S. Almería

Bovine piroplasms in Minorca (Balearic Islands, Spain): a comparison of PCR-based and light microscopy detection.

The present study provides the first epidemiological data regarding infection by Theileria and Babesia piroplasms in cattle in Minorca. More than 94% of the studied animals were positive for the presence of Theileria sp., and of those, 41.3% were positive for the presence of Theileria annulata. These results indicate that the prevalence of Mediterranean theileriosis caused by T. annulata is very high in Minorcan dairy farms and that other Theileria sp. are also present in the area. The prevalence of infection was similar throughout the study indicating an endemic situation in this island. The use of PCR resulted in significantly higher efficacy of detection of Theileria sp. compared to microscopical observation (MO) of blood smears and allowed the specific discrimination between pathogenic and non-pathogenic theilerias which cannot be accomplished by traditional diagnosis by MO. Babesia infection in the area was mainly due to Babesia bigemina (6.0% of the studied animals were infected), while one animal (0.75%) was found to be infected by Babesia bovis. It was observed that 31% of animals infected with B. bigemina had a concurrent infection of T. annulata. PCR also resulted in a significantly higher efficacy of detection of Babesia sp. compared to MO when infection levels were higher, towards the end of the study period. The results clearly demonstrate that parasitic infection by piroplasms, especially Theileria sp. is common and endemic in the island of Minorca and that PCR is the optimal approach for the detection and discrimination of these important parasites.

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.

Disease threats to the endangered Iberian lynx (Lynx pardinus)

Abstract The Iberian lynx, (Lynx pardinus), is the most endangered felid in the world. To determine whether sympatric carnivores are reservoirs of pathogens posing a disease risk for the lynx, evidence of exposure to 17 viral, bacterial and protozoan agents was investigated in 176 carnivores comprising 26 free-living lynx, 53 domestic cats, 28 dogs, 33 red foxes (Vulpes vulpes), 24 Egyptian mongooses (Herpestes ichneumon), 10 common genets (Genetta genetta) and 2 Eurasian badgers (Meles meles) in the areas inhabited by the last two populations of Iberian lynx, both in Andalusia (South-Western Spain). The results indicated that the lynx had low rates of contact with viral pathogens, with one seropositive finding each for feline leukemia virus, parvovirus and canine adenovirus-1, whereas contact with bacteria and protozoa appeared more frequent. Active infections with parvovirus, Ehrlichia spp., Mycobacterium bovis, Leptospira interrogans and Cytauxzoon spp. were confirmed. In contrast, 53% of the domestic cats were exposed to some infectious agent (prevalence range 4.5–11.4%). Antibodies to canine distemper virus and parvovirus were frequently found in dogs (32% and 42%, respectively) and foxes (30% and 12%). Past or present infections with parvovirus, Ehrlichia spp., Chlamydophila spp., M. bovis, Salmonella enterica, L. interrogans, Toxoplasma gondii, and Neospora caninum were also detected in these and other species surveyed. Questionnaires to owners revealed that 14% of the dogs but none of the cats had been vaccinated, and no cat had been neutered. Based on the apparent absence of acquired immunity of the lynx against infectious agents, the frequent detection of agents among sympatric carnivores, and the reported lack of immunocompetence of the Iberian lynx, a disease outbreak among the local abundant carnivores may pose a serious disease risk for lynx conservation.

Seroprevalence of Toxoplasma gondii Antibodies in Domestic Cats from Barcelona, Spain

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally resistant oocysts. Antibodies to T. gondii were determined in serum samples from 220 domestic cats (Felis catus) from Barcelona, Spain, using the modified agglutination test (MAT). Antibodies to T. gondii were found in 99 (45%) of 220 cats, with MAT titers of 1:25 in 26, 1:50 in 57, and ≥1:500 in 16 cats. Seropositivity (MAT 1:25 or more) was significantly higher in adult (≥1 yr old, 49.7% of 153) than in juvenile (<1 yr old, 34.3% of 67) cats, in feral (51.9% of 131) than in domiciled (34.8% of 89) cats, and in cats living in a group (community) of more than 5 cats (50.7% of 142) than in cats living alone (28.0% of 50). These seropositive cats are likely to have already shed T. gondii oocysts in the environment around Barcelona.

Disseminated Toxoplasmosis in a Mediterranean Pregnant Risso's Dolphin (Grampus griseus) with Transplacental Fetal Infection

Fatal disseminated toxoplasmosis was diagnosed in a Rissos dolphin (Grampus griseus) dam and its fetus on the basis of pathologic findings, immunohistochemistry, and structure of the parasite. The dolphin was stranded alive on the Spanish Mediterranean coast and died a few hours later. At necropsy the dam was in good condition. From the standpoint of pathology, however, it had generalized lymphadenomegaly and splenomegaly, enlargement of and multifocal hemorrhage in the adrenal glands, diffuse mucosal hemorrhage of the glandular and pyloric stomach, ulcerative glossitis and stomatitis, focal erosions and reddening of the laryngeal appendix, and severe paraotic sinusitis with intralesional nematodes Crassicauda grampicola. The dolphin was pregnant, most probably in the first gestational trimester. The most prominent microscopic lesions were multifocal granulomatous encephalomyelitis, diffuse subacute interstitial pneumonia, mild multifocal necrotizing hepatitis and nonsuppurative cholangiohepatitis, gastritis and adrenalitis, mild lymphoid depletion, medullary sinus and follicular histyocitosis, and systemic hemosiderosis. The fetus had foci of coagulative and lytic necrosis in the kidneys, the lung, and the heart. Most lesions were associated with tachyzoites and tissue cysts of Toxoplasma gondii. The diagnosis was confirmed immunohistochemically. This is the first report on toxoplasmosis in a Rissos dolphin (G. griseus) and on transplacental transmission to an early-stage fetus in any cetaceans.

Cytokine gene expression in dams and foetuses after experimental Neospora caninum infection of heifers at 110 days of gestation

Neospora caninum is a major cause of abortion in cattle. An essential role for Th1 cytokines, such as IFN‐gamma and IL‐12 in protective immunity against N. caninum in murine models has been indicated. However, little is known about immunity to Neospora in pregnant cattle where a considerable level of immunomodulation may exist. In this study, the immune response of heifers infected early in the second trimester of pregnancy by intravenous inoculation of N. caninum tachyzoites was compared with immune responses in uninfected pregnant heifers. Animals were killed 3 weeks after infection. No abortion was observed in any infected dam, however, transplacental infection was shown to have already taken place. Infection with N. caninum during pregnancy induced significant immune responses in both dams and their foetuses. Infected dams showed significant changes in lymphocyte subpopulations compared with uninfected pregnant animals and these changes were compartmentalized. Increased levels of T lymphocytes were observed in the infected foetuses. Cytokine gene expression analysed by real time RT‐PCR showed increased expression of both Th1 and Th2 cytokines in N. caninum infected animals. This cytokine expression could have a role in the transplacental transmission of the parasite and/or mediate tissue damage.

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.

Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infection in equids.

A cross-sectional study was carried out on equids (horses, mules and donkeys) in Andalusia, Southern Spain, to assess the level of exposure to equine piroplasmosis and to investigate risk factors associated with these infections. At least one animal seropositive for Theileria equi and/or Babesia caballi was detected in 222/380 (58.4%) herds sampled by competitive inhibition ELISAs. The seroprevalences for B. caballi and T. equi were 13.2% and 56.1%, respectively; there was serological evidence of co-circulation of both piroplasms in 10.8% of herds. Antibodies against equine piroplasms were detected in 286/537 (53.3%) animals; 61 (11.4%) were seropositive for B. caballi, 270 (50.3%) were seropositive for T. equi and 24 (8.4%) were seropositive for both T. equi and B. caballi. There was a significantly higher seroprevalence of B. caballi in mules (32.1%) compared with donkeys (17.0%) and horses (7.9%), and a significantly higher seroprevalence of T. equi in mules (66.1%) in comparison with horses (48.6%), but not donkeys (47.2%). There were significant differences in prevalence of both piroplasms among locations; the seroprevalence of B. caballi ranged from 0 to 22.5%, while the seropositivity to T. equi ranged from 26.7 to 63.3%. A multiple logistic regression model indicated that the risk factors associated with a higher T. equi seroprevalence were increased age, presence of ticks and vaccination against other diseases. Risk factors associated with a higher seroprevalence of B. caballi were species (mules compared to horses), entry of horses in the last 6months, presence of ticks and presence of shelter. The findings indicate widespread exposure to equine piroplasmosis in Southern Spain.

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.