Prevalence of vertically transmitted Neospora caninum amongst beef cattle in Phayao, Thailand.

Neospora caninum, the causative agent of neosporosis, is recognised as a significant trigger of abortion and productivity losses in cattle worldwide. Current information regarding to the prevalence of N. caninum in Thailand is limited due to the limitations of detection methods and the difficulty of recovering of viable parasite. Vertical transmission is the main route of N. caninum infection in cattle. Therefore, detection of N. caninum DNA in placental tissue could be a possible means of laboratory diagnosis of neosporosis in live animals, particularly in the context of transplacental transmission. The aim of this study was to investigate the prevalence of transplacentally transmitted N. caninum infection in female beef cattle in the northern Thai province of Phayao by detection of N. caninum DNA in bovine placenta by PCR. A total of 96 bovine placentas were collected from 7 districts of Phayao. Our result indicated that overall PCR prevalence of N. caninum in cattle in this area was 36.5% varying from 16.7-50.0% between districts. The districts with the highest prevalence of infection were Muang (50.0%) and Mae Chai (44.7%). The proportion of N. caninum infection was quite high suggesting that newborn calves were at risk of congenital infection. This study provides a current snapshot of the status of bovine neosporosis in Phayao which could lead to the development of effective strategies for prevention and control this disease.

Neospora caninum: Chronic and congenital infection in consecutive pregnancies of mice.

Neospora caninum, the causative agent of bovine neosporosis is the major cause of abortion in cattle worldwide. The principal route of transmission is via in utero infection of the offspring. Congenitally-infected dams remain persistently infected for life and might undergo abortions in consecutive pregnancies. In the present study, the effect of N. caninum in chronic and congenital infection was examined. CD1 mice were infected intra-peritoneally with live tachyzoites of the NcIs491 isolate, while non-infected mice served as a control. There were no clinical signs of infection observed following inoculation, but high titers of specific anti- N. caninum antibodies were detected. A month after infection, when chronic-infection was established, mice were mated. Fertility, litter size and mortality rate were monitored within two generations of four consecutive pregnancies. During a nine months period of the study all females maintained high level of antibodies, while the non- infected control mice remained seronegative. There was no difference in the fertility rate of the dams, or in the litter size of infected and control mice. Mortality of offspring of the first and second generations of the infected dams was observed within the two first weeks of life. The vertical transmission was analyzed by PCR assay of offspring brains. PCR positive results were found in all 13 litters of the first generation tested during four consecutive pregnancies. The rate of vertical transmission slightly decreased in successive pregnancies being 74.2%, 59.5%, 48.1% and 40% for the first to fourth pregnancies respectively. In the second generation 21 out of 28 litters were found positive and the overall rate of vertical transmission was 28.5%. In chronically and congenitally infected dams N. caninum infection was maintained during all successive pregnancies for about 9 months. The results show that CD-1 outbred mice are a suitable model for studying chronic and congenital neosporosis.

Mice congenitally infected with low-to-moderate virulence Neospora caninum isolates exhibited clinical reactivation during the mating period without transmission to the next generation.

Endogenous transplacental transmission (EnTT) is the major transmission route of Neospora caninum in cattle. Thus, the development of a standardised experimental model of EnTT is needed for more appropriate testing of parasite biology and control strategies. A recent study reported up to 40-50% EnTT rates in chronically infected dams with either high or low-to-moderate virulence isolates, although low fertility rates were observed in dams inoculated with the high virulence isolate. Therefore, low-to-moderate virulence N. caninum isolates (Nc-Spain 3H; G1 and Nc-Spain 8; G2) that previously showed high TT rates versus low mortality and morbidity rates in a congenital mouse model were inoculated into BALB/c dams (first generation). The new approach followed in the present study aimed to start with a high number of congenitally infected mice (second generation), which allowed a more efficient EnTT from congenitally infected dams to their progeny (third generation). Interestingly, a reactivation of infection occurred in several congenitally infected non-pregnant females (second generation) from both infected groups. This fact was evidenced by neosporosis-associated clinical signs after mating accompanied by an increase of specific antibody levels (IgG1, IgG2a and specific antibodies against rNcGRA7) (P<0.0001; one-way ANOVA). Moreover, a higher number of PCR-positive mice compared to pregnant females were observed (P<0.05; Fisher's exact test). These results support the hypothesis that only mice without clinical signs and with a low parasite burden in the brain became pregnant, which may explain the posterior failure in inducing EnTT from the second to the third generation. These findings confirm that this mouse model is not a suitable experimental EnTT model for testing the efficacy of drugs and vaccine candidates against EnTT. The employment of other putative suitable species with a similar placenta structure, such as small ruminants, should be taken into consideration.

Infected dendritic cells facilitate systemic dissemination and transplacental passage of the obligate intracellular parasite Neospora caninum in mice.

The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites.

Congenital neosporosis in goats from the State of Minas Gerais, Brazil.

Congenital Neospora caninum infection was diagnosed in two Saanen goat kids from two distinct herds with a history of abortion and weak newborn goat kids in the Southern region of the State of Minas Gerais, Brazil. The first kid was weak at birth, had difficulty to rise and was unable to nurse. Gross lesions of porencephaly and hydrocephalus ex vacuo were seen. Multifocal necrosis, gliosis and non-supurative encephalitis were observed in the brain. Several parasitic cysts with a thick wall that reacted strongly only with polyclonal antiserum to Neospora caninum were seen in the cerebral cortex, brain stem and cerebellum. The second kid was born from a Neospora caninum seropositive mother that aborted in the last pregnancy. It was born without clinical signs. The diagnosis of neosporosis was based on antibody titer of 1:800 to N. caninum by indirect fluorescence antibody test obtained from blood collected before the goat kid ingested the colostrum and Neospora caninum DNA was detected by polymerase chain reaction and sequenced from placenta. This is the first report of neosporosis in goats in the southeast region of Brazil.

Congenital Neosporosis in Goats from the State of Minas Gerais, Brazil

Congenital Neospora caninum infection was diagnosed in two Saanen goat kids from two distinct herds with a history of abortion and weak newborn goat kids in the Southern region of the State of Minas Gerais, Brazil. The first kid was weak at birth, had difficulty to rise and was unable to nurse. Gross lesions of porencephaly and hydrocephalus ex vacuo were seen. Multifocal necrosis, gliosis and non-supurative encephalitis were observed in the brain. Several parasitic cysts with a thick wall that reacted strongly only with polyclonal antiserum to Neospora caninum were seen in the cerebral cortex, brain stem and cerebellum. The second kid was born from a Neospora caninum seropositive mother that aborted in the last pregnancy. It was born without clinical signs. The diagnosis of neosporosis was based on antibody titer of 1:800 to N. caninum by indirect fluorescence antibody test obtained from blood collected before the goat kid ingested the colostrum and Neospora caninum DNA was detected by polymerase chain reaction and sequenced from placenta. This is the first report of neosporosis in goats in the southeast region of Brazil.

Evidence of congenital transmission of Neospora caninum in naturally infected water buffalo (Bubalus bubalis) fetus from Brazil.

The aim of this study was to determine the congenital infection by Neospora caninum in the water buffalo (Bubalus bubalis), a natural intermediate host. Nine pregnant water buffalos, raised under free-grazing condition, were slaughtered, and their fetuses were collected. Samples of brain and thoracic fluid were obtained from those fetuses, with gestational ages ranging from 2 to 5 months. The DNA of N. caninum was detected and identified in the brain of one of those fetuses, using two PCR assays, one directed to the Nc5 gene and the other, to the common toxoplasmatiid ITS1 sequence. The DNA fragments produced on PCR were sequenced, and N. caninum was confirmed in the samples. No antibodies to N. caninum were detected on any sample of thoracic fluid by immunofluorescent antibody test (IFAT < 25). This is the first confirmation of congenital transmission of N. caninum in water buffalos.

Immunization with native surface protein NcSRS2 induces a Th2 immune response and reduces congenital Neospora caninum transmission in mice.

NcSRS2, a tachyzoite surface protein of Neospora caninum, is an immunodominant protein with respect to induction of antibody production and has a role in attachment and invasion of host cells. Native NcSRS2 was isolated from whole tachyzoite lysate antigen by affinity chromatography using NcSRS2 specific monoclonal antibody and used to immunize BALB/c mice in a congenital transmission study. NcSRS2 was a highly conserved protein as indicated by comparison of deduced amino acid sequence obtained from NcSRS2 gene sequences of 10 geographically distinct N. caninum isolates. Mice immunized with purified native NcSRS2 produced antigen-specific antibody, primarily of IgG 1 subtype. Following challenge during gestation with 10(7) tachyzoites, immunized mice had a statistically significant decreased frequency of congenital transmission compared to non-immunized mice (P

Seronegative conversion in four Neospora caninum-infected cows, with a low rate of transplacental transmission.

Four Neospora-seropositive pregnant cows (prebreeding indirect fluorescent antibody (IFA) titers between 1:400 and 1:1600) were confined and observed until parturition. All cows gave birth to normal calves. Selected tissues were tested for NC by histopathology, immunohistochemical (IHC) and polymerase chain reaction (PCR). Parasite isolation was attempted in vero cell cultures. At parturition, all cows were seronegative at 1:200 and two of four cows had a titer of 1:100 when further tested. Three of four calves were not infected, as determined by negative results of precolostral serology (1:25 cut-off), histopathology, IHC and PCR. One calf was congenitally infected, as shown by the presence of a thick-walled cyst labelled by IHC in its brain, positive PCR of brain and a precolostral IFA titer of 1:100. It was concluded that NC antibody titers may drop or convert to seronegative status in chronically infected cows by the time of parturition and this finding in four of four cows indicates that this could be a common occurrence. Similarly, the finding of an infected calf with a low antibody titer indicates that precolostral serology may not be a fool-proof means of identifying calves with congenital Neospora caninum infections. These findings call into question conclusions of other studies that have estimated rates of congenital transmission of this parasite based on serological tests at calving. This study is the first confirmed report of congenital NC infection in a calf in Thailand.

Isolation of Neospora caninum from a calf in Malaysia.

In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.

Vertical transmission of Neospora caninum in BALB/c mice in both acute and chronic infection.

To examine the frequency of congenital infection by Neospora caninum, BALB/c mice were inoculated intraperitoneally with tachyzoites of N. caninum either during pregnancy (Group 1) or 4 weeks or more before pregnancy (Group 2). Further, the mice inoculated during pregnancy were bred at 4 weeks or more after delivery to form Group 3. Congenital transmission was observed in 76% of the neonates of the mice in Group 1 and in 50% of the neonates of the mice in Group 2. Interestingly, congenital transmission was observed in 86% of the neonates from Group 3. These results suggest that chronically-infected BALB/c mice efficiently transmit N. caninum infection to their offspring.

Inclusion of CpG adjuvant with plasmid DNA coding for NcGRA7 improves protection against congenital neosporosis.

The present study showed that incorporation of CpG adjuvant into plasmid DNA coding for NcGRA7 antigen resulted in a twofold increase in the level of protection against congenital transfer of Neospora caninum. The level of protection was considerably higher than that observed in pups born from dams immunized with nonrecombinant plasmid.

Additional evaluation of undiagnosed bovine abortion cases may reveal fetal neosporosis.

Diagnosis of Neospora caninum associated bovine abortion is often made by histological examination of fetal tissues, although this procedure is not very sensitive. One hundred and forty-four undiagnosed bovine abortion cases were evaluated for N. caninum associated bovine abortion using a revised diagnostic protocol. As a result, 12 (8.3%) of these previously undiagnosed bovine abortion cases were definitively diagnosed as N. caninum positive. The 12 new positive cases included 7 that had exhibited histological lesions, and 5 that had not exhibited histological lesions when examined prior to this study. None of the 12 cases had been immunohistochemically (IHC) stained prior to this study. Also, IHC staining implemented during this research revealed tachyzoites without associated histological lesions in at least 1 tissue from 69 (47.9%) of 144 aborted bovine fetuses, and positive IHC staining of cytoplasmic N. caninum antigen in macrophages in at least 1 tissue from 44 (30.6%) of 144 aborted bovine fetuses. These results demonstrate the necessity for more aggressive evaluation of bovine fetuses for neosporosis.

Granulomatous encephalitis in a neurologically impaired goat kid associated with degeneration of Neospora caninum tissue cysts.

Congenital Neospora caninum infection was diagnosed in a Saanen goat from a farm in southern Brazil. The kid was unable to nurse and had difficulty rising, ataxia, and opistothotonos. The neurologic signs became more severe 3 days after birth, when it was euthanized. No gross lesions were observed at necropsy. Multifocal infiltrates primarily of mononuclear cells, nodular microgliosis, and perivascular cuffs of lymphocytes, plasma cells, and few neutrophils were observed in the brain, mostly in the cortex and adjacent to ventricles. Rare multinucleate giant cells were observed adjacent to inflammatory foci. Several tissue cysts with a thick wall that reacted strongly with polyclonal antiserum to N. caninum were in the cerebral cortex and medulla oblongata. Lesions were also present in heart, lungs, and liver, but N. caninum tachyzoites were not found. Distinguishing features in this goat kid included neurologic impairment resulting from congenital infection with N. caninum and the presence of granulomatous inflammation with rare giant cells associated with degeneration of tissue cysts.

Evidence of post-natal transmission of Neospora caninum in Dutch dairy herds.

Eighteen dairy herds with neosporosis-associated abortions were analysed for antibodies against Neospora caninum. Blood samples of all cows, heifers and calves were collected on the same day for each farm. A total of 2430 heads of cattle were examined. For each herd, the seropositive and seronegative animals were plotted against month of birth. Analysis of seroprevalence in relation to age showed an equal distribution of seropositives in all age-groups in 10 herds. In contrast, in eight herds an age-group could be identified which had a significantly higher seroprevalence than the other animals in the herd. Most seropositive animals in the high seroprevalence age-groups had either seronegative dams or seronegative offspring, whereas there was a strong relationship between the serostatus of dams and offspring in the other animals in the herd. Aborting animals were mainly part of the high seroprevalence age-group. These findings strongly indicate a post-natal infection of the animals in the high seroprevalence age-groups, probably due to a point source exposure to N. caninum.

Isolation of Neospora caninum from the brain of a pregnant sheep.

Neospora caninum was isolated from the brain of a naturally infected pregnant sheep by inoculation of immunodeficient mice with a homogenate of the brain tissue. The ewe showed no clinical signs. Tachyzoites were observed in the tissues of the nu/nu mice injected with the brain tissue homogenate and the diagnosis was confirmed by immunohistochemical staining with anti-N. caninum antibodies and by detecting N. caninum-specific DNA by polymerase chain reaction.

Neutralization of maternal IL-4 modulates congenital protozoal transmission: comparison of innate versus acquired immune responses.

IL-4 levels were modulated in mice to test the hypothesis that induction of a maternal type 1 response would decrease the frequency of congenital Neospora caninum transmission. This hypothesis tested the relationship between IL-4 and both innate and adaptive immunity utilizing two basic experimental designs. In the first, maternal IL-4 was neutralized with mAb during pregnancy in naive mice concomitant with initial, virulent infection. In the second, maternal IL-4 was neutralized before pregnancy concomitant with a priming inoculation consisting of live, avirulent N. caninum tachyzoites followed by virulent challenge during subsequent gestation. In mice that were naive before pregnancy, neutralization of IL-4 during gestational challenge did not result in decreased congenital transmission as measured by PCR performed on 1-day-old neonatal mice. In mice that were primed and modulated before pregnancy, congenital transmission from gestational challenge was significantly decreased compared with control mice. Reduction in transmission constituted a decrease in the numbers of mice transmitting N. caninum and a lower frequency of transmission by individual dams (p < 0.05). Decreased congenital transmission was associated with significantly lower levels of maternal splenocyte IL-4 secretion, lower IL-4 mRNA levels, and higher levels of IFN-gamma secretion. Protected mice had significantly decreased Neospora-specific IgG1 compared with nonmodulated mice. These studies define a relationship between maternal Ag-specific immunity and the frequency of congenital transmission and demonstrate that modulation of type 2 cytokine responses can change the frequency of congenital protozoal transmission.

Postnatal Neospora caninum transmission and transient serologic responses in two dairies.

Cattle on two typically managed drylot dairies were serologically monitored from birth through year 1 to year 4 of life to characterise congenital and postnatal Neospora caninum transmission. Of the 456 calves enrolled, 284 were classified as N. caninum negative and 172 were classified as N. caninum positive. Ninety-six percent of congenitally infected calves were seropositive for all samples tested. Seven (4%) of the 172 congenitally infected animals had a period that persisted for 9 to 18 months when they were seronegative; however, all returned to seropositive status by 25 months of age. In N. caninum-negative calves, colostral antibody decayed by 128 days, with an estimated half-life of 19.6 +/- 5.2 days. Of the 284 calves classified as negative, 18% had sporadic, isolated responses to N. caninum, typically between 29 and 35 months of age, without subsequent seroconversion or infection. During the study, 17 animals seroconverted and remained seropositive throughout the follow-up. Thirteen of the seroconversions occurred in the neonatal period; however, in nine of 10 where dam status was available, the dam was N. caninum positive, suggesting late gestation congenital infection rather than postnatal infection. Seroconversion was detected in an additional four animals, between 13 and 22 months of age. The estimate of postnatal infection rate was less than 1% per year despite a high N. caninum seroprevalence in the herds, and the presence of potential definitive and intermediate hosts on the dairy throughout the study. The extremely low rate of postnatal infection, as well as the lifelong persistence of congenital infection, emphasises the importance of congenital transmission in maintaining N. caninum infection in dairies.

Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle.

OBJECTIVES: To estimate the extent to which abortion risk in dairy cattle during subsequent pregnancies was associated with congenitally-acquired Neospora caninum infection and previous abortions. ANIMALS: 468 Holstein cattle. PROCEDURE: Newborn heifer calves were tested for evidence of congenital infection attributable to N caninum and examined repeatedly until the completion of their second lactation for serologic status and evidence of abortion. RESULTS: Compared with noninfected cows, congenitally infected cows had a 7.4-fold higher risk of abortion during their initial pregnancy and a 1.7-fold higher risk of aborting the first pregnancy during their first lactation. During the first pregnancy of their second lactation, congenitally infected cows that had aborted previously had a 5.6-fold higher risk of abortion, compared with cows that had not previously aborted and that were seronegative. The fetal risk period for N caninum-associated death began sooner and extended later during the initial pregnancy compared with subsequent pregnancies. CONCLUSION: Congenitally acquired N caninum infection can cause a substantial number of abortions during the initial pregnancy of heifers, with abortion risk attributable to N caninum decreasing in subsequent pregnancies, possibly because of selective culling. Subsequent abortions can be expected in congenitally infected cows that have aborted previously.