Neospora caninum and Toxoplasma gondii infections and their relationship with reproductive losses in farmed red deer (Cervus elaphus).

The aims of the present study were to determine the Neospora caninum and Toxoplasma gondii seropositivity rates in farmed red deer hinds from Argentina and their relationship with reproductive losses. Over a 2-year period, 449 hinds from 4 commercial farms were serologically tested at late gestation for N. caninum and T. gondii by IFAT. During the first year, a sequential serological analysis was carried out at 3 different time points to analyze antibody dynamics from mating until the end of the gestation period. Fetal and postnatal mortality rates were estimated by 3 successive ultrasound scannings (us) annually and a breeding control carried out after the calving period. Ultrasound fetal measurements were used to estimate conception date and gestational age of abortions. The seropositivity rate for N. caninum was 25.5% (37/145) for the yearlings and 34.2% (104/304) for the adults, while for T. gondii was 64.3% (93/145) and 78.3% (238/304), respectively. Abortions detected at us1 and us2 were 13/21 (61.9%) with a range of gestational age of 30-87 days, while abortions detected at us3 were 8/21 (38.1%) with a range of gestational age of 49-209 days. The fetal mortality rate was 4% and 5.8%, while the postnatal mortality rate was 18.8% and 4.1% of 101 yearlings and 294 adult pregnant hinds, respectively. Most seropositive hinds to both protozoans showed a stable antibody titer pattern from mating to the end of gestation, and a lower proportion developed an increase in titers suggesting infection recrudescence. Seroconversion during the gestational period was demonstrated in 6 and 50 hinds for N. caninum and T. gondii, respectively. Hinds with fetal mortality were more likely to be seropositive to N. caninum (OR = 3.1) or have N. caninum titers ≥400 (OR = 27.4) than hinds that weaned a fawn. No statistical associations were detected for T. gondii seropositivity and reproductive losses. The pregnancy rate was not affected by N. caninum or T. gondii infection, while the serological evidence of N. caninum causing postnatal mortality was marginal. Based on serological evidence, N. caninum would be a potential abortigenic agent in red deer hinds.

Global prevalence of Toxoplasma gondii infection in the aborted fetuses and ruminants that had an abortion: A systematic review and meta-analysis.

Toxoplasma gondii (T. gondii) is a zoonotic apicomplexan protozoan that can cause reproductive losses in ruminants across the world. Therefore, a meta-analysis was conducted to estimate the worldwide prevalence of T. gondii infection in the aborted fetuses and stillbirths of sheep, goat, and cattle. Moreover, it attempted to evaluate the prevalence rate of T. gondii infection in ruminants that had abortions using serological methods. Based on the keywords, a systematic search of six databases was conducted to retrieve cross-sectional articles in English-language. Data were synthesized to calculate the overall prevalence of T. gondii infection worldwide using the random-effects model with a 95 % confidence interval (CI). Moreover, the present study includes sensitivity analysis, publication bias test, and quality assessment of the studies. The final analyses included 37, 19, and 8 studies conducted on sheep (4383 aborted fetuses and stillbirths as well as 1940 abortive sheep), goat (248 aborted fetuses and stillbirths and 533 abortive goats), and cattle (460 aborted fetuses and stillbirths and 165 abortive cattle). The overall prevalence rates of T. gondii infection in aborted fetuses and stillbirths globally were 42 % (95 % CI: 17 %-67 %), and 31 % (95 % CI: 11 %-51 %) using molecular methods for sheep and goats, as well as 16 % (95 % CI: 11 %-22 %) and 27 % (95 % CI: 1 %-54 %) using serological tests for sheep and goats, respectively. In addition, the overall prevalence rates of T. gondii infection in sheep and goats that had abortions were 56 % (95 % CI: 35 %-76 %) and 50 % (95 % CI: 6 %-94 %), respectively. These analyses could not be performed in cattle due to the small number of studies and the small number of animals under study. According to the high prevalence of T. gondii infection in sheep and goats that had an abortion, T. gondii could be a potential factor of causing reproductive failures in ruminants worldwide. So, effective control measures and strategies are needed to reduce the rate of abortion in sheep and goats as well as reduce the economic damage to the livestock industry.

Experimental infection of tachyzoites of the NC1 strain of Neosporacaninum in female swine.

Neospora caninum is a protozoan that can cause reproductive problems in several animal species. Although N. caninum infection has been reported in swine, the pathogenesis and clinical signs are not fully known in this species. The objective of this work was to evaluate the effect of experimental infection with tachyzoites of the N. caninum strain Nc1 in swine matrices at different stages of gestation. For that purpose, 12 gilts, seronegative for N. caninum and T. gondii, were selected and allocated into four groups of three animals each. Animals in group A were not inoculated (control) and animals in groups B, C, and D were inoculated intravenously with of 2.9 × 107 tachyzoites, 30 days before conception, and at 45 and 90 days of gestation, respectively. Temperature, heart rate, blood, saliva, and vaginal mucus samples from the animals were collected periodically until the time of delivery for the investigation of IgG and IgM antibodies against N. caninum using IFAT and PCR to detect the parasite DNA. All gilts sero-converted from 5 and 7 DPI (days postinoculation) to IgM and IgG, respectively. Two gilts showed hypothermia on the 5th and 7th DPI, and five inoculated animals had leukocytosis on the 7th DPI. It was possible to detect DNA of N. caninum in samples of saliva (33/84), vaginal mucus (17/84), and blood (2/84). Based on serology (IgM) and PCR, three animals in group B showed evidence of reappearance of the infection during pregnancy. It is concluded that N. caninum can cause clinical signs in infected swine females, in addition to indicating saliva as a suitable diagnostic biological material for the detection of N. caninum DNA in this animal species.

Isolation of Neospora caninum from a beef cattle fetus from Argentina: Immunopathological and molecular studies.

Neospora caninum is an important abortifacient agent affecting mainly cattle worldwide. The aim of the present work was to describe the histopathological findings in a naturally infected beef cow and its midterm fetus caused by a genetically defined N. caninum isolate in Argentina. A N. caninum seropositive multiparous Aberdeen Angus pregnant cow and its fetus in the sixth month of gestation were submitted for histopathological, immunohistochemical, serological, and molecular studies and parasite isolation. The cow belonged to a beef herd under extensive management, with a N. caninum seroprevalence of 11%, and low level of annual abortion rate (≤ 5%). The dam had mild lymphocytic infiltrate in CNS, heart and uterus and no parasites were detected by Immunohistochemistry (IHC). No parasitic DNA was detected in the dam's brain, and gamma interferon knockout mice inoculated with brain material did not become infected. Clusters of tachyzoites and parasitic DNA were detected in the placenta by IHC and PCR, respectively. However, isolation from the placenta was unsuccessful. The fetus developed specific antibodies and an inflammatory response was detected in multiple organs. Furthermore, in vitro and in vivo isolation was achieved from gamma interferon knockout mice inoculated with CNS from the fetus. Multilocus-microsatellite typing revealed a genetically defined N. caninum isolate similar to the previously reported as MLG 72. We report the first N. caninum isolate from beef cattle in Argentina.

Endogenous transplacental transmission of Neospora caninum in successive generations of congenitally infected goats.

Neospora caninum is a protozoan that is considered an important agent of reproductive disorders in ruminants worldwide, and vertical transmission is the main form of infection and maintenance of neosporosis in herds. In goats, there have been no studies that have evaluated the transmission of N. caninum between successive generations. Thus, the objective of this study was to evaluate, through IFAT and PCR, the endogenous transplacental transmission of N. caninum in up to five generations of six families of dairy goats naturally infected by the parasite and whether it was possible for dairy goats to become free of infection over successive generations. Ninety-five serum samples from positive animals and 75 samples from negative animals were analyzed for N. caninum. Of the 95 samples analyzed, 93 contained anti-N. caninum antibodies (97.8 %). Titers of anti-N. caninum antibodies varied (increasing or decreasing) in the offspring; however, with an increase in the number of the goat generations, the offspring tended to have lower titers (p = 0.021) at the day of birth. Reproductive disorders such as abortions, stillbirth or fetal retention occurred at a rate of 10.4 % and were not influenced by the mother's titer of anti-N. caninum antibodies at the day of parturition or abortion. The results showed that infection by N. caninum persists throughout generations in congenitally infected goats.

Molecular Diagnosis and Pathological Study of Toxoplasma gondii in Aborted Caprine and Ovine Fetuses in Borderline of Iran-Iraq.

BACKGROUND: Infection with the protozoan parasite Toxoplasma gondii is widely prevalent in animals and humans. In goats and sheep this infection has a high economic impact as it causes abortions and lamb losses. Although there are several studies reporting seroprevalence in small ruminants in Iran, molecular-based information is scarce. PURPOSE: This study aimed to screen caprine and ovine aborted fetuses for T. gondii infection by PCR and histopathology. METHODS: Brains of 121 aborted fetuses (10 caprine and 111 ovine) were collected from different parts of the Kordestan province, bordering with Iraq. Gestational age and the general status of each fetus such as freshness, autolysis, mummification and presence of macroscopic lesions in the fetus and foetal membranes was recorded. Individual brain tissues of fetuses were subjected to nested-PCR targeting the B1 gene, and histopathological sections prepared from brains were examined microscopically. RESULTS: PCR results revealed T. gondii-associated abortion in one caprine and nine ovine fetuses (8.3%). Microscopically, pathological lesions included non-purulent meningitis associated with gliosis, focal necrosis, and occasionally tissue cyst. CONCLUSION: This paper reports for the first time T. gondii-associated abortion in goats in Iran. As organs from aborted fetuses do not necessarily show lesions, molecular confirmation is the unique diagnostic method and should be used in situations of an abortion epidemic.

Does Neospora caninum cause reproductive problems in pigs?

Neospora caninum is known to cause reproductive disturbances in several animal species, such as cattle, sheep, and goats. However, research on the effects of N. caninum on reproduction in pigs is limited. The objective of this study was to verify the transplacental transmission of N. caninum in pigs during several gestational stages. Twelve healthy Toxoplasma gondii and N. caninum seronegative female pigs were selected and separated into four groups of three animals each. Group A was maintained as a control group. Groups B, C, and D were inoculated intravenously with 2.9 × 107 tachyzoites of the N. caninum strain Nc1, 30 days before conception and at 45 and 90 days of gestation, respectively. Blood samples were collected from females periodically through IFAT for IgG and IgM screening to confirm the infection. At birth, after blood samples were collected from the piglets, they were then euthanized for the collection of the brain, heart, lung, liver, and diaphragm, which were then subjected to PCR. All inoculated gilts seroconverted (IgG) from the seventh day after inoculation. Nine of the 12 females expelled 24 mummified fetuses at the time of delivery, two in group A (eight), two in group B (four), three in group C (nine), and two in group D (three). Of the 24 mummified fetuses, nine were positive for N. caninum (one (25%) fetus of group B, seven (77.8%) of group C, and one (33.3%) of group D). A total of 126 live piglets were born. When the organs of the piglets from the inoculated females were analyzed by PCR for N. caninum, 88 (93.61%) were positive. All gilts inoculated produced at least one positive piglet. This demonstrates that there is transplacental transmission of N. caninum in all phases of gestation, regardless of the time of infection.

Effects of challenge dose and inoculation route of the virulent Neospora caninum Nc-Spain7 isolate in pregnant cattle at mid-gestation.

Parameters such as pathogen dose and inoculation route are paramount in animal models when studying disease pathogenesis. Here, clinical findings, including foetal mortality, parasite transmission rates and lesion severity, and immune responses were evaluated in Asturiana pregnant heifers at day 110 of gestation challenged with a virulent (Nc-Spain7) Neospora caninum isolate. Four different doses of parasite tachyzoites were inoculated intravenously (IV1, 107 parasites, n = 6; IV2, 105, n = 6; IV3, 103, n = 6; and IV4, 102, n = 5), and the subcutaneous (SC) inoculation route was also assessed for the dose of 105 tachyzoites (SC, n = 6). In addition, a control group (n = 4 pregnant heifers) was evaluated. Foetal death was observed in all infected groups from 25 to 62 days post-infection, varying with the dose (IV1:4/6, IV2:3/6; IV4:2/5, IV3:1/6), and was three times less frequently associated with the SC route than IV inoculation (1/6 vs. 3/6). A dose-dependent effect for parasite loads in placental and foetal brain tissues was also detected. After SC challenge, a reduced number of tachyzoites were able to reach foetal brain tissues, and no lesions were observed. In calves, specific IgG responses in precolostral sera were mainly associated with high-dose groups (IV1 [100.0%] and IV2 [66.7%]), and cerebral parasite DNA detection was scarce (3/18). In dams, IFN-γ production and the dynamics of anti-N. caninum IgG antibodies varied with the dose, and the cell-mediated immune response was also found to be route-dependent. Our results confirm the influence of parasite dose and inoculation route on the outcome and dynamics of bovine neosporosis at mid-gestation.

Ovine Abortion by Neospora caninum: First Case Reported in Argentina.

PURPOSE: The aim of this study was to describe for the first time a natural case of ovine abortion associated with Neospora caninum in a flock with reproductive losses in Argentina. MATERIALS AND METHODS: The analyzed flock consisted of 256 Texel sheep, of which 134 had been mated. A single blood sample was obtained by jugular vein puncture from 220 ewes (116 adult ewes, 104 yearling ewes) and 93 lambs. Serum samples and fetal fluid were tested using the indirect fluorescence antibody test to detect antibodies against N. caninum and T. gondii. Fetal and placental tissues from aborted fetus were examined by standard gross pathology procedures and were tested using histopathology and immunohistochemistry. Moreover, DNA from fetal and placental tissues was isolated and a PCR assay to detect N. caninum, T. gondii and Chlamydia spp. was used. RESULTS AND DISCUSSION: The pregnancy rate was 89% (119/134), the abortion rate was 8.4% (10/119) whereas the perinatal mortality rate was 15% (16/109). Out of 116 adult ewes sampled, 34.48% presented anti-N. caninum antibodies. Ten ewes had aborted, and one fetus was directly submitted to the diagnostic laboratory for further processing. Antibodies against N. caninum were detected in fetal fluid and in the aborted dam. Histopathological analysis in fetal tissues showed multifocal lymphohistiocytic glossitis, diffuse mild lymphohistiocytic endocarditis, pericarditis and focally extensive myocarditis. Severe multifocal necrotizing placentitis and diffuse mild lymphohistiocytic placentitis with the presence of lymphohistiocytic vasculitis were observed in placenta. N. caninum was immunolabeled in the placenta and fetal tongue. In addition, N. caninum DNA was detected in placenta, central neural system, lung and heart of the aborted fetus. There was no evidence of other infectious abortifacients in the aborted fetus. CONCLUSION: The present study described for the first time an ovine abortion caused by N. caninum in Argentina. Further investigations at a larger scale are required to establish the role of N. caninum as an important cause of reproductive losses in sheep flocks from the region.

Early Neospora caninum infection dynamics in cattle after inoculation at mid-gestation with high (Nc-Spain7)- or low (Nc-Spain1H)-virulence isolates.

Early Neospora caninum infection dynamics were investigated in pregnant heifers intravenously inoculated with PBS (G-Control) or 107 tachyzoites of high (G-NcSpain7)- or low (G-NcSpain1H)-virulence isolates at 110 days of gestation. Serial culling at 10 and 20 days post-infection (dpi) was performed. Fever was detected at 1 dpi in both infected groups (P < 0.0001), and a second peak was detected at 3 dpi only in G-NcSpain7 (P < 0.0001). At 10 dpi, Nc-Spain7 was detected in placental samples from one animal related to focal necrosis, and Nc-Spain7 transmission was observed, although no foetal lesions were associated with this finding. The presence of Nc-Spain1H in the placenta or foetuses, as well as lesions, were not detected at 10 dpi. At 20 dpi, G-NcSpain7 animals showed almost 100% positive placental tissues and severe focal necrosis as well as 100% transmission. Remarkably, foetal mortality was detected in two G-NcSpain7 heifers. Only one animal from G-NcSpain1H presented positive placental samples. No foetal mortality was detected, and lesions and parasite transmission to the foetus were not observed in this group. Finally, 100% of G-NcSpain7 heifers at 20 dpi presented specific antibodies, while only 60% of G-NcSpain1H animals presented specific antibodies at 20 dpi. In addition, earlier seroconversion in G-Nc-Spain7 was observed. In conclusion, tachyzoites from Nc-Spain7 reached the placenta earlier and multiplied, leading to lesion development, transmission to the foetus and foetal mortality, whereas Nc-Spain1H showed delayed infection of the placenta and no lesional development or transmission during early infection.

A probe-based real-time PCR assay for the detection of Neospora caninum in clinical samples from cattle.

Neospora caninum is an apicomplexan protozoan parasite that is a leading cause of abortion in cattle. Detection of parasite-specific DNA by PCR is a highly sensitive method for identifying the presence of N. caninum in a variety of tissues. We developed and validated a probe-based real-time PCR assay targeting the conserved Nc5 gene of N. caninum. Using N. caninum strain Nc-1 genomic DNA and a synthetic gene fragment as amplification standards, we determined the PCR amplification efficiency and the limit of detection to be 95.60% and 3 copies, respectively. Five pathogens frequently associated with bovine abortions, namely bovine viral diarrhea virus types I and II, bovine alphaherpesvirus-1, Chlamydia, and Leptospira, were tested to ensure analytical exclusivity. A total of 103 clinical samples from aborted fetuses were tested concurrently with a standard conventional PCR and the new probe-based real-time PCR assay. All tested samples showed 100% agreement between these two assays. In conclusion, the probe-based real-time PCR assay facilitates accurate and rapid detection of N. caninum from abortions in cattle.

Evidence of reduced vertical transmission of Neospora caninum associated with higher IgG1 than IgG2 serum levels and presence of IFN-γ in non-aborting chronically infected cattle under natural condition.

Neospora caninum infection of cattle can be vertically transmitted, resulting in abortion or birth of infected calves. Vertical transmission occurs both in acutely or chronically infected cattle. There is little information on the immune response needed to prevent endogenous transplacental transmission, particularly from chronically infected cattle to their offspring in a natural environment. In this study, N. caninum seropositive pregnant cattle from three different farms with high avidity antibodies and low IgM titers were selected and their newborn colostrum-deprived calves were tested for anti-N. caninum antibodies. Based on these results, dams were grouped according to their congenital transmission status. The analysis of the immune profile of the chronically-infected pregnant cattle revealed that higher ratio between IgG1 and IgG2 anti-N. caninum serum titers and higher levels of systemic IFN-γ were associated with diminished vertical transmission rates, compared to dams with the opposite profile. Our results evidenced an association between the immune profile and vertical transmission in non-aborting chronically infected dams, and confirm that vertical transmission, even when not leading to abortion, is related to a defined immune profile. This is important information to accomplish successful vaccine development efforts.

Prevalence and Risk Factors of Brucellosis, Toxoplasmosis, and Neosporosis Among Yanbian Yellow Cattle in Jilin Province, China.

Brucellosis and Toxoplasmosis are important zoonotic diseases, and Neospora caninum is a parasite causing disease in cattle and other animals. Brucella spp. and N. caninum can cause abortions in cattle, and Toxoplasmosis is a relevant cause of abortion for small ruminants, resulting in economic loses to farmers. In this study, from July 2015 to April 2017, a total of 535 Yanbian yellow cattle blood samples were collected from 3 cities in Jilin Province, China. We detected the frequency of N. caninum, Toxoplasma gondii, and Brucella spp. using enzyme-linked immunosorbent assays, indirect hemagglutination assay, and real-time PCR methods, respectively. The frequency of Brucella was 7.7% (41/535), and the seroprevalences of N. caninum and T. gondii were 6.2% (33/535) and 5.0% (27/535), respectively. The region, gender, and age of Yanbian yellow cattle were considered as risk factors when analyzed using a logistic regression model. These findings provide the foundation for the prevention and control of infection by these pathogens in Yanbian yellow cattle and humans.

Endogenous transplacental transmission of Neospora caninum during successive pregnancies across three generations of naturally infected sheep.

Endogenous transplacental transmission, which occurs during pregnancy as the result of reactivation of a latent infection in the dam, is the main mechanism of propagation of Neospora caninum within cattle herds. However, the importance of this propagation mechanism has not yet been evaluated in relation to ovine neosporosis. In this study, involving three generations of ewes naturally infected by N. caninum, we demonstrated that endogenous transplacental transmission may also be highly efficient in the ovine host since transmission of infection occurred in 96.6% of gestations and the congenital infection rate ranged between 66.7 and 93%. Nevertheless, parasite burdens decreased gradually in consecutive generations. Reactivation of latent infections had a strong impact on the pregnancy outcome, with high mortality rates recorded in the offspring of the two first generations of ewes (21.4-46.1%). Histological examination of the brain revealed that all aborted foetuses had characteristic lesions of neosporosis (necrotic glial foci) and a few parasite cysts, whereas most stillborn and newborn lambs that died shortly after birth had non-specific lesions (mild glial foci without necrosis) and parasite cysts were more frequent. Microsatellite analysis revealed scarce genetic variability in the N. caninum population, in accordance with a scenario in which infections were of a single origin and were exclusively maintained by clonal propagation through endogenous transplacental transmission.

Influence of the physiological stage of Blackbelly sheep on immunological behaviour against gastrointestinal nematodes.

The influence of the physiological stage of a Blackbelly flock against natural infection by gastrointestinal parasitic nematodes (GIN) was investigated. Data on serum and saliva IgA levels and peripheral cellular immune response were recorded in 51 sheep for 6 months. The flock was divided based on their physiological stage as follows: a) pregnant ewes, b) lactating ewes, c) non-pregnant ewes, d) pre-weaning lambs, e) growing lambs and f) replacement sheep. Additionally, sheep were classified based on the parasite infection as stabled or grazing management system as follows: g) non-infected stabled sheep, h) infected and stabled sheep and i) infected grazing sheep. Faeces and blood samples were collected every fifteen days. The percentage of packed cell volume (PCV), plasma protein, (PP) and leucocyte differential counts (LDC) were recorded. Likewise, serum and saliva IgA levels were measured by the indirect ELISA technique. The number of GIN eggs excreted per g of faeces (EPG) was also determined using the McMaster technique. Data were analysed using the SAS program to identify fixed effect of physiological stages, infection time and interactions. Results showed high EPG values in stabled lactating ewes (2764) and in growing lambs (2950-4550 EPG). Pregnant ewes had higher PCV values (27%) than lactating ewes (23.5%). Infected lambs showed low PCV values (28.5%) and high PP values (5.9 g/dL) compared with non-infected lambs (31% PCV and 5.6 g/dL PP). The two most abundant GIN species identified were Haemonchus contortus (91%) and Trichostrongylus colubriformis (81%). Pregnant and non-pregnant ewes showed 29% and <20% of IgA values, respectively. High EPG, low IgA and eosinophils counts were observed until 180 days of age in infected lambs. In conclusion, the group of infected lactating ewes and lambs showed susceptibility to GIN than the other groups, including the pregnant ewes.

Influence of dose and route of administration on the outcome of infection with the virulent Neospora caninum isolate Nc-Spain7 in pregnant sheep at mid-gestation.

Experimental infections in pregnant sheep have been focused on studying the effect of the time of challenge on the outcome of N. caninum infection, whereas the impact of the dose and route of challenge has not been studied in depth. Therefore, clinical outcome, immune responses, parasite detection and burden, and lesion severity in placental tissues and foetal brains were investigated in 90-day-pregnant sheep inoculated intravenously with 105 (G1), 104 (G2), 103 (G3), or 102 (G4) tachyzoites or subcutaneously with 104 (G5) tachyzoites of the virulent Nc-Spain7 isolate and an uninfected group (G6). Comparing challenge doses, G1 was the only group that had 100% abortion. Likewise, IFNγ levels in G1 increased earlier than those in other intravenously infected groups, and IgG levels on day 21 post-infection (pi) were higher in G1 than those in other intravenously infected groups. Concerning vertical transmission, G1 shows a higher parasite burden in the foetal brain than did G2 and G3. Comparing routes of administration, no differences in foetal survival rate or parasite load in the foetal brain were found. Although G2 had higher IFNγ levels than G5 on day 10 pi, no differences were found in humoral immune responses. Because the outcome after intravenous infection with 105 tachyzoites was similar to that observed after intravenous infection with 106 tachyzoites used in a previous work (100% abortion and vertical transmission), we conclude that it may be reasonable to use 105 tachyzoites administered by the intravenous route in further experiments when assessing drugs or vaccine candidates.

Microsatellite pattern analysis of Neospora caninum from a naturally infected goat fetus.

Neospora caninum is an apicomplexan protozoan responsible for abortion in ruminants. The present study aimed to diagnose an abortion from an Anglo Nubian goat from a dairy herd located in Buenos Aires province, Argentina. The goat delivered a fetus of approximately 3 months gestation that was studied by indirect fluorescent antibody test (IFAT), Immunoblot (IB), histopathology (HP), immunohistochemistry (IHC), and molecular assays (PCR, sequencing and microsatellite genotyping). Interferon gamma knock-out mice were inoculated with a pool of tissues for isolation attempts. The mother had IFAT titers of 1:3200 and 1:400 for N. caninum and Toxoplasma gondii, respectively, as well as positive IB reactions, whereas the fetus was seronegative to both parasites by IFAT and IB. The fetus had severe multifocal necrotizing myocarditis and hepatitis, moderate interstitial pneumonia, and nephritis. Myocardium sample resulted positive by IHC, evidencing clusters of N. caninum tachyzoites within myocardiocytes associated with histopathological lesions. Neospora caninum-DNA was detected by PCR in heart, liver, lungs, kidney, and muscle from the fetus, and was negative for T. gondii by PCR. NC-5 and 18 S rRNA gene fragment sequences showed 100% identity with N. caninum. Inoculated mice bled 30 days post-inoculation resulted seronegative to N. caninum and T. gondii by IFAT, and showed no clinical signs. Multilocus-microsatellite genotyping revealed a genetic profile that differed from previously reported N. caninum genotypes, with unique MS21 and MS10 alleles. These findings indicate that N. caninum was efficiently transmitted from the mother to the fetus. We report the first case of direct detection of N. caninum in a goat fetus in Argentina and N. caninum microsatellite genotyping in naturally infected goat.

Presumptive vertical transmission of Neospora caninum in related Bernese Mountain dogs.

Neospora caninum is a tissue cyst-forming coccidium capable of causing spinal cord or skeletal muscle disease in dogs. Infected bitches can transmit the parasite to their pups in utero. Seroprevalence of N. caninum was studied in naturally-infected, privately owned Bernese Mountain dogs, using antibody detection via an indirect fluorescent antibody test (IFAT) to identify infected individuals. A retrospective study was undertaken on available dogs from 14 litters. Five of eight dams tested seropositive. The index case was a bitch with a titre of 1:3200 by IFAT. Only one offspring from her first litter was seropositive. The frequency of putative congenital transmission in the breeding kennel was variable. Our results reiterate the importance of serological testing of all dams and littermates in a breeding kennel when clinical neosporosis is suspected in neonatal puppies.

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.