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

ABSTRACT. 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
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  Medical and Veterinary Entomology (1988) 2 351-355 Serologic diagnosis of Oestrus ovis (Diptera: Oestridae) in naturally infested sheep CARLOS R. BAUTISTA-GARFIAS, ROSA M. ANGULO-CONTRERAS and ENRIQUE GARAY-GARZON Laboratorio de Inmunoparasitologia, Centro Nacional de Investigaciones Disciplinarias en Macrobiologia (CENID-Macrobiologia), Instituto Nacional de Investigaciones Forestales y Agropecuarias (INIFAP), SARH, Morelos, Mkxico ABSTRACT. 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 0.ovis infestations were tested to detect anti-Oestrus antibodies by double immunodiffusion (DD) and indirect haemagglutination (IH) tests with somatic crude antigens from first (Ll), second (L2) and third (L3) instar of 0.ovis larvae. At necropsy, eighty-eight out of 100 sheep from the 0.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 0.ovis or with L2 larvae in IH tests. Key words. Oestrus ovis larval antigens, serologic tests, indirect haemag- glutination, double immunodiffusion. Introduction The sheep nasal bot, Oestrus ovis Linnaeus, 1761, occurs from temperate to tropical areas (Harwood & James, 1979), producing con- siderable economic losses (Steelman, 1976), predisposing sheep to bacterial infections (Lloyd, 1985) and sometimes causing ophthal- momyiasis in man (Dar et al., 1980; Le Fichoux et al., 1981). Diagnosis of infestation with 0.ovis larvae can be made tentatively Correspondence: Dr Carlos R. Bautista-Garfias, Laboratorio de lnmunoparasitologia, CENID- Macrobiologia, INIFAP, SARH, Apdo. Postal 206, C.P. 62500, CIVAC, Morelos, Mexico. from clinical signs such as observing mature larvae exiting the nostrils (Lloyd, 1985) or at necropsy finding larvae in the nasal passages and sinuses (Horak, 1977). However, observa- tional diagnosis is difficult, because not all sheep in a herd show signs which can be differentiated from those of other diseases of the respiratory tract (Soulsby, 1968). Also, animals with the parasitic larvae overwintering in the sino-nasal area of the host, do not show any clinical signs (Cobbett & Mitchell, 1941; Pandey & Ouhelly, 1984). It is possible to detect parasitism by rnyiasis- causing Diptera larvae, such as Hypoderma in cattle (Boulard, 1975, 1979, 1985; Robertson, 35 1  352 C. R. BautiJta-Garfias R. M. Atrgiiio-Cotitreras and E. Garay-Garzon 1980). Liiciliu clcprirru in sheep (O’Donnell er ul.. 1980) and Oesrrrrs ovis in goats (Bautista- Garfias et ul.. 1982) and sheep (Ilchmann & IIiepe. 1985). by serological tests. This study was performed to test detection of antibodies to O.ovi.7 larvae in sheep serum using somatic crude extracts from the first. second and third instar of O.ovis larvae in double immunodiffusion and indirect haemag- glutination tests. Materials and Methods Ar7itnal.c. One hundred crossbred sheep. 3 months to 6 years old. from an area where O.o~~is nfestations are common (San Felipe del Progreso. State of Mexico, Mexico) and eighteen Pelibuey sheep. 6-18 months old. from an 0.ovis free area (Hueytamalco. State of Puebla. Mexico) were examined. The latter animals were transported to the Central Unit of INIFAP at Mexico City. Sampling. Seven to ten sheep were ex- amined weekly at the San Felipe del Progreso abattoir, between July and September. Before slaughter, blood was obtained from the jugular vein. allowed to clot and the serum harvested. A11 samples were stored at -70°C until tested. After slaughter, the head of each animal was severed from the carcass and a sagital section trough was cut. All larvae present on the mucosa of the nasal septum. nasal passages and conchae were removed. counted. and identified as to stage of development. There- after the septum and conchae were removed for closer examination. the sinuses opened. and larvae recovered as above. Antigens. Oestrio ovis larvae were collected from sheep killed at the Ferreria abattoir in Mexico City. From each of the three larval instars a crude somatic antigen was prepared as described by Bautista-Garfias er al. (1982). Briefly. 10 g of lyophilized second or third instar larvae were added to cold. 3°C. phosphate-buffered saline (PBS). pH 7.2. con- taining 100 IUirnl penicillin and 100 pg/ml streptomycin. and macerated in a tissue grin- der in an ice bath. First instar larvae were used fresh hecause it was only possible to collect 1 g of larvae which were macerated in 5 ml of cold PBS, JJH 7.2. as above. The homogenates were then gently stirred with a magnetic stirrer for 18 h at 4°C. The extracts were centrifuged, 1200 g for 30 min, at 4°C. The supernatants were recovered and stored at -70°C until used. The protein content for each antigen was determined by the Biuret reaction (Garvey et al.. 1977) using bovine serum albumin as the standard. Serologic assays Double diffusion in gel (DD) (Williams & Chase, 1971) using 100 pg of antigeniwell and 15 p1 of serumiwell, and indirect haemagglutination (IH) tests (Kagan & Norman, 1976) using 180 pg of antigeniml to sensitize the erythrocytes, were used for each antigen. For IH all the sera which reacted above the titre of the non-infested sheep from the O.or*is nfested area were considered posi- tive. The sensitivity, defined as the capacity to detect 0.ovis parasitism in sheep, was calcu- lated for both methods (Palmer & Cavallaro, 1980; Tizard, 1982). Two New Zealand white rabbits (1.5-2 kg) were used to prepare hyperimmune serum for each crude antigen. Each rabbit was immu- nized over a month with a total of 6 mg antigen in four inoculations. Freund’s com- plete adjuvant was used for the first inocula- tion and Freund’s incomplete adjuvant for the second inoculation. no adjuvant was used for the subsequent inoculations (Williams & Chase. 1Y67). The serum obtained was used as the positive control serum in the tests. Results Eighty-eight of 100 sheep from San Felipe del Progreso were parasitized by 0.ovis larvae. Of these, seventy-four had first, forty-six second, and sixteen third instar larvae. Therefore some sheep had mixed instar infestations. Also, thirty-seven sheep were parasitized by Fasciola hepatica Linnaeus. 1758. and five by Thysano- soma actinioides Diesing, 1835. Animals were not examined for nematodes. All control sheep were free from 0.ovis larvae, but they were parasitized by adult gastrointestinal nematodes, of the following genera, Haemonchus Trichostrongylus Cooperia Strongyloides, Oesophagostornum and Trickuris. The protein content for each crude antigen was: first instar larval antigen (Ll): 6.1 mgiml; second instar larval antigen (L2): 16.2 mgiml,  Serologic diagnosis of Oestrus ovis 353 4 9G 2 48 1 24 512 256 TABLE 1. Number of sheep from the infested area with or without Oestrus ovis larvae at necropsy and positive or negative anti-Oesfrus antibody titres as determined by double immunodiffusion or indirect haemagglutination. Crude extracts from the first Ll), second L2) and third (L3) instar of Oestrus ovis larvae were used as antigens. Presence of Oestrus ovis: + 88 12 __ Antigen: L1 L2 L3 L1 L2 L3 - - Positive serology by DD 37 52 16 0 0 0 IH* 88 88 86 0 0 2 DD 42 59 18 IH 100 100 97.7 of sensitivity 4 96 2048 1 24 512 256 DD = Double immunodiffusion. IH = Indirect haemagglutination. Total number of O.ovis infested sheep n=88) Number of 0.ovis infested sheep, positive to the test of sensitivity = x 100 Threshold of positivity = 1:16 for L1; 1:32 for L2; 1:8 for L3. and third instar larval antigen L3): 27.2 mg/ ml When sera from sheep were screened by DD, with L1, thirty-seven out of eighty-eight parasitized sheep (42 ), with L2, fifty-two out of eighty-eight infested sheep (59 ) and with 4 2 L3, sixteen out of eighty-eight parasitized animals (18 ) were detected (Table 1). Sera from control sheep parasitized by gastrointes- tinal nematodes and sera from non-infested ovines in the 0.0vis infested area did not react in DD with the three antigens. L 64 ........ ....... ....... 0 .... ...... ......... ........ ........ ....... 2 0000 I I CONTROL SHEEP INFESTED SHEEP NON INFESTEDSHEE INFESTED AREA INFESTEDAREA N=18) FROM 0.OVIS FROM 0.OVIS “=88) N-12) FIG. 1. Distribution of the antibody titres in sera from control sheep, infested sheep and non-infested sheep from the O.ovis infested area by the indirect haemagglutination test using a crude somatic antigen from first instar of 0.ovis larvae. IG ...... ............. =lib ~ ct ttt I ~~ COhTROL ShEEP INFESTED SHEEP NON INFESTED WEE IhFESTED AREA INFESTED AREA N=18) FROM 0.OvlS FROM 0.OVIS N=88) N=lZ) FIG. 2. Distribution of the antibody titres in sera from control sheep, infested sheep and non-infested sheep from the 0.0vis infested area by the indirect haemagglutination test using a crude somatic antigen from second instar of 0.0vis larvae.  75.1 c . R. Bautista-G<irfias, R. iM Angitlo-Contreras and E. Garay-Garzon c 4090 2048 - - 1024, 512 25G - 4 2 .... ... y . .. . .... .......... CONTROL SHEEP INESTEDSMEEP NON INFESTED SH P IN=181 FROM O.OVIS FROM aovis INFESTEDAREA INFESTED AREA (N-881 N=121 PIC;. .?. Distribution of the antibody titres in sera from control 5heep. infested sheep and non-infested sheep from the 0.oi'is infested area by the indirect 1iaem;igglutination teht using a crude somatic antigen from third instar of O.oiir larvae. When sheep sera were tested by IH all parasitized sheep were detected with minimum dilutions of 1:16 with L1 and 1:32 with L2 antigens (Figs 1 and 2). At a minimum dilution of 1:s with L3 antigen. eighty-six out of eighty-eight parasitized sheep were detected (Fig. 3). Sera from control sheep were nega- tive with L 2 as the antigen (Fig. 2). Three control sheep with L1 as antigen and seventeen control sheep with L3 as antigen were false positives (Figs 1 and 3). iscussion The results indicate that it is possible to detect. with varying sensitivity depending on the na- ture of the antigen. antibodies to 0.ovis larvae in serum from sheep naturally infested by 0.ovis. Better sensitivity was obtained by DD and best results by IH were with L1 and L2 as compared with L3 (Table 1). These are also better than results obtained previously for path by DD and IH with L2 and L3 antigens (Bautista-Garfias et al., 1982). In human and bovine hypodermosis, using DD and IH re- spectively, first instar larvae of Hypoderma can be detected similarly (Petithory & Boulard, 1979: Boulard, 1985). The results observed in sera by DD and IH, demonstrate that 0.ovis larvae elicit an im- mune response in the host and that Oestrus parasitism can be diagnosed. The sensitivity of detection using 1H was similar for L1 and L2 and slightly lower for L3 (Table 1). Three control sheep and one non-infested animal showed positive reactions using IH with the defined threshold of 1:16 for L1. It is not clear if these were false positive results or whether there might have been antibodies from a previous infestation. There were no false posi- tives using the threshold of 1:32 for L2. The false positives might have resulted from cross reactions with antibodies to other parasites like nematodes. The results indicate that more studies on cross immunity to different parasites of sheep. are needed. These tests with the aforementioned antigen could be of practical use in preliminary seroepidemiological surveys, because slaughter of animals is not required. References Bautista-Garfias. C.R.. Ruiz-Navarrete, M.A.. Morales M.. F. & Morilla, G.A. (1982) Anti- cuerpos circulantes contra larvas de Oestrus ovis (Diptera: Oestridar) en cabras infestadas natur- almente. Foliu Entonioldgicu Mexicana. 52 75- 86. Boulard, C. (1975) Evolution des anticorps circu- lants chez les bovins trait contre l'hypo- derrnose. Annales de Recherches Vitirinairrs, 6, 143-154. Boulard. C (1979) Circulating antibodies and blood histamine in cattle after treatment against hypodermosis. Veterinury Parasitology, 5 379- 387. Boulard. C. (1985) Avantages de I'irnmunodiagnos- tic de I'hypodermnse bovine etabli par hemaygiu- tination passive et par ELISA, partir du serum et du IactosCrum, sur la numkration des varons. Annales de Recherches Vt2erinaire.7, 16 335-343. Cobbett, N.G. & Mitchell, W.C. (1941) Further observations on the life cycle of the sheep bot fly. Oestrus ovis. in New Mexico and Texas. Amer- ic n Joicrnal of Veterinary Research, 2 358-366. Dar. M.S., Ben Arner. M., Dar, F.K. & Papazotos, V. (1980) Ophthalmornyiasis caused by the sheep nasal bot, Oestrus ovis (Oestridae) larvae, in the Benghazi area o€ Eastern Libya. Transactions of  Serologic diagnosis of Oestrus ovis 355 the Royal Society of Tropical Medicine and Hygiene, 74, 303-306. Garvey, J.S., Cremer, N.E. & Sussdorf, D.H. (1977) Methods in Immunology, 3rd edn, pp. 84-86. W.A. Benjamin Inc., Reading, Mass. Harwood, R.F. & James, M.T. (1979) Entomology in Human and Animal Health, 7th edn, pp. 296318. Macmillan Publishing Co. Inc., New York. Horak, I.G. (1977) Parasites of domestic and wild animals in South Africa. I. Oestrus ovis in sheep. Onderstepoort Journal of Veterinary Research, Ilchmann, G. & Hiepe, T. (1985) Immunologische Untersuchungen zur Intravital-diagnostik der Oestrose. Mh. Vet.-Med. 40, 304-307. Kagan, I.G. & Norman, L. (1976) Serodiagnosis of parasitic diseases. Manual of Clinical Immuno- logy (ed. by N. R. Rose and H. Friedman), pp. 382-409. American Society for Microbiology, Washington, D.C. Le Fichoux, Y., Marty, P., Denis, G., Couturier, P. & Dellamonica, P. (1981) Un cas d’ophtalmomy- iase externe a Oestrus ovis, LinnC, 1758 contrac- tCe sur la plage de Nice. Acta Tropica, 38 Lloyd, J.E. (1985) Arthropod pests of sheep. Live- stock Entomology (ed. by R. E. Williams, R. D. Hall, A. B. Broce and P. J. Scholl), pp. 263-267. John Wiley & Sons, New York. O’Donnell, I.J., Green, P.E., Connell, J.A. & Hopkins, P.S. (1980) Immunoglobulin G anti- bodies to the antigens of Lucilia cuprina in the sera of fly-struck sheep. Australian Journal of Biological Sciences, 33 27-34. Palmer, D.F. & Cavallaro, J.J. (1980) Some con- cepts of quality control in immunoserology. 44 5-64. 461-468. Manual of Clinical Immunology, 2nd edn (ed. by N. R. Rose and H. Friedman), pp. 1078-1082. American Society for Microbiology, Washington, D.C. Pandey, V.S. & Ouhelly, H. (1984) Epidemiology of Oestrus ovis infection of sheep in Morocco. Tropical Animal Health and Production, 16 246252. Petithory, J. & Boulard, C. (1979) Etude compara- tive des antigenes H.bovis et H.lineatum dans le diagnostic serologique de l’hypodermose humaine. Mtdecine et Maladies Infectieuses, 9 Robertson, R.H. (1980) Antibody production in cattle infected with Hypoderma spp. Canadian Journal of Zoology, 58, 245-251. Soulsby, E.J.L. (1968) Helminths, arthropods and protozoa of domesticated animals. Mdnnig’s Veterinary Helminthology and Entomology, 6th edn, pp. 444-445. Williams & Wilkins Co., Baltimore. Steelman, C.D. (1976) Effects of external and inter- nal arthropod parasites of domestic livestock production. Annual Review of Entomology, 21, Tizard, I. (1982) Serologic assays. Journal of the American Veterinary Medical Association, 181 Williams, C.A. & Chase, M.W. (eds.) (1967) Methods in Immunology and Immunochemistry , Vol. I, pp. 209-224. Academic Press, New York. Williams, C.A. & Chase, M.W. (eds.) (1971) Methods in Immunology and Immunochembtry Vol. 111 pp. 103-374. Academic Press, New York. 393-396. 155-178. 1162-1 165. Accepted 29 February 1988
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