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Occurrence of Neospora caninum in dogs and its correlation with visceral leishmaniasis in the urban area of Campo Grande, Mato Grosso do Sul, Brazil
Veterinary Parasitology 135 (2006) 375–379 www.elsevier.com/locate/vetpar
Short communication
Occurrence of Neospora caninum in dogs and its correlation with visceral leishmaniasis in the urban area of Campo Grande, Mato Grosso do Sul, Brazil Renato Andreotti a,*, Jacqueline Marques Oliveira b, Elaine Araujo e Silva c, Leandra Marla Oshiro d, Maria de Fatima Cepa Matos e a
Embrapa Gado de Corte, Animal Health, Rodovia BR 262, km 4, Caixa postal 154, Campo Grande, MS 79002-970, Brazil b Ageˆncia de Defesa Sanita´ria Animal e Vegetal (IAGRO), Campo Grande, MS 79074-460, Brazil c Centro de Controle de Zoonoses, Prefeitura Municipal de Campo Grande. Campo Grande, MS 79074-460, Brazil d CNPq-grant recipient, Embrapa Gado de Corte, Campo Grande, MS, Brazil e Universidade Federal de Mato Grosso do Sul, UFMS, Campo Grande, MS 79070-900, Brazil Received 17 April 2005; received in revised form 22 September 2005; accepted 4 October 2005
Abstract Neospora caninum is an obligate intracellular protozoan that can infect domestic and wild canids, as well as ruminants and equines, and is described as causing neuromuscular alteration and death in dogs. Visceral leishmaniasis (VL) is an infectious disease that affects both humans and animals, being caused by protozoan parasites of the genus Leishmania, of which Leishmania (Leishmania) chagasi is found in Brazil – transmitted by sand flies, such as Lutzomyia longipalpis, in most of the American continent. The immunosuppression caused by VL can promote the occurrence of co-infections with other agents. In order to determine the frequency of N. caninum and its relationship to VL in Campo Grande, MS, Brazil, 345 blood sera were collected from dogs. The sera were submitted to an indirect fluorescent antibody test (IFAT) for detection of anti-N. caninum antibodies and VL antibodies. N. caninum was found in 26.5% of VL-negative dogs and in 29% of VL-positive ones. Among males, it was found in 30.7% of VL-negative animals and in 30.4% of VL-positive ones; among females, in 21% of VL-negative animals and in 27.7% of VL-positive ones. Among juvenile dogs (under 1 year), N. caninum was detected in 10.5% of VLnegative animals and in 11.2% of VL-positive ones. For adult dogs (1 year and older) the results were 31.4% for VL-negative animals and 28.8% of VL-positive ones. The study revealed a statistically significant association with age (x2 = 9.76, P < 0.05) in the N. caninum results for VL-negative animals. No significant correlation in N. caninum seroprevalence was found when VL-positive or VL-negative dogs were compared (x2 = 0.21, P = 0.64). The findings suggest that in Campo Grande N. caninum and VL co-infection is common in dogs, though VL does not appear to enhance susceptibility to N. caninum. # 2005 Elsevier B.V. All rights reserved. Keywords: Neospora caninum; Visceral leishmaniasis; Dogs; Brazil
* Corresponding author. Tel.: +55 67 368 2173; fax: +55 67 368 2150. E-mail address: [email protected] (R. Andreotti). 0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.10.011
376
R. Andreotti et al. / Veterinary Parasitology 135 (2006) 375–379
1. Introduction Neospora caninum is a protozoan of the phylum Apicomplexa, class Sporozoea, family Sarcocystidae, subfamily Toxoplasmatinae, that can infect domestic and wild canids. It was first described as causing neuromuscular alterations and death in dogs (Dubey et al., 1988). Domestic dogs, which have been considered the only definitive hosts of the parasite, shed oocysts in their feces after ingesting tissue cysts of N. caninum (Lindsay et al., 1999; Dijkstra et al., 2001). Clinical and subclinical infections with N. caninum in dogs are epidemiologically relevant because domestic dogs (Canis familiaris) are the principal definitive hosts of the protozoan, being capable of shedding oocysts in the environment and thus constituting a risk factor for the occurrence of miscarriages associated with N. caninum in bovines (Pare´ et al., 1998). Dogs of any age can develop clinical neosporosis, which may be generalized – when several organs, including the skin, are involved – or localized (Dubey, 2003). Congenital and postnatal neosporosis have been experimentally induced in dogs, but the typical signs of congenital neosporosis, observed in naturally occurring cases, have not been reproduced (Buxton et al., 2002). Dog sera were submitted to an indirect fluorescence antibody test (IFAT) for detection of anti-N. caninum antibodies in dogs living in the urban area of Campo Grande, MS, Brazil, and a frequency of 30% was found (Silva et al., 2001). Visceral leishmaniasis (VL) is an infectious disease that affects both humans and animals, caused by a protozoan parasite of the genus Leishmania, such as Leishmania (Leishmania) donovani, Leishmania (Leishmania) infantum, and Leishmania (Leishmania) chagasi. The last one is found in Brazil (Feitosa et al., 2000; Brasil, 2003) and is transmitted by sand flies, such as Lutzomyia longipalpis, in most of the American continent, from northern Argentina to southern Mexico (Arias et al., 1996). During the past few years, many countries have witnessed a re-emergence of VL, not only in the number of cases, but also in geographic spread. Given the usual proximity between humans and dogs and the urbanization of the disease, dogs are viewed as the main urban reservoir of the parasite (Arias et al., 1996).
The first report on VL in dogs in the urban area of Campo Grande dates back from 2000, with 29 positive dogs out of 1303 surveyed (Silva et al., 2000). The numbers rose every year and reached 104 in 2003, making that an endemic area (SESAU/MS, 2005). Recently, however, skin co-infection with L. infantum and N. caninum has been reported in young dog (Tarantino et al., 2001) and high levels of N. Caninum and L. Infantum co-infection was observed in Italy (Cringoli et al., 1996). Cringoli et al. (2002) reported the co-presence of antibodies of N. Caninum and L. Infantum is very common in dogs in the Campania region of southern Italy, and that infeccion by one protozoan seems to enhance the susceptibility to the other one. The immunosuppression caused by VL can promote the occurrence of co-infections with other agents such as Ehrlichia, Babesia, and Dirofilaria in endemic regions (Feitosa et al., 2000). As an emergent disease, VL may be an influential factor in the enhancement of neosporosis. The purpose of this study was to determine if the seroprevalence of N. caninum in dogs is influenced by VL in Campo Grande, MS, Brazil.
2. Materials and methods Blood samples were collected from 345 dogs that were brought by their owners to the Center for Zoonosis Control of Campo Grande for VL diagnosis in response to the publicizing in the media of the risks posed by the disease. The samples were also used for detection of anti-N. caninum antibodies. Data on the animals’ age and sex were provided by the owners. Dogs younger than 1 year were categorized as juveniles; older ones, as adults. The sample size (310) was calculated (Thrusfield, 1994) with a 95% confidence interval and a maximum error of 5%. An expected prevalence of 20% was adopted, which corresponds to the mean value found in previous epidemiological studies (Silva et al., 2001). Antigen production and the IFAT technique for N. caninum basically followed the guidelines provided by Locatelli-Dittich (2002). Tachyzoites of N. caninum, strain NC-1 (Dubey et al., 1988), were transferred into an established cell line originated from the American
R. Andreotti et al. / Veterinary Parasitology 135 (2006) 375–379
Type Culture Collection: Vero (African three green monkey kidney, ATCC-CCL 81). Cell monolayers were used after 48–72 h of incubation, as described by Hemphill and Gottstein (1996). The sera were tested for N. caninum evaluation at an initial dilution of 1:50 and considered as positive for titers 1:50. Anti-canine IgG conjugate (VMDR Inc., WA, USA) was added to the slides. Tachyzoite fluorescence was viewed under a Nikon microscope (model BX50) equipped with an ultraviolet source. Samples displaying complete peripheral fluorescence of parasites at a 1:50 dilution were considered reactive (Pare´ et al., 1998). Antibodies against canine leishmaniasis were detected by IFAT using a Bio-Manguinhos antigen (Bio-Manguinhos, RJ, BR). A titer of at least 1:40 was considered a positive result. The results were grouped by sex and age for VL and Neospora-positive and negative sera, and submitted to a chi-square test to compare N. caninum results in VL-positive and VL-negative dogs, with one degree of tolerance and adopting a significance level of 0.05.
3. Results and discussion Of the 345 dog sera evaluated, 29% were positive for VL and 27.2% for neosporosis. Table 1 shows the distribution of the results for neosporosis in Table 1 Distribution of Neospora caninum IFAT results in dog sera negative for canine visceral leishmaniasis, by sex and age Variables
N. caninumpositive
N. caninumnegative
Total
N
%
N
%
N
Sex Males Females
43 22
30.7 21.0
97 83
69.3 79.0
140 105
Total
65
26.5
180
73.5
245
6 59
10.5 31.4
51 129
89.5 68.6
57 188
65
26.5
180
73.5
245
Age Juveniles (<1 year) Adults (1 year) Total
N. caninum-positive sera:titers 1:50. Regarding age, the prevalence was 10.5% for juveniles and significantly higher for adults: 31.4% (x2 = 9.76, P < 0.05).
377
Table 2 Distribution of N. caninum IFAT results in dog sera positive for canine visceral leishmaniasis, by sex and age Variables
N. caninumpositive
N. caninumnegative
Total
N
%
N
%
N
Sex Males Females
14 15
30.4 27.7
32 39
69.6 72.2
46 54
Total
29
29.0
71
71.0
100
1 21
11.2 28.8
8 52
88.8 71.2
9 73
22
26.8
60
73.2
82
Age Juveniles (<1 year) Adults (1 year) Total
Leishmania-positive sera:titers 1:40. N. caninum-positive sera:titers 1:50. Information on the age of 18 dogs was not provided by owners.
VL-negative dogs, and in Table 2 the results for VL positive dogs. In VL-negative dogs, the prevalence of neosporosis was 26.5%. In regard to sex, the prevalence was 30.7% for males and 21% for females, but no significant difference was found between these two subgroups (x2 = 2.93, P = 0.086). Regarding age, the prevalence was 10.5% for juveniles and significantly higher for adults: 31.4% (x2 = 9.76, P < 0.05) (Table 1). In VL-positive dogs, the prevalence of neosporosis was 29%. In regard to sex, the prevalence was 30.4% for males and 27.7% for females, but no significant difference was found between these two subgroups (x2 = 0.085, P = 0.77). Among VL-positive dogs of known age, the prevalence was 11.2% for juveniles and 28.8% for adults, but the difference between these two groups was not significant (x2 = 1.27, P = 0.26) (Table 2). No significant correlation was found when VL-positive and VL-negative dogs were compared considering N. caninum seroprevalence (x2 = 0.21, P = 0.64), confirming that N. caninum antibody titres in dogs do not change in spite of the status of L. infantum antibody titres (Cringoli et al., 2002). The frequency was similar to that found in a preliminary study conducted in the urban area of Campo Grande (Silva et al., 2001) comprising 40 dogs. The findings suggest that in Campo Grande N. caninum and Leishmania co-infection is very common
378
R. Andreotti et al. / Veterinary Parasitology 135 (2006) 375–379
in dogs, and infection by Leishmania does not seem to enhance susceptibility to N. caninum. Although the results found in the present study are preliminary, they add to the information available on N. caninum in Brazil. Souza et al. (2002) found anti-N. caninum antibodies in 29 (21.6%) out of 134 dogs living on dairy–cattle farms in the Brazilian state of Parana´; Gennari et al. (2002) found a frequency of 10% for domiciled dogs and 25% for stray ones in the state of Sa˜o Paulo. Regarding the variables sex (males, x2 = 0.001, P = 0.97; females, x2 = 0.93, P = 0.33) and age (juveniles, x2 = 0.002, P = 0.95; adults, x2 = 0.17, P = 0.68), no statistically significant differences were found in our study—a result also obtained by Varandas et al. (2001) and Souza et al. (2002) and explained by the fact that both male and female dogs are exposed to the same risk conditions. Age, however, played a role in the results: the frequency found for adult dogs was higher than that for juvenile ones. Souza et al. (2002) also detected an increase in seroprevalence with age. These findings are suggestive of postnatal exposure to the parasite. Barber and Trees (1998) reported that the rate of vertical transmission of neosporosis in dogs was low, since 80% of the offspring of seropositive mothers had not been infected until birth time, indicating that postnatal infection is significant in this species. Varandas et al. (2001), however, found no significant difference regarding the parameter age, suggesting that dogs of any age are subject to the same risk of infection. Additional studies on this matter are needed. Further studies should be developed with dogs in the rural area of the country, since epidemiological studies conducted in Canada, Japan, and the Netherlands have reported a positive correlation between canine and bovine neosporosis (Pare´ et al., 1998, Wouda et al., 1999), thus drawing attention to the role of these animals in the epidemiology of N. caninum. References Arias, J.R., Monteiro, P.S., Zicker, F., 1996. The re-emergence of visceral leishmaniasis in Brazil. Emerg. Infect. Diseases 2 (2), 145–146. Barber, J.S., Trees, A.J., 1998. Naturally occurring vertical transmission of Neospora caninum in dogs. Int. J. Parasitol. 28, 57–64.
Brasil. 2003. Ministe´rio da Sau´de. Secretaria de Vigilaˆncia em Sau´de. Manual de vigilaˆncia e controle da leishmaniose visceral, 122p. Buxton, D., McAllister, M.M., Dubey, J.P., 2002. The comparative pathogenesis of neosporosis. Trends Parasitol. 18, 546–552. Cringoli, G., Capuano, F., Landolfi, M.C., Veneziano, V., Barber, J.S., Trees, A.J., 1996. Anticorpi verso Neospora caninum in cani della Campania. Acta Medica Veterinaria 42, 197–202. Cringoli, G., Rinaldi, L., Capuano, F., Baldi, L., Veneziano, V., Capelli, G., 2002. Serological survey of Neospora caninum and Leishmania infantum co-infection in dogs. Vet. Parasitol. 106, 307–313. Dijkstra, T., Eysker, M., Schares, G., Conraths, F.J., Wouda, W., Barkema, H.W., 2001. Dogs shed Neospora caninum oocysts after ingestion of naturally infected bovine placenta but not after ingestion of colostrum spiked with Neospora caninum tachyzoites. Int. J. Parasitol. 8, 747–752. Dubey, J.P., Hatel, A.L., Lindsay, D.L., Topper, M.J., 1988. Neonatal Neospora caninum infection in dogs: Isolation of the causative agent and experimental transmission. J. Am. Vet. Med. Assoc. 193, 1259–1263. Dubey, J.P., 2003. Neospora caninum and neosporosis in animals. Korean J. Parasitol. 41, 1–16. Feitosa, M.M., Ikeda, F.A., Luvizotto, M.C.R., Perri, S.H.V., 2000. Aspectos clı´nicos de ca˜es com leishmaniose visceral no municı´pio de Arac¸atuba, Sa˜o Paulo (Brasil). Clı´n. Me´dica 28, 34–44. Gennari, S.M., Yai, L.E., D’Auria, S.N., Cardoso, S.M., Kwok, O.C., Jenkins, M.C., Dubey, J.P., 2002. Occurrence of Neospora caninum antibodies in sera from dogs of the city of Sao Paulo. Brazil. Vet. Parasitol. 106, 177–179. Hemphill, A., Gottstein, B., 1996. Identification of a major surface protein on Neospora caninum tachyzoites. Parasitol. Res. 82, 497–504. Lindsay, D.S., Dubey, J.P., Duncan, R.B., 1999. Confirmation that dogs is a definitive host for Neospora caninum. Vet. Parasitol. 82, 327–333. Locatelli-Dittich, R., 2002. Diagno´stico sorolo´gico, isolamento, cultivo e caracterizac¸a˜o molecular de Neospora caninum em bovinos leiteiros e em equ¨inos no estado do Parana´, Doctoral dissertation, Universidade Federal do Parana´. Curitiba, Brasil, 184 p. Pare´, J., Fecteau, G., Fortin, M., Marsolais, G., 1998. Seroepidemiologic study of Neospora caninum in dairy herds. J. Am. Vet. Med. Assoc. 213, 1595–1598. SESAU/MS. Boletim Informativo. Leishmaniose visceral Americana (Calazar). Available from: http://www.saude.ms.gov.br/ Saude/Outros/LeishmanioseVisceralAmericana.htm. Access: 12 April 2005. Silva, E.S., Carvalho, F.G., Silva, E.A., Fiozi, E., Oliveira, A.G., Brazil, R.P., 2000. Primeiro relato de leishmaniose visceral canina em a´rea urbana do municı´pio de Campo Grande, Mato Grosso do Sul. In: Congresso Brasileiro de Medicina Tropical, vol. 31. Sa˜o Luis. Anais. . .Sa˜o Luı´s, Brazil. Silva, E.A., Barros, J.C., Santos, F., Andreotti, R., 2001. Sorologia de Neospora caninum em gado de corte e em ca˜es na regia˜o de Campo Grande, MS. In: Congresso Brasileiro de Buiatria, vol. 5. Campo Grande. Anais. . .Campo Grande, CBB, 1 CD-ROM.
R. Andreotti et al. / Veterinary Parasitology 135 (2006) 375–379 Souza, S.L.P., Guimara˜es, J.S., Ferreira, F., Dubey, J.P., Gennari, S.M., 2002. Prevalence of Neospora caninum antibodies in dogs from dairy cattle farms in Parana´. J. Parasitol. 88, 408–409. Tarantino, C., Rossi, G., Kramer, L.H., Perucci, S., Cringole, G., Machionni, G., 2001. Leishmania infantum and Neospora caninum: simultaneous skin infection in a young dog in Italy. Vet. Parasitol. 102, 77–83. Thrusfield, M., 1994. Veterinary Epidemiology. Blackwell Science, Edinburgh, p. 412.
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Varandas, N.P., Rached, P.A., Costa, G.H.N., Souza, L.M., Castagnolli, K.C., Costa, A.J., 2001. Frequ¨eˆncia de anticorpos antiNeospora caninum e anti-Toxoplasma gondii em ca˜es da regia˜o nordeste do estado de Sa˜o Paulo: correlac¸a˜o com neuropatias. Semina: Ciencias Agrarias 22, 105–111. Wouda, W., Djekstra, T., Kramer, A.M.H., Maanen, C.V., Brinkhof, J.M.A., 1999. Seroepidemiological evidence for a relationship between Neospora caninum infections in dogs and cattle. Int. J. Parasitol. 29, 1677–1682.
Short communication
Occurrence of Neospora caninum in dogs and its correlation with visceral leishmaniasis in the urban area of Campo Grande, Mato Grosso do Sul, Brazil Renato Andreotti a,*, Jacqueline Marques Oliveira b, Elaine Araujo e Silva c, Leandra Marla Oshiro d, Maria de Fatima Cepa Matos e a
Embrapa Gado de Corte, Animal Health, Rodovia BR 262, km 4, Caixa postal 154, Campo Grande, MS 79002-970, Brazil b Ageˆncia de Defesa Sanita´ria Animal e Vegetal (IAGRO), Campo Grande, MS 79074-460, Brazil c Centro de Controle de Zoonoses, Prefeitura Municipal de Campo Grande. Campo Grande, MS 79074-460, Brazil d CNPq-grant recipient, Embrapa Gado de Corte, Campo Grande, MS, Brazil e Universidade Federal de Mato Grosso do Sul, UFMS, Campo Grande, MS 79070-900, Brazil Received 17 April 2005; received in revised form 22 September 2005; accepted 4 October 2005
Abstract Neospora caninum is an obligate intracellular protozoan that can infect domestic and wild canids, as well as ruminants and equines, and is described as causing neuromuscular alteration and death in dogs. Visceral leishmaniasis (VL) is an infectious disease that affects both humans and animals, being caused by protozoan parasites of the genus Leishmania, of which Leishmania (Leishmania) chagasi is found in Brazil – transmitted by sand flies, such as Lutzomyia longipalpis, in most of the American continent. The immunosuppression caused by VL can promote the occurrence of co-infections with other agents. In order to determine the frequency of N. caninum and its relationship to VL in Campo Grande, MS, Brazil, 345 blood sera were collected from dogs. The sera were submitted to an indirect fluorescent antibody test (IFAT) for detection of anti-N. caninum antibodies and VL antibodies. N. caninum was found in 26.5% of VL-negative dogs and in 29% of VL-positive ones. Among males, it was found in 30.7% of VL-negative animals and in 30.4% of VL-positive ones; among females, in 21% of VL-negative animals and in 27.7% of VL-positive ones. Among juvenile dogs (under 1 year), N. caninum was detected in 10.5% of VLnegative animals and in 11.2% of VL-positive ones. For adult dogs (1 year and older) the results were 31.4% for VL-negative animals and 28.8% of VL-positive ones. The study revealed a statistically significant association with age (x2 = 9.76, P < 0.05) in the N. caninum results for VL-negative animals. No significant correlation in N. caninum seroprevalence was found when VL-positive or VL-negative dogs were compared (x2 = 0.21, P = 0.64). The findings suggest that in Campo Grande N. caninum and VL co-infection is common in dogs, though VL does not appear to enhance susceptibility to N. caninum. # 2005 Elsevier B.V. All rights reserved. Keywords: Neospora caninum; Visceral leishmaniasis; Dogs; Brazil
* Corresponding author. Tel.: +55 67 368 2173; fax: +55 67 368 2150. E-mail address: [email protected] (R. Andreotti). 0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.10.011
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R. Andreotti et al. / Veterinary Parasitology 135 (2006) 375–379
1. Introduction Neospora caninum is a protozoan of the phylum Apicomplexa, class Sporozoea, family Sarcocystidae, subfamily Toxoplasmatinae, that can infect domestic and wild canids. It was first described as causing neuromuscular alterations and death in dogs (Dubey et al., 1988). Domestic dogs, which have been considered the only definitive hosts of the parasite, shed oocysts in their feces after ingesting tissue cysts of N. caninum (Lindsay et al., 1999; Dijkstra et al., 2001). Clinical and subclinical infections with N. caninum in dogs are epidemiologically relevant because domestic dogs (Canis familiaris) are the principal definitive hosts of the protozoan, being capable of shedding oocysts in the environment and thus constituting a risk factor for the occurrence of miscarriages associated with N. caninum in bovines (Pare´ et al., 1998). Dogs of any age can develop clinical neosporosis, which may be generalized – when several organs, including the skin, are involved – or localized (Dubey, 2003). Congenital and postnatal neosporosis have been experimentally induced in dogs, but the typical signs of congenital neosporosis, observed in naturally occurring cases, have not been reproduced (Buxton et al., 2002). Dog sera were submitted to an indirect fluorescence antibody test (IFAT) for detection of anti-N. caninum antibodies in dogs living in the urban area of Campo Grande, MS, Brazil, and a frequency of 30% was found (Silva et al., 2001). Visceral leishmaniasis (VL) is an infectious disease that affects both humans and animals, caused by a protozoan parasite of the genus Leishmania, such as Leishmania (Leishmania) donovani, Leishmania (Leishmania) infantum, and Leishmania (Leishmania) chagasi. The last one is found in Brazil (Feitosa et al., 2000; Brasil, 2003) and is transmitted by sand flies, such as Lutzomyia longipalpis, in most of the American continent, from northern Argentina to southern Mexico (Arias et al., 1996). During the past few years, many countries have witnessed a re-emergence of VL, not only in the number of cases, but also in geographic spread. Given the usual proximity between humans and dogs and the urbanization of the disease, dogs are viewed as the main urban reservoir of the parasite (Arias et al., 1996).
The first report on VL in dogs in the urban area of Campo Grande dates back from 2000, with 29 positive dogs out of 1303 surveyed (Silva et al., 2000). The numbers rose every year and reached 104 in 2003, making that an endemic area (SESAU/MS, 2005). Recently, however, skin co-infection with L. infantum and N. caninum has been reported in young dog (Tarantino et al., 2001) and high levels of N. Caninum and L. Infantum co-infection was observed in Italy (Cringoli et al., 1996). Cringoli et al. (2002) reported the co-presence of antibodies of N. Caninum and L. Infantum is very common in dogs in the Campania region of southern Italy, and that infeccion by one protozoan seems to enhance the susceptibility to the other one. The immunosuppression caused by VL can promote the occurrence of co-infections with other agents such as Ehrlichia, Babesia, and Dirofilaria in endemic regions (Feitosa et al., 2000). As an emergent disease, VL may be an influential factor in the enhancement of neosporosis. The purpose of this study was to determine if the seroprevalence of N. caninum in dogs is influenced by VL in Campo Grande, MS, Brazil.
2. Materials and methods Blood samples were collected from 345 dogs that were brought by their owners to the Center for Zoonosis Control of Campo Grande for VL diagnosis in response to the publicizing in the media of the risks posed by the disease. The samples were also used for detection of anti-N. caninum antibodies. Data on the animals’ age and sex were provided by the owners. Dogs younger than 1 year were categorized as juveniles; older ones, as adults. The sample size (310) was calculated (Thrusfield, 1994) with a 95% confidence interval and a maximum error of 5%. An expected prevalence of 20% was adopted, which corresponds to the mean value found in previous epidemiological studies (Silva et al., 2001). Antigen production and the IFAT technique for N. caninum basically followed the guidelines provided by Locatelli-Dittich (2002). Tachyzoites of N. caninum, strain NC-1 (Dubey et al., 1988), were transferred into an established cell line originated from the American
R. Andreotti et al. / Veterinary Parasitology 135 (2006) 375–379
Type Culture Collection: Vero (African three green monkey kidney, ATCC-CCL 81). Cell monolayers were used after 48–72 h of incubation, as described by Hemphill and Gottstein (1996). The sera were tested for N. caninum evaluation at an initial dilution of 1:50 and considered as positive for titers 1:50. Anti-canine IgG conjugate (VMDR Inc., WA, USA) was added to the slides. Tachyzoite fluorescence was viewed under a Nikon microscope (model BX50) equipped with an ultraviolet source. Samples displaying complete peripheral fluorescence of parasites at a 1:50 dilution were considered reactive (Pare´ et al., 1998). Antibodies against canine leishmaniasis were detected by IFAT using a Bio-Manguinhos antigen (Bio-Manguinhos, RJ, BR). A titer of at least 1:40 was considered a positive result. The results were grouped by sex and age for VL and Neospora-positive and negative sera, and submitted to a chi-square test to compare N. caninum results in VL-positive and VL-negative dogs, with one degree of tolerance and adopting a significance level of 0.05.
3. Results and discussion Of the 345 dog sera evaluated, 29% were positive for VL and 27.2% for neosporosis. Table 1 shows the distribution of the results for neosporosis in Table 1 Distribution of Neospora caninum IFAT results in dog sera negative for canine visceral leishmaniasis, by sex and age Variables
N. caninumpositive
N. caninumnegative
Total
N
%
N
%
N
Sex Males Females
43 22
30.7 21.0
97 83
69.3 79.0
140 105
Total
65
26.5
180
73.5
245
6 59
10.5 31.4
51 129
89.5 68.6
57 188
65
26.5
180
73.5
245
Age Juveniles (<1 year) Adults (1 year) Total
N. caninum-positive sera:titers 1:50. Regarding age, the prevalence was 10.5% for juveniles and significantly higher for adults: 31.4% (x2 = 9.76, P < 0.05).
377
Table 2 Distribution of N. caninum IFAT results in dog sera positive for canine visceral leishmaniasis, by sex and age Variables
N. caninumpositive
N. caninumnegative
Total
N
%
N
%
N
Sex Males Females
14 15
30.4 27.7
32 39
69.6 72.2
46 54
Total
29
29.0
71
71.0
100
1 21
11.2 28.8
8 52
88.8 71.2
9 73
22
26.8
60
73.2
82
Age Juveniles (<1 year) Adults (1 year) Total
Leishmania-positive sera:titers 1:40. N. caninum-positive sera:titers 1:50. Information on the age of 18 dogs was not provided by owners.
VL-negative dogs, and in Table 2 the results for VL positive dogs. In VL-negative dogs, the prevalence of neosporosis was 26.5%. In regard to sex, the prevalence was 30.7% for males and 21% for females, but no significant difference was found between these two subgroups (x2 = 2.93, P = 0.086). Regarding age, the prevalence was 10.5% for juveniles and significantly higher for adults: 31.4% (x2 = 9.76, P < 0.05) (Table 1). In VL-positive dogs, the prevalence of neosporosis was 29%. In regard to sex, the prevalence was 30.4% for males and 27.7% for females, but no significant difference was found between these two subgroups (x2 = 0.085, P = 0.77). Among VL-positive dogs of known age, the prevalence was 11.2% for juveniles and 28.8% for adults, but the difference between these two groups was not significant (x2 = 1.27, P = 0.26) (Table 2). No significant correlation was found when VL-positive and VL-negative dogs were compared considering N. caninum seroprevalence (x2 = 0.21, P = 0.64), confirming that N. caninum antibody titres in dogs do not change in spite of the status of L. infantum antibody titres (Cringoli et al., 2002). The frequency was similar to that found in a preliminary study conducted in the urban area of Campo Grande (Silva et al., 2001) comprising 40 dogs. The findings suggest that in Campo Grande N. caninum and Leishmania co-infection is very common
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in dogs, and infection by Leishmania does not seem to enhance susceptibility to N. caninum. Although the results found in the present study are preliminary, they add to the information available on N. caninum in Brazil. Souza et al. (2002) found anti-N. caninum antibodies in 29 (21.6%) out of 134 dogs living on dairy–cattle farms in the Brazilian state of Parana´; Gennari et al. (2002) found a frequency of 10% for domiciled dogs and 25% for stray ones in the state of Sa˜o Paulo. Regarding the variables sex (males, x2 = 0.001, P = 0.97; females, x2 = 0.93, P = 0.33) and age (juveniles, x2 = 0.002, P = 0.95; adults, x2 = 0.17, P = 0.68), no statistically significant differences were found in our study—a result also obtained by Varandas et al. (2001) and Souza et al. (2002) and explained by the fact that both male and female dogs are exposed to the same risk conditions. Age, however, played a role in the results: the frequency found for adult dogs was higher than that for juvenile ones. Souza et al. (2002) also detected an increase in seroprevalence with age. These findings are suggestive of postnatal exposure to the parasite. Barber and Trees (1998) reported that the rate of vertical transmission of neosporosis in dogs was low, since 80% of the offspring of seropositive mothers had not been infected until birth time, indicating that postnatal infection is significant in this species. Varandas et al. (2001), however, found no significant difference regarding the parameter age, suggesting that dogs of any age are subject to the same risk of infection. Additional studies on this matter are needed. Further studies should be developed with dogs in the rural area of the country, since epidemiological studies conducted in Canada, Japan, and the Netherlands have reported a positive correlation between canine and bovine neosporosis (Pare´ et al., 1998, Wouda et al., 1999), thus drawing attention to the role of these animals in the epidemiology of N. caninum. References Arias, J.R., Monteiro, P.S., Zicker, F., 1996. The re-emergence of visceral leishmaniasis in Brazil. Emerg. Infect. Diseases 2 (2), 145–146. Barber, J.S., Trees, A.J., 1998. Naturally occurring vertical transmission of Neospora caninum in dogs. Int. J. Parasitol. 28, 57–64.
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