Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

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).

Antioxidant enzymes in erythrocytes from goats seropositive to the sheep nose bot fly (Oestrus ovis L., Diptera: Oestridae) infection.

Oestrus ovis (Diptera: Oestridae) causes an important cosmopolitan parasitosis of the nasal and sinusal cavities of sheep and goats called oestrosis. Our objective was to analyze the participation of erythrocytes in the antioxidant system in goats seropositive to O. ovis infection under field conditions. Fifty female goats naturally exposed to O. ovis infection from Baja California Sur, México, were blood-sampled. Erythrocytic intracellular content was obtained from blood plasma. Oestrosis serodiagnosis was determined by ELISA. Protein, hemoglobin (Hb), superoxide dismutase (SOD), mieloperoxidase (MPO), catalase (CAT), glutathione-S-transferase (GST), and lipid peroxidation in erythrocytes were determined in both seropositive and seronegative goats. Overall seroprevalence of O. ovis infection in goats was 56%. Positive significant (P<0.05) associations were observed among systemic IgG level and protein (0.34), hemoglobin (0.43), SOD (0.32), and MPO (0.41) in erythrocytes. Protein and hemoglobin concentrations, as well as SOD and MPO activities in erythrocytes were found significantly higher (P<0.05) in seropositive than in seronegative goats. By contrast, enzymatic activities of CAT and GST and lipid peroxidation values were similar in seropositive and seronegative groups. In conclusion, there was a systemic stimulation of Reactive Oxygen Species which was efficiently scavenged by erythrocytic antioxidant enzymes in goats seropositive to O. ovis infection.

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.

IgG antibody response against salivary gland antigens from Oestrus ovis L. larvae (Diptera: Oestridae) in experimentally and naturally infected goats.

The aims of this study were to analyze the systemic IgG responses against third-instar salivary gland (L3SG) antigens by ELISA in Oestrus ovis experimentally infected kids (EIK) and in naturally exposed adult goats (NEG). Firstly, kids (n=4 per group) were assigned to receive intranasally 0, 12, 24, 36, and 48 first-instars in experimental infections. Blood samples were taken from EIK at Days 0, 14, 42 and 67 post-infection. At necropsy (Day 67), larval number and developmental instars were recorded. In an epidemiological study, blood serum samples were collected from 448 grazing NEG (n=20 flocks) in Baja California Sur, Mexico. Results showed that larval establishment rate was similar in EIK groups. Systemic IgG response reached the threshold after Day 42, but humoral response was not statistically different among EIK groups receiving experimental infections. In NEG, all surveyed flocks (100%) showed specific systemic IgG antibodies to L3SG antigens and the overall goat oestrosis prevalence was 59.2%. In conclusion, larval L3SG antigens were effective in detection of specific systemic IgG antibodies against O. ovis infected kids and goats by ELISA.

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.

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.

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.

Management of myiasis: current status and future prospects.

The management of myiasis in livestock has been an example of the success of modern chemical approaches for parasite control, yet in some cases remains extremely intractable, requiring the development of novel strategies. In addition, the growing and urgent need to develop integrated strategies that enhance the sustainability of livestock production systems drives the search for new techniques [see Int. J. Parasitol. 29 (1999) 7].The following summary represents a synthesis of a symposium presented at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology, New Orleans,USA, 10­14 August 2003. The coverage began with a review of the need for more subtle economic analysis of the impact of myiasis based on the use of the sterile insect technique (SIT) for control of bovine hypodermosis in North America. This was followed by a review of the status of chemical control with particular emphasis on the macrocyclic lactones. The outcome of the use of these compounds in a regulated control program for eradication of bovine hypodermosis in EU was surveyed. Similarly, the success of the screwworm eradication program, using the sterile insect technique has shown how effective this approach can be given the appropriate target. Several aspects of the development of newer approaches were surveyed in discussion of newer chemical control products, development of vaccines, use of host genetics, use of predictive simulation modelling and trapping for monitoring and control and the development of new diagnostic approaches for occult infestations. Finally, use of the latest molecular tools for identification of larvae causing myiasis and their use for the identification of species coming from different and distant geographical areas to colonize regions where they have been eradicated was reviewed.

Testicular maturation in the sheep bot fly Oestrus ovis.

The process of testicular maturation in relation to intrapuparial development was studied in the sheep nasal bot fly, Oestrus ovis L. (Diptera: Oestridae). After formation of the puparium during larval-pupal apolysis and the cryptocephalic pupal stage (approximately 24-72 h), spermatogonia had undergone mitotic divisions and sperm cysts had been formed. Five days after pupariation, spermatogonia transformed into primary spermatocytes during the phanerocephalic pupal stage, and secondary spermatocytes first appeared during the pupal-adult apolysis. Secondary spermatocytes began undergoing the second meiotic division by day 8 (transparent-eye pharate adult stage). By days 9 and 10, round spermatids were present and began to elongate by day 11. By day 12, the first bundles of tailed spermatozoa had appeared. By day 15 (the yellow-orange eye pharate adult stage), round, elongated, tailed and bundled spermatids were predominant and by day 17 differentiating spermatids occupied nearly 35% of the testicular cavity, and 60% was occupied by free sperm. By day 21 (the red-brown eye pharate adult stage), spermatozoa colonized the seminal vesicle. At emergence (approximately day 22), a complement of free sperm occupied the testis and the seminal vesicle, but groups of developing cells frequently remained in certain zones. Spermatogenesis was carried out after pupariation and spermiogenesis occurred during the pharate adult stage. After emergence, males possessed fully formed spermatozoa ready for ejaculation.

Immunisation of lambs with excretory secretory products of Oestrus ovis third instar larvae and subsequent experimental challenge.

Excretory-secretory products (ESP) of myiasis producing agents are involved in nutrition and development of larvae and are often immunogens. This study was carried out in order to define the antigenicity, the immunogenicity of Oestrus ovis ESP and the role of sheep immune response to ESP. Twenty-four six to eight month old female lambs were randomly allocated into two groups. The first one was immunised twice, four weeks apart, with excretory-secretory products of Oestrus ovis third instar larvae (L3ESP) in complete then incomplete Freund adjuvant. The second one served as a control, and received two injections of PBS plus complete and incomplete Freund adjuvant. Fifteen and twenty-eight days after the second immunisation, animals of both groups were experimentally challenged with O. ovis first instar larvae. Twelve days after the second experimental challenge, the twenty-four lambs were necropsied. The total number of O. ovis larvae, their stages of development, weights and sizes were recorded per animal and compared between the two groups. Establishment rates were very similar in both groups: 39% and 35% in control and vaccinated groups respectively but the percentage of developing stages was higher in the control group (13%) than in the vaccinated group (6%). It was concluded that the L3ESP immunisation of sheep did not protect against larval establishment but provided an inhibitory effect on larval growth.

Factors affecting the larvipositional activity of Oestrus ovis gravid females (Diptera: oestridae).

Observations of fly strikes or larvipositions (n=68 in 21 days of observation) were carried out in a herd of goats during the spring in Baja California Sur, Mexico in order to identify the climatic conditions favoring larviposition activity of gravid Oestrus ovis L. flies, as well as to investigate whether a mixture of some potentially useful compounds was involved in this behavior. Hand-caught, tethered flies (n=43) were either exposed or unexposed to a combination of carbon dioxide, humidity, 1-octen-3-ol, butyric, propionic, acetic acid and acetone released from movable sheep and goat dummies under open field and cage conditions. Fly strikes occurred at temperatures greater than 20 degrees C, but mainly between 25 and 28 degrees C and from 116 to 838W m(-2) of solar irradiance. Few or no strikes were seen under moderate or strong wind, but did occur in a wide range of relative humidity. The chemicals applied did not improve the capacity of animal dummies to induce the flies to larviposit, but very irregular behavior was observed. Fourteen larvipositions were made on the dummies lacking chemical stimuli, so visual ability and movement by the dummies was very important in stimulation of the flies. Temperature appeared to be the main factor determining fly activity, but wind and solar irradiance also played important roles. Characteristics of O. ovis larviposition are discussed.

Expected effects of reducing Oestrus ovis L. mature larval weight on adult populations.

In order to estimate the effects of eventual reductions in larval weight (LW) of Oestrus ovis L. as a measure of control, the correlation between mature LW and adult fly length (AL) in laboratory specimens (n=150) was calculated. The regression equation AL=5.62+10.65LW (r(2)=0.76) was obtained. This equation was then applied to estimate the mature LW of wild larvipositing females (n=51) to predict the minimum mature LW at which fly viability would be compromised. The critical weight, 0.28 g (standard error limits 0. 235, 0.323), was obtained from a small fly measuring 8.6mm in length. Data from 383 mature third instars were used to estimate, by statistical analysis, the expected effects of decreasing the mature LW on subsequent fly population size. A considerable mean reduction (38%) in adult populations might be achieved by a 40% reduction of mature LW, but this eventual reduction may be temporary due to the high reproductive rate in this species. Sex differences in mature LW and fly size are also reported.

Intra-puparial development in Oestrus ovis (Diptera: Oestridae).

The phenology of intrapuparial development in Oestrus ovis L. is described, based on 302 specimens collected from the head cavities of goats and reared in the laboratory at a photoperiod of 12:12 (L:D) h and 32 and 16 degrees C. Dissection and histology of puparia at pupariation and at 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42, 48, 66, and 72 h after pupariation and every day of the intra-puparial period showed that pupariation was achieved in approximately 12 h in heavily pigmented larvae (range, 2-46 h in postfeeding period). Larval-pupal apolysis began immediately after pupariation and was completed by 18-36 h after pupariation (prepupal period). The cryptocephalic pupa was found from this time to the 5th d, when head eversion occurred. Pupal-adult apolysis was initiated before head eversion and completed by day 7. The pharate adult presented progressive coloration in compound eyes (transparent, white, yellow, orange, red, brown, silver) while integumental pigmentation and sclerotization were in progress. Adult emergence occurred at 22 and 23 d in males and females, respectively. Changes in the weekly puparial weight of specimens reared under both field and laboratory conditions was described. It was concluded that although the intra-puparial development of O. ovis displayed some unique characteristics, it was essentially similar to other cyclorraphous flies. The actual pupal period of O. ovis lasted from the 2-7 d post-pupariation, whereas approximately two-thirds of the intra-puparial period was used for the maturation of the pharate adult.

Gonotrophic development in Oestrus ovis (Diptera: Oestridae).

Microanatomical characteristics and the size of the ovaries of Oestrus ovis L. during development were related to the intrapuparial-phenological stadia. Mature 3rd instars were collected from the head cavities of slaughtered goats, and pupae were reared under laboratory conditions. The length of freshly dissected ovaries and follicles were measured daily after pupal-adult apolysis to emergence. Ovarian tissue was stained using the PAS-Picroindigocarmine techniques. Oocyte development was classified according to a six-stage scale previously used in oestrid species. Shortly after pupal-adult apolysis, the single primary follicle is still unseparated from the germarium. In early white-eyed pharate adults, the primary follicle of stage 1 separates from the germarium, the nurse cells and oocyte are surrounded by a layer of cuboidal follicular cells, and the remaining oogonia degenerate. Oocytes in stage 2 initiate yolk deposition becoming ovoid, and this occurs when pharate adults have white-yellow to orange eyes. Oocytes in stage 3 are mainly vitellogenic, during the orange to red-eye stage. In stage 4, oocytes complete vitellogenesis and nurse cells degenerate when pupae achieve 90-96% of development. Mature oocytes of stage 5 can be found at emergence. Ovaries and ovarioles increase in size because of yolk deposition. O. ovis begins oogenesis as pharate adults, whereas vitellogenesis occurs during 55-96% of pupal development. Females emerge with one life-long complement of eggs ready to be fertilized, similar to other species of the Family Oestridae.

Estimation of the growth patterns of Oestrus ovis L. larvae hosted by goats in Baja California Sur, Mexico.

To estimate parameters for the variation in larval weight and dimension in Oestrus ovis L. and to describe the approximate pattern of growth, 2041 larvae were collected from 431 slaughtered goats and classified on a scale of 12 identified physiological ages, according to spiracular and integumental pigmentation. Larval live weight and measures of length, width, and thickness at the seventh segment were obtained. Weight increased from 0.23 mg in L1 larvae to 49 mg in late L2 larvae; however, the highest increases in weight were found to occur after the L2-L3 molt, especially during the early L3 period, when larvae acquired about 45% of the average mature weight (518 mg). Average larval length increased almost linearly from about 2 mm in L1 larvae to reach full length (21.3 mm) just before initiating integumental pigmentation, then it tended to stabilize until full maturation. Except for higher increases during the early L3 period, larval width and thickness increased linearly to reach mean maximum values (6.0 and 5.0 mm, respectively) at full maturity. Weight and measurement data were fitted by monophasic, s-shaped growth functions. The y = 0.25 [1 + tanh[0.504 (12-6.65)]] function (R2 = 0.99) indicated that L3 larvae began to have a decrease in weight gain rates before the beginning of integumental pigmentation. The patterns of growth of O. ovis described from individual measurements in larvae may be suitable for descriptive purposes.