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Epidemiological survey of Neospora caninum infection in dogs from Romania
Veterinary Parasitology 188 (2012) 382–385
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Epidemiological survey of Neospora caninum infection in dogs from Romania R. Gavrea, V. Mircean, A. Pastiu, V. Cozma ∗ University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Veterinary Medicine, Department of Parasitology, Calea M˘an˘an˘atur 3-5, 400372 Cluj-Napoca, Romania
a r t i c l e
i n f o
Article history: Received 8 February 2012 Received in revised form 21 March 2012 Accepted 23 March 2012 Keywords: Dogs Neosporosis Romania Epidemiology IFAT
a b s t r a c t The aim of this study was to evaluate the seroprevalence of Neospora caninum infection in dogs from Romania at a national level and to detect possible correlations between the value of seroprevalence and a series of factors like sex, age, breed category and area. The prevalence of anti-N. caninum antibodies was determined in sera samples from 1114 dogs located in different regions of Romania using the IFAT method. Antibodies to N. caninum were found in 364 dogs (32.7%). The titers of anti-N. caninum antibodies varied from 1:50 to 1:800. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Neospora caninum is a protozoan parasite that causes abortions in cattle worldwide. In dogs, infection with this parasite can cause a severe disease characterized by various clinical signs as ataxia, ascending paralysis and other general nervous symptoms (Dubey and Lindsay, 1996). The infection can also induce myocardial, pulmonary and dermatological symptoms (Lindsay et al., 1999). In cattle neosporosis can persist in raising farms for many generations (Pare et al., 1996) that can serve as a reservoirs for N. caninum infection in dogs (Wouda et al., 1999). Dogs (Canis familiaris) have an important role in the epidemiology of N. caninum infection as they are, together with coyotes (Canis latrans) and wolves (Canis lupus) the definitive hosts that shed oocysts in the environment (Gondim et al., 2004; Dubey et al., 2011). Some epidemiological studies presented a positive correlation between presence of dogs and cattle abortions
∗ Corresponding author. Tel.: +40 264 596 384x165; fax: +40 264 593 792. E-mail address: [email protected] (V. Cozma). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.03.044
induced by N. caninum infection (Pare et al., 1998). Some other studies showed that dogs raised near farms that presented or not episodes of abortion in their history, had a higher seroprevalence than those raised in urban areas (Ferroglio et al., 2004). N. caninum has a wide distribution and the presence of specific antibodies is mentioned both in healthy dogs and in cattle (Dubey et al., 2007). For their detection various types of serological tests have been used: ELISA (Ghalmi et al., 2007), DAT (Packham et al., 1998) and IFAT (Dubey et al., 1988) tests. A large number of studies showed that IFAT expressed the fewest cross-reactivity reactions with other coccidian or non-coccidian parasites like T. gondii or H. heydorni (Dubey et al., 2007) and because of that is considered the golden-standard test used for detection of anti-N. caninum antibodies (Capelli et al., 2006).
2. Materials and methods A number of 1114 sera samples were collected from dogs located in different regions of Romania. Most of the samples were collected from Transylvania (511), Oltenia (173) and Dobrogea (132) (Table 1).
R. Gavrea et al. / Veterinary Parasitology 188 (2012) 382–385 Table 1 Number of samples collected from each region. Region
Number of samples
Banat Cris¸ana Maramures¸ Transilvania Oltenia Dobrogea Bucovina Moldova
32 92 37 511 255 132 37 18
The majority of samples were collected from males (645) and 469 samples were collected from females. 932 pure breed dogs and 182 cross-breed dogs were sampled. Depending on their category the dogs were classified in: dogs raised in kennels, pets, farm dogs and hunting dogs. According to age the dogs were divided in three categories: under one year old (257), between 1 and 5 years old (572) and over 5 years old (285 samples). 641 samples were collected from dogs originated from urban areas and 473 from rural areas. All these sera samples were analyzed by IFAT method using MegaScreen FLUONEOSPORA commercial kit (Megacor Diagnostik, Austria). Briefly all sera samples were diluted at 1:50 dilution and 10 l from each one were distributed in slides in different wells. Positive and negative control were used undiluted and in volume of 20 l. After distribution in wells the slides were incubated for 30 min at 37 ◦ C in humid chamber. Rinsing with PBS and adding of one drop of FITC anti-dog conjugate followed. Next, incubation at 37 ◦ C for 30 min, adding two drops of mounting medium and reading the results at UV light at 400×. For positive samples obtained at the cut-off dilution of 1:50, serial dilutions were made until negativity was reached. All the obtained results were statistically analyzed using Epi Info Program, Version 3.5.3. All activities have been performed according to European Animal Welfare Legislation (Directive 2010/63/EU).
3. Results The interpretation of results was made at a cut-off dilution of 1:50. For assessing the intensity of N. caninum infection for positive samples serial solutions were made until they reached negativity. In some cases the positivity for N. caninum infection persisted until a dilution of 1:800. After reading the results, a number of 364 samples were positive indicating a seroprevalence for neosporosis of 32.7%. Doubtful samples were considered as being negative. At a dilution of 1:100 the seroprevalence reached a value of 14.5% (95% CI 12.6–16.8%), at 1:200 it dropped at 8.9% (95 CI 7.3–10.8%), at 1:400 was 4.1% (95% CI 3.1–5.5%). At the dilution of 1:800 the positivity reached a value of 1.9%. All these differences were statistically significant, p < 0.0001. At 1:50 dilution out of 932 samples collected from mixed breed dogs, 299 were positive, indicating a seroprevalence of neosporosis in this category of 32.1%. Regarding pure breed dogs, a similar value of 35.7%
383
(65/182) was obtained. The difference was not statistically significant (p > 0.05). Males were affected in 29.6% (95% CI 26.1–33.3%) of cases (dilution 1:50), value that decreases until 1.7% (95% CI 0.9–3.1%) at 1:800 dilution. Females were more affected than males, reaching a value of infection of 36.9% (95% CI 32.5–41.5%) that dropped to 2.1% (95% CI 1.1–4%) at the dilution of 1:800. At the initial dilution of 1:50, the highest seroprevalence of N. caninum infection was detected in hunting dogs (54.5%) and none of the dogs kept in kennels were positive for N. caninum infection (Table 2). Most of the infected dogs (36.0%) were included in the second age group (1–5 years old), compared to the ones with age under 1 year old (23.0%) and the third group (older than 5 years old, 34.7%). These differences were statistically significant (p < 0.001), and they remained significant even at next dilutions (1:100, 1:200). These aspects that can be observed in Table 3. 34.5% (95% CI 30.2-39%) of the dogs that originated from rural areas and 31.4% (95% CI 27.8-35.1%) from urban areas presented anti-N. caninum specific antibodies at a dilution of 1:50. 4. Discussions This study aimed for detection of anti-N. caninum specific antibodies in dogs from different regions of Romania. A number of 1114 sera samples were collected from 26 counties. After testing of these sera samples by IFAT method, 32.7% of them were positive for neosporosis. This percentage indicates a higher rate of infection than the ones obtained in other European countries like Sweden 0.5% (Bjorkman et al., 1994); Austria 3.6% (Wanha et al., 2005), Germany 4% (Klein and Muller, 2001) and Czech Republic 5.9% (Vaclavek et al., 2002). Pure breed animals were more affected (35.7%) than mixed breed dogs (32.1%) aspect that can be explained by the fact that the number of samples collected from purebreed animals was much lower than that of mixed breed animals. The similar aspect was noticed by Vaclavek et al. (2007), Wanha et al. (2005), Fernandes et al. (2004) and Ghalmi et al. (2009). 36.9% of females were positive for neosporosis, a higher percentage than the one obtained in males (29.6). The same differences between males and females was obtained by Wouda et al. (1999), unlike Ghalmi et al. (2009) and Vaclavek et al. (2007) who have not noticed any significant differences between infection in both sexes. The highest percentage of infection was obtained in hunting dogs (54.5%) compared to pets (34.8%). This difference can be explained by dog feeding habits: the ones that have outdoor access have more chances to come in contact with bovine infected tissues or with other intermediate hosts of N. caninum. Hunting dogs can also get infected with N. caninum especially given the fact that most often hunted animals (like deers) that can be intermediate hosts for the parasite are eviscerated in the field and dogs have access to them. Increased seroprevalence of anti-N. caninum antibodies in dogs from farms (50%) (Table 2) can be caused by
384
R. Gavrea et al. / Veterinary Parasitology 188 (2012) 382–385
Table 2 Percentage of positive animals from each service. Category of animals
1:50 Serological titers % a
1:100 Serological titers % a
1:200 Serological titers % a
1:400 Serological titers % a
1:800 Serological titers % a
Kennel Pet Shelter Guard Farms Hunting p
0. (0/4) 34.8 (174/500) 31.2 (119/381) 29.6 (64/216) 50. (1/2) 54.5 (6/11) 0.22
0. (0/4) 16.2 (81/500) 10.8 (41/381) 17.1 (37/216) 0. (0/2) 27.3 (3/11) 0.10
0. (0/4) 8.8 (44/500) 7.6 (29/381) 11.6 (25/216) 0. (0/2) 9.1 (1/11) 0.65
0. (0/4) 4.2 (21/500) 2.6 (10/381) 6.9 (15/216) 0. (0/2) 0. (0/11) 0.20
0. (0/4) 1.8 (9/500) 1.0 (4/381) 3.7 (8/216) 0. (0/2) 0. (0/11) 0.34
a
Positive samples/total of collected samples from each category.
Table 3 The results obtained at successive dilutions 1:100–1:800. Age category
1:100 Serological titers (95% CI)
1:200 Serological titers (95% CI)
1:400 Serological titers (95% CI)
1:800 Serological titers (95% CI)
Less than 1 year old 1–5 years old Over 5 years old p
7.4 (4.5–11.3) 15.9 (13.1–19.2) 18.2 (13.9–23.2) 0.0007
4.7 (2.4–8) 9.6 (7.4–12.4) 11.2 (7.8–15.5) 0.018
2.3 (0.9–5) 3.8 (2.5–5.9) 6.3 (3.8–9.8) 0.059
1.6 (0.4–3.9) 1.7 (0.9–3.3) 2.5 (1–5) 0.70
a higher chance of them being fed with bovine fetuses or placentas infected with tachyzoites or parasitic cysts. Another possible explanation for this high seroprevalence is the close contact between them and other wild animals (foxes, wolves) or other domestic animals (horses, sheep or goats). Dogs from rural areas (34.5%) were more affected than dogs from urban areas (31.4%), but there was no significant statistical difference (p > 0.05). A possible explanation of this phenomenon is the fact that animals from urban areas come in contact with infected tissues or other possible intermediate hosts much less than those from rural areas. The latter may also present a higher percentage of infection due to their feeding habits: their owners do not cook the meat offered as food. The highest seroprevalence was obtained for dogs with age between 1 and 5 years old (36.0%) followed by dogs over 5 years old (34.7%). In similar studies (Barber and Trees, 1996; Wouda et al., 1999; De Souza et al., 2002; ˜ Canón-Franco et al., 2003), authors mentioned a significantly higher seroprevalence in older dogs.
5. Conclusions Even if other studies have been performed in Romania using IFAT in order to detect the seroprevalence of neosporosis in dogs (S¸uteu et al., 2005, 2008) they were performed on a small number of animals (56 dogs in 2005 and 57 in 2008) and were limited to a small region of one county. This is the first large scale study on this disease an involved a large number of animals (1114) that originated from 26 counties of the country. In first studies the seroprevalence was 12.5% in 2005 (S¸uteu et al.) and 12.3 in 2008 (S¸uteu et al.), but in our study the value increased to 32.7%. This new obtained value shows us that in Romania neosporosis is present in a larger number of animals than we expected and prevention and control measures must be implemented.
Acknowledgment This study was supported by the Ministry of Education, Research and Innovation and National Center for Programme Management from Romania, Grant PNII PC 52177/2008. References Barber, J.S., Trees, A.J., 1996. Clinical aspects of 27 cases of neosporosis in dogs. Vet. Rec. 139, 439–443. Bjorkman, C., Lunden, A., Uggla, A., 1994. Prevalence of antibodies to Neospora caninum and Toxoplasma gondii in Swedish dogs. Acta Vet. Scand. 35, 445–447. ˜ W.A., Bergamaschi, D.P., Labruna, M.B., Camargo, L.M.A., Canón-Franco, Souza, S.L.P., Silva, J.C.R., Pinter, A., Dubey, J.P., Gennari, S.M., 2003. Prevalence of antibodies to Neospora caninum in dogs from Amazon. Brazil. Vet. Parasitol. 115, 71–74. Capelli, G., Natale, A., Nardelli, S., Frangipane Di Regalbono, A., Pietrobelli, M., 2006. Validation of a commercially available cELISA test for canine neosporosis against an indirect fluorescent antibody test (IFAT). Prev. Vet. Med. 73, 315–320. De Souza, S.L.P., Guimaraes, J.S., Ferreira, F., Dubey, J.P., Gennari, S.M., 2002. Prevalence of Neospora caninum antibodies in dogs from dairy cattle farms in Parana. Brazil. J. Parasitol. 88, 408–409. Dubey, J.P., Carpenter, J.L., Speer, C.A., Topper, M.J., Uggla, A., 1988. Newly recognized fatal protozoan disease of dogs. J. Am. Vet. Med. Assoc. 192, 1269–1285. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Dubey, J.P., Schares, G., Ortega-Mora, L.M., 2007. Epidemiology and Control of Neosporosis and Neospora caninum. Clin. Microbiol. Rev. 20, 323–367. Dubey, J.P., Jenkins, M.C., Rajendran, C., Miska, K., Ferreira, L.R., Martins, J., Kwok, O.C.H., Choudhar, S., 2011. Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum. Vet. Parasitol. 181, 382–387. Fernandes, B.C.T.M., Gennari, S.M., de Souza, S.L.P., Carvalho, J.M., Oliveira, W.G., Cury, M.C., 2004. Prevalence of anti-Neospora caninum antibodies in dogs from urban, periurban and rural areas of the city of Uberlandia, Minas Gerais—Brazil. Vet. Parasitol. 123, 33–40. Ferroglio, E., Guiso, P., Pasino, M., Accossato, A., Trisciuoglio, A., 2004. Antibodies to Neospora caninum in stray cats from north Italy. Vet. Parasitol. 131, 31–34. Ghalmi, F., China, B., Losson, B., 2007. Diagnostic et surveillance epidemiologique de Neospora caninum. Ann. Med. Vet. 151, 123–149. Ghalmi, F., China, B., Kaidi, R., Losson, B., 2009. First epidemiological study on exposure to Neospora caninum in different canine populations in the Algiers District (Algeria). Parasitol. Int. 58, 444–450.
R. Gavrea et al. / Veterinary Parasitology 188 (2012) 382–385 Gondim, L.F.P., McCallister, M.M., Pitt, W.C., Zemlick, D.E., 2004. Coyotes (Canis latrans) are definitive hosts of Neospora caninum. Int. J. Parasitol. 34, 159–161. Klein, B.U., Muller, E., 2001. Seroprävalenz von Antikörpern gegen Neospora caninum bei Hunden mit und ohne klinischem Neosporoseverdacht in Deutschland. Prakt. Tierarzt 82, 437–440. Lindsay, D.S., Dubey, J.P., Duncan, R.B., 1999. Confirmation that the dog is a definitive host for Neospora caninum. Vet. Parasitol. 82, 327–333. Packham, A.E., Sverlow, K.W., Conrad, P.A., Loomis, E.F., Rowe, J.D., Anderson, M.L., Marsh, A.E., Cray, C., Barr, B.C., 1998. A modified agglutination test for neospora caninum: development optimization, and comparison to the indirect fluorescent-antibody test and enzymelinked immunosorbent assay. Clin. Diagn. Lab. Immunol. 5, 467–473. Pare, J., Thurmond, M.C., Hietala, S.K., 1996. Congenital Neospora caninum infection in dairy cattle and associated calf hood mortality. Can. J. Vet. Res. 60, 133–139. Pare, J., Thurmond, M.C., Hietala, S.K., 1998. Neospora caninum antibodies in cows during pregnancy as a predictor of congenital infection and abortion. J. Parasitol. 83, 82–87.
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S¸uteu, O., Losson, B., Oltean, M., Cozma, V., 2005. First serological survey for canine neosporosis in Romania. USAMV-CN Bull. 62, 591–592. S¸uteu, O., Tititlincu, A., Cozma, V., 2008. Seroepidemiological study in neosporosis in carnivores (Canis lupus familiaris) and bovines from Cluj county in 2005. Rev. Rom. Parazitol. XIII, 217–218. Vaclavek, P., Koudela, B., Sedlak, K., Sebesta, R., 2002. Seroprevalence of antibodies against Neospora caninum in cattle and dogs in the Czech Republic. In: Society of Protozoologists, Czech Section, 32nd Annual Meeting. Vaclavek, P., Sedlak, K., Hurkova, L., Vodrazka, P., Sebesta, R., Koudela, B., 2007. Serological survey of Neospora caninum in dogs in the Czech Republic and a long-term study of dynamics of antibodies. Vet. Parasitol. 143, 35–41. Wanha, K., Edelhofera, R., Gabler-Eduardo, C., Prosl, H., 2005. Prevalence of antibodies against Neospora caninum and Toxoplasma gondii in dogs and foxes in Austria. Vet. Parasitol. 128, 189–193. Wouda, W., Bartels, C.J.M., Moen, A.R., 1999. Characteristics of Neospora caninum-associated abortion storms in dairy herds in the Netherlands (1995–1997). Theriogenology 52, 233–245.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Epidemiological survey of Neospora caninum infection in dogs from Romania R. Gavrea, V. Mircean, A. Pastiu, V. Cozma ∗ University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Veterinary Medicine, Department of Parasitology, Calea M˘an˘an˘atur 3-5, 400372 Cluj-Napoca, Romania
a r t i c l e
i n f o
Article history: Received 8 February 2012 Received in revised form 21 March 2012 Accepted 23 March 2012 Keywords: Dogs Neosporosis Romania Epidemiology IFAT
a b s t r a c t The aim of this study was to evaluate the seroprevalence of Neospora caninum infection in dogs from Romania at a national level and to detect possible correlations between the value of seroprevalence and a series of factors like sex, age, breed category and area. The prevalence of anti-N. caninum antibodies was determined in sera samples from 1114 dogs located in different regions of Romania using the IFAT method. Antibodies to N. caninum were found in 364 dogs (32.7%). The titers of anti-N. caninum antibodies varied from 1:50 to 1:800. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Neospora caninum is a protozoan parasite that causes abortions in cattle worldwide. In dogs, infection with this parasite can cause a severe disease characterized by various clinical signs as ataxia, ascending paralysis and other general nervous symptoms (Dubey and Lindsay, 1996). The infection can also induce myocardial, pulmonary and dermatological symptoms (Lindsay et al., 1999). In cattle neosporosis can persist in raising farms for many generations (Pare et al., 1996) that can serve as a reservoirs for N. caninum infection in dogs (Wouda et al., 1999). Dogs (Canis familiaris) have an important role in the epidemiology of N. caninum infection as they are, together with coyotes (Canis latrans) and wolves (Canis lupus) the definitive hosts that shed oocysts in the environment (Gondim et al., 2004; Dubey et al., 2011). Some epidemiological studies presented a positive correlation between presence of dogs and cattle abortions
∗ Corresponding author. Tel.: +40 264 596 384x165; fax: +40 264 593 792. E-mail address: [email protected] (V. Cozma). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.03.044
induced by N. caninum infection (Pare et al., 1998). Some other studies showed that dogs raised near farms that presented or not episodes of abortion in their history, had a higher seroprevalence than those raised in urban areas (Ferroglio et al., 2004). N. caninum has a wide distribution and the presence of specific antibodies is mentioned both in healthy dogs and in cattle (Dubey et al., 2007). For their detection various types of serological tests have been used: ELISA (Ghalmi et al., 2007), DAT (Packham et al., 1998) and IFAT (Dubey et al., 1988) tests. A large number of studies showed that IFAT expressed the fewest cross-reactivity reactions with other coccidian or non-coccidian parasites like T. gondii or H. heydorni (Dubey et al., 2007) and because of that is considered the golden-standard test used for detection of anti-N. caninum antibodies (Capelli et al., 2006).
2. Materials and methods A number of 1114 sera samples were collected from dogs located in different regions of Romania. Most of the samples were collected from Transylvania (511), Oltenia (173) and Dobrogea (132) (Table 1).
R. Gavrea et al. / Veterinary Parasitology 188 (2012) 382–385 Table 1 Number of samples collected from each region. Region
Number of samples
Banat Cris¸ana Maramures¸ Transilvania Oltenia Dobrogea Bucovina Moldova
32 92 37 511 255 132 37 18
The majority of samples were collected from males (645) and 469 samples were collected from females. 932 pure breed dogs and 182 cross-breed dogs were sampled. Depending on their category the dogs were classified in: dogs raised in kennels, pets, farm dogs and hunting dogs. According to age the dogs were divided in three categories: under one year old (257), between 1 and 5 years old (572) and over 5 years old (285 samples). 641 samples were collected from dogs originated from urban areas and 473 from rural areas. All these sera samples were analyzed by IFAT method using MegaScreen FLUONEOSPORA commercial kit (Megacor Diagnostik, Austria). Briefly all sera samples were diluted at 1:50 dilution and 10 l from each one were distributed in slides in different wells. Positive and negative control were used undiluted and in volume of 20 l. After distribution in wells the slides were incubated for 30 min at 37 ◦ C in humid chamber. Rinsing with PBS and adding of one drop of FITC anti-dog conjugate followed. Next, incubation at 37 ◦ C for 30 min, adding two drops of mounting medium and reading the results at UV light at 400×. For positive samples obtained at the cut-off dilution of 1:50, serial dilutions were made until negativity was reached. All the obtained results were statistically analyzed using Epi Info Program, Version 3.5.3. All activities have been performed according to European Animal Welfare Legislation (Directive 2010/63/EU).
3. Results The interpretation of results was made at a cut-off dilution of 1:50. For assessing the intensity of N. caninum infection for positive samples serial solutions were made until they reached negativity. In some cases the positivity for N. caninum infection persisted until a dilution of 1:800. After reading the results, a number of 364 samples were positive indicating a seroprevalence for neosporosis of 32.7%. Doubtful samples were considered as being negative. At a dilution of 1:100 the seroprevalence reached a value of 14.5% (95% CI 12.6–16.8%), at 1:200 it dropped at 8.9% (95 CI 7.3–10.8%), at 1:400 was 4.1% (95% CI 3.1–5.5%). At the dilution of 1:800 the positivity reached a value of 1.9%. All these differences were statistically significant, p < 0.0001. At 1:50 dilution out of 932 samples collected from mixed breed dogs, 299 were positive, indicating a seroprevalence of neosporosis in this category of 32.1%. Regarding pure breed dogs, a similar value of 35.7%
383
(65/182) was obtained. The difference was not statistically significant (p > 0.05). Males were affected in 29.6% (95% CI 26.1–33.3%) of cases (dilution 1:50), value that decreases until 1.7% (95% CI 0.9–3.1%) at 1:800 dilution. Females were more affected than males, reaching a value of infection of 36.9% (95% CI 32.5–41.5%) that dropped to 2.1% (95% CI 1.1–4%) at the dilution of 1:800. At the initial dilution of 1:50, the highest seroprevalence of N. caninum infection was detected in hunting dogs (54.5%) and none of the dogs kept in kennels were positive for N. caninum infection (Table 2). Most of the infected dogs (36.0%) were included in the second age group (1–5 years old), compared to the ones with age under 1 year old (23.0%) and the third group (older than 5 years old, 34.7%). These differences were statistically significant (p < 0.001), and they remained significant even at next dilutions (1:100, 1:200). These aspects that can be observed in Table 3. 34.5% (95% CI 30.2-39%) of the dogs that originated from rural areas and 31.4% (95% CI 27.8-35.1%) from urban areas presented anti-N. caninum specific antibodies at a dilution of 1:50. 4. Discussions This study aimed for detection of anti-N. caninum specific antibodies in dogs from different regions of Romania. A number of 1114 sera samples were collected from 26 counties. After testing of these sera samples by IFAT method, 32.7% of them were positive for neosporosis. This percentage indicates a higher rate of infection than the ones obtained in other European countries like Sweden 0.5% (Bjorkman et al., 1994); Austria 3.6% (Wanha et al., 2005), Germany 4% (Klein and Muller, 2001) and Czech Republic 5.9% (Vaclavek et al., 2002). Pure breed animals were more affected (35.7%) than mixed breed dogs (32.1%) aspect that can be explained by the fact that the number of samples collected from purebreed animals was much lower than that of mixed breed animals. The similar aspect was noticed by Vaclavek et al. (2007), Wanha et al. (2005), Fernandes et al. (2004) and Ghalmi et al. (2009). 36.9% of females were positive for neosporosis, a higher percentage than the one obtained in males (29.6). The same differences between males and females was obtained by Wouda et al. (1999), unlike Ghalmi et al. (2009) and Vaclavek et al. (2007) who have not noticed any significant differences between infection in both sexes. The highest percentage of infection was obtained in hunting dogs (54.5%) compared to pets (34.8%). This difference can be explained by dog feeding habits: the ones that have outdoor access have more chances to come in contact with bovine infected tissues or with other intermediate hosts of N. caninum. Hunting dogs can also get infected with N. caninum especially given the fact that most often hunted animals (like deers) that can be intermediate hosts for the parasite are eviscerated in the field and dogs have access to them. Increased seroprevalence of anti-N. caninum antibodies in dogs from farms (50%) (Table 2) can be caused by
384
R. Gavrea et al. / Veterinary Parasitology 188 (2012) 382–385
Table 2 Percentage of positive animals from each service. Category of animals
1:50 Serological titers % a
1:100 Serological titers % a
1:200 Serological titers % a
1:400 Serological titers % a
1:800 Serological titers % a
Kennel Pet Shelter Guard Farms Hunting p
0. (0/4) 34.8 (174/500) 31.2 (119/381) 29.6 (64/216) 50. (1/2) 54.5 (6/11) 0.22
0. (0/4) 16.2 (81/500) 10.8 (41/381) 17.1 (37/216) 0. (0/2) 27.3 (3/11) 0.10
0. (0/4) 8.8 (44/500) 7.6 (29/381) 11.6 (25/216) 0. (0/2) 9.1 (1/11) 0.65
0. (0/4) 4.2 (21/500) 2.6 (10/381) 6.9 (15/216) 0. (0/2) 0. (0/11) 0.20
0. (0/4) 1.8 (9/500) 1.0 (4/381) 3.7 (8/216) 0. (0/2) 0. (0/11) 0.34
a
Positive samples/total of collected samples from each category.
Table 3 The results obtained at successive dilutions 1:100–1:800. Age category
1:100 Serological titers (95% CI)
1:200 Serological titers (95% CI)
1:400 Serological titers (95% CI)
1:800 Serological titers (95% CI)
Less than 1 year old 1–5 years old Over 5 years old p
7.4 (4.5–11.3) 15.9 (13.1–19.2) 18.2 (13.9–23.2) 0.0007
4.7 (2.4–8) 9.6 (7.4–12.4) 11.2 (7.8–15.5) 0.018
2.3 (0.9–5) 3.8 (2.5–5.9) 6.3 (3.8–9.8) 0.059
1.6 (0.4–3.9) 1.7 (0.9–3.3) 2.5 (1–5) 0.70
a higher chance of them being fed with bovine fetuses or placentas infected with tachyzoites or parasitic cysts. Another possible explanation for this high seroprevalence is the close contact between them and other wild animals (foxes, wolves) or other domestic animals (horses, sheep or goats). Dogs from rural areas (34.5%) were more affected than dogs from urban areas (31.4%), but there was no significant statistical difference (p > 0.05). A possible explanation of this phenomenon is the fact that animals from urban areas come in contact with infected tissues or other possible intermediate hosts much less than those from rural areas. The latter may also present a higher percentage of infection due to their feeding habits: their owners do not cook the meat offered as food. The highest seroprevalence was obtained for dogs with age between 1 and 5 years old (36.0%) followed by dogs over 5 years old (34.7%). In similar studies (Barber and Trees, 1996; Wouda et al., 1999; De Souza et al., 2002; ˜ Canón-Franco et al., 2003), authors mentioned a significantly higher seroprevalence in older dogs.
5. Conclusions Even if other studies have been performed in Romania using IFAT in order to detect the seroprevalence of neosporosis in dogs (S¸uteu et al., 2005, 2008) they were performed on a small number of animals (56 dogs in 2005 and 57 in 2008) and were limited to a small region of one county. This is the first large scale study on this disease an involved a large number of animals (1114) that originated from 26 counties of the country. In first studies the seroprevalence was 12.5% in 2005 (S¸uteu et al.) and 12.3 in 2008 (S¸uteu et al.), but in our study the value increased to 32.7%. This new obtained value shows us that in Romania neosporosis is present in a larger number of animals than we expected and prevention and control measures must be implemented.
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