, distinctive features as the spines-crown or the currycomb-shaped spines. The third larval instar shows many differences with Oestrus ovis, mostly in the ventral and dorsal spines. The imagos of the two species have closely related morphologies. This study is a contribution to a revision of phylogeny of Oestridae family.