Elsevier

Veterinary Parasitology

Volume 173, Issues 3–4, 29 October 2010, Pages 200-205
Veterinary Parasitology

In vitro isolation and identification of the first Neospora caninum isolate from European bison (Bison bonasus bonasus L.)

https://doi.org/10.1016/j.vetpar.2010.06.038Get rights and content

Abstract

Peripheral blood from European bison (Bison bonasus bonasus L.) living in Białowieża Forest, north-east Poland, were investigated for the presence of antibodies to Neospora caninum and isolation of parasite. Out of 23 animals three of them showed a strong positive response to N. caninum (13%).

The white blood cells from two positive and two negative bison were loaded on monolayer Vero cells culture. The first viable tachyzoites were detected only in positive samples at days 60 and 70 after incubation.

For the purpose of the identification the isolates, tachyzoites were evaluated by PCR and sequence analysis of fragment of the Nc-5 region.

The sequences of N. caninum-specific Nc5 region were found to be identical to those of other N. caninum isolates found in the public database. The isolate was subsequently named NC-PolBb1 and NC-PolBb2.

This is the first record of in vitro isolation of N. caninum from naturally infected European bison (Bison bonasus bonasus L.) and the first isolate obtained from the peripheral blood.

Introduction

Neospora caninum is an important an obligate intracellular protozoan that causes abortion and economic losses in the cattle industry. The parasite has also been detected in other livestock species, companion animals and wildlife animals (Dubey, 2003, Gondim, 2006). There is much current interest in wild animal reservoirs for the parasite.

Neospora caninum infection was confirmed in many wild ruminant species such as black-tailed deer (Odocoileus hemionus columbianus), white-tailed deer (O. virginianus), moose (Alces alces), mule deer (O. hemionus hemionus), caribou (Rangifer tarandus), American bison (Bison bison), chamois (Rupicapra rupicapra), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) (Dubey et al., 1999, Ferroglio et al., 2003, Dubey, 2003, Dubey and Thulliez, 2005, Wolf et al., 2005, Gondim, 2006, Almeria et al., 2007). In Europe, the presence of the parasite has been serologically shown in free-living and captive red deer (C. elaphus), roe deer (C. capreolus), fallow deer (Dama dama), sika deer (C. nippon) and also in free-living and captive European bison (Bison bonasus bonasus L.) (Ferroglio and Rossi, 2001, Cabaj et al., 2005, Almeria et al., 2007, Bartova et al., 2007, Goździk et al., 2010). European bison (B. bonasus bonasus L.) is the largest herbivorous animal in Europe. Historically, the species was distributed throughout western, central, and south-eastern Europe and the Caucasus. In 1951, the Lowland (Białowieża) bison were reintroduced successfully on wild grounds of the Polish part of the Białowieża Forest. In 1966 the Union of International for the Conservation of the Nature and Natural Resources (IUCN) formed the Red List of Threatened Species which classified the bison as the endangered species. European bison in Białowieża Forest play an important role in the restitution program and protection of the species.

The aim of the study was to monitor European bison in terms of the presence of antibodies against N. caninum and to isolate the parasite and to characterize it using PCR techniques and sequencing analysis.

Section snippets

Animals and source of samples

The Białowieża Forest (the last ancient primeval woodland in Europe) is a contiguous forest complex covering 1500 km2. The western part this complex is located in Poland and covers 650 km2. The eastern (Belarusian) part is larger covering 875 km2. The Forest extends for 55 km in the east-west direction and 51 km from north to south. The area is delineated by the coordinates 23°31′–24°21′E and 52°29′–52°37′N (Sokołowski, 2004).

In this study, 23 blood samples were collected from the jugular vein

ELISA and Western blot

In the population of 23 European bison eliminated in Białowieża Forest in 2009, three of them showed a strong positive response to N. caninum (13%). These positive sera were tested also by Western blot. The results confirmed the presence of antibodies to Neospora tachyzoites antigens. The antibody response was directed against a wide range proteins (9.5, 17, 21, 27, 31, 36.5, 38,40,43,47, 48.5, 53.5 and 58 kDa). The most heavily stained bands had molecular weights of 9.5, 17, 27 and 58 kDa.

The

Discussion

Previously, Cabaj et al. (2005) reported for the first time the presence of specific antibodies in 13 (7.3%) among 320 examined European bison (B. bonasus bonasus L.) living in free and fenced areas in Białowieża Forest. The first record of antibodies against N. caninum in European bison was found in a 16 years old cow, lived in the Białowieża Forest in freedom, and shot on December 14th, 1988 (Cabaj et al., 2005). It is worth noticing, that the earliest detection of European bison positive

Acknowledgements

Grateful thanks are due to Dr. Katarzyna Goździk for help in sequence analysis of the Polish bison isolate N. caninum.

The research is financed by The Ministry of Sciences and Higher Education, Poland, grant no. N303 062 32/2263.

References (38)

  • M.C.B. Vianna et al.

    Isolation of Neospora caninum from naturally infected white-tailed deer (Odocoileus virginianus)

    Vet. Parasitol.

    (2005)
  • D. Wolf et al.

    Detection of specific antibodies to Neospora caninum and Toxoplasma gondii in naturally infected alpacas (Lama pacos), Ilamas (Lama glama), and vicuñas (Lama vicugna) from Peru and Germany

    Vet. Parasitol.

    (2005)
  • I. Yamane et al.

    In vitro isolation and characterisation of a bovine Neospora species in Japan

    Res. Vet. Sci.

    (1997)
  • J.S. Barber et al.

    Characterization of the first European isolate of Neospora caninum (Dubey, Carpenter, Speer Topper and Uggla)

    Parasitology

    (1995)
  • W. Basso et al.

    First isolation of Neospora caninum from the feces of a naturally infected dog

    J. Parasitol.

    (2001)
  • E. Bartova et al.

    Prevalence of Neospora caninum and Toxoplasma gondii antibodies in wild ruminants from the countryside or captivity in Czech Republic

    J. Parasitol.

    (2007)
  • A.L. Bierly et al.

    Dendritic cells expressing plasmacytoid marker PDCA-1 are Trojan horses during Toxoplasma gondii infection

    J. Immunol.

    (2008)
  • C. Björkman et al.

    Neospora caninum in dogs: detection of antibodies by ELISA using an iscom antigen

    Parasite Immunol.

    (1994)
  • C. Björkman et al.

    Characterization of Neospora caninum iscom antigens using monoclonal antibodies

    Parasite Immunol.

    (1998)
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