Neosporosis in Argentina: Past, present and future perspectives.

Neosporosis, caused by the protozoan Neospora caninum, was first diagnosed in Argentinean cattle in the 90's. With a national bovine stock of approximately 53 million head, the cattle industry is socially and economically relevant. Severe economic losses have been estimated at US$ 33 and 12 million annually in dairy and beef cattle, respectively. Approximately 9% of bovine abortions in the Buenos Aires province are caused by N. caninum. In 2001, the first isolation of N. caninum oocysts from feces of a naturally infected dog was performed in Argentina and named as NC-6 Argentina. Further strains were isolated from cattle (NC-Argentina LP1, NC-Argentina LP2) and axis deer (Axis axis, NC-Axis). Epidemiological studies revealed a high distribution of Neospora-infections not only in dairy but also in beef cattle, with seroprevalence rates of 16.6-88.8% and 0-73%, respectively. Several experimental infection studies in cattle have been carried out, as well as attempts to develop effective vaccines to avoid Neospora-abortions and transmission. However, no vaccine has proven successful for its use in daily practice. Reduction of seroprevalence, vertical transmission and Neospora-related abortions have been achieved in dairy farms by the use of selective breeding strategies and embryo transfer. Neospora-infections have been also detected in goats, sheep, deer, water buffaloes (Bubalus bubalis) and gray foxes (Lycalopex griseus). Moreover, Neospora-related reproductive losses were reported in small ruminants and deer species and could be more frequent than previously thought. Even though diagnostic methods have been improved during the last decades, control of neosporosis is still not optimal. The development of new strategies including new antiprotozoal drugs and vaccines is highly needed. This paper reviews the information from the previous 28 years of research of N. caninum in Argentina, including seroprevalence and epidemiological studies, available diagnostic techniques, experimental reproduction, immunization strategies, isolations and control measures in domestic and non-domestic animals from Argentina.

Risk factors associated with Toxoplasma gondii seroprevalence in domestic pig farms in Argentina.

Toxoplasmosis is a worldwide parasitic zoonosis caused by Toxoplasma gondii. Pigs can become infected by consuming water or food contaminated with sporulated oocysts, or by carnivorism (like the consumption of infected rodents). In pigs most infections are asymptomatic. In certain countries, pig meat containing tissue cysts is a major source of infection for human beings. The aims of this study were to estimate the seroprevalence of toxoplasmosis and to identify which factors were related with the increase of the risk of infection in Argentina. The seroprevalence of T. gondii was determined in 240 pigs from 27 farms in the central-western area of Buenos Aires province, Argentina. Serum samples were analyzed using indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) techniques. Prevalence determined was 53.33% and 32.08% by IFAT and ELISA, respectively. Results showed that 81.5% (22/27) of the farms were seropositive to T. gondii. Seropositivity for T. gondii was related with the following risk factors (p value ≤0.05): presence of felids and rodents in the farms, feeding with waste of human food and storage of food outdoors with free access to felids and to the reservoirs when applying both serological techniques. Our results strongly suggest that the risk of infection with T. gondii in pigs is related to the outdoor/extensive type of production system with low infrastructure conditions, which allows both felids and rodents to have free access to pigs and stored food. Also, the high seroprevalence detected in the present study could indicate a potential role of pork in human infections in the region.

Eleven years of Toxoplasma gondii serological follow-up in a goat herd and association of toxoplasmosis with reproductive losses.

Toxoplasmosis is considered one of the most important causes of abortion in small ruminants. The aim of this study was to evaluate the relationship between Toxoplasma gondii antibody titres and reproductive losses over an 11 year period in a goat farm located in Buenos Aires province, Argentina. Blood samples were obtained from 85 goats, representing three breeds, during the last third of gestation (n = 165 gestations), in consecutive pregnancies (2008-2019), and from 51 goats during kidding to analyze seroconversion. Serum was evaluated by IFAT with T. gondii antigen, using 1:100 dilution as the cut-off titre and processed to end titre. An overall reproductive loss of 31% (51/165) was detected, including 16.4% (27/165) abortions and 14.6% (24/165) perinatal deaths. The seropositivity to T. gondii was 100% (85/85) with all animals positive in successive samplings and, therefore, considered chronically infected. Antibody titres showed average values greater than 1100 in each year and breed group. Differences in antibody levels were associated with breed and were lower in those that were predominately Creole and higher in those that were predominately Saanen. Seroconversion was detected in 16.2% (6/37) and 57.1% (8/14) of goats from the Creole and Sannen breed groups, respectively. There were no significant differences in the antibody titre average between goats with reproductive losses and those with healthy kids, although the goats with perinatal deaths had a significantly higher titre average. These results suggest reinfection or reactivation, although no association with reproductive losses was observed. Higher antibody titres were associated with perinatal deaths. The high T. gondii antibody titres in a farm with 100% seroprevalence did not allow for association with reproductive losses, particularly abortion, to be assessed.

ROP18 and ROP5 alleles combinations are related with virulence of T. gondii isolates from Argentina.

The allelic combination of ROP18/ROP5 genes of Toxoplasma gondii has been shown to be highly predictive of mouse virulence in canonical isolates and strains. The aims of this study were to analyze the alleles present in the ROP18/ROP5 genes from T. gondii isolates obtained in Argentina, to associate the results with the virulence registered in mouse model, and to compare with other isolates and reference strains using a phylogenetic network. Fourteen T. gondii isolates from Argentina were analyzed by nPCR-RFLP for ROP18/ROP5. Phylogenetic network analysis was inferred using the ToxoDB genotypes and the ROPs molecular markers. All isolates and reference strains were categorized as lethal or non-lethal. As results, combinations 2/2, 3/3 and 4/3 for ROP18/ROP5 were detected in 12 isolates, whereas only alleles 1 and 2 of ROP5 were detected in 2 isolates. The majority of the isolates had a mouse virulence matching to that predicted by the ROP18/ROP5 allele combination. The 3 isolates that differed from the expected virulence presented non-clonal genotypes. ROPs incorporation increased the accuracy of the phylogenetic network relations among the T. gondii samples, prevailing the clustering according to regions. Our results indicate a predominance of type 3 allele in both ROP18 and ROP5 markers and an association of allelic profiles 3/3 and 4/3 of non-clonal genotypes from Argentina, both with virulent and avirulent profiles in mice.

Toxoplasma gondii genotyping from free-range chickens (Gallus gallus domesticus) in a rural area of Rio Grande do Sul, Brazil / [Genotipagem de Toxoplasma gondii em galinhas domésticas em uma área rural do Rio Grande do Sul, Brasil]

Free-range chickens may ingest oocysts of T. gondii present in the environment and consequently harbor virulent strains of this parasite in different tissues, without any clinical signs. Isolation of T. gondii through bioassays on mice and cats from naturally infected chicken tissues has been described in several countries, demonstrating the importance of free-range chickens in the transmission of this parasite. The aim of this study was the genotypic characterization of T. gondii isolates obtained from naturally infected free-range chickens in a rural area of the state of Rio Grande do Sul, Brazil. Brain and heart tissue from 12 chickens seropositive for T. gondii were processed using peptic digestion technique for parasite isolation. From 12 samples subjected to mouse bioassay, nine isolates were obtained. RFLP-PCR genotypic characterization was performed using 11 genetic markers: SAG1, 5'-3'SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genetic characterization of the isolates revealed the presence of five atypical genotypes according to ToxoDB (# 11, # 55, # 64, # 140 and # 163). Our results showed a wide genetic diversity of T. gondii in free-range chickens in this region.(AU)

Galinhas criadas ao ar livre podem ingerir oocistos de T. gondii presentes no ambiente e, com isso, albergar cepas virulentas desse parasita em diferentes tecidos, sem sinais clínicos. O isolamento de T. gondii por meio de bioensaios em camundongos e gatos, a partir de tecidos de galinhas naturalmente infectadas, tem sido descrito em vários países. Isso demonstra a importância das galinhas caipiras na epidemiologia desse parasita. O objetivo deste trabalho foi caracterizar genotipicamente isolados de T. gondii obtidos de galinhas caipiras naturalmente infectadas em uma área rural do município de Santa Maria, estado do Rio Grande do Sul, Brasil. Fragmentos de cérebro e de coração, de 12 galinhas soropositivas para T. gondii, foram processados pela técnica de digestão péptica para isolamento do parasita. Das 12 amostras submetidas a bioensaio com camundongos, nove isolados foram obtidos. A caracterização genotípica por RFLP-PCR foi realizada utilizando-se 11 marcadores genéticos: SAG1, 5'-3'SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 e Apico e revelou a presença de cinco genótipos atípicos de acordo com o ToxoDB (# 11, # 55, # 64, # 140 e # 163). Os resultados mostraram uma ampla diversidade genética de T. gondii em galinhas caipiras nessa região.(AU)

Toxoplasma gondii genotyping from free-range chickens (Gallus gallus domesticus) in a rural area of Rio Grande do Sul, Brazil / [Genotipagem de Toxoplasma gondii em galinhas domésticas em uma área rural do Rio Grande do Sul, Brasil]

Free-range chickens may ingest oocysts of T. gondii present in the environment and consequently harbor virulent strains of this parasite in different tissues, without any clinical signs. Isolation of T. gondii through bioassays on mice and cats from naturally infected chicken tissues has been described in several countries, demonstrating the importance of free-range chickens in the transmission of this parasite. The aim of this study was the genotypic characterization of T. gondii isolates obtained from naturally infected free-range chickens in a rural area of the state of Rio Grande do Sul, Brazil. Brain and heart tissue from 12 chickens seropositive for T. gondii were processed using peptic digestion technique for parasite isolation. From 12 samples subjected to mouse bioassay, nine isolates were obtained. RFLP-PCR genotypic characterization was performed using 11 genetic markers: SAG1, 5'-3'SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genetic characterization of the isolates revealed the presence of five atypical genotypes according to ToxoDB (# 11, # 55, # 64, # 140 and # 163). Our results showed a wide genetic diversity of T. gondii in free-range chickens in this region.(AU)

Galinhas criadas ao ar livre podem ingerir oocistos de T. gondii presentes no ambiente e, com isso, albergar cepas virulentas desse parasita em diferentes tecidos, sem sinais clínicos. O isolamento de T. gondii por meio de bioensaios em camundongos e gatos, a partir de tecidos de galinhas naturalmente infectadas, tem sido descrito em vários países. Isso demonstra a importância das galinhas caipiras na epidemiologia desse parasita. O objetivo deste trabalho foi caracterizar genotipicamente isolados de T. gondii obtidos de galinhas caipiras naturalmente infectadas em uma área rural do município de Santa Maria, estado do Rio Grande do Sul, Brasil. Fragmentos de cérebro e de coração, de 12 galinhas soropositivas para T. gondii, foram processados pela técnica de digestão péptica para isolamento do parasita. Das 12 amostras submetidas a bioensaio com camundongos, nove isolados foram obtidos. A caracterização genotípica por RFLP-PCR foi realizada utilizando-se 11 marcadores genéticos: SAG1, 5'-3'SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 e Apico e revelou a presença de cinco genótipos atípicos de acordo com o ToxoDB (# 11, # 55, # 64, # 140 e # 163). Os resultados mostraram uma ampla diversidade genética de T. gondii em galinhas caipiras nessa região.(AU)

Galectin-3 Expression Correlates with Post-surgical Survival in Canine Oral Melanomas.

Malignant melanomas (MMs) represent 7% of all malignant neoplasms in dogs. Oral melanocytic neoplasms are often malignant and associated with poor prognosis. There are no universally accepted prognostic markers for canine oral melanoma. Galectin (Gal)-3 is a prognostic marker for human neoplasms such as thyroid, gastric, colorectal and prostate cancers. The protein is related to processes that favour cancer progression, such as angiogenesis, proliferation and apoptosis. The aim of the present study was to characterize the immunohistochemical expression of Gal-3 in canine oral melanomas and to compare it with post-surgical survival, the expression of apoptosis-related proteins and other known prognostic tools. Twenty-seven samples of canine oral melanomas were evaluated for Gal-3, B-cell lymphoma (BCL) 2, caspase (CASP) 3 and Ki67 expression, mitotic index and degree of nuclear atypia. Gal-3 cytoplasmic positivity was correlated positively, while nuclear positivity was correlated negatively, with survival. The intensity of BCL2 labelling was also correlated positively with Gal-3 cytoplasmic positivity. Higher nuclear atypia was observed in dogs with melanoma that died due to the tumour, as well as in dogs that survived for <1 year after surgery. We have confirmed the importance of nuclear atypia for MMs and suggest that Gal-3 is a valuable prognostic indicator for this neoplasm. More in-depth studies are needed to unveil Gal-3 functions in canine MMs using larger sample sizes.

First case report of Toxoplasma gondii-induced abortions and stillbirths in sheep in Argentina.

The aim of this study is to report an episode of reproductive losses due to toxoplasmosis in a sheep flock in Argentina. A total of 15 abortions and 9 stillbirths were recorded in a flock of 190 Texel ewes. The affected ewes were more likely to be seropositive for Toxoplasma gondii (15/24) than ewes that delivered normal lambs (5/34, OR=9.6, 95%CI=2.7-34.0, p=0.0004). A pair of aborted twins was recovered for diagnostic investigation. One of these fetuses and its dam were seropositive for T. gondii. Histological examination of the two fetuses revealed non-suppurative myocarditis and epicarditis, portal hepatitis and multifocal necrotizing encephalitis with protozoal cysts in the brain. T. gondii was detected intralesionally by immunohistochemistry in one fetus and by PCR in both. Further investigations are necessary to evaluate the economic losses due to T. gondii in the Argentinean ovine industry.

Cryptosporidium varanii infection in leopard geckos (Eublepharis macularius) in Argentina.

Cryptosporidiosis is observed in reptiles with high morbidity and considerable mortality. The objective of this study was to achieve the molecular identification of Cryptosporidium spp. in pet leopard geckos (Eublepharis macularius) from a breeder colony in Buenos Aires, Argentina. Oocysts comparable to those of Cryptosporidium spp. were detected in three geckos with a history of diarrhea, anorexia and cachexia. Molecular identification methods confirmed the presence of Cryptosporidium varanii (syn. C. saurophilum). This agent was considered to be the primary cause of the observed clinical disease. This is the first description of C. varanii infection in pet reptiles in Argentina.

Variation in cassava germplasm for tolerance to post-harvest physiological deterioration.

Tolerant varieties can effectively control post-harvest physiological deterioration (PPD) of cassava, although knowledge on the genetic variability and inheritance of this trait is needed. The objective of this study was to estimate genetic parameters and identify sources of tolerance to PPD and their stability in cassava accessions. Roots from 418 cassava accessions, grown in four independent experiments, were evaluated for PPD tolerance 0, 2, 5, and 10 days post-harvest. Data were transformed into area under the PPD-progress curve (AUP-PPD) to quantify tolerance. Genetic parameters, stability (Si), adaptability (Ai), and the joint analysis of stability and adaptability (Zi) were obtained via residual maximum likelihood (REML) and best linear unbiased prediction (BLUP) methods. Variance in the genotype (G) x environment (E) interaction and genotypic variance were important for PPD tolerance. Individual broad-sense heritability (hg(2)= 0.38 ± 0.04) and average heritability in accessions (hmg(2)= 0.52) showed high genetic control of PPD tolerance. Genotypic correlation of AUP-PPD in different experiments was of medium magnitude (ȓgA = 0.42), indicating significant G x E interaction. The predicted genotypic values o f G x E free of interaction (û + gi) showed high variation. Of the 30 accessions with high Zi, 19 were common to û + gi, Si, and Ai parameters. The genetic gain with selection of these 19 cassava accessions was -55.94, -466.86, -397.72, and -444.03% for û + gi, Si, Ai, and Zi, respectively, compared with the overall mean for each parameter. These results demonstrate the variability and potential of cassava germplasm to introduce PPD tolerance in commercial varieties.

Sarcocystis masoni, n. sp. (Apicomplexa: Sarcocystidae), and redescription of Sarcocystis aucheniae from llama (Lama glama), guanaco (Lama guanicoe) and alpaca (Vicugna pacos).

There is considerable confusion concerning the species of Sarcocystis in South American camelids (SAC). Several species names have been used; however, proper descriptions are lacking. In the present paper, we redescribe the macroscopic sarcocyst forming Sarcocystis aucheniae and describe and propose a new name, Sarcocystis masoni for the microscopic sarcocyst forming species. Muscles samples were obtained from llamas (Lama glama) and guanacos (Lama guanicoe) from Argentina and from alpacas (Vicugna pacos) and llamas from Peru. Individual sarcocysts were processed by optical and electron microscopy, and molecular studies. Microscopic sarcocysts of S. masoni were up to 800 µm long and 35-95 µm wide, the sarcocyst wall was 2·5-3·5 µm thick, and had conical to cylindrical villar protrusions (vp) with several microtubules. Each vp had 11 or more rows of knob-like projections. Seven 18S rRNA gene sequences obtained from sarcocysts revealed 95-96% identity with other Sarcocystis spp. sequences reported in the GenBank. Sarcocysts of S. aucheniae were macroscopic, up to 1·2 cm long and surrounded by a dense and laminar 50 µm thick secondary cyst wall. The sarcocyst wall was up to 10 µm thick, and had branched vp, appearing like cauliflower. Comparison of the 11 sequences obtained from individual macroscopic cysts evidenced a 98-99% of sequence homology with other S. aucheniae sequences. In conclusion, 2 morphologically and molecularly different Sarcocystis species, S. masoni (microscopic cysts) and S. aucheniae (macroscopic cysts), were identified affecting different SAC from Argentina and Peru.

Investigação de anticorpos contra Sarcocystis neurona e Sarcocystis cruzi em equinos / Investigation of antibodies against Sarcocystis neurona and Sarcocystis cruzi in equines

Sarcocystis neurona is the primary agent for Equine Protozoal Myeloencephalitis (EPM), important neurological disease characterized by behavior or muscular changes, that impairs animal performance and husbandry. Sarcocystis cruzi is a pathogen related to myositis in cattle. Although related the life cycles of the parasites are distinct. S. neurona has opossums (Didelphis spp.) and S. cruzi, dogs as definitive hosts. However, S. neurona and S. cruzi may undergo cross-reactivity in serological tests, interfering on results of EPM ante-mortem diagnostic tests. In the present study, serology of 189 mares was performed by indirect immunofluorescence antibody test, using antigens of S. neurona and S. cruzi in order to assess the exposure degree of animals to antigens. Analyzing the results, it was observed that most of the animals (84.13%) reacted with at least one protozoal species and the number of animals which showed antibodies against S. cruzi was greater than S. neurona (80.42% and 33.86%, respectively) and a third of seropositive animals reacted to antigens of both species.(AU)

Investigação de anticorpos contra Sarcocystis neurona e Sarcocystis cruzi em equinos / Investigation of antibodies against Sarcocystis neurona and Sarcocystis cruzi in equines

Sarcocystis neurona is the primary agent for Equine Protozoal Myeloencephalitis (EPM), important neurological disease characterized by behavior or muscular changes, that impairs animal performance and husbandry. Sarcocystis cruzi is a pathogen related to myositis in cattle. Although related the life cycles of the parasites are distinct. S. neurona has opossums (Didelphis spp.) and S. cruzi, dogs as definitive hosts. However, S. neurona and S. cruzi may undergo cross-reactivity in serological tests, interfering on results of EPM ante-mortem diagnostic tests. In the present study, serology of 189 mares was performed by indirect immunofluorescence antibody test, using antigens of S. neurona and S. cruzi in order to assess the exposure degree of animals to antigens. Analyzing the results, it was observed that most of the animals (84.13%) reacted with at least one protozoal species and the number of animals which showed antibodies against S. cruzi was greater than S. neurona (80.42% and 33.86%, respectively) and a third of seropositive animals reacted to antigens of both species.(AU)

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.

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.

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.

Immune response and protection provided by live tachyzoites and native antigens from the NC-6 Argentina strain of Neospora caninum in pregnant heifers.

The aim of the present study was to compare the immunogenicity and protective efficacy of live tachyzoites and native antigen extract obtained from the NC-6 Argentina strain against vertical transmission of Neospora caninum, following experimental challenge in pregnant heifers with the NC-1 strain. Sixteen pregnant heifers were divided in 4 groups of 4 animals, each receiving different inoculation before mating: group A animals were intravenously (iv) inoculated with 6.25×10(7) live tachyzoites of the NC-6 strain, group B heifers were inoculated twice subcutaneously (sc) with N. caninum native antigen extract formulated with ISCOMs, group C heifers were sc injected with sterile phosphate-buffered saline (PBS) and group D heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv challenged with the NC-1 strain at 70 days of gestation. Serum and heparinized blood samples were collected eight times on weeks 0, 2, 3, 5, 9, 13, 16 and 17 post-inoculation. Dams were slaughtered at the 17th week of experiment (104 days of pregnancy) and placental and fetal tissue samples were collected. Specific antibody responses in heifers were tested by indirect enzyme linked immunosorbent assay (iELISA). The cellular immune response in dams was assessed by quantifying IFN-γ production and the percentages of T-cells (CD4(+), CD8(+) and γδ(+)) and monocytes in peripheral blood mononuclear cells (PBMC). Fetal fluids and tissue samples were tested using the indirect fluorescence antibody test, western blot, histopathology, immunohistochemistry and nested-PCR. A significant increase in N. caninum antibody response was detected in heifers of groups A and B from week 3 after inoculation (P<0.001). IFN-γ production was similar in groups A and B at week 13 (P>0.05). All fetuses were viable at necropsy. Specific IgG against N. caninum was detected in 1/4 fetal fluids recovered from groups A, C and D heifers and 3/4 fetal fluids from group B. Transplacental transmission could be determined in one fetus from group A and three fetuses from group B by nPCR. All fetuses from groups C and D were positive by nPCR. It is noteworthy that dams with higher CD4(+)/CD8(+) ratios in PBMC, regardless of the experimental group, had lower pathology scores. The results of this study confirm that inoculation with live parasites pre-mating may provide at least partial protection against vertical transmission of N. caninum following challenge in heifers at early gestation.

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.

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.