Nuno Canada

Isolation of viable Toxoplasma gondii from naturally infected aborted bovine fetuses.

Neospora caninum and Toxoplasma gondii are related parasites. The former is a common cause of abortion in dairy cattle. The latter has not been conclusively demonstrated in bovine fetuses. During the course of attempts to isolate N. caninum from aborted fetuses, T. gondii was isolated from 2 aborted fetuses, 1 from Portugal and 1 from the United States. Both isolates were made by bioassay of fetal brains in mice. The fetus from Portugal was about 5 mo in gestational age, and the fetus from the United States was a full-term stillborn.

Genotype analysis of Giardia isolated from asymptomatic children in northern Portugal.

ANDRE A. ALMEIDA, MARIA L. DELGADO, SONIA C. SOARES, ANTONIO O. CASTRO, MARIA J. MOREIRA, CARLA M. MENDONCA, NUNO B. CANADA and JOSE M. CORREIA DA COSTA Centro de Imunologia e Biologia Parasitaria—INSA, 4000-509 Porto, Portugal, and Centro de Estudo de Ciencia Animal—ICETA, Universidade do Porto, Porto, Portugal, and Abel Salazar Institute for Biomedical Sciences—ICBAS, Universidade do Porto, Portugal

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.

Characterization of the B-cell immune response elicited in BALB/c mice challenged with Neospora caninum tachyzoites.

Activation of B cells occurring in hosts infected with protozoan parasites has been implicated either in protective or parasite‐evasion immune‐mediated mechanisms. Intraperitoneal inoculation of Neospora caninum tachyzoites into BALB/c mice induces an acute response characterized by a rapid increase in the numbers of CD69‐expressing peritoneal and splenic B cells. This early B‐cell stimulatory effect preceded an increase in the numbers of total and immunoglobulin‐secreting splenic B cells and a rise in serum levels of N. caninum‐specific immunoglobulins, predominantly of the immunoglobulin G2a (IgG2a) and IgM isotypes. Increased numbers of B cells expressing the costimulatory molecules CD80 and CD86 were also observed in the N. caninum‐infected mice. The B‐cell stimulatory effect observed in mice challenged with N. caninum tachyzoites was reduced in mice challenged with γ‐irradiated parasites. Contrasting with the peripheral B‐cell expansion, a depletion of B‐lineage cells was observed in the bone‐marrow of the N. caninum‐infected mice. Intradermal immunization of BALB/c mice with diverse N. caninum antigenic preparations although inducing the production of parasite‐specific antibodies nevertheless impaired interferon‐γ (IFN‐γ) mRNA expression and caused lethal susceptibility to infection in mice inoculated with a non‐lethal parasitic inoculum. This increased susceptibility to N. caninum was not observed in naïve mice passively transferred with anti‐N. caninum antibodies. Taken together, these results show that N. caninum induces in BALB/c mice a parasite‐specific, non‐polyclonal, B‐cell response, reinforce previous observations made by others showing that immunization with N. caninum whole structural antigens increases susceptibility to murine neosporosis and further stress the role of IFN‐γ in the host protective immune mechanisms against this parasite.

Serotyping of naturally Toxoplasma gondii infected meat-producing animals

Serotyping was previously described as a promising method for typing strains of Toxoplasma gondii. The majority of precedent studies utilized serum samples collected from human patients with different T. gondii-associated pathologies. The aim of this work was to study the applicability of the same procedure for serotyping naturally infected meat-producing animals. An ELISA test based on GRA6 and GRA7 C-terminal polymorphic peptides was used. Peptide GRA6II has polymorphisms specific for the archetypal strains type II, GRA6I/III for strains type I and III, GRA7I for strains type I and GRA7III for strains type III. As reference material, and to validate this approach, serum samples from eleven free-range chickens and fifteen pigs used for Toxoplasma genotypes isolation were selected. These strains integrate the Biological Resource Centre (BRC) ToxoBS Bank. Three serum samples from chickens and two from pigs had serotyping results in agreement with genotyping. Thirty-five serum samples from chickens, twenty-nine from pigs and fifty from sheep, seropositive for T. gondii, from which no isolate was obtained, were also serotyped. Serotype III appeared significantly more frequent among sheep. Our results show that serotyping still need refinement, but may become a valuable tool for typing Toxoplasma strains from animal origin.

First isolation of Neospora caninum from an aborted bovine fetus in Spain.

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti–N. caninum antibodies and not with anti–Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum–specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.

Determination of the more adequate modified agglutination test cut-off for serodiagnosis of Toxoplasma gondii infection in sheep.

Toxoplasmosis is one of the most common food borne disease world‐wide. Among food animals, sheep seems to having higher prevalence of Toxoplasma gondii infection. However, there is no consensus about the best cut‐off for serodiagnosis in sheep. To estimate the more adequate cut‐off value of Modified Agglutination Test (MAT) for serodiagnosis in sheep, a commercial ELISA kit was used as a golden standard. Evaluation of the optimal sensitivity and specificity was calculated using Youden’s J‐statistics. Values obtained were used to estimate the prevalence of sheep toxoplasmosis. One thousand four hundred and sixty seven blood samples were collected randomly from 160 farms from northern Portugal, representing approximately 10% of the ovine population from the region. All sera were tested for anti‐T. gondii antibodies using the MAT. One hundred nine sheep (7.4%) presented a MAT titer ≥1 : 80; 45 (3.0%) a MAT titer of 1 : 40; 97 (6.6%) a MAT titer of 1 : 20 and 1216 (83.0%) a MAT titer ≤1 : 20. The best Youden’s J‐statistic was obtained at 1 : 20 titer (0.752), with 86.15% of sensitivity and 89.09% of specificity with negative and positive predictive values of 90.32% and 84.48% respectively, suggesting that the 1 : 20 was the most appropriate cut‐off for serodiagnosis of toxoplasmosis in sheep. Assuming this cut‐off, the prevalence of toxoplasmosis in the studied population was 17.1% and 92 (57.5%) of the 160 studied flocks having one or more positive sheep. Those results indicate that toxoplasmosis in Portugal should be considered in the differential diagnosis of abortions in sheep and neurological signs in lambs. Furthermore, while Portugal produces ovine meat for internal consumption and for exportation, isolation of T. gondii from ovine meat and further characterization of the isolates will be needed to understand the risk that ovine toxoplasmosis may represent for human health.

Genetic characterization of Cryptosporidium isolates from humans in northern Portugal.

ANDRE A. ALMEIDA, MARIA L. DELGADO, SONIA C. SOARES, ANTONIO O. CASTRO, MARIA J. MOREIRA, CARLA M. MENDONCA, NUNO B. CANADA, JOSE M. CORREIA DA COSTA and HELENA G. COELHO Centro de Imunologia e Biologia Parasitaria, INSA, Rua de S. Luis n. 16, 4000-509 Porto, Portugal and Centro de Estudo de Ciencia Animal, ICETA, Universidade do Porto, and Abel Salazar Institute for Biomedical Sciences, ICBAS, Universidade do Porto, Portugal, and Department of Infectious Diseases, Hospital of Joaquim Urbano, Rua Câmara Pestana, Porto

Dilated Cardiomyopathy and Sinoatrial Dysfunction in an Estrela Mountain Dog

A 1 yr old male Estrela mountain dog was evaluated as a part of a screening program for dilated cardiomyopathy. The dog came from a family with a history of dilated cardiomyopathy but was asymptomatic. Occult dilated cardiomyopathy and sino-atrial dysfunction were diagnosed based on echocardiography and electrocardiography. These two disorders may be associated given that related dogs have been diagnosed with the same disorders. The dog has remained asymptomatic for 4 years following initial evaluation.