Analysis of larval antigens of Oestrus ovis for the diagnosis of oestrosis by enzyme-linked immunosorbent assay

A serodiagnostic test for the diagnosis of infestation by the sheep nasal bot fly, Oestrus ovis (Linné) was examined. The enzyme-linked immunosorbent assay (ELISA) technique was used to analyze and compare the production of immunoglobulin G (IgG) antibodies against excretory-secretory products (ESP) and crude extract (CE) antigens from all the different larval stages of O. ovis in the sera of 276 adult sheep sampled in summer (n = 135) and winter (n = 141). ESP from first stage larvae was the most sensitive, coating antigen in winter and ESP from second stage larvae during summer. The most specific values were obtained by ESP against L1 in winter and by CE against L3 in summer. These results show that the stage of larval development has a significant impact on the humoral immune response over the course of a season. A significant correlation (P < 0.001) was found between the number of O. ovis larvae and the serum antibody levels using all differents antigens, except L3 CE. In Spain, where a long favourable period exists for the evolution and development of the different stage larvae between March and November, the ELISA test using L1 ESP antigen during winter and L2 ESP antigen in summer may be used for ovine oestrosis immunodiagnosis.     

Analysis of somatic and salivary gland antigens of third stage larvae of Rhinoestrus spp. (Diptera, Oestridae)

Larvae of Rhinoestrus spp. (Diptera, Oestridae) infect nasal and sinus cavities of horses, causing a nasal myiasis characterized by severe respiratory distress. Presently, the diagnosis of horse nasal botfly relies on the observation of clinical signs, on the post mortem retrieval of larvae or on molecular assays performed using pharyngeal swabs. The present study was carried out to characterize larval somatic proteins and salivary glands of Rhinoestrus spp. in a preliminary assessment towards the immunodiagnosis of equine rhinoestrosis. Out of the 212 necropsied horses 13 were positive for the presence of Rhinoestrus spp. larvae. The analysis of the sera from the infected animals by Western blotting assay showed the presence of a specific host humoral immune response against Rhinoestrus spp. larvae and proved that the salivary glands are the major immunogens in horse nasal botflies.     

Serologic diagnosis of Oestrus ovis (Diptera: Oestridae) in naturally infested sheep

Sera from eighteen control sheep supposed to be free from parasitism by Oestrus ovis Linnaeus, 1761, and from 100 sheep raised in an enzootic area of O.ovis infestations were tested to detect anti-Oestrus antibodies by double immunodiffusion (DD) and indirect haemagglutination (IH) tests with somatic crude antigens from first (L1), second (L2) and third (L3) instar of O.ovis larvae. At necropsy, eighty-eight out of 100 sheep from the O.ovis infested area were found to be parasitized while the eighteen control ovines did not show Oestrus larvae. Examination of the sera from the parasitized sheep by DD showed positive results of 42% for L1, 59% for L2 and 18% for L3. Screening the sera with IH gave sensitivities of 100% for L1, 100% for L2 and 97.7% for L3. Sheep, naturally parasitized by gastrointestinal nematodes, presented no cross immune reactions in DD tests with the three larval stages of O.ovis or with L2 larvae in IH tests.     

Trichinella spiralis: a 76-kDa excretory/secretory larval antigen identified by a monoclonal antibody

Spleen cells from BALB/c mice were fused with myeloma cells following infection of the mice with Trichinella spiralis larvae and an ip booster injection with larval homogenate antigen. A monoclonal antibody (Mab), designated as TS 3G6 which did not react with sera or tissue extracts from noninfected mice, rats, and guinea pigs, was selected for further studies because of its high activity and specificity. When tested in ELISA TS 3G6 did not cross-react with Ascaris suum, A. lumbricoides, Toxocara canis, E. granulosus (larvae), Trichiuris suis, or T. ovis. Western blot analysis showed that Mab 3G6 recognized an antigen of 76 kDa located in the stichosome of the larvae as well as on the surface of the larval cuticle. Digestion of a larval extract with different enzymes suggests that the Mab TS 3G6 corresponding epitope is a polypeptide. The TS 3G6 antigen was detected in culture supernatants of Trichinella muscle larvae and in sera of experimentally infected animals using a sensitive ELISA assay. This secretory antigen also seemed to induce a specific immune response in the host since sera from infected animals could block the binding of Mab TS 3G6 to its target antigen when tested in a competitive ELISA.     

Prevalence and intensity of Oestrus ovis in Akkaraman sheep in the Konya region of Turkey

Slaughterhouse surveys to determine the prevalence and intensity of larval Oestrus ovis Linnaeus (Diptera: Oestridae) in sheep, were conducted monthly for 1 year in Konya, Turkey. A total of 624 sheep, selected at random, were examined and 59% were found to be infested by O. ovis. A total of 8801 larvae were collected, of which 68.9% were first-stage, 19.1% second-stage and 12% third-stage larvae. All three larval stadia were seen in each month of the year. The larval intensity for infected sheep was 23.9, with 16.48 L(1), 4.55 L(2) and 2.87 L(3). The monthly prevalence ranged from 34.6% in January to 76.9% in October. The largest number of larvae (180) was obtained from a sheep in August (122 L(1), 52 L(2) and 6 L(3)). The infestation rate was higher in 4 - 6-year-old sheep, at 72.6%. The infestation rates were 64.4% in female and 47.5% in male sheep.     

The kinetics of specific serum antibodies in sheep infested with sheep bot larvae]

The kinetics of specific antibodies of the blood serum of sheep experimentally infested with 80, 160 and 1000 specimens of Oestrus ovis larvae was examined. The affinity pure serum IgG and the immunoferment analysis (ELISA) were used for qualitative estimation of specific antibodies. It has been shown that the level of specific antibodies correlates with the larval biomass and is connected with ontogenesis of this parasite. The younger animals, which were infested for the first time, are characterized by more intensive production of specific IgG than adult reinfested ones. The ways of immunity response formation in animals infested with Oestrus ovis larvae are considered.     

Epidemiology of Oestrus ovis infection of sheep in Argentina's Western Pampas

Seasonal population trends and effects of Oestrus ovis in naturally infected sheep were studied over 13 months, in the Western of the Pampeana region. At weaning, 140 growing lambs were randomly allocated to two groups: UG, untreated group and TG treated every 4 weeks with closantel (10 mg/kg). Successive Oestrus free tracer lambs (TL) by previous treatment (n = 65) were slaughtered after 20-30 exposition days for larval counts. Likewise, other group PL of 117 permanent untreated lambs was slaughtered from four to 17 months of age. Weighing and assessment of health signs of UG and TG and blood samples were monthly carried out. The prevalence of infection in permanent group varied from 33% to 100%. Mean number of larvae in PL was 6.1 with 3 L1, 1.4 L2 and 1.6 L3 during spring-summer and 17.9 with 16.9 L1, 0.5 L2 and 0.4 L3 during autumn-winter months. In PL the proportions of larvae in each of the different larval stages was similar during spring and summer, but a significant (P < 0.01) increase of L1 was detected during autumn and winter. The prevalence in tracer lambs was 100% during summer time and larvae were absent from 25-May to 25-October. Mean larval burdens of positive TL varied from 6.4 to one Oestrus and a significant peak (P < 0.05) of larvae was seen from December to March. Since March to November only L1 was recovered from TL. TG group showed a reduction in nasal discharge and in antibody ELISA levels, but no difference was observed in live weight gain between TG and UG. These results show a high prevalence during summer and that the perpetuation of Oestrus is ensured by an autumn period of arrested development and the over wintering larvae in the sheep heads.     

Survival of the larvae of the ship botfly Oestrus ovis L. depending on the function of the immune system of the host's body

In the experiments on artificial infection the survival of larvae of Oestrus ovis L. in sheep with depressed, normal and stimulated immune system was studied. The maximum number of larvae survived in immune depressed animals (62.9%), the minimum number survived in immune stimulated animals (0.4%). For the estimation of specific immune response the reaction of indirect hemagglutination (IHA), the reaction of diffused precipitation (RDP) and the immune ferment analysis (ELISA) were used.     

Initial characterisation of the sheep immune response to infections of Lucilia cuprina

Assays for total serum antibody, histamine sensitivity and the presence of reaginic antibodies were carried out on sheep repeatedly infected with first stage larvae of Lucilia cuprina. Effects of the sheep response on the larvae were monitored at the final infection and compared with control animals by the recovery of larvae and measurement of the wound caused by the larvae. Overall larval survival was not significantly different in the pre-infected group though wound sizes were smaller. Histamine sensitivity appeared to correlate with wound size only in the control group. Thus recent infection experience may lead to immune responses which override non-specific inflammatory events and cause smaller wound sizes. A sub-group of the pre-infected sheep had lower larval survival and smaller wound sizes than the other animals and this correlated with increased levels of reaginic antibody and lower total antibody levels. The results suggest a genetic basis for resistance to fly strike and the possible involvement of reaginic antibody in protective responses.     

The mouse antibody response to Trichinella spiralis defines a single, immunodominant epitope shared by multiple antigens.

Immunoblot analysis was used to characterize the Trichinella spiralis L1 larval Ag recognized by antisera from T. spiralis-infected AKR/J mice. Antisera were analyzed for reactivity with crude worm extract, excretory/secretory proteins and cuticle proteins from L1 larvae. The response was biphasic; antibodies against one set of Ag were detected 13 days after infection (group I Ag), and antibodies against a different set of Ag were detected 35 days after infection (group II Ag). Excretory/secretory and cuticle proteins were recognized only by antibodies produced late in infection. The predominant isotype in day 42 antiserum was IgG1, and 80% of these IgG1 antibodies reacted with a stage-specific determinant shared by virtually all group II Ag. A mAb reactive with the shared determinant was used to purify the group II Ag from L1 larval extract. Such affinity-purified Ag were protective when used to immunize mice against subsequent infection, and T cells from infected mice proliferated when cultured with these Ag in vitro. Other mouse strains also made a strong serum antibody response to the group II Ag. We hypothesize that immune responses to the shared epitope play an important role in determining the outcome of the host-parasite interaction during infection.     

Three cases of ophthalmomyiasis externa by sheep botfly Oestrus ovis in Italy

Human infection with the sheep nasal botfly Oestrus ovis is sporadic and is often the consequence of an accidental deposit of the larvae by an adult botfly in the eye. This infestation results in external ophthalmomyiasis that, although a very rare condition, is more common among people living close to farming communities. We report three cases of O. ovis infestation which occurred in Italy in a limited area of La Spezia province (Le Cinque Terre), Italy during summer 2004. None of the patients had contact with wild or farm animals.     

Reactions of cells of nasal and sinusoidal mucosa of goats and sheep naturally infected by Oestrus ovis Linné 1758 (Diptera: Oestridae)]

OEstrosis is a very common myiasis of sheep and goats in mediterranean and tropical countries. Goats are suitable hosts for OEstrus ovis but the parasitic burden remain lower than in sheep. Cellular responses (mucous and serous mast cells, eosinophis and globules leucocytes) were measured in 30 infected and 30 non infected sheep and in 23 infected and 24 non infected goats. The presence of OEstrus ovis larvae led to an important inflammatory response in sheep and in goats as well. Furthermore, the intensity of the cellular response correlated with the larva burden, specially with globules leucocytes and eosinophils. Nevertheless, huge differences occurred between sheep and goats responses even in similar larval burden range. Infected sheep showed larger counts of mucous mast cells than goats, the differences were smaller in serous mast cells and eosinophils and no difference was detected in globules leucocytes (intraepithelial mast cells) counts between the two hosts species. These results were compared with those obtained in gastro-intestinal strongyles infections.     

Epidemiology of sheep infection by Oestrus ovis in Algeria

313 sheep were examined in 1996 to assess the importance and seasonal evolution of Oestrus ovis infection in the Algerian region of El-Tarf. Prevalence was found to be 67.4%. The larval burden was 18 larvae by infected sheep. The prevalence was higher in older sheep than in lambs; intensity was similar. The different larval stages were found all along the year in sheep with prevalence ranging from 33.1 to 80.5% for L1, 9.7 to 43.9% for L2 and 8.4 to 23.0% for L3. The sheep were the least infected in winter (prevalence from 35.7 to 44% and intensity seven to ten larvae per sheep). The highest infection was found during the warm season (spring to autumn, prevalence from 62 to 90% and intensity ranging from 15 to 25). This larval evolution profile suggested the existence of one long cycle (November-April) and possibly two shorts cycles (May-October). This epidemiological pattern is similar to that in Morocco but was slightly different from the situation in Tunisia where the winter cycle was apparently of lesser importance.     

Interactions between an entomopathogenic microsporidium and the endoparasitoid Glyptapanteles liparidis within their host, the gypsy moth larva

Interactions in the host-parasitoid-pathogen system, Lymantria dispar L. (Lep., Lymantriidae)-Glyptapanteles liparidis (Bouché) (Hym., Braconidae)-Vairimorpha sp. (Protista, Microspora), were investigated. Host selection experiments revealed that G. liparidis females did not discriminate between infected and uninfected host larvae for oviposition. Transmission of the microsporidium from infected to uninfected hosts by stinging female wasps could not be ascertained. Females that developed in infected L. dispar larvae did not transmit the pathogen via oviposition. Vairimorpha infection of the host negatively affected the performance of the braconid, when inoculation took place either before or after parasitization. Microsporidiosis of the host caused delayed development, reduced pupation and adult eclosion, reduction in size and weight, and reduction of adult longevity of G. liparidis. Parasitoids themselves were not systemically infected by Vairimorpha sp., but braconid larvae did ingest microsporidian spores at the end of their endoparasitic development and accumulated the undigested and ungerminated spores in the blind midgut. Negative effects of host infection on parasitoid larvae were detectable from the beginning of parasitoid larval development. Lethal time was reduced when L. dispar larvae were infected and parasitized, often at the expense of the parasitoid when G. liparidis were unable to complete endoparasitic development before the host died. Intensity of infection, measured as number of spores produced per milligram fresh weight of L. dispar larva, was slightly higher in parasitized and infected hosts than in unparasitized and infected hosts.     

Effects of Glyptapanteles liparidis (Hym.: Braconidae) parasitism, polydnavirus, and venom on development of microsporidia-infected and uninfected Lymantria dispar (Lep.: Lymantriidae) larvae

Effects of parasitism, polydnavirus, and venom of the endoparasitoid Glyptapanteles liparidis on Lymantria dispar larvae infected with the microsporidium Vairimorpha sp. and uninfected hosts were studied. We tested the impact on growth and development of hosts, as well as on microsporidian infection. Both parasitism and polydnavirus/venom treatment alone caused a slight increase in growth rate and relative growth rate in uninfected fourth instar hosts. This effect was more pronounced with the addition of Vairimorpha infection. With no parasitism, however, infection reduced host growth markedly. Microsporidiosis delayed larval molts of L. dispar, and additional polydnavirus/venom treatment or parasitization induced significantly earlier molting. Polydnavirus/venom treatment of uninfected L. dispar resulted in prolonged larval development due to supernumerary molts and in higher pupal mortality. Infected larvae treated with polydnavirus/venom died earlier than infected larvae that were not treated and produced more Vairimorpha spores per unit fresh mass of the host.     

Comparison of percutaneous vs oral infection of hamsters with the hookworm Ancylostoma ceylanicum: Parasite development,pathology and primary immune response

BackgroundHundreds of millions of people in poor countries continue to suffer from disease caused by bloodfeeding hookworms. While mice and rats are not reliably permissive hosts for any human hookworm species, adult Golden Syrian hamsters are fully permissive for the human and animal pathogen Ancylostoma ceylanicum. Similar to humans, hamsters may be infected with A. ceylanicum third-stage larvae orally or percutaneously. Oral infection typically leads to consistent worm yields in hamsters but may not accurately reflect the clinical and immunological manifestations of human infection resulting from skin penetration.Methodology/Principal findingsIn this study we compared host responses following percutaneous infection to those utilizing an established oral infection protocol. Infected hamsters exhibited a dose-dependent pathology, with 1000 percutaneous larvae (L3) causing anemia and adult worm recovery comparable to that of 50 orally administered L3. A delayed arrival and maturity of worms in the intestine was observed, as was variation in measured cellular immune responses. A long-term study found that the decline in blood hemoglobin was more gradual and did not reach levels as low, with the nadir of disease coming later in percutaneously infected hamsters. Both groups exhibited moderate growth delay, an effect that was more persistent in the percutaneously infected group. Fecal egg output also peaked later and at lower levels in the percutaneously infected animals. In contrast to orally infected hamsters, antibody titers to larval antigens continued to increase throughout the course of the experiment in the percutaneous group.Conclusions/SignificanceThese results demonstrate that the route of infection with A. ceylanicum impacts disease pathogenesis, as well as humoral and cellular immune responses in an experimental setting. These data further validate the utility of the Golden Syrian hamster as a model of both oral and percutaneous infection with human hookworms.     

Active and passive immunization of mice against larval Dirofilaria immitis

The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.     

Immune reactions and allergy in experimental anisakiasis

The third-stage larvae (L3) of the parasitic nematode, Anisakis simplex, have been implicated in the induction of hyperimmune allergic reactions in orally infected humans. In this work, we have conducted a review of an investigation into immune reactions occurring in animals experimentally infected with A. simplex L3. The patterns of serum antibody productions in the experimental animals against excretory-secretory products (ESP) of A. simplex L3 contributed to our current knowledge regarding specific humoral immune reactions in humans. In our review, we were able to determine that L3 infection of experimental animals may constitute a good model system for further exploration of immune mechanisms and allergy in anisakiasis of humans.     

Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.