Toxoplasma gondii and Neospora caninum infections in South American camelids in Switzerland and assessment of serological tests for diagnosis

Abstract

Background: Little is known about the epidemiology of Toxoplasma gondii and Neospora caninum infections in alpacas (Vicugna pacos) and llamas (Lama glama) outside South America. The study aimed to estimate the seroprevalence of T. gondii and N. caninum infections in South American camelids (SAC) in Switzerland, to optimize serological tests for SAC and to identify risk factors, which may favour infection.

Methods: A total of 571 sera from 132 Swiss farms (374 alpacas and 197 llamas, mean 4.3 animals/farm) were obtained. Four commercial enzyme-linked immunosorbent assays (ELISA) for detecting antibodies against T. gondii (ID Screen^® Toxoplasmosis Indirect (TOXO-MS)) or N. caninum (i.e. ID Screen^® Neospora caninum Indirect Multi-species (NCS-MS); ID Screen^® Neospora caninum Competition (NCC) and ID Screen^® Neospora caninum Indirect (NCS)) were first assessed for their use on SAC comparing their results with those in immunoblot, and optimizing cut-offs. Subsequently, two kits (TOXO-MS and NCS-MS) were selected for seroprevalence estimation. Additionally, a risk factor analysis for infection was performed on 41 farms, which agreed to participate in a web-based survey.

Results: Three kits (TOXO-MS, NCS-MS and NCC) showed almost perfect agreement (kappa > 0.901) with immunoblot results when the cut-offs were optimized, and one kit (NCS) proved not to be useful for detecting N. caninum seropositive SAC. By TOXO-MS ELISA, 82.3% (308/374) of the alpacas and 84.8% (167/197) of the llamas were seropositive for T. gondii, and 131/132 (99.2%) farms had seropositive animals. By NCS-MS ELISA, 3.5% (13/374) of the alpacas and 2.5% (5/197) of the llamas evidenced antibodies against N. caninum, and 9.1% (12/132) of the farms had seropositive animals. The variables "age" and "female sex" were identified as risk factors for T. gondii infection and "absence of cats in the farm during the last two years" as a protective factor. No risk or protective factors for N. caninum infection could be identified.

Conclusions: This nationwide cross-sectional study demonstrated for the first time the presence of antibodies against T. gondii and N. caninum in the Swiss SAC population, highlighting a high seroprevalence for T. gondii, the presence of cats as a risk factor and suggesting that SAC meat might represent an additional infection source for humans.

Keywords: Alpaca; ELISA; Immunoblot; Lama glama; Llama; Neosporosis; Toxoplasmosis; Vicugna pacos.

Fig. 1

Map of Switzerland showing the distribution of South American camelid (SAC) breeding farms (n = 132) sampled in the study (all dots) and the farms in which N. caninum seropositive SAC were detected (red dots)

Fig. 2

Immunoblot results for antibodies against T. gondii in Swiss South American camelids (SAC). Abbreviations: M, molecular marker; Neg, seronegative Swiss SAC field samples; Pos, seropositive Swiss SAC field samples; PC, positive control (Llama); NC, negative control (Llama); >, 30 kDa band corresponding to SAG-1 T. gondii tachyzoite surface antigen. Samples recognizing the 30 kDa antigenic band were considered positive

Fig. 3

ELISA results for antibodies against N. caninum on serum samples from a llama experimentally inoculated with 4.8 × 10E6 cell culture-derived N. caninum tachyzoites (Llama 1) and from a control llama inoculated with 5 × 10E4 Vero cells (Llama 2) [30]. a ID Screen® Neospora caninum indirect multi-species, S/P% = sample-to-positive ratio, calculated based on the OD (optical density) of the sample and the positive and negative controls of the kit according to the formula S/P% = (OD_sample − OD_{negative control}/OD_{positive control} − OD_{negative control}) × 100. b ID Screen® Neospora caninum Competition, S/N% = competition percentage, calculated according to the formula: S/N% = (OD_sample/OD_{negative control}) × 100

Fig. 4

Immunoblot results for antibodies against N. caninum on field serum samples from Swiss South American camelids (SAC). Abbreviations: M, marker; PB, positive control (bovine); PC, positive control (Llama 1); NC, negative control (Llama 2); samples 8–28, Swiss SAC field serum samples; +, positive immunoblot result; −, negative immunoblot result. Bands corresponding to immunodominant N. caninum antigens (17, 29, 30, 33 and 37 kDa) are indicated with *. Samples recognizing two or more immunodominant antigens were considered positive

Fig. 5

Immunoblot results for antibodies against N. caninum on serum samples from a llama experimentally inoculated with 4.8 × 10E6 cell culture-derived N. caninum tachyzoites (Llama 1) and from a seronegative control llama inoculated with 5 × 10E4 Vero cells (Llama 2). Abbreviations: M, marker; kDa, kilodaltons; 2–52 (indicated above), days post-inoculation (dpi). Bands corresponding to immunodominant N. caninum antigens (17, 29, 30, 33 and 37 kDa) are indicated with an asterisk. In Llama 1, reaction against the 29 kDa antigen is observed at 12 dpi. From 16 to 52 dpi also further immunodominant antigens are recognized. Samples recognizing two or more immunodominant antigens were considered positive. Llama 2 did not recognize any of the N. caninum immunodominant antigens during the observation period