M. Pabón

Red foxes (Vulpes vulpes) are a natural intermediate host of Neospora caninum

The present study was undertaken to determine if red foxes are natural intermediate and/or definitive host for Neospora caninum and to study the importance of infection of N. caninum in this species in North-eastern Spain. Faecal samples and brain tissues were obtained from 122 foxes from 21 rural areas of Catalonia. Faeces collected were examined for parasite eggs and coccidian oocysts using sucrose flotation. For PCR-based diagnosis of N. caninum in brain tissues, the specific genomic Nc5 region was selected as the target sequence for DNA amplification. To control for PCR failure and facilitate identification of truly negative samples, the competitor pNc5C molecule was added to all negative samples in a second round of PCR reactions. Of the 122 foxes analysed, 13 (10.7%) were positive by PCR for N. caninum. Signal intensities of all positive samples were relatively weak with the exception of one sample from a 3-month male animal, that also showed the highest repeatability. No differences were observed by sex, age or area of sampling analysis. Detection of stages of N. caninum in brain from naturally infected red foxes demonstrated that red foxes are a natural intermediate host for N. caninum. Faecal samples were analysed for the presence of N. caninum oocysts, however, no oocysts compatible with N. caninum were found. A widespread latent infection of red foxes in North-eastern Spain found in the present study indicates that red foxes could have a very important role in the epidemiology of neosporosis in our area.

Neospora caninum antibodies in wild carnivores from Spain

Serum samples from 251 wild carnivores from different regions of Spain were tested for antibodies to Neospora caninum by the commercial competitive screening enzyme linked immunosorbent assay (c-ELISA) and confirmed by Neospora agglutination test (NAT) and/or by indirect fluorescent antibody test (IFAT). Samples with antibodies detected by at least two serological tests were considered seropositive. Antibodies to N. caninum were found in 3.2% of 95 red foxes (Vulpes vulpes); in 21.4% of 28 wolves (Canis lupus); in 12.0% of 25 Iberian lynx (Lynx pardinus); in 16.7% of 6 European wildcats (Felis silvestris); in 6.4% of 31 Eurasian badgers (Meles meles); in 21.4% of 14 stone martens (Martes foina); in 66.7% of 3 pine martens (M. martes) and in 50% of 2 polecats (Mustela putorius). Antibodies to N. caninum in common genets (Genetta genetta) and Egyptian mongooses (Herpestes ichneumon) were only observed by c-ELISA but were not confirmed by IFAT and/or NAT. No antibodies were detected in 5 Eurasian otters (Lutra lutra) by any technique. Statistically significant differences were observed among species and among geographical areas. The highest seroprevalence of N. caninum infection was observed in the Cantabric Coastal region characterized by high humidity. To our knowledge, this is the first report of antibodies to N. caninum in free ranging wild carnivores, other than wild canids, in Europe. The existence of a possible sylvatic cycle could have important implications in both sylvatic and domestic cycles since they might influence the prevalence of infection in cattle farms in those areas.

Presence of Toxoplasma gondii and Neospora caninum DNA in the brain of wild birds

Toxoplasma gondii infections are prevalent in many avian species and can cause mortality in some bird hosts. Although T. gondii has been isolated from various species of birds, the role of many different species of wild birds in the epidemiology of T. gondii remains unknown. Neospora caninum, a closely related parasite to T. gondii, has been recently confirmed to infect domestic chickens and wild birds such as house sparrows (Passer domesticus). The present study reports the presence of T. gondii and N. caninum DNA by PCR in brain tissues of 14 species of wild birds from Spain. From a total of 200 samples analyzed, 12 samples (6%) were positive for T. gondii [5 Eurasian jays (Garrulus glandarius), 5 magpies (Pica pica), 1 black kite (Milvus migrans) and 1 Griffon vulture (Gyps fulvus)], while 3 samples (1.5%) were positive for N. caninum [2 magpies and 1 common buzzard (Buteo buteo)]. This is the first report of detection of T. gondii in magpies, griffon vulture and black kite and of N. caninum in common buzzard and magpies, extending the list of natural intermediate hosts for T. gondii and N. caninum infections to these species.

Prevalence of antibodies against Toxoplasma gondii in roe deer from Spain.

Roe deer (Capreolus capreolus) is an important game animal in Spain. Sera from 278 roe deer from eight areas in mainland Spain were assayed for antibodies to Toxoplasma gondii by modified agglutination test (MAT). Titers of 1:25 or higher were found in 109 (39.2%) of 278 deer. No significant differences in antibody prevalence were found between sex or age categories. In contrast, significant differences in seroprevalence between locations were evident. Roe deer from the Northern coastal habitats (high humidity and roe deer density) had the highest prevalence, compared with low prevalence in Central Spain (arid areas and low roe deer density). There was a positive correlation between antibody prevalence and mean annual rainfall (r(s)=0.85, n=8, P<0.01). These findings have environmental and/or public health implications because venison can be an important meat source of T. gondii infections for humans and feral cats.

Seroprevalence of Toxoplasma gondii and Neospora caninum in feral cats (Felis silvestris catus) in Majorca, Balearic Islands, Spain

Felids are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete environmentally resistant oocysts. Antibodies to T. gondii and Neospora caninum were determined in serum samples from 59 feral cats (Felis silvestris catus) captured in baited traps during authorized predator control campaigns in Majorca, Balearic Islands, Spain. Antibodies to T. gondii assayed by the modified agglutination test (MAT) were found in 50 (84.7%) of 59 cats with MAT titers of 1:25 in one cat, 1:200 in four cats, 1:500 in seven cats, 1:1000 in nine cats, and >or=1:2000 in 29 cats. Seroprevalence (MAT, 1:25 or more) was significantly higher in adults (94.6%) than in juveniles (<6 months old; 40.0%). Seroprevalence to N. caninum, assayed by cELISA (VMRD) and confirmed by an indirect fluorescent antibody test, was low (6.8%, 4 of 59). The prevalence of T. gondii observed in wild cats in Majorca is one of the highest reported worldwide in this species and the highest observed in Europe to date. The results suggest that feral cats in Majorca have a high rate of T. gondii infection with important implications for public health on the island since the seropositive cats are likely to have already shed T. gondii oocysts in the environment.

High seroprevalence of Toxoplasma gondii and Neospora caninum in the Common raven (Corvus corax) in the Northeast of Spain

In recent years, multiple cases of aggressive behavior of Common ravens (Corvus corax) have been reported by farmers in Catalonia (NE Spain), including attacking of newborn animals and consumption of dead foetuses. In the present study, seroprevalence of Toxoplasma gondii and Neospora caninum was determined from 113 legally trapped and released Common ravens. T. gondii antibodies were found in 91 (80.5%; CI 95%:72-87) of 113 sera tested by the modified agglutination test. Antibodies to N. caninum were found in 24 (35.8%; IC 95%: 24.5-48.5) of 67 Common ravens tested by an indirect fluorescence antibody test with titers ranging from 1:50 (n=18) to ≥1:100 (n=6). To the authors knowledge, this is the first report of the presence of T. gondii and N. caninum antibodies in C. corax. The seroprevalence detected is one of the highest reported worldwide in wild birds, suggesting an important role for this species in the epidemiology of both parasites.

LOW SEROPREVALENCE OF NEOSPORA CANINUM INFECTION ASSOCIATED WITH THE LIMOUSIN BREED IN COW-CALF HERDS IN ANDORRA, EUROPE

Neospora caninum seroprevalence and risk factors affecting seroprevalence in beef cattle in Andorra were investigated. Antibodies to N. caninum were evaluated by enzyme-linked immunosorbent assay performed on a yearly basis in 1,758 animals older than 6 mo, belonging to 26 herds. Mean seroprevalence of antibodies to N. caninum for the herds was 7.4 ± 1.2% (130/1,758). Logistic regression analyses were performed on data from each animal, considering N. caninum seropositivity as the dependent variable, and herd, grazing area, year of sampling, repeat-test animal (animals sampled twice or more), sex, breed, age (animals <4 yr old or ≥5 yr old), and country of birth as possible risk factors. Based on the odds ratio, the prevalence of infection was 2.1 times higher (P < 0.01) in animals from the Ordino grazing area, 1.64 times higher in animals older than 5 yr (P < 0.01), and 6.7 times (1/0.15) lower in Limousin–mixed Limousin cattle (P < 0.002). The results suggest that the particular grazing location could promote the horizontal transmission of this parasite and that certain breeds are less susceptible to N. caninum infection than others.

Different humoral mechanisms against Neospora caninum infection in purebreed and crossbreed beef/dairy cattle pregnancies

The antigen-specific IgG subclass response may be a convenient indicator of the underlying nature of T helper cell regulation. The aim of the present study was to identify possible differences in Neospora caninum-specific total plasma IgG, IgG1 and IgG2 antibody levels in purebreed and crossbreed pregnancies throughout gestation in beef and dairy cattle chronically infected with N. caninum. Comparisons were also made between aborting and non-aborting dams. The population examined comprised 96 pregnant parous cows seropositive for N. caninum. Plasma antibodies were determined on Days 90, 120, 150, 180 and 210 of gestation or until abortion. Of the 96 pregnancies examined, 12 ended in abortion. None of the 14 Holstein-Friesian (HF) cows inseminated with HF semen (HF-HF group) aborted, whereas 6 (11.0%) of the 54 HF cows inseminated with Limousin semen (HF-L group) and 6 (21.4%) of the 28 Rubia Gallega (RG) beef cows inseminated with RG semen (RG-RG group) aborted. In the 84 non-aborting cows, a significant positive effect of gestation day was observed on total IgG, IgG1 and IgG2 antibodies levels (P<0.0001 for the three variables). In RG-RG cows, significantly higher levels of IgG (P=0.003; d.f.=2; F-value=6.41), IgG1 (P<0.001; d.f.=2; F-value=10.55) and IgG2 (P=0.004; d.f.=2; F-value=5.82) antibodies against N. caninum were recorded throughout gestation compared to the other groups, whereas the levels of these antibodies were significantly lower in HF-HF on Days 180 and 210 of gestation. In aborting cows, significantly lower IgG (P=0.001; d.f.=1; F-value=25.21) and IgG2 (P=0.001; d.f.=1; F-value=20.39) antibody levels were observed in the RG-RG cows compared to the HF-L cows, whereas no significant effect on IgG1 antibody levels was detected in the two groups with aborting animals (RG-RG and HF-L). Our findings indicate that humoral mechanisms against N. caninum infection and abortion differ in purebreed pregnancies and crossbreed pregnancies in beef/dairy cattle.

Seroprevalence of Toxoplasma gondii in North-eastern Atlantic harbor seal (Phoca vitulina vitulina) and grey seal (Halichoerus grypus)

Antibodies to Toxoplasma gondii were determined in serum samples from 47 grey seals (Halichoerus grypus) and 56 harbor seals (Phoca vitulina vitulina) from the Atlantic coasts of United Kingdom and France. Antibodies to T. gondii assayed by the modified agglutination test (MAT) were found in 14 (13.6%; IC(95%): 7.0-20.2) of 103 seals tested, with titres of 1:25 in 13 seals and 1:50 in 1 seal. Seroprevalence against T. gondii (MAT 1:25 or higher) was significantly higher in grey seals (23.4%) compared to harbor seals (5.4%). No significant differences were found between seroprevalence against T. gondii and sex, age or geographical locations. These results show natural exposure of European harbor and grey seals to T. gondii oocysts in the Atlantic Ocean. To the best of our knowledge, this is the first serological survey of T. gondii in European grey and harbor seals.

Prevalence of Toxoplasma gondii and Neospora caninum antibodies in Spanish ibex (Capra pyrenaica hispanica).

A cross-sectional study was carried out on Spanish ibex populations in Southern Spain to assess the seroprevalence of Toxoplasma gondii and Neospora caninum and to investigate the risk factors associated with these infections. Using the modified agglutination test, the seroprevalence to T. gondii was 27.5% (146/531; CI(95%), 23.7-31.3), and this seropositivity significantly increased with age. Among adults, statistically significant differences were observed between geographical locations and over different sampling years. Thirty of 531 (5.6%) ibex had antibodies to N. caninum using a competitive ELISA, of which 27/30 (5.1%; CI(95%), 3.1-7.1) were confirmed as seropositive by the indirect fluorescent antibody test. This study is the first to report the presence of N. caninum antibodies in Spanish ibex and also indicates widespread exposure of this species to T. gondii. The findings indicate that ibex are more exposed to T. gondii than to N. caninum in their natural environment and there is little evidence of co-infection with both parasites. The seroprevalence levels reported suggest a role for ibex in the sylvatic cycle of both parasites with potentially important environmental and public health implications.