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Seroprevalence of Neospora caninum in cats from the Czech Republic. Kamil Sedlák1, Eva Bartova2* and Tereza Machacova2. 1State Veterinary Institute ...
DOI: 10.2478/s11686-014-0246-y © W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2014, 59(2), 359–361; ISSN 1230-2821

RESEARCH NOTE

Seroprevalence of Neospora caninum in cats from the Czech Republic Kamil Sedlák1, Eva Bartova2* and Tereza Machacova2 1 2

State Veterinary Institute Prague, Department of Virology and Serology, Sidlistni 136/24, 165 03 Prague 6, Czech Republic University of Veterinary and Pharmaceutical Sciences, Faculty of Veterinary, Department of Biology and Wildlife Diseases, Hygiene and Ecology, Palackého tř. 1/3, 612 42 Brno, Czech Republic

Abstract Sera of 414 cats coming from different parts of the Czech Republic were tested for N. caninum antibodies. Sera samples were collected during years 2002–2011. N. caninum antibodies were detected by a commercial competitive-inhibition enzyme-linked immunosorbent assay (cELISA) with cut off ≥30% inhibition. Samples positive in cELISA were confirmed by an indirect fluorescence antibody test (IFAT); titre ≥50 was considered positive. In total, 137 (33%) cats reacted positively in cELISA; N. caninum antibodies in IFAT were detected in 16 (3.86%) cats with titres 50 and 100. In 6 cats, positive for N. caninum antibodies, T. gondii antibodies were also detected by IFAT. It is the first report of N. caninum antibodies in domestic cats from the Czech Republic and third report in Europe.

Keywords Neosporosis, antibodies, ELISA, IFAT, feline

Neospora caninum have been confused with Toxoplasma gondii for long time. Both parasites are very similar cyst-forming coccidian of the phylum Apicomplexa with indirect life cycle, with carnivores as definitive hosts (Dubey et al. 2007). A wide range of domestic and wild animals have been experimentally exposed to N. caninum infection. However, viable Neospora sp. has been isolated only from a few hosts as summarized by Dubey and Schares (2011). The cats could be sensitive to neospora infection, but clinical symptoms such as myositis and encephalitis are not so specific. According to the experimental studies, the infection with N. caninum is more serious in case of cats infected in neonatal and prenatal period or in case of nursing cats (Dubey 1992). Only some studies focused on detection of N. caninum antibodies in cats. The aim of this study was to test sera of cats from the Czech Republic for N. caninum antibodies by two different serological tests. In this study, domestic cats without evident clinical symptoms of any diseases coming from different parts of the Czech Republic (especially from Prague, Central Bohemia and North Moravia) were tested for N. caninum antibodies. Blood was taken by puncture of the saphenous vein from 414 cats during

years 2002–2011. Serum obtained by centrifugation was stored at –20°C until examination at State Veterinary Institute in Prague. Sera were tested for the presence and level of IgG antibodies against N. caninum by a commercial competitive-inhibition enzyme-linked immunosorbent assay (cELISA, VMRD, Pullman, USA) with cut off ≥ 30% inhibition. Serum samples were also tested by indirect fluorescent antibody test (IFAT) that is used as a reference method. Commercially available antigen (VMRD, Pullman, USA) and species-specific conjugates anticat IgG immunoglobulin (Sigma Aldrich, Czech Republic) were used. The sera were diluted in a two-fold series starting at 1:50 as a basic dilution; the titre ≥50 was considered positive. All serum samples were also examined for T. gondii antibodies by IFAT using a commercially available antigen (VMRD) and anti-cat IgG immunoglobulin (Sigma Aldrich). The sera were diluted in a two-fold series starting at 1:50 as a basic dilution; the titre ≥50 was considered positive. Neospora caninum antibodies were detected in 137 (33%) and 16 (3.86%) of 414 cats by cELISA and IFAT, respectively. In IFAT, we found 11 and 5 positive cats with titre 50 and 100,

*Corresponding author: [email protected]

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Table I. Neospora caninum antibodies in domestic cats in cELISA and IFAT Method

Positive/n (%)

cELISA

137/414 (33%)

Inhibition (%)

30–40

40.1–50

50.1–60

60.1–70

70.1–80

80.1–90

Positive

81

44

4

5

2

1

IFAT

16/414 (3.86%)

Titre 50*

4

2

0

4

1

0

Titre 100*

1

1

2

0

1

0

cELISA – competitive-inhibition enzyme-linked immunosorbent assay (cut-off 30% inhibition), IFAT – indirect fluorescent antibody test (cut-off titre 50). *Samples positive in IFAT (titre 50 or 100) and simultaneously positive in cELISA (% inhibition 30–80)

respectively and with 30–90% inhibition in cELISA. The results are summarized in Table I. This is the first serologic survey for N. caninum antibodies performed in cats in the Czech Republic and third report in Europe. N. caninum antibodies were found by IFAT with cut-off titre 40 in 0.6% of 330 cats from Hungary (Hornok et al. 2008) and in 24.8% of 282 stray cats from Italy by latex-agglutination test (Ferroglio et al. 2005). The same methods cELISA and/or IFAT were used also in Brazil; N. caninum antibodies were found in 25% of 400 (Bresciani et al. 2007) and in none of 70 (Coelho et al. 2011) cats, respectively. In Thailand, 36 tested cats were negative for N. caninum antibodies in both IFAT and cELISA (Arunvipas et al. 2011). The discrepancy in N. caninum prevalence found in our study in cELISA and IFAT could be explained by different sensitivity and specifity of these tests. The method cELISA was primarily developed and validated for cattle sera (Baszler et al. 2001). Till yet, it has not been validated for testing of cat sera that it is why this method could be used as a screening method since it has high sensitivity. However it is recommended to confirm the results with reference method IFAT that is more specific. There are also some studies focusing on prevalence of N. caninum antibodies in feral and zoo felids. Antibodies to N. caninum were found in 19% of 100 feral cats (Felis catus) by cELISA in Iran (Hamidine et al. 2011) and in 19% of 26 Eurasian lynx (Lynx lynx), 6.8%–16.7% European wild cat (Felis silvestris) and 12% of 25 Iberian lynx (Lynx pardinus) from Spain by cELISA and confirmed by IFAT (Sobrino et al. 2008; Millan et al. 2009a, b). Andre et al. (2010) used IFAT to test captive wild felids in zoos in Brazil and found 11% –71% prevalence in different felids (lion, jaguarondi, puma, jaguar, tiger and ocelot). Domestic cat and other felids are definitive hosts of T. gondii that is why there is usually higher prevalence of T. gondii compared to N. caninum in cats. T. gondii and N. caninum antibodies were detected in 43.6% and 6.9% zoo felids from U.S.A (Spencer et al. 2003) and in 92% and 12% zoo felids from the Czech Republic (Sedlak and Bartova 2006), respectively. In both studies, the same method IFAT was used but with different cut-off titre (50 and 40, respectively). In our study, we found only 6 cats (1.4%) positive for T. gondii antibodies with titres 400, 1600 and 3200 in 3, 2 and

1 cats, respectively. These cats were simultaneously infected with N. caninum with titre 50. Acknowledgements. This study was funded by the grant no. MSM6215712402 from the Ministry of Education, Youth and Sports of the Czech Republic. We would like to thank Barbora Zahradnickova for help with sample examinations.

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Received: December 12, 2013 Revised: March 5, 2014 Accepted for publication: March 17, 2014

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