D. Bacigalupe

Diagnosis of Sarcocystis cruzi , Neospora caninum , and Toxoplasma gondii infections in cattle

The aim of the study was to diagnose Sarcocystis sp. infections in cattle and to detect coinfections by Toxoplasma gondii and/or Neospora caninum. Blood, diaphragm, esophagus, and myocardium from 90 beef cattle from Argentina were collected. Histopathological, immunohistochemical, polymerase chain reaction assays, and direct microscopical examination were carried out. Sarcocysts from myocardium were measured and counted. Indirect fluorescent antibody test (IFAT) for the three protozoans was performed. Sarcocystis cruzi sarcocysts were found in 100% of myocardium samples. Sarcocysts per gram ranged from 8 to 380 with higher values found in adult cattle (p < 0.001). T. gondii and N. caninum were not detected by immunohistochemistry. T. gondii DNA was found in myocardium of 2/20 seropositive animals, while N. caninum DNA was not found. Antibodies against S. cruzi were detected in all samples, those against N. caninum in 73% and against T. gondii in 91% of the samples (IFAT titer ≥25). It is concluded that serology by IFAT is a suitable method to diagnose these protozoan infections due to its specific IgG detection; therefore, IFAT may be a useful tool to evaluate the impact of each protozoan infection in coinfected animals.

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.

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.

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.

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.

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.

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.

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.

Serologic profiles for Sarcocystis sp. and Neospora caninum and productive performance in naturally infected beef calves

Sarcocystis sp. and Neospora caninum infections affect cattle worldwide causing important economic losses. The objective of the present study was to trace serologic profiles for Sarcocystis sp. and N. caninum in naturally infected beef calves and analyze their relationship with transmission routes and productive performance. Samples were collected in two cow-calf operations located in Buenos Aires province, Argentina. In farm 1, 43 calves were bled and weighed three times. In farm 2, 69 calves were bled and weighed six times. Sarcocystis sp. and N. caninum immunofluorescence antibody test (IFAT) titers were averaged for each sampling point in order to trace serologic profiles for each infection. Categories were created to evaluate differences in daily weight gain. For S. cruzi antigen, animals were separated in a low-titer (≤200) and high-titer group (>200); for N. caninum, animals were grouped as infected and uninfected. Sarcocystis sp. antibody titer as well as the number of infected animals increased gradually over time in both farms. In farm 2 the low-titer group had significantly higher daily weight gain than the high-titer group. For N. caninum 44% (farm 1) and 65% (farm 2) of calves were considered infected, and the serological profile was horizontal or decreasing over time. However, seroprevalence increased in both farms and vertical and horizontal transmission frequency were estimated between 18.5%–29% and 22–25.5%, respectively. No differences were detected in daily weight gain between N. caninum groups from both farms. This is the first report of serological profiles for Sarcocystis sp. and N. caninum by IFAT in naturally infected beef calves and their relationship to different transmission routes and productive performance.

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.