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Seroprevalence of hypodermosis in cattle in some provinces of Turkey
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Research in Veterinary Science 84 (2008) 246–249 www.elsevier.com/locate/rvsc
Seroprevalence of hypodermosis in cattle in some provinces of Turkey Sami Simsek *, Armagan Erdem Utuk, Ergun Koroglu, Nazir Dumanli Department of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey Accepted 7 May 2007
Abstract The aim of this study was to determine the seroprevalence of hypodermosis in cattle in the east and southeast of Turkey. For this purpose, a total of 634 sera samples of cattle were collected from Malatya, Elazig and Diyarbakir provinces of east and southeast of Turkey from November 2005 to February 2006. The sera were analyzed using a Hypodermin C antigen by means of indirect ELISA. One hundred and forty eight (23.3%) out of 634 cattle were seropositive for hypoderma antibodies. The highest percentage of seropositivity were detected at Elazig province (26.3%) followed by Malatya (22.3%) and Diyarbakir provinces (22.1%). The seropositivity rate was higher in female (31%) than male (14.1%). When the mean is considered by animal breed, the highest seropositivity was detected at local breed (27.7%) following crossbreed (26.8%) and purebreed (19.7%). There was a positive relation between age and seropositivity. Seropositivity rate was 15.9% in 2 and under ages while these rates were 38.1% and 30.4% in 3–4 ages and 5 and up ages, respectively. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Hypodermosis; Cattle; ELISA; Prevalence; Turkey
1. Introduction Hypodermosis is a parasitic disease due to the development of the larval stages of insects (Diptera) inducing myiasis and belonging to the Hypoderma genus (Boulard, 2002). Cattle hypodermosis is a myiasis caused by larvae of Hypoderma bovis and Hypoderma lineatum characterized by the presence of subcutaneous warbles of animals in the dorsal and lumbar region (Otranto et al., 2005). Hypoderma spp. cause economic losses by reducing milk production, increasing meat trim and damaging hide (Boulard, 2002). In Turkey, hypodermosis is prevalent in the eastern regions of country in which there are large pastures and animal breeding potential. There is moderate infestation rate of Hypoderma spp. and the average infestation rate was 41.9% for eastern region while, the rate was 47.8% for southeast region of Turkey (Sayin et al., 2000). How*
Corresponding author. Tel.: +90 424 2370000/3967; fax: +90 424 238 81 73. E-mail address: ssimsek@firat.edu.tr (S. Simsek). 0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.05.007
ever, researches usually based on the palpation and abattoir examinations (Celep and Gursoy, 1987; Zeybek, 1988; Tasci et al., 1994; Gulanber et al., 2000; Sayin et al., 2000; Kara et al., 2005). In the last year, immunodiagnostic techniques for cattle hypodermosis have been improved so that increase diagnostic sensitivity, and avoid visual examinations, palpation of warbles or postmortem examination of the oesophagus (H. lineatum) or of the spinal canal (H. bovis) (Boulard, 1975; Otranto, 2001). Therefore a simple, reliable and accurate early sero-diagnostic method for surveillance of the disease is fundamental for use in an eradication procedure (Guan et al., 2005). ELISA methods have been widely used in many countries to detect hypodermosis prior to larval arrival in the back. This provides for a diagnosis earlier than that defined by the warbles emergence during spring and allows to treat cattle with active compounds when larvae are still migrating and have not yet caused economic losses (Boulard et al., 1996; Frangipane di Regalbono et al., 2003). Anti-hypoderma antibodies appear within 4–8 weeks post infection during the migration of first instar larvae and persist at diagnostic levels for 3 or 4 months after
S. Simsek et al. / Research in Veterinary Science 84 (2008) 246–249
the emergence of third instar larvae (Otranto, 2001). They peak between November and March in Europe. This represents the optimal period for the collection of sera in order to obtain an early diagnosis (Boulard, 1975). The object of this work was to estimate the seroprevalance of hypodermosis in some regions of Turkey, using an ELISA technique on serum samples, collected from three different provinces. 2. Material and methods 2.1. Antigen Hypodermin C was used as antigen (Boulard, 1970; Boulard et al., 1970). 2.2. Serum samples Turkey is divided into seven geographical regions and a total of 81 provinces. Malatya, Elazig and Diyarbakir provinces in the east and southeast Turkey are the major animal (mainly cattle and sheep) breeding area of the country and have the large pasture. A total of 634 sera samples of cattle were collected from Malatya, Elazig and Diyarbakir provinces of east and southeast of Turkey from November 2005 to February 2006. All samples were collected from local abattoirs and blood samples were collected just before slaughter and transferred at +4 °C to laboratory, where they were centrifuged, aliquoted, and stored at 20 °C until analyzed. Sex, age and breed characteristics were recorded. Cattle age was determined via oral herd records and dentition patterns. All samples were obtained from purebreed (Holstein, Simmental and Brown Swiss), local (Anatolian Black and East Anatolian Red) and crossbreed cattle. Positive and negative control sera were provided by abroad. Additional positive and negative control sera were obtained from cattle in Elazig province of Turkey. 2.3. ELISA This technique was performed according to Boulard (1985) with little modifications. ELISA plates (Dynatech Laboratories, IA, USA) were coated with l00 ll of 2.5 lg/ml of antigens in 0.1 M carbonate/bicarbonate buffer (pH 9.6) per well. Following overnight incubation the plates were washed twice with PBS containing 0.01% Tween-20 (PBS/Tween) and blocked with 130 ll per well of a solution containing 5% skimmed-milk powder in 0.01 M PBS (pH 7.4) for 1.5 h at 37 °C. After blocking, the plates were washing three times with PBS/Tween, 100 ll sera diluted 1:200 in PBS containing 0.05% Tween20 were added to the wells and incubated for 1.5 h at 37 °C. The plate was again washed five times and 100 ll of a 1:7500 anti-bovine IgG conjugated to rabbit peroxidase (Sigma Co. St. Louis, MO. USA Cat no: A7414) were added to the wells and the plate incubated at 37°C for 2 h.
247
Finally, after five washes, 100 ll of substrate at a containing of O-phenylene diamine (OPD) and hydrogen peroxide (H2O2) in citrate/phosphate buffer were added to each well stained for 15 min before reading at the spectrophotometer. Enzymatic reaction was stopped with 50 ll per well of 1 N sulphiric acid and the plate was read out 450 nm on an ELISA reader (Bio-Tek Instruments, USA). The results expressed as the mean of the optical density (OD). All samples were worked duplicate and repeated which sera were different up to 10% between. Cut-off value was calculated as the mean of the negative control sera absorbance values plus 3 standard deviations. 2.4. Data analysis For the data management and statistical analysis, Pearson’s chi-square and binary logistic regression model (forward condition) was performed by using SPSS 15.0 software for Windows. 3. Results The results of the present study were summarized in Table 1. Hypodermosis was ubiquitous in all three provinces. One hundred and forty eight (23.3%) out of 634 cattle were seropositive for hypoderma antibodies. In particular, the cattle from Elazig province presented the highest percentage of seropositivity (26.3%) followed by the cattle from Malatya (22.3%). The cattle from Diyarbakir province presented a lower seropositivity rate (22.1%) during this period. The seropositivity rate was higher in female (31%) than male (14.1%) and this difference was important as statistically and according to logistic regression analysis the prevalence of hypodermosis was found to be 1.96 times higher in females than males (P < 0.01; OR: 1.96 (1.20–3.21) CI: %95). When the mean is considered by animal breed, the highest seropositivity was detected at local breed (27.7%) following crossbreed (26.8%) and purebreed (19.7%) however there was no statistically difference among these group (P > 0.05; CI:%95). There was a positive relation between age and seropositivity. Seropositivity rate was 15.9% in 2 and under ages while these rates were 38.1% and 30.4% in 3–4 ages and 5 and up ages, respectively and infection risk was 2.10 times higher in 3–4 ages group than 2 and under group according to logistic regression analysis (P < 0.05; OR: 2.11 (1.14–3.89), CI: %95). The results of logistic regression analysis were showed in Table 2. 4. Discussion Bovine hypodermosis is widely distributed in Turkey, and the average infestation rate reaches 84% in some regions (Sayin et al., 2000). Sayin et al. (2000) reported that examination of cattle by palpation in field indicated that warble infestation rate (caused by either H. bovis or H. lineatum or both) was 28.3% in Blacksea, 28% in Marmara,
248
S. Simsek et al. / Research in Veterinary Science 84 (2008) 246–249
Table 1 Seroprevalence of hypodermosis in three different provinces of Turkey and relationship with sex, breed and ages Malatya
Elazig 2
Diyarbakir 2
Total 2
Ex. (n)
Inf. (n)
Prev. (%)
X values
Ex. (n)
Inf. (n)
Prev. (%)
X values
Ex. (n)
Inf. (n)
Prev. (%)
X values
Ex. (n)
Inf. (n)
Prev. (%)
X2 values
Sex Male Female
216 192
35 56
16.2b 29.1a
9.857
40 127
3 41
7.5b 32.2a
9.62
33 26
3 10
9.1b 38.4a
7.30
289 345
41 107
14.1b 31a
24.88
Breed Purebreed Local Crossbreed
226 136 46
39 43 9
17.2b 31.6a 19.5b
10.32
96 15 56
25 1 18
26.1a 6.6a 32.1a
3.96
13 40 6
2 9 2
15.4a 22.5a 33.3a
0.78
335 191 108
66 53 29
19.7a 27.7a 26.8a
5.29
Age 62 3–4 P5 Total
294 16 98 408
51 5 35 91
17.3b 31.2b 35.7a 22.3
15.07
45 56 66 167
4 22 18 44
8.8a 39.2b 23.8b 26.3
11.92
12 4 43 59
1 2 10 13
8.3a 50a 13.6a 22.1
3.17
351 76 207 634
56 29 63 148
15.9a 38.1b 30.4b 23.3
25.84
Values with the same letters within the same column are not significantly different (P > 0.05); values with the different letters within the same column are significantly different (P < 0.01). Abbreviations: Ex. (Examined), Inf. (Infected), Prev. (Prevalence).
Table 2 Explanatory variables with odds ratio (OR) and 95% confidence interval of the binary logistic regression model (forward condition) for hypodermosis seroprevalence in cattle Variable
Parameter
OR
95% CI
P
Sex: male (ref.) Age: 62 (ref.)
Female 3–4
1.96 2.11
1.20–3.21 1.14–3.89
0.007 0.017
41.6% in Agean, 33% in Mediterranean, 38.9% in Central Anatolia, 41.9% in East Anatolia and 47.8% in Southeast Anatolia. On the other hand, Gulanber et al. (2000) detected 3.56% prevalence in cattle in Thrace region. Above data have often been obtained from animals slaughtered in abattoirs. Detection of warbles fly infestation in live cattle is based either upon the observation of clinical signs on the palpation of second and third stage larvae. But, the precise data are difficult to obtain in slight infection regions, so that a serological method was developed for hypodermosis (Boulard, 1970). However, Ozer and Saki (unpublished data) did abattoir examination in Elazig province between February and May 2006. They examined 2346 cattle and detected 4% (94/2346) Hypoderma spp. larvae. We believe that there may be some overlooked larvae and this rate might be more high. ELISA was an extremely useful tool for the detection of mass serological survey in blood an milk serum in developing countries (Boulard and Villejoubert, 1991). It was also used in a large-scale survey of hypodermosis in several countries. ELISA is a serodiagnosis method that is usually more specific and sensitive than others, it is also convenient for automated sample handling and dispensing and the test can be automatically read (Guan et al., 2004). Specificity and sensitivity of Hypodermin C and secretory antigen were better than crude soluble antigen in ELISA (Boulard and Villejoubert, 1991). Therefore, we used
Hypodermin C antigen in ELISA for detection of seroprevalence. In Turkey, there is only one publish about seroprevalence of hypodermosis in cattle by ELISA (Ozkutlu and Sevgili, 2005). Ozkutlu and Sevgili (2005) collected a total of 300 cattle sera from Sanliurfa province of southeast Turkey. They used a commercial ELISA kit for the analyzing of the sera. As a result, out of 300 cattle, 116 (38.6%) were found to be seropositive. They detected the statistically difference between pure and crossbreed animals and no significant difference were found between seropositivity rates as a result of sex differences. In the present study, we detected 23.3% average seropositivity in three different provinces of Turkey. However, the seropositivity rate was 22.1% in Diyarbakir province where belong to Southeast region of Turkey. Both serological examinations (Ozkutlu and Sevgili, 2005) and abattoir one (Sayin et al., 2000) showed that southeast region of Turkey has more seroprevalence rate than the other regions. This may be related to climatic factors of this region. In this region, summer is very hot and dry, winter is warm and rainy. Therefore the fly activities are high. The reason of low degree of our seroprevalence results in Diyarbakir province may be related to position of this province. Because Diyarbakir is upper side of Southeast region and close to east region. Our study was based on only serological investigation. It was expected that there would be a higher seroprevalence in purebreed animals, than in the local and crossbreeds, but cattle without imported (local and crossbreed) showed a significantly higher seropositivity than cattle with imported (purebreed). In Turkey, because of the high yield and cost of purebreed cattle, these animals are kept and fed indoors, thereby avoiding warble flies. Thus, purebreed cattle have lower anti-hypoderma antibodies than extensive breeding cattle (generally local and crossbreed cattle). On the contrary, Kara et al. (2005) recorded a lower prevalence
S. Simsek et al. / Research in Veterinary Science 84 (2008) 246–249
in local animals, with lower larval counts than in the purebreed and crossbreeds. Young animals were less infested with warble larvae than were mature cattle (P < 0.05) because calves born after the fly season and animals kept indoors during the summer in these regions. However, Kara et al. (2005) detected more heavily infestation at young animals than mature milking cows and they stressed that older animals develop a degree of immunity to warble larvae. We could not obtaine any report about the treatment history of cattle. According to animal breeders report already lots of them couldn’t use any antiparasitic agent against this parasite. At least, we know that especially for cow, there was no parasitic drug using in out of dry season. The results of this survey indicate that hypodermosis spreads in east and southeast regions of Turkey and that it is necessary to perform further studies in order to detect national prevalence. As a result of these investigations a national eradication programme should be conducted. Acknowledgements The authors would like to thank Dr. Chantal Boulard from Institue National de la Recherche Agronomique (France) for gifting the Hypodermin C antigen and control sera and also for valuable comments. We thank Veterinary Medicines Hilmi Tasß and Eyu¨p Arıog˘ul for their kind help during sample collection and also Dr. Gulcihan Simsek and Dr. Umit Celen for providing the statistical consultation and analysis of data. This study was supported by a Grant from The Scientific and Technical Research Council of Turkey (TUBITAK). References Boulard, C., 1970. Preliminary study of crude collagenase extracted from the 1st stage larva of Hypoderma lineatum (de Villers). C.R. Acad. Sci. Hebd. Seances. Acad. Sci. D. 270, 1349–1351 (in French). Boulard, C., 1975. Evolution des anticorps circulants chez les bovins traites centre l’hypodermose. Ann. Rech. Vet. 6, 143–151. Boulard, C., 1985. Advantages of the immunodiagnosis of bovine hypodermosis established by positive hemagglutination and by ELISA, using serum and lactoserum, over the warble. Count. Ann. Rech. Vet. 16, 335–343.
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Boulard, C., 2002. Durably controlling bovine hypodermosis. Vet. Res. 33, 455–464. Boulard, C., Villejoubert, C., 1991. Use of pooled serum or milk samples for the epidemiological surveillance of bovine hypodermosis. Vet. Parasitol. 39, 171–183. Boulard, C., Soria, J., Soria, C., 1970. Possibility to use passive hemagglutination test for diagnosis of hypodermosis, using as antigen a raw collagenase extracted from 1st stage larvae of Hypoderma lineatum. C.R. Acad. Sci. Hebd. Seances. Acad. Sci. D. 270, 1965–1968 (in French). Boulard, C., Villejoubert, C., Moire, N., Losson, B., Lonneux, JF., 1996. Sero-surveillance of hypodermosis in a herd under therapeutic control. Effect of a low level of infestation. Vet. Parasitol. 66, 109–117. Celep, A., Gursoy, S., 1987. Researches on the scattering ratio of hypodermosis on the cows in Samsun and Amasya regions and their visiting time on the cows according to the terms (periods) of hypoderms spp. larvae. Etlik Vet. Mikrob. Derg. 6, 143–150 (in Turkish). Frangipane di Regalbono, A., Capelli, G., Otranto, D., Pietrobelli, M., 2003. Assessment of cattle grub (Hypoderma spp.) prevalence in Northeastern Italy: an immunoepidemiological survey on bulk milk samples using ELISA. Vet. Parasitol. 111, 343–350. Guan, G., Luo, J., Ma, J., Yuan, G., Boulard, C., Hong, Y., 2004. An enzyme-linked immunosorbent assay for serodiagnosis of hypodermosis in cattle. Chin. J. Vet. Parasitol. 12, 1–5. Guan, G., Luo, J., Ma, J., Yaung, D., Wang, Y., Gao, J., Sun, H., Liu, Z., Liu, A., Dang, Z., Boulard, C., Yin, H., 2005. Sero-epidemiological surveillance of hypodermosis in yaks and cattle in North China by ELISA. Vet. Parasitol. 129, 133–137. Gulanber, A., Tuzer, E., Gargili, A., Toparlak, M., Efil, I., Keles, V., Ulutas, M., 2000. A survey of hypodermosis in cattle slaughtered in Thrace (Trakya) Turkey. Tr. J. Vet. Anim. Sci. 24, 429–430. Kara, M., Arslan, M.O., Gicik, Y., 2005. The prevalence of bovine hypodermosis in Kars province, Turkey. Trop. Anim. Health Prod. 37, 617–622. Otranto, D., 2001. The immunology of myiasis: parasite survival and host defense strategies. Trends Parasitol. 17, 176–182. Otranto, D., Zalla, P., Testini, G., Zanaj, S., 2005. Cattle grub infestation by Hypoderma sp. in Albania and risks for European countries. Vet. Parasitol. 128, 157–162. Ozkutlu, Z., Sevgili, M., 2005. Seroprevalence of hypodermosis in cattle in the province of Sanliurfa (Turkey). T. Parazitol. Derg. 29, 275–279 (in Turkish). Sayin, F., Kalkan, A., Karaer, Z., 2000. Epidemiological studies on cattle ¨ . Sag˘lık Bil. Derg. 14, 115–127 (in hypodermosis in Turkey. F.U Turkish). Tasci, S., Deger, S., Akgul, Y., 1994. Hypodermosis in Van and around. ¨ . Vet. Fak. Derg. 5, 143–153 (in Turkish). Y.Y.U Zeybek, H., 1988. The prevalence of Hypoderma spp. in cattle and angora goats in Ankara province. Etlik Vet. Mikrob. Derg. 6, 45–56 (in Turkish).
Research in Veterinary Science 84 (2008) 246–249 www.elsevier.com/locate/rvsc
Seroprevalence of hypodermosis in cattle in some provinces of Turkey Sami Simsek *, Armagan Erdem Utuk, Ergun Koroglu, Nazir Dumanli Department of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey Accepted 7 May 2007
Abstract The aim of this study was to determine the seroprevalence of hypodermosis in cattle in the east and southeast of Turkey. For this purpose, a total of 634 sera samples of cattle were collected from Malatya, Elazig and Diyarbakir provinces of east and southeast of Turkey from November 2005 to February 2006. The sera were analyzed using a Hypodermin C antigen by means of indirect ELISA. One hundred and forty eight (23.3%) out of 634 cattle were seropositive for hypoderma antibodies. The highest percentage of seropositivity were detected at Elazig province (26.3%) followed by Malatya (22.3%) and Diyarbakir provinces (22.1%). The seropositivity rate was higher in female (31%) than male (14.1%). When the mean is considered by animal breed, the highest seropositivity was detected at local breed (27.7%) following crossbreed (26.8%) and purebreed (19.7%). There was a positive relation between age and seropositivity. Seropositivity rate was 15.9% in 2 and under ages while these rates were 38.1% and 30.4% in 3–4 ages and 5 and up ages, respectively. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Hypodermosis; Cattle; ELISA; Prevalence; Turkey
1. Introduction Hypodermosis is a parasitic disease due to the development of the larval stages of insects (Diptera) inducing myiasis and belonging to the Hypoderma genus (Boulard, 2002). Cattle hypodermosis is a myiasis caused by larvae of Hypoderma bovis and Hypoderma lineatum characterized by the presence of subcutaneous warbles of animals in the dorsal and lumbar region (Otranto et al., 2005). Hypoderma spp. cause economic losses by reducing milk production, increasing meat trim and damaging hide (Boulard, 2002). In Turkey, hypodermosis is prevalent in the eastern regions of country in which there are large pastures and animal breeding potential. There is moderate infestation rate of Hypoderma spp. and the average infestation rate was 41.9% for eastern region while, the rate was 47.8% for southeast region of Turkey (Sayin et al., 2000). How*
Corresponding author. Tel.: +90 424 2370000/3967; fax: +90 424 238 81 73. E-mail address: ssimsek@firat.edu.tr (S. Simsek). 0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.05.007
ever, researches usually based on the palpation and abattoir examinations (Celep and Gursoy, 1987; Zeybek, 1988; Tasci et al., 1994; Gulanber et al., 2000; Sayin et al., 2000; Kara et al., 2005). In the last year, immunodiagnostic techniques for cattle hypodermosis have been improved so that increase diagnostic sensitivity, and avoid visual examinations, palpation of warbles or postmortem examination of the oesophagus (H. lineatum) or of the spinal canal (H. bovis) (Boulard, 1975; Otranto, 2001). Therefore a simple, reliable and accurate early sero-diagnostic method for surveillance of the disease is fundamental for use in an eradication procedure (Guan et al., 2005). ELISA methods have been widely used in many countries to detect hypodermosis prior to larval arrival in the back. This provides for a diagnosis earlier than that defined by the warbles emergence during spring and allows to treat cattle with active compounds when larvae are still migrating and have not yet caused economic losses (Boulard et al., 1996; Frangipane di Regalbono et al., 2003). Anti-hypoderma antibodies appear within 4–8 weeks post infection during the migration of first instar larvae and persist at diagnostic levels for 3 or 4 months after
S. Simsek et al. / Research in Veterinary Science 84 (2008) 246–249
the emergence of third instar larvae (Otranto, 2001). They peak between November and March in Europe. This represents the optimal period for the collection of sera in order to obtain an early diagnosis (Boulard, 1975). The object of this work was to estimate the seroprevalance of hypodermosis in some regions of Turkey, using an ELISA technique on serum samples, collected from three different provinces. 2. Material and methods 2.1. Antigen Hypodermin C was used as antigen (Boulard, 1970; Boulard et al., 1970). 2.2. Serum samples Turkey is divided into seven geographical regions and a total of 81 provinces. Malatya, Elazig and Diyarbakir provinces in the east and southeast Turkey are the major animal (mainly cattle and sheep) breeding area of the country and have the large pasture. A total of 634 sera samples of cattle were collected from Malatya, Elazig and Diyarbakir provinces of east and southeast of Turkey from November 2005 to February 2006. All samples were collected from local abattoirs and blood samples were collected just before slaughter and transferred at +4 °C to laboratory, where they were centrifuged, aliquoted, and stored at 20 °C until analyzed. Sex, age and breed characteristics were recorded. Cattle age was determined via oral herd records and dentition patterns. All samples were obtained from purebreed (Holstein, Simmental and Brown Swiss), local (Anatolian Black and East Anatolian Red) and crossbreed cattle. Positive and negative control sera were provided by abroad. Additional positive and negative control sera were obtained from cattle in Elazig province of Turkey. 2.3. ELISA This technique was performed according to Boulard (1985) with little modifications. ELISA plates (Dynatech Laboratories, IA, USA) were coated with l00 ll of 2.5 lg/ml of antigens in 0.1 M carbonate/bicarbonate buffer (pH 9.6) per well. Following overnight incubation the plates were washed twice with PBS containing 0.01% Tween-20 (PBS/Tween) and blocked with 130 ll per well of a solution containing 5% skimmed-milk powder in 0.01 M PBS (pH 7.4) for 1.5 h at 37 °C. After blocking, the plates were washing three times with PBS/Tween, 100 ll sera diluted 1:200 in PBS containing 0.05% Tween20 were added to the wells and incubated for 1.5 h at 37 °C. The plate was again washed five times and 100 ll of a 1:7500 anti-bovine IgG conjugated to rabbit peroxidase (Sigma Co. St. Louis, MO. USA Cat no: A7414) were added to the wells and the plate incubated at 37°C for 2 h.
247
Finally, after five washes, 100 ll of substrate at a containing of O-phenylene diamine (OPD) and hydrogen peroxide (H2O2) in citrate/phosphate buffer were added to each well stained for 15 min before reading at the spectrophotometer. Enzymatic reaction was stopped with 50 ll per well of 1 N sulphiric acid and the plate was read out 450 nm on an ELISA reader (Bio-Tek Instruments, USA). The results expressed as the mean of the optical density (OD). All samples were worked duplicate and repeated which sera were different up to 10% between. Cut-off value was calculated as the mean of the negative control sera absorbance values plus 3 standard deviations. 2.4. Data analysis For the data management and statistical analysis, Pearson’s chi-square and binary logistic regression model (forward condition) was performed by using SPSS 15.0 software for Windows. 3. Results The results of the present study were summarized in Table 1. Hypodermosis was ubiquitous in all three provinces. One hundred and forty eight (23.3%) out of 634 cattle were seropositive for hypoderma antibodies. In particular, the cattle from Elazig province presented the highest percentage of seropositivity (26.3%) followed by the cattle from Malatya (22.3%). The cattle from Diyarbakir province presented a lower seropositivity rate (22.1%) during this period. The seropositivity rate was higher in female (31%) than male (14.1%) and this difference was important as statistically and according to logistic regression analysis the prevalence of hypodermosis was found to be 1.96 times higher in females than males (P < 0.01; OR: 1.96 (1.20–3.21) CI: %95). When the mean is considered by animal breed, the highest seropositivity was detected at local breed (27.7%) following crossbreed (26.8%) and purebreed (19.7%) however there was no statistically difference among these group (P > 0.05; CI:%95). There was a positive relation between age and seropositivity. Seropositivity rate was 15.9% in 2 and under ages while these rates were 38.1% and 30.4% in 3–4 ages and 5 and up ages, respectively and infection risk was 2.10 times higher in 3–4 ages group than 2 and under group according to logistic regression analysis (P < 0.05; OR: 2.11 (1.14–3.89), CI: %95). The results of logistic regression analysis were showed in Table 2. 4. Discussion Bovine hypodermosis is widely distributed in Turkey, and the average infestation rate reaches 84% in some regions (Sayin et al., 2000). Sayin et al. (2000) reported that examination of cattle by palpation in field indicated that warble infestation rate (caused by either H. bovis or H. lineatum or both) was 28.3% in Blacksea, 28% in Marmara,
248
S. Simsek et al. / Research in Veterinary Science 84 (2008) 246–249
Table 1 Seroprevalence of hypodermosis in three different provinces of Turkey and relationship with sex, breed and ages Malatya
Elazig 2
Diyarbakir 2
Total 2
Ex. (n)
Inf. (n)
Prev. (%)
X values
Ex. (n)
Inf. (n)
Prev. (%)
X values
Ex. (n)
Inf. (n)
Prev. (%)
X values
Ex. (n)
Inf. (n)
Prev. (%)
X2 values
Sex Male Female
216 192
35 56
16.2b 29.1a
9.857
40 127
3 41
7.5b 32.2a
9.62
33 26
3 10
9.1b 38.4a
7.30
289 345
41 107
14.1b 31a
24.88
Breed Purebreed Local Crossbreed
226 136 46
39 43 9
17.2b 31.6a 19.5b
10.32
96 15 56
25 1 18
26.1a 6.6a 32.1a
3.96
13 40 6
2 9 2
15.4a 22.5a 33.3a
0.78
335 191 108
66 53 29
19.7a 27.7a 26.8a
5.29
Age 62 3–4 P5 Total
294 16 98 408
51 5 35 91
17.3b 31.2b 35.7a 22.3
15.07
45 56 66 167
4 22 18 44
8.8a 39.2b 23.8b 26.3
11.92
12 4 43 59
1 2 10 13
8.3a 50a 13.6a 22.1
3.17
351 76 207 634
56 29 63 148
15.9a 38.1b 30.4b 23.3
25.84
Values with the same letters within the same column are not significantly different (P > 0.05); values with the different letters within the same column are significantly different (P < 0.01). Abbreviations: Ex. (Examined), Inf. (Infected), Prev. (Prevalence).
Table 2 Explanatory variables with odds ratio (OR) and 95% confidence interval of the binary logistic regression model (forward condition) for hypodermosis seroprevalence in cattle Variable
Parameter
OR
95% CI
P
Sex: male (ref.) Age: 62 (ref.)
Female 3–4
1.96 2.11
1.20–3.21 1.14–3.89
0.007 0.017
41.6% in Agean, 33% in Mediterranean, 38.9% in Central Anatolia, 41.9% in East Anatolia and 47.8% in Southeast Anatolia. On the other hand, Gulanber et al. (2000) detected 3.56% prevalence in cattle in Thrace region. Above data have often been obtained from animals slaughtered in abattoirs. Detection of warbles fly infestation in live cattle is based either upon the observation of clinical signs on the palpation of second and third stage larvae. But, the precise data are difficult to obtain in slight infection regions, so that a serological method was developed for hypodermosis (Boulard, 1970). However, Ozer and Saki (unpublished data) did abattoir examination in Elazig province between February and May 2006. They examined 2346 cattle and detected 4% (94/2346) Hypoderma spp. larvae. We believe that there may be some overlooked larvae and this rate might be more high. ELISA was an extremely useful tool for the detection of mass serological survey in blood an milk serum in developing countries (Boulard and Villejoubert, 1991). It was also used in a large-scale survey of hypodermosis in several countries. ELISA is a serodiagnosis method that is usually more specific and sensitive than others, it is also convenient for automated sample handling and dispensing and the test can be automatically read (Guan et al., 2004). Specificity and sensitivity of Hypodermin C and secretory antigen were better than crude soluble antigen in ELISA (Boulard and Villejoubert, 1991). Therefore, we used
Hypodermin C antigen in ELISA for detection of seroprevalence. In Turkey, there is only one publish about seroprevalence of hypodermosis in cattle by ELISA (Ozkutlu and Sevgili, 2005). Ozkutlu and Sevgili (2005) collected a total of 300 cattle sera from Sanliurfa province of southeast Turkey. They used a commercial ELISA kit for the analyzing of the sera. As a result, out of 300 cattle, 116 (38.6%) were found to be seropositive. They detected the statistically difference between pure and crossbreed animals and no significant difference were found between seropositivity rates as a result of sex differences. In the present study, we detected 23.3% average seropositivity in three different provinces of Turkey. However, the seropositivity rate was 22.1% in Diyarbakir province where belong to Southeast region of Turkey. Both serological examinations (Ozkutlu and Sevgili, 2005) and abattoir one (Sayin et al., 2000) showed that southeast region of Turkey has more seroprevalence rate than the other regions. This may be related to climatic factors of this region. In this region, summer is very hot and dry, winter is warm and rainy. Therefore the fly activities are high. The reason of low degree of our seroprevalence results in Diyarbakir province may be related to position of this province. Because Diyarbakir is upper side of Southeast region and close to east region. Our study was based on only serological investigation. It was expected that there would be a higher seroprevalence in purebreed animals, than in the local and crossbreeds, but cattle without imported (local and crossbreed) showed a significantly higher seropositivity than cattle with imported (purebreed). In Turkey, because of the high yield and cost of purebreed cattle, these animals are kept and fed indoors, thereby avoiding warble flies. Thus, purebreed cattle have lower anti-hypoderma antibodies than extensive breeding cattle (generally local and crossbreed cattle). On the contrary, Kara et al. (2005) recorded a lower prevalence
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in local animals, with lower larval counts than in the purebreed and crossbreeds. Young animals were less infested with warble larvae than were mature cattle (P < 0.05) because calves born after the fly season and animals kept indoors during the summer in these regions. However, Kara et al. (2005) detected more heavily infestation at young animals than mature milking cows and they stressed that older animals develop a degree of immunity to warble larvae. We could not obtaine any report about the treatment history of cattle. According to animal breeders report already lots of them couldn’t use any antiparasitic agent against this parasite. At least, we know that especially for cow, there was no parasitic drug using in out of dry season. The results of this survey indicate that hypodermosis spreads in east and southeast regions of Turkey and that it is necessary to perform further studies in order to detect national prevalence. As a result of these investigations a national eradication programme should be conducted. Acknowledgements The authors would like to thank Dr. Chantal Boulard from Institue National de la Recherche Agronomique (France) for gifting the Hypodermin C antigen and control sera and also for valuable comments. We thank Veterinary Medicines Hilmi Tasß and Eyu¨p Arıog˘ul for their kind help during sample collection and also Dr. Gulcihan Simsek and Dr. Umit Celen for providing the statistical consultation and analysis of data. This study was supported by a Grant from The Scientific and Technical Research Council of Turkey (TUBITAK). References Boulard, C., 1970. Preliminary study of crude collagenase extracted from the 1st stage larva of Hypoderma lineatum (de Villers). C.R. Acad. Sci. Hebd. Seances. Acad. Sci. D. 270, 1349–1351 (in French). Boulard, C., 1975. Evolution des anticorps circulants chez les bovins traites centre l’hypodermose. Ann. Rech. Vet. 6, 143–151. Boulard, C., 1985. Advantages of the immunodiagnosis of bovine hypodermosis established by positive hemagglutination and by ELISA, using serum and lactoserum, over the warble. Count. Ann. Rech. Vet. 16, 335–343.
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