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Seasonal variation of Oestrus ovis-specific antibodies in sheep and goats mixed flocks in Greece
Veterinary Parasitology 95 (2001) 73–77
Short communication
Seasonal variation of Oestrus ovis-specific antibodies in sheep and goats mixed flocks in Greece E. Papadopoulos a , F. Prevot b , Ph. Jacquiet b , C. Duranton b , J.P. Bergeaud b , E. Kalaitzakis a , Ph. Dorchies b,∗ a
b
Faculty of Veterinary Medicine, Laboratory of Parasitology and Parasitic Diseases, Aristotelian University, Thessaloniki 540 06, Greece UMR INRA/DGER 959, Physiopathologie Infectieuse et Parasitaire des Ruminants, Ecole Nationale Vétérinaire, 23 Chemin des Capelles, F 31 076 Toulouse 03, France
Received 26 April 2000; received in revised form 14 September 2000; accepted 14 September 2000
Abstract The aim of this survey was to investigate the year-round epidemiological patterns of Oestrus ovis ELISA sero-prevalence in sheep and goats kept together under the same husbandry system in an endemic area of Greece. Twenty-five adult female sheep and 25 adult female goats, coming from a large mixed flock, were randomly selected, eartaged and monthly blood sampled during 1 year period (November 1998–October 1999). Serological prevalence in sheep was 100% all around the year. Mean intensities of specific O. ovis antibodies follow a seasonal evolution with higher mean titers between March and July than in winter. In contrast, the serological prevalences in goats were low specially in winter months (from October to January). No significant difference were noticed in goats antibody levels during the year period. The possible reasons of this difference of O. ovis sero-prevalence between sheep and goats are discussed. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Sheep; Goat; Oestrus ovis; ELISA; Epidemiology
Oestrus ovis (Linné 1761) larvae are obligatory parasites of nasal and sinus cavities of sheep and goats (Zumpt, 1965). This parasite is widespread in Mediterranean countries occasionally causing severe problems in small ruminants (Caracappa et al., 2000; Yilma and Dorchies, 1991). The clinical signs and lesions of oestrosis are usually more severe in ∗ Corresponding author. Tel.: +33-5-61-19-3871; fax: +33-5-61-19-3944. E-mail address: [email protected] (Ph. Dorchies).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 4 1 4 - 3
74
E. Papadopoulos et al. / Veterinary Parasitology 95 (2001) 73–77
sheep than in goats. This difference could be due to the most important eosinophilic and mastocytic responses in sheep upper respiratory tract than in goats (Nguyen van Khanh et al., 1999). Artificial and natural O. ovis infestations elicit an antibody response in both sheep and goats (Yilma, 1992; Duranton, 1997) and ELISA tests are used to study the serological prevalence in both host species (Dorchies et al., 1999). Moreover, the natural prevalence of O. ovis and parasitic burdens are usually higher in sheep than in goats in slaughterhouse surveys (Horak and Butt, 1977a; Dorchies et al., 2000), however, there is no information about O. ovis prevalence in sheep and goats natural mixed flocks. Therefore, the aim of this survey was to study the seasonal pattern of ELISA sero-prevalence in sheep and goats kept together under the same husbandry system in an endemic area of Greece. The survey was carried out in a mixed flock of 300 Chios cross sheep and 500 Chalkidiki goats grazing together in the mountainous area of Chalkidiki (40◦ 290 N, 23◦ 170 E) which is located in a northern Mediterranean climate (mean temperature 25◦ C during the summer and 7◦ C during the winter). The animals were kept in a fenced area at night and were at pasture during the day. They were milked twice daily morning and evening. The lambing period is during November and December with the goats giving birth to kids in January and February. Lambs and kids are by-products of the milk production and are sold for Easter, weighing up to 15 kg body weight. Adult female animals, 2–4 years old, were selected randomly in order to make one group of 25 sheep and another of 25 goats. No males were selected because they were drastically less numerous than females. All the selected animals were individually marked with a numbered eartag and they did not receive any antiparasitic treatment during the whole experimental period. Blood samples were taken monthly from each eartaged animal from November 1998 until October 1999. Sera were stored at −20◦ C until use. The ELISA method used was that described previously (Papadopoulos et al., 1997). Briefly, second instar larvae crude antigens were diluted in carbonate buffer (pH 9.6) at 2 g/ml for sheep sera examination and 10 g/ml for goats sera examination and distributed in 96 well plates (Nunclon, Polylabo), incubated for 1 h at 37◦ C, then overnight at 4◦ C. The wells were washed three times with PBST (0.01 M phosphate, 0.15 M sodium chloride, pH 7.2 and 0.1% Tween 20). The antigen-coated wells were then incubated for 30 min with a 10% skimmed milk solution at 37◦ C before blotting dry. Triplicate serum samples diluted in 1 1 for sheep and 100 for goats) were incubated for 60 min at 37◦ C. The plates were PBST ( 200 washed three times with PBST before addition of (i) a horseradish peroxidase-conjugated donkey anti-sheep IgG (SIGMA A3415) diluted (1:2000) in carbonate buffer (60 min of incubation) for sheep sera, or (ii) a peroxidase-conjugate anti-goat IgG (SIGMA A5420) diluted (1:1000) in carbonate buffer (60 min of incubation) for goats sera. Three final washes with PBST were carried out before addition and incubation at 37◦ C of 100 l per well of the chromogen (2,20 -azino-bis(2-ethylbenzthiazoline-6-sulfonic acid)diammonium). The reaction was stopped after 1 h and the optical densities determined with a spectrophotometer by measuring the absorbance at 405 nm. An antibody percentage was calculated for each serum sample by comparison with positive reference serum (artificially infected sheep and goats) and negative reference serum (young sheep and goats kept indoor to avoid O. ovis infections) as follows: % of antibodies =
OD (serum sample) − OD (negative control) OD (positive control) − OD (negative control)
E. Papadopoulos et al. / Veterinary Parasitology 95 (2001) 73–77
75
Fig. 1. Seasonal pattern of intensity of specific O. ovis antibodies in sheep.
The cut-off value between negative and positive ELISA results was calculated as the average plus three times the standard deviation of the optical density at 405 nm of sera from O. ovis-free sheep (N = 75) and O. ovis-free goats (N = 46). Analysis of prevalence data has been done with chi-square test (STAT-ITCF software) and comparison of monthly antibody mean titers within a host species by the non-parametric Kruskall–Wallis test (SIMSTAT software). Serological prevalence in sheep was 100% all around the year (data not shown in Fig. 1). Mean intensities of specific O. ovis antibodies follow a seasonal evolution (Fig. 1) with higher mean titers between March and July than in winter time (P < 0.001). In contrast, the serological prevalences in goats (Fig. 2) were low (8–28%) in winter time (from October to January), higher (50–75%) from February to September without reaching the prevalence in sheep. There was a significant difference between monthly serological prevalences in goats (P < 0.05) but no significant differences (P = 0.135) in goat’s antibody levels all around the year. The results of this survey confirm that O. ovis is a widespread parasite of sheep and goats in Greece. In a precedent survey (Papadopoulos et al., 1997), more than 95% of goats in Macedonia were positive in ELISA when sampled in summer. The striking point of interest of the sheep and goats mixed flock survey is the difference of serological prevalence between sheep and goats. This was previously shown in various countries such as France (Dorchies et al., 2000), South Africa (Horak and Butt, 1977a,b) or Libya (Gabaj et al., 1993), but these data came from slaughterhouse examinations with no reference about the animals origin. Moreover, this difference is greater in winter season, i.e. during the hypobiosis period as in the French slaughterhouse survey (Dorchies et al., 2000). In the present serological survey, it is not possible to exclude some false negative results in goats and comparative necropsies should be done in the near future to definitively demonstrate the difference of prevalence between sheep and goats in mixed flock. Nevertheless,
76
E. Papadopoulos et al. / Veterinary Parasitology 95 (2001) 73–77
Fig. 2. Seasonal pattern of prevalence and intensity of specific O. ovis antibodies in goats.
this putative difference is of great interest because (i) goats are more susceptible than sheep to other common parasites such as gastro-intestinal strongyles (Hoste and Chartier, 1998) and in contrast (ii) goats could represent a less suitable environment for hypobiotic O. ovis first stage larvae.
Acknowledgements We thank Dr. Dermot O’Brien (Dublin, Ireland) for his very kind help. This study was partly supported by COST 833 “Mange and Myiasis of Livestock”.
References Caracappa, S., Rilli, S., Zanghi, P., Di Marco, V., Dorchies, Ph., 2000. Epidemiology of ovine oestrosis (Oestrus ovis Linné 1761, Diptera: Oestridae) in Sicily. Vet. Parasitol. 92, 233–237. Dorchies, Ph., Prevot, F., Duranton, C., Bergeaud, J.P., Akakpo, J., Pangui, L.J., Missohou, A., Deconinck, P., Ouattara, L., Roger, F., Achi-Yaba, L., Dia, M.L., Jacquiet, Ph., 1999. Oestrose du mouton et de la chèvre (Oestrus ovis Linné 1761) en Afrique: résultats d’une enquête sur 3204 sérums provenant de neuf pays. Rev. Méd. Vét. 150, 463–466. Dorchies, Ph., Bergeaud, J.P., Tabouret, G., Duranton, C., Prevot, F., Jacquiet, Ph., 2000. Prevalence and larval burden of Oestrus ovis (Linné 1761) in sheep and goats in northern Mediterranean region of France. Vet. Parasitol. 88, 269–273. Duranton, C., 1997. Comparaison de l’infestation par Oestrus ovis (Linné 1761) chez la chèvre et le mouton. Thèse de l’Université paul Sabatier de Toulouse, 208 pp. Gabaj, M.M., Beesley, W.N., Awan, M.A., 1993. Oestrus ovis myiasis in Libyan sheep and goats. Trop. Anim. Health Prod. 25, 65–68.
E. Papadopoulos et al. / Veterinary Parasitology 95 (2001) 73–77
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Horak, I.G., Butt, M.J., 1977a. Parasites of domestic and wild animals in South Africa I. Oestrus ovis in sheep. Onderst. J. Vet. Res. 44, 55–64. Horak, I.G., Butt, M.J., 1977b. Parasites of domestic and wild animals in South Africa II. Oestrus ovis in goats. Onderst. J. Vet. Res. 44, 65–68. Hoste, H., Chartier, C., 1998. Résistance des chèvres aux strongyloses gastro-intestinales: différences avec les moutons. Le Point Vétérinaire 29 (189), 69–74. Nguyen van Khanh, Jacquiet, Ph., Bergeaud, J.P., Duranton, C., Prevot, F., Dorchies, Ph., 1999. Réactions cellulaires des muqueuses nasales et sinusales des chèvres et des moutons à l’infestation naturelle par Oestrus ovis Linné 1761 (Diptera: Oestridae). Parasite 6, 141–149. Papadopoulos, E., Prevot, F., Himonas, C., Dorchies, Ph., 1997. Prévalence d’Oestrus ovis (Linné 1761) chez la chèvre en Grèce: enquête sérologique par ELISA. Rev. Méd. Vét. 148, 721–724. Yilma, J.M., 1992. Contribution à l’étude de l’épidémiologie, du diagnostic immunologique et de la physiopathologie de l’oestrose ovine (Oestrus ovis Linné 1761). Thèse de l’Institut National Polytechnique de Toulouse, 219 pp. Yilma, J.M., Dorchies, Ph., 1991. Epidemiology of Oestrus ovis in southwest France. Vet. Parasitol. 40, 315–323. Zumpt, P., 1965. Myiasis in Man and Animals in the Old World. Butterworths, London, 257 pp.
Short communication
Seasonal variation of Oestrus ovis-specific antibodies in sheep and goats mixed flocks in Greece E. Papadopoulos a , F. Prevot b , Ph. Jacquiet b , C. Duranton b , J.P. Bergeaud b , E. Kalaitzakis a , Ph. Dorchies b,∗ a
b
Faculty of Veterinary Medicine, Laboratory of Parasitology and Parasitic Diseases, Aristotelian University, Thessaloniki 540 06, Greece UMR INRA/DGER 959, Physiopathologie Infectieuse et Parasitaire des Ruminants, Ecole Nationale Vétérinaire, 23 Chemin des Capelles, F 31 076 Toulouse 03, France
Received 26 April 2000; received in revised form 14 September 2000; accepted 14 September 2000
Abstract The aim of this survey was to investigate the year-round epidemiological patterns of Oestrus ovis ELISA sero-prevalence in sheep and goats kept together under the same husbandry system in an endemic area of Greece. Twenty-five adult female sheep and 25 adult female goats, coming from a large mixed flock, were randomly selected, eartaged and monthly blood sampled during 1 year period (November 1998–October 1999). Serological prevalence in sheep was 100% all around the year. Mean intensities of specific O. ovis antibodies follow a seasonal evolution with higher mean titers between March and July than in winter. In contrast, the serological prevalences in goats were low specially in winter months (from October to January). No significant difference were noticed in goats antibody levels during the year period. The possible reasons of this difference of O. ovis sero-prevalence between sheep and goats are discussed. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Sheep; Goat; Oestrus ovis; ELISA; Epidemiology
Oestrus ovis (Linné 1761) larvae are obligatory parasites of nasal and sinus cavities of sheep and goats (Zumpt, 1965). This parasite is widespread in Mediterranean countries occasionally causing severe problems in small ruminants (Caracappa et al., 2000; Yilma and Dorchies, 1991). The clinical signs and lesions of oestrosis are usually more severe in ∗ Corresponding author. Tel.: +33-5-61-19-3871; fax: +33-5-61-19-3944. E-mail address: [email protected] (Ph. Dorchies).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 4 1 4 - 3
74
E. Papadopoulos et al. / Veterinary Parasitology 95 (2001) 73–77
sheep than in goats. This difference could be due to the most important eosinophilic and mastocytic responses in sheep upper respiratory tract than in goats (Nguyen van Khanh et al., 1999). Artificial and natural O. ovis infestations elicit an antibody response in both sheep and goats (Yilma, 1992; Duranton, 1997) and ELISA tests are used to study the serological prevalence in both host species (Dorchies et al., 1999). Moreover, the natural prevalence of O. ovis and parasitic burdens are usually higher in sheep than in goats in slaughterhouse surveys (Horak and Butt, 1977a; Dorchies et al., 2000), however, there is no information about O. ovis prevalence in sheep and goats natural mixed flocks. Therefore, the aim of this survey was to study the seasonal pattern of ELISA sero-prevalence in sheep and goats kept together under the same husbandry system in an endemic area of Greece. The survey was carried out in a mixed flock of 300 Chios cross sheep and 500 Chalkidiki goats grazing together in the mountainous area of Chalkidiki (40◦ 290 N, 23◦ 170 E) which is located in a northern Mediterranean climate (mean temperature 25◦ C during the summer and 7◦ C during the winter). The animals were kept in a fenced area at night and were at pasture during the day. They were milked twice daily morning and evening. The lambing period is during November and December with the goats giving birth to kids in January and February. Lambs and kids are by-products of the milk production and are sold for Easter, weighing up to 15 kg body weight. Adult female animals, 2–4 years old, were selected randomly in order to make one group of 25 sheep and another of 25 goats. No males were selected because they were drastically less numerous than females. All the selected animals were individually marked with a numbered eartag and they did not receive any antiparasitic treatment during the whole experimental period. Blood samples were taken monthly from each eartaged animal from November 1998 until October 1999. Sera were stored at −20◦ C until use. The ELISA method used was that described previously (Papadopoulos et al., 1997). Briefly, second instar larvae crude antigens were diluted in carbonate buffer (pH 9.6) at 2 g/ml for sheep sera examination and 10 g/ml for goats sera examination and distributed in 96 well plates (Nunclon, Polylabo), incubated for 1 h at 37◦ C, then overnight at 4◦ C. The wells were washed three times with PBST (0.01 M phosphate, 0.15 M sodium chloride, pH 7.2 and 0.1% Tween 20). The antigen-coated wells were then incubated for 30 min with a 10% skimmed milk solution at 37◦ C before blotting dry. Triplicate serum samples diluted in 1 1 for sheep and 100 for goats) were incubated for 60 min at 37◦ C. The plates were PBST ( 200 washed three times with PBST before addition of (i) a horseradish peroxidase-conjugated donkey anti-sheep IgG (SIGMA A3415) diluted (1:2000) in carbonate buffer (60 min of incubation) for sheep sera, or (ii) a peroxidase-conjugate anti-goat IgG (SIGMA A5420) diluted (1:1000) in carbonate buffer (60 min of incubation) for goats sera. Three final washes with PBST were carried out before addition and incubation at 37◦ C of 100 l per well of the chromogen (2,20 -azino-bis(2-ethylbenzthiazoline-6-sulfonic acid)diammonium). The reaction was stopped after 1 h and the optical densities determined with a spectrophotometer by measuring the absorbance at 405 nm. An antibody percentage was calculated for each serum sample by comparison with positive reference serum (artificially infected sheep and goats) and negative reference serum (young sheep and goats kept indoor to avoid O. ovis infections) as follows: % of antibodies =
OD (serum sample) − OD (negative control) OD (positive control) − OD (negative control)
E. Papadopoulos et al. / Veterinary Parasitology 95 (2001) 73–77
75
Fig. 1. Seasonal pattern of intensity of specific O. ovis antibodies in sheep.
The cut-off value between negative and positive ELISA results was calculated as the average plus three times the standard deviation of the optical density at 405 nm of sera from O. ovis-free sheep (N = 75) and O. ovis-free goats (N = 46). Analysis of prevalence data has been done with chi-square test (STAT-ITCF software) and comparison of monthly antibody mean titers within a host species by the non-parametric Kruskall–Wallis test (SIMSTAT software). Serological prevalence in sheep was 100% all around the year (data not shown in Fig. 1). Mean intensities of specific O. ovis antibodies follow a seasonal evolution (Fig. 1) with higher mean titers between March and July than in winter time (P < 0.001). In contrast, the serological prevalences in goats (Fig. 2) were low (8–28%) in winter time (from October to January), higher (50–75%) from February to September without reaching the prevalence in sheep. There was a significant difference between monthly serological prevalences in goats (P < 0.05) but no significant differences (P = 0.135) in goat’s antibody levels all around the year. The results of this survey confirm that O. ovis is a widespread parasite of sheep and goats in Greece. In a precedent survey (Papadopoulos et al., 1997), more than 95% of goats in Macedonia were positive in ELISA when sampled in summer. The striking point of interest of the sheep and goats mixed flock survey is the difference of serological prevalence between sheep and goats. This was previously shown in various countries such as France (Dorchies et al., 2000), South Africa (Horak and Butt, 1977a,b) or Libya (Gabaj et al., 1993), but these data came from slaughterhouse examinations with no reference about the animals origin. Moreover, this difference is greater in winter season, i.e. during the hypobiosis period as in the French slaughterhouse survey (Dorchies et al., 2000). In the present serological survey, it is not possible to exclude some false negative results in goats and comparative necropsies should be done in the near future to definitively demonstrate the difference of prevalence between sheep and goats in mixed flock. Nevertheless,
76
E. Papadopoulos et al. / Veterinary Parasitology 95 (2001) 73–77
Fig. 2. Seasonal pattern of prevalence and intensity of specific O. ovis antibodies in goats.
this putative difference is of great interest because (i) goats are more susceptible than sheep to other common parasites such as gastro-intestinal strongyles (Hoste and Chartier, 1998) and in contrast (ii) goats could represent a less suitable environment for hypobiotic O. ovis first stage larvae.
Acknowledgements We thank Dr. Dermot O’Brien (Dublin, Ireland) for his very kind help. This study was partly supported by COST 833 “Mange and Myiasis of Livestock”.
References Caracappa, S., Rilli, S., Zanghi, P., Di Marco, V., Dorchies, Ph., 2000. Epidemiology of ovine oestrosis (Oestrus ovis Linné 1761, Diptera: Oestridae) in Sicily. Vet. Parasitol. 92, 233–237. Dorchies, Ph., Prevot, F., Duranton, C., Bergeaud, J.P., Akakpo, J., Pangui, L.J., Missohou, A., Deconinck, P., Ouattara, L., Roger, F., Achi-Yaba, L., Dia, M.L., Jacquiet, Ph., 1999. Oestrose du mouton et de la chèvre (Oestrus ovis Linné 1761) en Afrique: résultats d’une enquête sur 3204 sérums provenant de neuf pays. Rev. Méd. Vét. 150, 463–466. Dorchies, Ph., Bergeaud, J.P., Tabouret, G., Duranton, C., Prevot, F., Jacquiet, Ph., 2000. Prevalence and larval burden of Oestrus ovis (Linné 1761) in sheep and goats in northern Mediterranean region of France. Vet. Parasitol. 88, 269–273. Duranton, C., 1997. Comparaison de l’infestation par Oestrus ovis (Linné 1761) chez la chèvre et le mouton. Thèse de l’Université paul Sabatier de Toulouse, 208 pp. Gabaj, M.M., Beesley, W.N., Awan, M.A., 1993. Oestrus ovis myiasis in Libyan sheep and goats. Trop. Anim. Health Prod. 25, 65–68.
E. Papadopoulos et al. / Veterinary Parasitology 95 (2001) 73–77
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Horak, I.G., Butt, M.J., 1977a. Parasites of domestic and wild animals in South Africa I. Oestrus ovis in sheep. Onderst. J. Vet. Res. 44, 55–64. Horak, I.G., Butt, M.J., 1977b. Parasites of domestic and wild animals in South Africa II. Oestrus ovis in goats. Onderst. J. Vet. Res. 44, 65–68. Hoste, H., Chartier, C., 1998. Résistance des chèvres aux strongyloses gastro-intestinales: différences avec les moutons. Le Point Vétérinaire 29 (189), 69–74. Nguyen van Khanh, Jacquiet, Ph., Bergeaud, J.P., Duranton, C., Prevot, F., Dorchies, Ph., 1999. Réactions cellulaires des muqueuses nasales et sinusales des chèvres et des moutons à l’infestation naturelle par Oestrus ovis Linné 1761 (Diptera: Oestridae). Parasite 6, 141–149. Papadopoulos, E., Prevot, F., Himonas, C., Dorchies, Ph., 1997. Prévalence d’Oestrus ovis (Linné 1761) chez la chèvre en Grèce: enquête sérologique par ELISA. Rev. Méd. Vét. 148, 721–724. Yilma, J.M., 1992. Contribution à l’étude de l’épidémiologie, du diagnostic immunologique et de la physiopathologie de l’oestrose ovine (Oestrus ovis Linné 1761). Thèse de l’Institut National Polytechnique de Toulouse, 219 pp. Yilma, J.M., Dorchies, Ph., 1991. Epidemiology of Oestrus ovis in southwest France. Vet. Parasitol. 40, 315–323. Zumpt, P., 1965. Myiasis in Man and Animals in the Old World. Butterworths, London, 257 pp.