Isolation and molecular characterization of a new Neospora caninum isolate from cattle in Argentina.

Neospora caninum is one of the most important causes of bovine abortion, but isolation of live parasites from infected tissue is difficult. The aims of the present study were to obtain new isolates of N. caninum from congenitally infected asymptomatic newborn cattle in Argentina and to perform characterization by multilocus-microsatellite analysis. Five clinically normal born calves, with demonstrable N. caninum antibodies in precolostrum serum by indirect fluorescent antibody test, were euthanized and their brain samples were processed for histopathological, immunohistochemical, polymerase chain reaction (PCR) analysis, and for bioassay in γ-interferon knockout (GKO) mice. Although N. caninum DNA was detected in brain from all the calves by PCR, viable N. caninum was isolated in GKO mice from only one calf. Neospora caninum tachyzoites of this Argentinean isolate, designated NC-Argentina LP1, were propagated in VERO cell cultures seeded with tachyzoites from the infected GKO mice tissues. Multilocus-microsatellite typing on DNA derived from cell cultured tachyzoites revealed a unique genetic pattern, different from reported isolates. This is the first bovine isolation and genetic characterization of N. caninum in Argentina.

Neospora caninum NC-6 Argentina induces fetopathy in both serologically positive and negative experimentally inoculated pregnant dams.

Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108 ± 2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.

Frequency of horizontal and vertical transmission for Sarcocystis cruzi and Neospora caninum in dairy cattle.

Sarcocystis cruzi and Neospora caninum infections in cattle are common throughout the world, and cause important economical losses. N. caninum can be transmitted horizontally by ingestion of oocysts or vertically from the infected dam to the fetus via the placenta. Vertical transmission for S. cruzi is infrequent and horizontal transmission is considered the most important route of infection. The objectives of this study were to evaluate the frequency of horizontal and vertical transmission for S. cruzi and N. caninum in a dairy cattle herd and to analyze IFAT titers as predictors of vertical transmission. Serum samples (n = 173) were collected from dairy calves at birth prior to colostrum ingestion and from their dams. In addition, 12 calves were also sampled after ingestion of colostrum, 25 female calves were sampled at 7 months, and 81 of the dams were also sampled at breeding. Sera were evaluated for S. cruzi and N. caninum antibodies by IFAT starting at 1:25 dilution. For S. cruzi, vertical transmission frequency was 1.7%, and all female calves evaluated at 7 months and cows were seropositive. Seroprevalence for N. caninum was 80.9% in cows and 30% in precolostrum calves. Vertical transmission frequency was 37.1%. Cows with high antibody titers (> or = 400) showed higher vertical transmission frequency (94.8%) than cows with low antibody titers (between 25 and 200) (14.8%). Negative precolostrum calves (7/12) had postcolostrum N. caninum titers 2-8 times higher than their dams. Estimated horizontal transmission frequency was 51 and 47%, based on differences of seroprevalences in calves and dams, and on the seroconversion of 9/19 negative precolostrum female calves when retested at 7 months, respectively. Average N. caninum titers of cows at breeding and calving were 120.6 and 320.9, respectively. Cows with a high titer at breeding had a high titer at calving. Therefore, N. caninum IFAT titers at breeding and calving could potentially be used as predictors of vertical transmission.

Isolation and molecular characterization of Toxoplasma gondii from captive slender-tailed meerkats (Suricata suricatta) with fatal toxoplasmosis in Argentina.

In this study, the diagnosis of fatal disseminated toxoplasmosis in three captive slender-tailed meerkats (Suricata suricatta) in the zoo of La Plata, Argentina and the invitro isolation and molecular characterization of Toxoplasma gondii are reported. The animals showed depression, dyspnea and hypothermia, and also ataxia in one case, and died within 1-5 days. The main histopathological lesions included interstitial pneumonia, non-suppurative inflammatory changes and focal necrosis in liver, spleen, kidney and brain. Tachyzoites or tissue cysts were present in lung, liver, spleen, brain, striated muscle, kidney, intestine and mesenteric lymph node sections, and stained strongly with T. gondii antiserum in immunohistochemical analysis. T. gondii was isolated in Swiss mice and in bovine monocytes cultures from tissues of one of the meerkats. The isolate was cryopreserved and it was named TG-Suricata-1. T. gondii DNA was demonstrated in tissues of all three animals and in tachyzoites isolated in cell cultures. The PCR-RFLP analysis of markers based in the loci 3'-SAG2, 5'-SAG2, BTUB, GRA6, SAG3, c22-8, L358, PK1, c29-2 and Apico of T. gondii produced patterns corresponding to the clonal type III. Type III strains of T. gondii possess no or only little virulence in the mouse model, however their association with virulence in other animal species is uncertain. In the present case, T. gondii of the clonal lineage III was responsible for fatal cases in S. suricatta. To our knowledge, this is the first report of isolation and genotyping of T. gondii from S. suricatta.

Seroprevalence of Neospora caninum, Toxoplasma gondii and Sarcocystis sp. in llamas (Lama glama) from Jujuy, Argentina.

Llamas (Lama glama) are South American camelids described as intermediate hosts of Neospora caninum, Toxoplasma gondii and Sarcocystis aucheniae. Due to the potential role of these protozoan infections as a cause of economic losses, the aim of this study was to determine the seroprevalence for T. gondii, N. caninum and Sarcocystis sp. in llamas from Argentina. Serum samples from 308 llamas (>2 years old) were collected between 2005 and 2007. A total of 55 farms located in six departments of Jujuy province, Argentina were sampled. Presence of antibodies to N. caninum, T. gondii and Sarcocystis sp. was determined by the indirect fluorescent antibody test (IFAT). For Sarcocystis, 2 different bradyzoites-based antigens were prepared using S. aucheniae and S. cruzi. Sera were tested at dilutions 1:25 and 1:50. Antibodies to N. caninum were found in 4.6% serum samples. Fifty percent of departments and 14.5% of farms had positive animals. Antibodies to T. gondii were found in 30% of samples, distributed in 66% of departments and 43.6% of farms. Antibodies to Sarcocystis sp. were detected in 96% of samples and all departments and farms had positive animals, suggesting frequent contact between llamas and canids. Co-infection with N. caninum, T. gondii and Sarcocystis sp. was also recorded. Low seroprevalence of N. caninum in llamas detected in this study could be related to climatic and geographical conditions that limit cattle breeding activity, reducing the source of infection for definitive hosts. Seroprevalence of T. gondii and the positive animal distribution suggest frequent contamination of grass with felid faeces. In conclusion, this is the first report of combined seroprevalence for N. caninum, T. gondii and Sarcocystis sp. in llamas. Further studies are needed to determine the potential role of these protozoan infections as cause of abortion in Argentina as well as presence of these protozoans in llama meat used for human consumption.

Toxoplasmosis in captive Bennett's wallabies (Macropus rufogriseus) in Argentina.

Wallabies and other Australian marsupials are among the most susceptible species to Toxoplasma gondii. Fatal generalized toxoplasmosis was diagnosed in two captive 3 year-old female Bennett's wallabies (Macropus rufogriseus) from Argentina (w 1 and w 2) with a history of sudden death. Both animals had internal joeys which died 2 days after their mothers. Serologically, both females and one adult male without clinical signs from the same enclosure (w 3) had antibody titers for T. gondii>or=800 by the modified agglutination test (MAT); another adult male (w 4) was negative (MAT titer<25). Microscopically, tachyzoites were observed associated to non-suppurative meningoencephalitis, hepatitis, myositis, myocarditis and severe enteritis in hematoxylin and eosin stained sections from both w 1 and w 2. Immunohistochemically, parasites in heart, brain and liver sections of both female wallabies reacted with T. gondii antiserum. T. gondii was isolated from brain tissues of w 1 and w 2 by bioassay in mice and by culture in bovine monocytes and both isolates were cryopreserved. Genomic DNA was isolated from tachyzoites grown in cultures derived from both animals. The primer pair B22/B23 specific for T. gondii produced 115bp amplicons on poliacrylamide electrophoretic gels. Stray cats were suspected as the possible source of infection. Not all infected macropods were ill, showing that the infection may be asymptomatic and is not always fatal. A vertical infection could not be proved in the joey from w 2. As far as we know, this is the first confirmed report of toxoplasmosis in Bennet's wallabies in Argentina.

Nanomolar cationic dendrimeric sulfadiazine as potential antitoxoplasmic agent.

The high doses of sulfadiazine (SDZ), used in synergistic combination with pyrimethamine, are mainly responsible for severe side effects and discontinuation of toxoplasmosis treatments. In the search for new strategies that improve the efficacy of treatments with reduced doses of SDZ, we have determined the performance of cationic G4 (DG4) and anionic G4.5 (DG4.5) poly(amidoamine) (PAMAM) dendrimers to act as SDZ nanocarriers. Both dendrimers could efficiently load SDZ (SDZ-DG4 and SDZ-DG4.5) up to a ratio of 30 molecules SDZ per dendrimer molecule. The MTT assay on Vero and J774 cells showed no cytotoxicity for DG4.5 and its SDZ complex incubated between 0.03 and 33 microM of dendrimer concentration. On the other hand, DG4 and its SDZ complex resulted cytotoxic when incubated at dendrimer concentrations higher than 3.3 microM. Finally, complexes and empty dendrimers were in vitro tested against Vero cells infected with RH strain of Toxoplasma gondii along 4h of treatment. For SDZ-DG4.5 and DG4.5 to cause an infection decrease between 25 and 40%, respectively, a dendrimer concentration of 33 microM was required; however, SDZ-DG4 produced the highest infection decrease of 60% at 0.03 microM. These preliminary results, achieved with nanomolar doses of SDZ-DG4 as unique active principle, point to this complex as a suitable potential candidate for antitoxoplasmic therapy.

Confirmed clinical Neospora caninum infection in a boxer puppy from Argentina.

Generalized neosporosis was diagnosed in a 2-month-old boxer puppy. The dog had a history of progressive paralysis and muscle atrophy, followed by cervical weakness, stiff jaws and dysphagia. The dog had a 1:12,800 antibody titer for Neospora caninum and was negative for antibodies to Toxoplasma gondii by the indirect fluorescent antibody test (IFAT). After euthanasia a complete necropsy was carried out. The puppy had a megaesophagus. Microscopically, tachyzoites and tissue cysts were observed in histologic brain sections. Severe myositis was observed in esophagus and striated muscle sections and several groups of tachyzoites were associated with these lesions. Immunohistochemically, parasites in the brain and striated muscle reacted to anti-N. caninum antiserum. Western blot analysis allowed the identification of three major and four minor antigens of N. caninum tachyzoites corresponding to 30, 37, 45-kDa and 28, 29, 43, 47 and 67-kDa bands, respectively. Cerebral homogenate of the dog was inoculated into four Mongolian gerbils (Meriones unguiculatus). Forty-nine days after inoculation, all the gerbils had positive IFAT titers to N. caninum (1:200, 1:400, 1:100 and 1:400). Genomic DNA was isolated from the brain, lung and striated muscle from the puppy and from the brain of one of the inoculated gerbils. The N. caninum specific primer pair Np 6/21 produced 328 bp amplicons on electrophoretic gels. This is the first confirmed clinical case of generalized canine neosporosis in Argentina.

Isolation and molecular characterization of Toxoplasma gondii in a colony of captive black-capped squirrel monkeys (Saimiri boliviensis).

Toxoplasmosis is commonly asymptomatic; however, it can be a fatal multisystemic disease in some animal species, such as New World monkeys. An outbreak of acute fatal toxoplasmosis was reported in a colony of black-capped squirrel monkeys (Saimiri boliviensis) from the zoo of La Plata, Argentina. Post-mortem examination of two monkeys revealed macroscopical and microscopical lesions compatible with acute toxoplasmosis. The presence of Toxoplasma gondii was confirmed by immunohistochemistry on monkey tissues, bioassay in mice and PCR using the specific primers B22-B23. By PCR-RFLP analysis, T. gondii isolated in mice, deriving from both monkeys, showed the same restriction pattern, with most markers showing a type III restriction pattern, except for C22-8 (type II) and C29-2 (type I). To our knowledge this is the first report of fatal toxoplasmosis in S. boliviensis caused by a non-canonical or atypical genotype of T. gondii.

Neospora caninum infections in bovine foetuses and dairy cows with abortions in Argentina.

Antibodies to Neospora caninum were measured in bovine foetuses, dairy cows and beef cows in Argentina using the IFAT, the N. caninum agglutination test, and the recombinant NCDG1 and NCDG2 ELISA. Serum antibodies (IFAT titre 1:80) were found in 20 of 82 (24.4%) dairy cow foetuses and one of 22 (4.5%) beef cow foetuses. Microscopic lesions suggestive of neosporosis were seen in brains of seven of eight foetuses with IFAT titres of 1:80. Antibodies (IFAT) were found in 122 of 189 (64.5%) dairy cows that aborted. Serum antibody titres (IFAT) of 189 dairy cows that aborted were: < 1:25 (67 cows), 1:25 (four cows), 1:50 (16 cows), 1:200 (seven cows), 1:> or = 800 (95 cows). Of the 87 sera with IFAT titres of < or = 1:50, 57 had no antibodies in 1:40 dilution and 30 had titres of 1:40 in the N. caninum agglutination test. Thus, sera from at least 56 dairy cows which had aborted were seronegative both in the N. caninum agglutination test and the IFAT. The distribution of positive and negative sera was similar when measured by ELISA, except that, depending on cut-off titre, the ELISA indicated a greater number of seropositive cows that were negative by the IFAT and N. caninum agglutination test. These results suggest that transplacental transmission of N. caninum in dairy cows in Argentina is frequent.

Detection of antibodies to Toxoplasma gondii in stillborn piglets in Argentina.

Fetal fluids of 738 stillborn piglets from three swine farms in Argentina were examined for antibodies to Toxoplasma gondii. Antibodies were detected in 15 samples at a 1:20 dilution in the indirect fluorescent antibody test and 10 samples were positive in the modified agglutination test (MAT) at a dilution of 1:25; four of these samples had a MAT titer of > or = 1:100. This survey indicates a low rate of congenital T. gondii infection in stillborn pigs in Argentina.

Natural Besnoitia sp. infection in domestic rabbits from Argentina.

Besnoitia sp. are apicomplexan coccidian parasites affecting several species of mammals and cold-blooded animals in several countries. Besnoitia sp. tissue cysts were seen in several tissues of five rabbits from a rabbit breeder in La Plata, Argentina. Bradyzoites released from macroscopic tissue cysts were inoculated onto bovine monocytes, and into interferon gamma gene knockout (KO) mice. Besnoitia sp. tachyzoites were seen in the peritoneal exudate of KO mice on day 10 pi and these tachyzoites were infective to other KO mice. Tachyzoites grown in cell culture were infective to gerbils (Meriones unguiculatus). This is the first report of Besnoitia sp. infection in any host in Argentina.

Prevalence of Neospora caninum infection in dogs from beef-cattle farms, dairy farms, and from urban areas of Argentina.

Prevalence of anti-Neospora caninum antibodies was determined in sera of 320 dogs from Argentina using the indirect fluorescent antibody test (IFAT). Antibodies to N. caninum were found in 121 of 320 (37.8%) sera with titers of 1:50 (21 dogs), 1:100 (23 dogs), 1:200 (23 dogs), 1:400 (17 dogs), 1:800 (23 dogs), and > or = 1:1.600 (14 dogs). The seropositivity (IFAT, > or = 1:50) was higher in dogs from dairy (48% of 125) and beef (54.2% of 35) farms than in dogs from urban areas (26.2% of 160). Prevalence of anti-N. caninum antibodies was higher in dogs more than 12 mo of age (47.7%, 105 of 222) versus in 12-mo-old or younger dogs (12.7% of 86), suggesting postnatal exposure of N. caninum infection in dogs.

Serological survey of selected pathogens of free-ranging foxes in southern Argentina, 1998--2001.

A serological survey was conducted in the Patagonia region of Argentina to estimate the prevalence of nine disease agents within the populations of free-ranging culpeo (Dusicyon culpaeus) and grey (Dusicyon griseus) foxes. The disease agents were Aujeszky's disease virus (ADV), Brucella, canine adenovirus (CAV), canine distemper virus (CDV), canine parvovirus (CPV), Encephalitozoon cuniculi, Leptospira, Neospora caninum, and Toxoplasma gondii. A total of 84 foxes were sampled (28 culpeo and 56 grey), and 73% of the sera had antibodies against one or more pathogens. Among these seropositive sera, 47% of them reacted to only one antigen, while the other 53% reacted to multiple antigens. The presence of antibodies to Toxoplasma (20%), Neospora (44%), Leptospira (30%) and Brucella (18%) suggests that these organisms actively circulate in the area. Antibodies against CDV, CAV and CPV were detected in 2%, 5% and 5% of foxes, respectively. Regarding Encephalitozoon cuniculi and ADV, no evidence of either was found.

Toxoplasma gondii and Neospora caninum infections in goat abortions from Argentina.

The aims of this study were to identify the occurrence of Toxoplasma gondii and Neospora caninum abortions in goats from Argentina by serological, macroscopical and microscopical examination and bioassay, and to characterize the obtained isolates by molecular techniques. For this purpose, 25 caprine fetal fluids, 18 caprine fetal brains and 10 caprine placentas from 8 dairy/meat goat farms from Argentina were analyzed. Gestational age of the aborted fetuses was determined in 18 cases. Protozoal infections were detected by at least one of the applied diagnostic techniques in 44% (11/25) of examined fetuses; specifically, 24% (6/25) were positive to T. gondii, 8% (2/25) were positive to N. caninum and 12% (3/25) were positive to both parasites. In this study IFAT titers were similarly distributed in younger and older fetuses. Macroscopical and microscopical examination of one placenta revealed chalky nodules in the fetal cotyledons and normal intercotyledonary areas, as well as necrosis and calcification of mesenchymal cells in villi. Tachyzoites were observed in peritoneal wash from 2 mice inoculated with brain and a pool of brain and placenta of two fetuses. Cell culture growth of tachyzoites was achieved from one inoculated mouse, and confirmed as T. gondii by PCR. The T. gondii isolate was identified as atypical or non-canonical by nested-PCR-RFLP. This is the first study that investigated the involvement of N. caninum and T. gondii in cases of goat abortion in Argentina.

Evaluation of an in-house TgSAG1 (P30) IgG ELISA for diagnosis of naturally acquired Toxoplasma gondii infection in pigs.

Toxoplasma gondii is an apicomplexan protozoan parasite which is able to infect a large variety of warm-blooded animals. Raw or undercooked pork has been regarded as an important source of infection for humans. The aim of this study was to evaluate an in-house enzyme-linked immunosorbent assay to diagnose natural T. gondii infection in swine using native affinity chromatography-purified T. gondii surface protein-1 (TgSAG1-ELISA) as antigen, comparing its performance to that of indirect fluorescent antibody test (IFAT) and immunoblotting (IB). To obtain a panel of sera showing the evolution of the antibody response in the time course 12 pigs were experimentally inoculated intravenously (iv) with tachyzoites of the T. gondii strains RH (clonal type I), ME49 (clonal type II) and NED (clonal type III) and serologically monitored for a period of 11 weeks. Both IFAT and ELISA showed a similar time course of antibody response to T. gondii; but by IFAT this response was characterized by rapidly rising titers with peaks at two weeks post inoculation (wpi), while the ELISA indices increased slowly and reached a maximum in most animals at five wpi. Three-hundred randomly selected sera from a total of 602 pigs of different ages derived from outdoor and indoor farms from Argentina were analyzed. Serum samples testing either positive or negative by both IFAT and IB were considered as "relative standards of comparison" (RSC). Sensitivity and specificity of TgSAG1-ELISA were obtained by a Receiver Operating Characteristics (ROC) analysis and statistical agreement among serological tests was evaluated. Antibodies to T. gondii were detected in 160 of 300 sera (53.3%) by IB, in 133 of 300 (44.3%) by IFAT and in 123 of 300 sera (41%) by TgSAG1-ELISA. One hundred and eleven sera tested positive and 118 sera tested negative by both IFAT and IB (RSC); 103 of 111 positive RSC sera tested positive by TgSAG1-ELISA, and 116 of 118 negative RSC sera tested negative by TgSAG1-ELISA. Agreement observed between RSC and TgSAG1-ELISA was almost perfect (κ=0.9124, p ≥ 0.05) and between IFAT and IB was moderate (κ=0.53, p ≥ 0.05). Relative sensitivity and specificity of the TgSAG1-ELISA using a cut-off index of 0.204 were of 92.8% and 98.3%, respectively. ROC analysis revealed that TgSAG1-ELISA was highly accurate (AUC=0.983) relative to the RSC. According to the results in this study, the ELISA based on affinity purified T. gondii surface antigen TgSAG1 was useful for the specific and sensitive detection of antibodies to this protozoan parasite in naturally infected pigs.

Toxoplasma gondii infection in sentinel and free-range chickens from Argentina.

This study aimed at isolating and genotyping Toxoplasma gondii from serologically positive free-range chickens from Argentina, and to evaluate the use of sentinel animals during a short time period of exposure to determine environmental contamination with T. gondii oocysts. Two groups of chickens on six farms were compared in this study: (i) young, 2-3 month-old broiler-type chickens reared as sentinel animals on the farms and (ii) adult chickens reared on the same farms for more than one year. Seroconversion rates of 7.0% or 5.7% were observed in sentinel broiler chickens reared for a period of 74 days (January-April 2010) or 88 days (August-November 2010) respectively, as shown by a T. gondii specific immunofluorescent antibody test. Fifty-three percent (17 of 32) of adult chickens were positive and showed higher titres than sentinel animals. Isolation of T. gondii from tissues (brain and heart) of serologically positive chickens was achieved from six of seven free-range adult birds with IFAT titres of 200 and higher. The isolated parasites were analysed by multi-locus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The isolated T. gondii showed three different genotypes: two genotypes consisted in atypical allele combinations, and the remaining genotype had exclusively clonal type II alleles. All isolates obtained at a single farm, corresponded to the same genotype. The T. gondii genotypes observed are identical to those described in cats, dogs, chickens and capybaras elsewhere in South America. Two isolates, which showed different allele combinations in PCR-RFLP, were characterized in a mouse virulence assay. While one isolate showed a low virulence a second isolate was of intermediate virulence to mice.

The Neospora caninum-Spain 7 isolate induces placental damage, fetal death and abortion in cattle when inoculated in early gestation.

The Nc-Spain 7 isolate of Neospora caninum, which was newly obtained from an asymptomatic congenitally infected calf, demonstrated a similar virulence as Nc-1 strain in mouse models. The aim of this study was to characterize the pathogenesis of Nc-Spain 7 isolate in cattle after experimental infection at 65 days of gestation. For this purpose, thirteen pregnant heifers were divided into three groups as follows: group A: 7 heifers inoculated with 1 × 10(8) tachyzoites of Nc-Spain 7 isolate; group B: 4 heifers inoculated with 1 × 10(8) tachyzoites of Nc 1 strain; and group C: 2 heifers received PBS. Serum samples were collected weekly and heparinized blood samples were collected three times (0, 28 and 42 days after inoculation) by jugular venipuncture. Placenta and fetal tissue samples were collected at time of necropsy. Specific antibody response in the dams was tested by IFAT, indirect ELISA, and rNcGRA7 and rNcSAG4 based-ELISA. Specific antibody response in fetal fluids was tested by IFAT. IFN-γ production was measured after in vitro culture of PBMC and the supernatant was assessed using a commercial kit (BOVIGAM). A significant increase in N. caninum antibody responses was detected in groups A and B by IFAT and by i-ELISA from day 14 after inoculation onwards. Besides, antibody response against rNCGra7 protein was also detected in all inoculated heifers by rNcGra7-based ELISA. Four fetuses from group A and one from group B were aborted between 3 and 5 weeks after infection. In the recovered fetuses, only 3 out of 4 fetal fluids from fetuses of group A and 1 out of 3 of group B were seropositive by IFAT, but all of them were positive by PCR. Transplacental transmission could be determined in all fetuses from groups A and B by PCR and/or IHC. Heifers of group C and their fetuses remained negative by all techniques. The results of this study demonstrate that the NC-Spain 7 isolate could be transmitted transplacentally, and produced fetal death and abortion in cattle.

Prevalence of Sarcocystis spp. in Argentinean cattle.

Sarcocystis cruzi, S. hirsuta and S. hominis are apicomplexan parasites that affect cattle worldwide with variable prevalence. The aim of the present study was to evaluate the prevalence of Sarcocystis spp. in Argentinean cattle comparing microscopic fresh examination and molecular methods. Blood, myocardium and loin samples were collected in five slaughterhouses from a total of 380 bovines. Origin of animals was representative of the major beef cattle production area of Argentina. Samples were analyzed by fresh microscopical examination, transmission electron microscopy (TEM), IFAT and PCR-RFLP. Thin walled sarcocysts corresponding with S. cruzi were found in 99.5% of heart samples. Sarcocysts were detected in 73.1% of loin samples; 71.5% had S. cruzi cysts and 23.1% had thick walled sarcocysts (S. hirsuta or S. hominis). TEM observation revealed the presence of characteristic S. hominis and S. hirsuta cyst walls in 7 and 1 loin samples respectively. Using IFAT, 379/380 animals had titers 25 or higher, showing a full agreement with fresh examination. Amplification products were detected in 35.5% (135/380) of loin samples; however Sarcocystis species could only be determined by RFLP in 29 samples. Agreement between fresh examination and PCR was low (Kappa value=0.262). This is the first report of S. hominis and S. hirsuta in Argentina. Further studies are needed to improve the sensitivity of molecular methods for species identification, especially for differentiation of S. cruzi and S. hirsuta from the zoonotic species S. hominis. The results of the present study and others focusing on sensitivity and specificity of Sarcocystis spp. diagnostic methods should contribute to improve food safety.

Toxoplasmosis and genotyping of Toxoplasma gondii in Macropus rufus and Macropus giganteus in Argentina.

Toxoplasma gondii infection is frequently asymptomatic; however, it can be severe or even fatal to some hosts. In this study, diagnosis of disseminated toxoplasmosis in one red kangaroo (Macropus rufus) and one great grey kangaroo (Macropus giganteus) from the La Plata Zoo, Argentina and the isolation and molecular characterization of T. gondii are reported. Both male kangaroos showed depression and sudden death. Toxoplasma gondii infection was diagnosed by fresh examination, histopathology, immunohistochemistry, PCR and bioassay in mice. During fresh examination many protozoan cysts were observed in diaphragm, heart and hind limb muscles of M. rufus. Cysts were also observed in samples from M. giganteus, although in lower number. Cysts from both kangaroos stained strongly with T. gondii anti-serum by immunohistochemistry. The M. rufus showed more considerable histopathological lesions like non-suppurative meningoencephalitis, myositis and myocarditis. All mice inoculated with tissues from both kangaroos developed IFAT titers to T. gondii (titer >or=800) and brain cysts at necropsy. Both T. gondii isolates were maintained by mice passages and the M. rufus isolate was also maintained in cell culture. Toxoplasma gondii DNA from tissue samples was analyzed by PCR-RFLP analysis using the markers 5'SAG2, 3'SAG2, BTUB, GRA6, SAG3, c22-8, L358, PK1, c29-2 and Apico. Genotyping revealed that the T. gondii isolate from M. rufus was clonal type III and the isolate from M. giganteus was clonal type II. This is the first report of disseminated toxoplasmosis in M. rufus and M. giganteus in Argentina caused by genotypes of T. gondii considered non-virulent in a mouse model.