- Email: [email protected]
Serological and molecular survey of Dirofilaria immitis infection in stray cats in Gyunggi province, South Korea
Veterinary Parasitology 130 (2005) 125–129 www.elsevier.com/locate/vetpar
Serological and molecular survey of Dirofilaria immitis infection in stray cats in Gyunggi province, South Korea J. Liu a, K.H. Song a,*, S.E. Lee a, J.Y. Lee b, J.I. Lee c, M. Hayasaki d, M.J. You e, D.H. Kim a a
Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, South Korea b Laboratory of Veterinary Internal Medicine, Department of Veterinary Clinical Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan c Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, South Korea d Veterinary Clinical Center, School of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan e Laboratory of Veterinary Parasitology, College of Veterinary Medicine and Bio-safty Research Center, Chonbuk National University, Jeonju 561-756, South Korea Received 1 September 2004; received in revised form 16 March 2005; accepted 16 March 2005
Abstract The aim of this study was to carry out a serological and molecular survey for the presence of Dirofilaria immitis infection in stray cats using an ELISA kit and PCR assay. One hundred and fifty-five stray cats (77 females and 78 males) in Gyunggi province in South Korea, were used in this study. Four (2.6%) tested with the ELISA kit showed a positive reaction, and all positive samples by the ELISA kit showed a positive reaction by PCR analysis. No significant difference was observed between the male (2.6%) and female (2.6%) cat groups by ELISA kit. The positive rates for dirofilariosis were 2.8% in the 4–6-year-old group, and 18.2% in the >6-year-old group by ELISA kit. With regard to the age element, older cats showed a higher prevalence of D. immitis infection in this study. A statistical analysis revealed that significant difference was observed in >6-year-old group ( p < 0.01). In conclusion, D. immitis infection in stray cats was present in Gyunggi province, although its incidence was low. Therefore, heartworm treatment and/or prophylaxis for stray cats captured are required in this area. # 2005 Elsevier B.V. All rights reserved. Keywords: Cat; Dirofilaria immitis; PCR; ELISA; Prevalence
* Corresponding author. Tel.: +82 42 821 6789; fax: +82 42 822 4216. E-mail address: [email protected] (K.H. Song). 0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.03.026
126
J. Liu et al. / Veterinary Parasitology 130 (2005) 125–129
1. Introduction Dirofilariosis, caused by the filarial nematode Dirofilaria immitis, has been reported in dogs (Kamiie et al., 2000; Araujo et al., 2003; Tarello, 2002), snow leopard (Murata et al., 2003), cats (Frank et al., 1998; Bazzocchi et al., 2000; Strickland, 1998), coyotes (Sacks and Caswell-Chen, 2003), ferret (McCall, 1998), ocelot (Pence et al., 2003), wolf, Canis lupus (Segovia et al., 2001), red foxes (Marks and Bloomfield, 1998) and even humans (Theis et al., 2001) in the USA (Nelson et al., 2003; Theis et al., 2001), Europe (Araujo et al., 2003; Tarello, 2002), Australia (Marks and Bloomfield, 1998) and Asia (Song et al., 2003; Murata et al., 2003; Retnasabapathy and San, 1976). In general, canines can easily be infected, but felines are not, however, feline infection is being diagnosed in clinics at an ever increasing rate. Unlike dogs, as few as two adult heartworms can cause cardiac enlargement and severe respiratory compromise in cats (Borgarelli et al., 1997), and these characteristics result in a low production of heartworm antigen and antibodies. Infected cats also exhibit different clinical signs to those of dogs. Anorexia, respiratory distress, coughing, chronic vomiting, weight loss or sudden death may be the only symptoms (Dillon, 1984; Strickland, 1998), whereas dogs typically show signs of right-sided heart failure. The prevalence in cats seems to parallel that in dogs with in the same area, but at a much lower level, with cats having approximately one-tenth (1/10) the prevalence seen in the unprotected dog population (Miller, 1998). In the U.S.A., dirofilariosis in cats has been estimated to be between 5 and 20% of the prevalence in local dog populations (Carleton and Tolbert, 2004). In Japan, the prevalence rate of D. immitis infection was 0.8%, but was 4.1% for animals aged 2 years and over (Nogami and Sato, 1997). In a report in Italy the prevalence in the feline population was as high as 23% (Genchi et al., 1992). In South Korea, only one study has reported feline dirofilariosis (Han, 2003), and the prevalence of this report was 0.5%, but the importance of D. immitis in cats remains to be fully recognized. Thus, examination of the status of D. immitis infection in cats is very important for the acquisition of information with regards feline dirofilariosis.
The purpose of this study was to examine the prevalence of feline dirofilariosis in Korean stray cats using an ELISA kit and PCR assays.
2. Materials and methods 2.1. Animals One hundred and fifty-five stray cats (77 females and 78 males) from Suwon and Gwachon cities in Gyunggi province were examined for D. immitis infection between 2001 and 2003. The age of the animals was estimated by the annual layers of teeth and bone (Ohtaishi and Hachiya, 1985). The age range of the felines was between 10 months and 7 years. 2.2. Blood collections Blood (4 ml) was drawn from the jugular vein of stray cats captured in Suwon and Guachon cities, Gyunggi province. One half of blood was collected in a tube containing EDTA as an anticoagulant, the other half of blood was collected in a tube without any anticoagulant, and this sample was used for examination of a modified Knott test and DNA isolation. 2.3. DNA isolation Three hundred microliters of whole blood were lysed in 0.1 M Tris–HCl (pH 8.0) containing 1% SDS, 0.1 M NaCl and 10 mM EDTA. The samples were then treated with proteinase K (100 m/ml), for 2 h, at 55 8C. The DNA was extracted with phenol/chloroform, precipitated by ethanol, and then dissolved in 50 ml of TE buffer (10 mM Tris–HCl (pH 8.0), 1 mM EDTA). The DNA samples were stored at 4 8C for later use. 2.4. PCR amplification The primers used in this study were used: 50 GCATCTTAGAACTTGGT CCATCC-30 (forward), and 50 -CAAAGGCGTATTTACC GCCAC-30 (reverse), which have been described by Watts et al. (1999) for the amplification of 440-bp 16 S rRNA gene segment. The
J. Liu et al. / Veterinary Parasitology 130 (2005) 125–129
assay specifically detects as little as 10 pg of D. immitis genomic DNA (Watts et al., 1999). PCR was performed in 50 ml reaction mixtures, containing 5 ml template DNA, 2 ml 20 pmol of each primer, 4 ml 200 mM of each dNTP, 1.25 U rTaq DNA polymerase (Takara, Japan), and 5 ml 10 PCR buffer (10 mM Tris–HCl, 1.5 mM MgCl2, and 0.001% gelatin). The thermal cycler was set at 94 8C for 3 min, to activate the polymerase, and then 30 cycles, each of denaturation for 30 s at 94 8C, annealing for 40 s at 55 8C and extension for 50 s at 72 8C. A 5-ml sample of the PCR products was then analyzed by electrophoresis in 0.8%agarose gel followed by ethidium-bromide staining and photography. 2.5. Modified Knott test and ELISA examination The modified Knott test (Newton and Wright, 1956) was performed for microfilaria detection. A feline heartworm antigen detecting kit (SNAP test, IDEXX Laboratories, Maine, USA) was used in this study; the protocol of this test was described in a booklet of directions supplied with this kit.
The data in Table 1 were analyzed with a database (SPSS v.10.0, K), and a statistical analysis of the prevalence carried out with the chi-squared test.
Table 1 Prevalence of D. immitis infection in stray cats using ELISA kit
Gender Male Female Age (years) <2 2–4 4–6 >6 Total *
3. Results 3.1. Modified Knott test, ELISA and PCR examination All cats examined were amicrofilaremic by the Modified Knott test and asymptomatic. The results of the ELISA examinations on the feline dirofilariaosis positive samples are shown in Table 1. In the 155 feline samples, 4 were positive using the ELISA kit test (2.6%). Of these 78 male and 77 female cats 2 (2.6 and 2.6%) each were positive. In the investigation of age, out of 72 feline samples (less than 4 years old), no positive tests were found for cats between 4 and 6 years, but there were 2 (2.8, 18.2%) positive tests each for the cats between 4 and 6 years and aged 6 years or more. With older age, the prevalence of D. immitis infection was shown to be higher in this study. A statistical analysis revealed that significant difference was observed in >6-year-old-group ( p < 0.01). All positive samples by the ELISA kit showed a positive reaction by PCR analysis.
4. Discussion
2.6. Statistical analysis
No. of examined
127
ELISA kit No. of positive
Positive rate (%)
78 77
2 2
2.6 2.6
9 63 72 11 155
0 0 2 2 4
0 0 2.8 18.2* 2.6
Significant statistical difference ( p < 0.01).
Feline dirofilariosis is now recognized as a potential cause of serious disease and can present both a clinical and diagnostic challenge to practicing veterinarians. Feline heartworm infection can be symptomatic in some cats. However, it is possible that the presence of only two adult worms in the lungs and/or heart may cause severe illness (Borgarelli et al., 1997). A cat may be totally asymptomatic and then die from heartworm disease within an hour (Genchi et al., 1995). The availability of a specific and sensitive test for the diagnosis of the infection would be very helpful for clinical management of infected cats. In Japan, Roncalli et al. (1998) reported that the prevalence of D. immitis by euthanasia varied from 0.5 to 9.5% for stray cats in several prefectures, and Nogami and Sato (1997) also reported that the prevalence of feline dirofilariosis by euthanasia in Saitama prefecture was 0.8% in abandoned and pound animals. Labarthe et al. (1997) reported that the incidence of feline dirofilariosis by euthanasia was 0.8% in stray cats in metropolitan Rio de Janeiro city, Brazil.
128
J. Liu et al. / Veterinary Parasitology 130 (2005) 125–129
In South Korea, Han (2003) first reported that the prevalence of feline dirofilariaosis by euthanasia was 0.5% in Seoul city, Kyunggi, Kangwon, Chungchong, Cholla and Kyongsang provinces. In this study, 4 (2.6%) of the 155 samples tested with the ELISA kit showed a positive reaction. This result was slightly higher than that of Han (2003), although surveyed areas and method in our study are different. Our study showed that the incidence in male (2.6% by ELISA kit) and female cats (2.6% ELISA kit) were similar, and no statistical difference was observed between the two groups. This result was different from those of Kramer and Genchi (2002), where the male cats had significantly higher seroprevalence than the female cats. The positive rates of dirofilariosis by ELISA kit were 2.8% in the 4–6-year-old group, 18.2% in those over 6-yearold, respectively. With older age, the prevalence of D. immitis infection was shown to be higher in this study. A statistical analysis revealed that significant difference was observed in >6-year-old-group ( p < 0.01). These results also were different from those of the report of Kramer and Genchi (2002), where incidence rate in the older-age group was no higher. Piche´ et al. (1998) evaluated the relationship between the antibody and antigen test results in a commercial laboratory, and reported that low Ab levels (<20 AbU/ml) were rarely associated with circulating antigens. The intensely positive reactions (>20 AbU/ml) may be more easily associated with the presence of adult worms in the heart/lungs (Piche´ et al., 1998; Genchi et al., 1998). D. immitis-specific PCR diagnostic method has become a valuable and highly dependable diagnostic tool. The assay specifically detects as little as 10 pg of D. immitis genomic DNA, and reliably and reproducibly detects the expected 440-bp long D. immitis-specific PCR product in all the D. immitis infected canine blood samples (Watts et al., 1999). All positive samples by the ELISA kit showed a positive reaction by PCR analysis in our study (Fig. 1). In conclusion, D. immitis infection in stray cats was present in Gyunggi province, although its incidence was low. Therefore, heartworm treatment and/or prophylaxis for captured stray cats is needed this area. More samples and areas will need to be studied for an accurate survey of feline dirofilariosis.
Fig. 1. The PCR-based detection of feline heartworm DNA in wild cats in South Korea. The amplified 440-bp product, from the positive sample control (lane 1) or positive test samples (lanes 2–6), was subjected to electrophoresis in 0.8%-agarose gel and stained with ethidium bromide. A 100-bp ladder (M) and a negative control free of DNA template (lane 7) were run in parallel.
References Araujo, R.T., Marcondes, C.B., Bastos, L.C., Sartor, D.C., 2003. Canine dirofilariasis in the region of Conceicao Lagoon, Florianopolis, and in the Military Police kennel, Sao Jose, State of Santa Catarina. Brazil. Vet. Parasitol. 113 (3–4), 239– 242. Bazzocchi, C., Ceciliani, F., McCall, J.W., Ricci, I., Genchi, C., Bandi, C., 2000. Antigenic role of the endosymbionts of filarial nematodes: IgG response against the Wolbachia surface protein in cats infected with Dirofilaria immitis. Proc. R. Soc. Lond. B. Biol. Sci. 267 (1461), 2511–2516. Borgarelli, M., Venco, L., Piga, P.M., Bonino, F., Ryan, W.G., 1997. Surgical removal of heartworms from the right atrium of a cat. J. Am. Vet. Med. Assoc. 211 (1), 68–69. Carleton, R.E., Tolbert, M.K., 2004. Prevalence of Dirofilaria immitis and gastrointestinal helminths in cats euthanized at animal control agencies in northwest Georgia. Vet. Parasitol. 119 (4), 319–326. Dillon, R., 1984. Feline dirofilariasis. Vet. Clin. N. Am. 14 (6), 1185–1199. Frank, G.R., Mondesire, R.R., Brandt, K.S., Wisnewski, N., 1998. Antibody to the Dirofilaria immitis aspartyl protease inhibitor homologue is a diagnostic marker for feline heartworm infections. J. Parasitol. 84 (6), 1231–1236. Genchi, C., Guerrero, J., Di Sacco, B., 1992. Prevalence of Dirofilaria immitis infection in Italian cats. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium 1992, Austin, TX, American Heartworm Society, Batavia, IL, pp. 97–102. Genchi, C., Kramer, L., Venco, L., Prieto, G., Simon, F., 1998. Comparison of antibody and antigen testing with echocardiography for the detection of heartworm (Dirofilaria immitis) in cats. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 1998. American Heartworm Society, Batavia, IL, pp. 173–177.
J. Liu et al. / Veterinary Parasitology 130 (2005) 125–129 Genchi, C., Venco, L., Vezzoni, A., 1995. Aggiornamento sulla filariosi cardiopolmonare del gatto. Veterinaria 9, 53–58. Han, D.U., 2003. Dirofilaria immitis infection in stray cat. Korean J. Vet. Public Health 27 (3), 167–170. Kamiie, J., Shirota, K., Yamaki, M., Kitagawa, H., Wasaki, M., Ooi, H.K., 2000. Abnormal distribution of anionic sites in the glomerular basement membrane in glomerulonephritis of dogs infected with Dirofilaria immitis. J. Vet. Med. Sci. 62 (11), 1193–1195. Kramer, L., Genchi, C., 2002. Feline heartworm infection: serological survey of asymptomatic cats living in northern Italy. Vet. Parasitol. 104 (1), 43–50. Labarthe, N., Ferreira, A.M.R., Guerrero, J., Newcomb, K., Paes-deAlmeida, E., 1997. Survey of Dirofilaria immitis (Leidy, 1856) in random source cats in metropolitan Rio de Janeiro, Brazil, with descriptions of lesions. Vet. Parasitol. 71, 301– 306. Marks, C.A., Bloomfield, T.E., 1998. Canine heartworm (Dirofilaria immitis) detected in red foxes (Vulpes vulpes) in urban Melbourne. Vet. Parasitol. 78 (2), 147–154. McCall, J.W., 1998. Dirofilariasis in the domestic ferret. Clin. Tech. Small Anim. Pract. 13 (2), 109–112. Miller, M.W., 1998. Feline dirofilariasis. Clin. Tech. Small Anim. Pract. 13 (2), 99–108. Murata, K., Yanai, T., Agatsuma, T., Uni, S., 2003. Dirofilaria immitis infection of a snow leopard (Uncia uncia) in a Japanese zoo with mitochondrial DNA analysis. J. Vet. Med. Sci. 65 (8), 945–947. Nelson, T.A., Gregory, D.G., Laursen, J.R., 2003. Canine heartworms in coyotes in Illinois. J. Wildl. Dis. 39 (3), 593– 599. Newton, W.L., Wright, W.H., 1956. The occurrence of a dog filariid other than Dirofilaria immitis in the United States. J. Parastol. 42, 246–256. Nogami, S., Sato, T., 1997. Prevalence of Dirofilaria immitis infection in cats in Saitama. Jpn. J. Vet. Med. Sci. 59 (10), 869–871.
129
Ohtaishi, N., Hachiya, N., 1985. Aging Techniques from Annual Layers in Teeth and Bone. Contemporary Mammalogy in China and Japan. Mammalogical Society of Japan, pp. 180–190. Pence, D.B., Tewes, M.E., Lack, L.L., 2003. Helminths of the ocelot from southern Texas. J. Wildl. Dis. 39 (3), 683–689. Piche´ , C.A., Cavanaugh, M.T., Donoghue, A.R., Redecki, S.V., 1998. Results of antibody and antigen testing for feline heartworm infection at Heska1 veterinary diagnostic laboratories. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 1998. American Heartworm Society, Batavia, IL, pp. 139–143. Retnasabapathy, A., San, K.T., 1976. Incidence of canine heartworm (Dirofilaria immitis) in Malaysia. Vet. Rec. 98 (4), 68–69. Roncalli, R.A., Yamane, Y., Nagata, T., 1998. Prevalence of Dirofilaria immitis in cats in Japan. Vet. Parasitol. 75, 81–89. Sacks, B.N., Caswell-Chen, E.P., 2003. Reconstructing the spread of Dirofilaria immitis in California coyotes. J. Parasitol. 89 (2), 319–323. Segovia, J.M., Torres, J., Miquel, J., Llaneza, L., Feliu, C., 2001. Helminths in the wolf, Canis lupus, from north-western Spain. J. Helminthol. 75 (2), 183–192. Strickland, K.N., 1998. Canine and feline caval syndrome. Clin. Tech. Small Anim. Pract. 13 (2), 88–95. Song, K.H., Lee, S.E., Hayasaki, M., Shiramizu, K., Kim, D.H., Cho, K.W., 2003. Seroprevalence of canine dirofilariosis in South Korea. Vet. Parasitol. 114 (3), 231–236. Tarello, W., 2002. Dermatitis associated with Dirofilaria (Nochtiella) repens microfilariae in dogs from central Italy. Acta Vet. Hung. 50 (1), 63–78. Theis, J.H., Gilson, A., Simon, G.E., Bradshaw, B., Clark, D., 2001. Case report: Unusual location of Dirofilaria immitis in a 28-yearold man necessitates orchiectomy. Am. J. Trop. Med. Hyg. 64 (5–6), 317–322. Watts, K.J., Courteny, C.H., Reddy, G.R., 1999. Development of a PCR- and probe-based test for the sensitive and specific detection of the dog heartworm, Dirofilaria immitis, in its mosquito intermediate host. Mol. Cell Probes 13 (6), 425–430.
Serological and molecular survey of Dirofilaria immitis infection in stray cats in Gyunggi province, South Korea J. Liu a, K.H. Song a,*, S.E. Lee a, J.Y. Lee b, J.I. Lee c, M. Hayasaki d, M.J. You e, D.H. Kim a a
Laboratory of Veterinary Internal Medicine, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, South Korea b Laboratory of Veterinary Internal Medicine, Department of Veterinary Clinical Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan c Laboratory of Veterinary Surgery, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, South Korea d Veterinary Clinical Center, School of Veterinary Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan e Laboratory of Veterinary Parasitology, College of Veterinary Medicine and Bio-safty Research Center, Chonbuk National University, Jeonju 561-756, South Korea Received 1 September 2004; received in revised form 16 March 2005; accepted 16 March 2005
Abstract The aim of this study was to carry out a serological and molecular survey for the presence of Dirofilaria immitis infection in stray cats using an ELISA kit and PCR assay. One hundred and fifty-five stray cats (77 females and 78 males) in Gyunggi province in South Korea, were used in this study. Four (2.6%) tested with the ELISA kit showed a positive reaction, and all positive samples by the ELISA kit showed a positive reaction by PCR analysis. No significant difference was observed between the male (2.6%) and female (2.6%) cat groups by ELISA kit. The positive rates for dirofilariosis were 2.8% in the 4–6-year-old group, and 18.2% in the >6-year-old group by ELISA kit. With regard to the age element, older cats showed a higher prevalence of D. immitis infection in this study. A statistical analysis revealed that significant difference was observed in >6-year-old group ( p < 0.01). In conclusion, D. immitis infection in stray cats was present in Gyunggi province, although its incidence was low. Therefore, heartworm treatment and/or prophylaxis for stray cats captured are required in this area. # 2005 Elsevier B.V. All rights reserved. Keywords: Cat; Dirofilaria immitis; PCR; ELISA; Prevalence
* Corresponding author. Tel.: +82 42 821 6789; fax: +82 42 822 4216. E-mail address: [email protected] (K.H. Song). 0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.03.026
126
J. Liu et al. / Veterinary Parasitology 130 (2005) 125–129
1. Introduction Dirofilariosis, caused by the filarial nematode Dirofilaria immitis, has been reported in dogs (Kamiie et al., 2000; Araujo et al., 2003; Tarello, 2002), snow leopard (Murata et al., 2003), cats (Frank et al., 1998; Bazzocchi et al., 2000; Strickland, 1998), coyotes (Sacks and Caswell-Chen, 2003), ferret (McCall, 1998), ocelot (Pence et al., 2003), wolf, Canis lupus (Segovia et al., 2001), red foxes (Marks and Bloomfield, 1998) and even humans (Theis et al., 2001) in the USA (Nelson et al., 2003; Theis et al., 2001), Europe (Araujo et al., 2003; Tarello, 2002), Australia (Marks and Bloomfield, 1998) and Asia (Song et al., 2003; Murata et al., 2003; Retnasabapathy and San, 1976). In general, canines can easily be infected, but felines are not, however, feline infection is being diagnosed in clinics at an ever increasing rate. Unlike dogs, as few as two adult heartworms can cause cardiac enlargement and severe respiratory compromise in cats (Borgarelli et al., 1997), and these characteristics result in a low production of heartworm antigen and antibodies. Infected cats also exhibit different clinical signs to those of dogs. Anorexia, respiratory distress, coughing, chronic vomiting, weight loss or sudden death may be the only symptoms (Dillon, 1984; Strickland, 1998), whereas dogs typically show signs of right-sided heart failure. The prevalence in cats seems to parallel that in dogs with in the same area, but at a much lower level, with cats having approximately one-tenth (1/10) the prevalence seen in the unprotected dog population (Miller, 1998). In the U.S.A., dirofilariosis in cats has been estimated to be between 5 and 20% of the prevalence in local dog populations (Carleton and Tolbert, 2004). In Japan, the prevalence rate of D. immitis infection was 0.8%, but was 4.1% for animals aged 2 years and over (Nogami and Sato, 1997). In a report in Italy the prevalence in the feline population was as high as 23% (Genchi et al., 1992). In South Korea, only one study has reported feline dirofilariosis (Han, 2003), and the prevalence of this report was 0.5%, but the importance of D. immitis in cats remains to be fully recognized. Thus, examination of the status of D. immitis infection in cats is very important for the acquisition of information with regards feline dirofilariosis.
The purpose of this study was to examine the prevalence of feline dirofilariosis in Korean stray cats using an ELISA kit and PCR assays.
2. Materials and methods 2.1. Animals One hundred and fifty-five stray cats (77 females and 78 males) from Suwon and Gwachon cities in Gyunggi province were examined for D. immitis infection between 2001 and 2003. The age of the animals was estimated by the annual layers of teeth and bone (Ohtaishi and Hachiya, 1985). The age range of the felines was between 10 months and 7 years. 2.2. Blood collections Blood (4 ml) was drawn from the jugular vein of stray cats captured in Suwon and Guachon cities, Gyunggi province. One half of blood was collected in a tube containing EDTA as an anticoagulant, the other half of blood was collected in a tube without any anticoagulant, and this sample was used for examination of a modified Knott test and DNA isolation. 2.3. DNA isolation Three hundred microliters of whole blood were lysed in 0.1 M Tris–HCl (pH 8.0) containing 1% SDS, 0.1 M NaCl and 10 mM EDTA. The samples were then treated with proteinase K (100 m/ml), for 2 h, at 55 8C. The DNA was extracted with phenol/chloroform, precipitated by ethanol, and then dissolved in 50 ml of TE buffer (10 mM Tris–HCl (pH 8.0), 1 mM EDTA). The DNA samples were stored at 4 8C for later use. 2.4. PCR amplification The primers used in this study were used: 50 GCATCTTAGAACTTGGT CCATCC-30 (forward), and 50 -CAAAGGCGTATTTACC GCCAC-30 (reverse), which have been described by Watts et al. (1999) for the amplification of 440-bp 16 S rRNA gene segment. The
J. Liu et al. / Veterinary Parasitology 130 (2005) 125–129
assay specifically detects as little as 10 pg of D. immitis genomic DNA (Watts et al., 1999). PCR was performed in 50 ml reaction mixtures, containing 5 ml template DNA, 2 ml 20 pmol of each primer, 4 ml 200 mM of each dNTP, 1.25 U rTaq DNA polymerase (Takara, Japan), and 5 ml 10 PCR buffer (10 mM Tris–HCl, 1.5 mM MgCl2, and 0.001% gelatin). The thermal cycler was set at 94 8C for 3 min, to activate the polymerase, and then 30 cycles, each of denaturation for 30 s at 94 8C, annealing for 40 s at 55 8C and extension for 50 s at 72 8C. A 5-ml sample of the PCR products was then analyzed by electrophoresis in 0.8%agarose gel followed by ethidium-bromide staining and photography. 2.5. Modified Knott test and ELISA examination The modified Knott test (Newton and Wright, 1956) was performed for microfilaria detection. A feline heartworm antigen detecting kit (SNAP test, IDEXX Laboratories, Maine, USA) was used in this study; the protocol of this test was described in a booklet of directions supplied with this kit.
The data in Table 1 were analyzed with a database (SPSS v.10.0, K), and a statistical analysis of the prevalence carried out with the chi-squared test.
Table 1 Prevalence of D. immitis infection in stray cats using ELISA kit
Gender Male Female Age (years) <2 2–4 4–6 >6 Total *
3. Results 3.1. Modified Knott test, ELISA and PCR examination All cats examined were amicrofilaremic by the Modified Knott test and asymptomatic. The results of the ELISA examinations on the feline dirofilariaosis positive samples are shown in Table 1. In the 155 feline samples, 4 were positive using the ELISA kit test (2.6%). Of these 78 male and 77 female cats 2 (2.6 and 2.6%) each were positive. In the investigation of age, out of 72 feline samples (less than 4 years old), no positive tests were found for cats between 4 and 6 years, but there were 2 (2.8, 18.2%) positive tests each for the cats between 4 and 6 years and aged 6 years or more. With older age, the prevalence of D. immitis infection was shown to be higher in this study. A statistical analysis revealed that significant difference was observed in >6-year-old-group ( p < 0.01). All positive samples by the ELISA kit showed a positive reaction by PCR analysis.
4. Discussion
2.6. Statistical analysis
No. of examined
127
ELISA kit No. of positive
Positive rate (%)
78 77
2 2
2.6 2.6
9 63 72 11 155
0 0 2 2 4
0 0 2.8 18.2* 2.6
Significant statistical difference ( p < 0.01).
Feline dirofilariosis is now recognized as a potential cause of serious disease and can present both a clinical and diagnostic challenge to practicing veterinarians. Feline heartworm infection can be symptomatic in some cats. However, it is possible that the presence of only two adult worms in the lungs and/or heart may cause severe illness (Borgarelli et al., 1997). A cat may be totally asymptomatic and then die from heartworm disease within an hour (Genchi et al., 1995). The availability of a specific and sensitive test for the diagnosis of the infection would be very helpful for clinical management of infected cats. In Japan, Roncalli et al. (1998) reported that the prevalence of D. immitis by euthanasia varied from 0.5 to 9.5% for stray cats in several prefectures, and Nogami and Sato (1997) also reported that the prevalence of feline dirofilariosis by euthanasia in Saitama prefecture was 0.8% in abandoned and pound animals. Labarthe et al. (1997) reported that the incidence of feline dirofilariosis by euthanasia was 0.8% in stray cats in metropolitan Rio de Janeiro city, Brazil.
128
J. Liu et al. / Veterinary Parasitology 130 (2005) 125–129
In South Korea, Han (2003) first reported that the prevalence of feline dirofilariaosis by euthanasia was 0.5% in Seoul city, Kyunggi, Kangwon, Chungchong, Cholla and Kyongsang provinces. In this study, 4 (2.6%) of the 155 samples tested with the ELISA kit showed a positive reaction. This result was slightly higher than that of Han (2003), although surveyed areas and method in our study are different. Our study showed that the incidence in male (2.6% by ELISA kit) and female cats (2.6% ELISA kit) were similar, and no statistical difference was observed between the two groups. This result was different from those of Kramer and Genchi (2002), where the male cats had significantly higher seroprevalence than the female cats. The positive rates of dirofilariosis by ELISA kit were 2.8% in the 4–6-year-old group, 18.2% in those over 6-yearold, respectively. With older age, the prevalence of D. immitis infection was shown to be higher in this study. A statistical analysis revealed that significant difference was observed in >6-year-old-group ( p < 0.01). These results also were different from those of the report of Kramer and Genchi (2002), where incidence rate in the older-age group was no higher. Piche´ et al. (1998) evaluated the relationship between the antibody and antigen test results in a commercial laboratory, and reported that low Ab levels (<20 AbU/ml) were rarely associated with circulating antigens. The intensely positive reactions (>20 AbU/ml) may be more easily associated with the presence of adult worms in the heart/lungs (Piche´ et al., 1998; Genchi et al., 1998). D. immitis-specific PCR diagnostic method has become a valuable and highly dependable diagnostic tool. The assay specifically detects as little as 10 pg of D. immitis genomic DNA, and reliably and reproducibly detects the expected 440-bp long D. immitis-specific PCR product in all the D. immitis infected canine blood samples (Watts et al., 1999). All positive samples by the ELISA kit showed a positive reaction by PCR analysis in our study (Fig. 1). In conclusion, D. immitis infection in stray cats was present in Gyunggi province, although its incidence was low. Therefore, heartworm treatment and/or prophylaxis for captured stray cats is needed this area. More samples and areas will need to be studied for an accurate survey of feline dirofilariosis.
Fig. 1. The PCR-based detection of feline heartworm DNA in wild cats in South Korea. The amplified 440-bp product, from the positive sample control (lane 1) or positive test samples (lanes 2–6), was subjected to electrophoresis in 0.8%-agarose gel and stained with ethidium bromide. A 100-bp ladder (M) and a negative control free of DNA template (lane 7) were run in parallel.
References Araujo, R.T., Marcondes, C.B., Bastos, L.C., Sartor, D.C., 2003. Canine dirofilariasis in the region of Conceicao Lagoon, Florianopolis, and in the Military Police kennel, Sao Jose, State of Santa Catarina. Brazil. Vet. Parasitol. 113 (3–4), 239– 242. Bazzocchi, C., Ceciliani, F., McCall, J.W., Ricci, I., Genchi, C., Bandi, C., 2000. Antigenic role of the endosymbionts of filarial nematodes: IgG response against the Wolbachia surface protein in cats infected with Dirofilaria immitis. Proc. R. Soc. Lond. B. Biol. Sci. 267 (1461), 2511–2516. Borgarelli, M., Venco, L., Piga, P.M., Bonino, F., Ryan, W.G., 1997. Surgical removal of heartworms from the right atrium of a cat. J. Am. Vet. Med. Assoc. 211 (1), 68–69. Carleton, R.E., Tolbert, M.K., 2004. Prevalence of Dirofilaria immitis and gastrointestinal helminths in cats euthanized at animal control agencies in northwest Georgia. Vet. Parasitol. 119 (4), 319–326. Dillon, R., 1984. Feline dirofilariasis. Vet. Clin. N. Am. 14 (6), 1185–1199. Frank, G.R., Mondesire, R.R., Brandt, K.S., Wisnewski, N., 1998. Antibody to the Dirofilaria immitis aspartyl protease inhibitor homologue is a diagnostic marker for feline heartworm infections. J. Parasitol. 84 (6), 1231–1236. Genchi, C., Guerrero, J., Di Sacco, B., 1992. Prevalence of Dirofilaria immitis infection in Italian cats. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium 1992, Austin, TX, American Heartworm Society, Batavia, IL, pp. 97–102. Genchi, C., Kramer, L., Venco, L., Prieto, G., Simon, F., 1998. Comparison of antibody and antigen testing with echocardiography for the detection of heartworm (Dirofilaria immitis) in cats. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 1998. American Heartworm Society, Batavia, IL, pp. 173–177.
J. Liu et al. / Veterinary Parasitology 130 (2005) 125–129 Genchi, C., Venco, L., Vezzoni, A., 1995. Aggiornamento sulla filariosi cardiopolmonare del gatto. Veterinaria 9, 53–58. Han, D.U., 2003. Dirofilaria immitis infection in stray cat. Korean J. Vet. Public Health 27 (3), 167–170. Kamiie, J., Shirota, K., Yamaki, M., Kitagawa, H., Wasaki, M., Ooi, H.K., 2000. Abnormal distribution of anionic sites in the glomerular basement membrane in glomerulonephritis of dogs infected with Dirofilaria immitis. J. Vet. Med. Sci. 62 (11), 1193–1195. Kramer, L., Genchi, C., 2002. Feline heartworm infection: serological survey of asymptomatic cats living in northern Italy. Vet. Parasitol. 104 (1), 43–50. Labarthe, N., Ferreira, A.M.R., Guerrero, J., Newcomb, K., Paes-deAlmeida, E., 1997. Survey of Dirofilaria immitis (Leidy, 1856) in random source cats in metropolitan Rio de Janeiro, Brazil, with descriptions of lesions. Vet. Parasitol. 71, 301– 306. Marks, C.A., Bloomfield, T.E., 1998. Canine heartworm (Dirofilaria immitis) detected in red foxes (Vulpes vulpes) in urban Melbourne. Vet. Parasitol. 78 (2), 147–154. McCall, J.W., 1998. Dirofilariasis in the domestic ferret. Clin. Tech. Small Anim. Pract. 13 (2), 109–112. Miller, M.W., 1998. Feline dirofilariasis. Clin. Tech. Small Anim. Pract. 13 (2), 99–108. Murata, K., Yanai, T., Agatsuma, T., Uni, S., 2003. Dirofilaria immitis infection of a snow leopard (Uncia uncia) in a Japanese zoo with mitochondrial DNA analysis. J. Vet. Med. Sci. 65 (8), 945–947. Nelson, T.A., Gregory, D.G., Laursen, J.R., 2003. Canine heartworms in coyotes in Illinois. J. Wildl. Dis. 39 (3), 593– 599. Newton, W.L., Wright, W.H., 1956. The occurrence of a dog filariid other than Dirofilaria immitis in the United States. J. Parastol. 42, 246–256. Nogami, S., Sato, T., 1997. Prevalence of Dirofilaria immitis infection in cats in Saitama. Jpn. J. Vet. Med. Sci. 59 (10), 869–871.
129
Ohtaishi, N., Hachiya, N., 1985. Aging Techniques from Annual Layers in Teeth and Bone. Contemporary Mammalogy in China and Japan. Mammalogical Society of Japan, pp. 180–190. Pence, D.B., Tewes, M.E., Lack, L.L., 2003. Helminths of the ocelot from southern Texas. J. Wildl. Dis. 39 (3), 683–689. Piche´ , C.A., Cavanaugh, M.T., Donoghue, A.R., Redecki, S.V., 1998. Results of antibody and antigen testing for feline heartworm infection at Heska1 veterinary diagnostic laboratories. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 1998. American Heartworm Society, Batavia, IL, pp. 139–143. Retnasabapathy, A., San, K.T., 1976. Incidence of canine heartworm (Dirofilaria immitis) in Malaysia. Vet. Rec. 98 (4), 68–69. Roncalli, R.A., Yamane, Y., Nagata, T., 1998. Prevalence of Dirofilaria immitis in cats in Japan. Vet. Parasitol. 75, 81–89. Sacks, B.N., Caswell-Chen, E.P., 2003. Reconstructing the spread of Dirofilaria immitis in California coyotes. J. Parasitol. 89 (2), 319–323. Segovia, J.M., Torres, J., Miquel, J., Llaneza, L., Feliu, C., 2001. Helminths in the wolf, Canis lupus, from north-western Spain. J. Helminthol. 75 (2), 183–192. Strickland, K.N., 1998. Canine and feline caval syndrome. Clin. Tech. Small Anim. Pract. 13 (2), 88–95. Song, K.H., Lee, S.E., Hayasaki, M., Shiramizu, K., Kim, D.H., Cho, K.W., 2003. Seroprevalence of canine dirofilariosis in South Korea. Vet. Parasitol. 114 (3), 231–236. Tarello, W., 2002. Dermatitis associated with Dirofilaria (Nochtiella) repens microfilariae in dogs from central Italy. Acta Vet. Hung. 50 (1), 63–78. Theis, J.H., Gilson, A., Simon, G.E., Bradshaw, B., Clark, D., 2001. Case report: Unusual location of Dirofilaria immitis in a 28-yearold man necessitates orchiectomy. Am. J. Trop. Med. Hyg. 64 (5–6), 317–322. Watts, K.J., Courteny, C.H., Reddy, G.R., 1999. Development of a PCR- and probe-based test for the sensitive and specific detection of the dog heartworm, Dirofilaria immitis, in its mosquito intermediate host. Mol. Cell Probes 13 (6), 425–430.