Neospora caninum tachyzoites inoculated by the conjunctival route are not vertically transmitted in pregnant cattle: A descriptive study

The aim of this study was to evaluate whether Neospora caninum tachyzoites (Nc-1) inoculated by the conjunctival route in pregnant cows were able to generate infection in their fetuses. Group 1 contained 2 naturally infected cows; group 2 contained two cows inoculated intravenously with 2.5 × 10⁸ tachyzoites, group 3 contained two cows inoculated with 2.5 × 10⁸ tachyzoites by the conjunctival route, and group 4 contained two uninfected control cows. The four inoculated cows from groups 2 and 3 were challenged at 23 weeks of gestation. An indirect fluorescent antibody test (IFAT), recombinant NcGRA7-based ELISA, ELISA for IgG subisotypes and Western blot analysis were assessed to characterize the humoral immune response in dams. Sera from their fetuses were tested also using Western blot analysis. Routine microscopic evaluation of H&E stained fetal tissues was made and any fetal tissues and placentas with lesions compatible with Neospora-infection were processed by immunohistochemistry (IHC). DNA extraction from fresh and formalin-fixed, paraffin-embedded fetal tissues were tested by nested PCR. All dams from groups 1, 2 and 3 were seropositive by IFAT, rNcGRA7-based-ELISA and Western blot. IgG₁/IgG₂ ratios were ≤1 at weeks 27 and 29 of gestation. Only fetuses from groups 1 and 2 developed N. caninum specific antibodies by Western blot. Histopathological lesions compatible with those caused by N. caninum were observed in fetuses from groups 1 and 2. N. caninum cysts and tachyzoites were observed by IHC on fetal tissues from groups 1 and 2. Only fetal samples from group 2 were positive by PCR. Further work is needed not only to characterize the cellular immune response but also to clarify the consequences on the dam after conjunctival inoculation of N. caninum tachyzoites. This study shows that N. caninum tachyzoites inoculated by the conjunctival route were not vertically transmitted in pregnant cows.     

Immune response to Neospora caninum in naturally infected heifers and heifers vaccinated with inactivated antigen during the second trimester of gestation

The objective of this study was to compare the immune response to Neospora caninum in naturally infected heifers and heifers inoculated with a killed whole N. caninum tachyzoite preparation during the second trimester of gestation. Nine Holstein heifers were used in this study; three naturally infected heifers were born from seropositive dams, and six seronegative heifers were born from seronegative dams. Four seronegative heifers were subcutaneously vaccinated with a killed whole N. caninum tachyzoite preparation at weeks 13, 15 and 17 of gestation. A killed whole N. caninum tachyzoite preparation containing 45 mg of protein/5 ml dose was formulated with 70% of mineral oil adjuvant (13% consisting of Arlacel C, 85% Marcol 52 and 2% Tween-80). Similarly, two seronegative heifers (negative controls) were inoculated with mock-infected bovine monocytes in oil adjuvant. Humoral immune responses were tested by using an indirect fluorescent antibody test (IFAT) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting isotype specific antibodies. Cellular immune responses were assessed by lymphocyte proliferation test (LPT) and IFN-gamma production. N. caninum-specific antibody responses increased in immunized cattle by week 15 of gestation (mean reciprocal antibody titers 450+/-252), peaked at week 23 (mean 16,000+/-6400). Maximum antibody response in naturally infected heifers was observed at week 19 of gestation (mean: 3467+/-2810). Mean serum IFAT titers were significantly higher in immunized heifers compared with those in naturally infected heifers from weeks 17 to 25 (P < 0.05). Analysis of isotype specific antibodies in naturally infected heifers revealed a predominant IgG1 response in one heifer and a predominant IgG2 response in the other two. Similar titers of IgG1 and IgG2 occurred in immunized heifers. Control heifers remained seronegative throughout the study by IFAT and ELISA. Significant antigen-specific proliferation responses were only detected in naturally infected heifers in week 19 of gestation. Peripheral mononuclear blood cells (PMBC) from immunized animals produced IFN-gamma in similar concentrations to those of infected animals (P > 0.05). No abortion was seen in any experimental group; however, one calf from a vaccinated heifer died due to dystocia. All calves from vaccinated and control heifers were seronegative by IFAT at 6 months of age; in contrast, calves born from naturally infected heifers remained seropositive with titers > or = 200. Killed vaccine induced similar immune responses to those found in chronically, naturally infected cattle which did not abort; however, different immune pathways may be followed in vaccinated and natural infected heifers with differences in degree of protective immunity.     

Immune response to Neospora caninum native antigens formulated with immune stimulating complexes in calves

The aim of this study was to compare the immune responses to live Neospora caninum tachyzoites and N. caninum native antigens formulated with immune stimulating complexes matrix (ISCOM-matrix) in calves. Fifteen calves were used in this study: 3 were intravenously inoculated with 1 × 10(8) live tachyzoites (Group A), 3 were inoculated twice with N. caninum native antigens formulated with ISCOMs (Group B); 3 with N. caninum native antigens in phosphate-buffered saline (PBS) (Group C); 3 received ISCOM-matrix (ISCOMs without antigen) (Group D) and 3 were negative controls receiving PBS (Group E). The last four groups were inoculated subcutaneously. The specific total IgG and its subtypes were analyzed by an indirect enzyme-linked immunosorbent assays (ELISAs) and by Western blot. IFN-γ levels in plasma was quantified using a commercial kit. All calves were challenged intravenously with 1 × 10(8) live tachyzoites at week 11 after receiving the first dose. Parasitemia was assessed in plasma samples by semi-nested PCR. Neospora-specific antibodies were detected in animals from Groups A and B in the week 2 after inoculation. The ELISA OD values were higher in Group B compared with Group A from weeks 6 to 11 (P<0.05). Analysis of the subisotype specific antibodies in experimentally infected calves revealed a predominant IgG(2) response; however, a predominant IgG(1) response was observed in animals inoculated with N. caninum native antigens formulated with ISCOM-matrix. Control calves remained seronegative until challenge infection. The pattern of bands by Western blot was similar when testing sera from animals in Groups A and B. The levels of IFN-γ production after respective immunization schedules were similar between Groups A and B. Neospora-DNA was detected in plasma samples shortly after intravenous challenge in calves from all groups including those receiving the experimental vaccine formulation. The duration of the parasitemia was similar in all groups.     

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

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

Intrauterine Neospora caninum inoculation of heifers

Here, we studied the potential of Neospora caninum tachyzoites to infect heifers when administered in utero by artificial insemination via contaminated semen. Eighteen primiparous cyclic heifers were hormonally synchronized and artificially inseminated. Nine of them, which were inseminated with semen containing 10(7) live N. caninum NC-1 isolate-tachyzoites, reacted with seroconversion and a specific IFN-gamma response. Moreover, N. caninum DNA was demonstrated by a nested-PCR in the blood of all nine heifers and in brain, lungs, liver and uterine horn of several of them. In contrast, nine heifers inseminated with tachyzoite-free semen developed no antibody or IFN-gamma responses, and no parasite DNA was detected in blood or organs. At necropsy, viable embryos were detected in one and six of the infected and non-infected heifers, respectively. No specific Neospora DNA was detected in any of the embryos. This study provides evidence that intrauterine inoculation via contaminated semen cause N. caninum infection in cattle.     

Transplacental Neospora caninum infection in dogs

A Beagle bitch was inoculated SC and IM with 1.5 million Neospora caninum tachyzoites on the 35th day of gestation. Eight pups were born alive 28 days after N caninum inoculation of the bitch. Pup 1 did not breathe and died while still enclosed in fetal membranes. Pup 2 died 2 days after birth (DAB). Pups 3, 4, and 5 were euthanatized on 2, 3, and 20 DAB, respectively, because they were hypothermic and not nursing. Pups 6, 7, and 8 remained clinically normal. Indirect fluorescent N caninum serum antibody titers were: less than 50 (pups 1 and 8 at 17 DAB, and the bitch before inoculation), 50 (pups 2 and 3 on 2 DAB), 200 (pups 6 and 7 on 17 DAB), and 800 (bitch on day 17 after parturition). Neospora caninum was recovered in cultured cells inoculated with placenta and tissues from all 5 pups necropsied, and N caninum was seen in histologic sections of the heart of pup 5. Results indicate that N caninum can be transplacentally transmitted in dogs.     

Vaccination of ewes for prevention of vertical transmission of Neospora caninum

OBJECTIVE: To evaluate the immunologic response of a killed tachyzoite vaccine against Neospora caninum and its effectiveness in preventing vertical transmission of N caninum in sheep. ANIMALS: 40 Dorset ewes seronegative for N caninum. PROCEDURE: Group-A ewes (n = 20) were vaccinated on days 1 and 126 with a killed N caninum tachyzoite preparation in a commercially available adjuvant. Group-B ewes (n = 20) were sham vaccinated. Blood samples were collected from ewes every 2 weeks and a recombinant ELISA (rELISA) was used to determine serum antibody titers against N caninum. During pregnancy, ewes were challenged with live N caninum tachyzoites. Precolostral serum was collected from lambs and tested for antibodies against N caninum by use of an indirect fluorescence antibody test and the rELISA. Tissue specimens from stillborn lambs or lambs that died within 2 weeks of birth were collected and examined for N caninum antigen and DNA by use of immunohistochemistry and polymerase chain reaction assay, respectively. RESULTS: Serum antibody titers against N caninum were significantly higher in group-A ewes, compared with group B ewes, following vaccination. Serum antibodies against N caninum were detected in 100% (33/33) of group-B lambs and 75% (18/24) of group-A lambs. In tissue specimens, N caninum DNA was detected in 9 of 11 group-B lambs and 0 of 10 group-A lambs. Histologically, N caninum tachyzoites were observed in 4 group-A lambs and 3 group-B lambs. CONCLUSIONS AND CLINICAL RELEVANCE: The killed tachyzoite vaccine against N caninum stimulated a humoral immune response in sheep and provided partial protection against vertical transmission.     

Associations between pregnancy outcome and serological response to Neospora caninum among a group of dairy heifers

AIM: To monitor pregnancy in a group of rising 2-year-old dairy heifers on a farm on which abortion due to Neospora caninum was known to occur in previous years. METHODS: A prospective cohort study group of 164 rising 2-year-old heifers was pregnancy-tested and blood-sampled at 4-5-week intervals throughout gestation. Sera were tested for antibodies to N. caninum at 3-4-month intervals, using an enzyme-linked immunosorbent assay (ELISA). When loss of pregnancy was detected, an N. caninum indirect fluorescent antibody test (IFAT) was conducted retrospectively on stored sera collected the month before abortion, the month abortion was detected, and for the following 2 months, from heifers that aborted. All fetal and placental material detected following abortion was subjected to gross post-mortem and histopathological examination. RESULTS: Eleven of 18 (61%) heifers that were seropositive and 4/146 (3%) heifers that were seronegative to N. caninum by ELISA, aborted. The relative risk for abortion among ELISA-positive heifers was 23.6. Abortion occurred predominantly between Days 120 and 152 gestation among the ELISA-positive heifers and throughout gestation among the ELISA-negative heifers. IFAT titres rose around the time of abortion in most of the heifers that were previously seropositive by ELISA, but dropped rapidly again in post-abortion samples. IFAT titres among 4/6 ELISA-positive heifers that did not abort increased, but later in gestation than the time other heifers aborted. IFAT titres remained negative in heifers that aborted that were ELISA negative. CONCLUSIONS: Heifers that were seropositive to N. caninum by ELISA had a much greater risk of abortion than seronegative heifers. Most seropositive heifers showed evidence of a reactivation of infection during pregnancy. High (> or =1:2,000) N. caninum IFAT titres also occurred in non-aborting heifers. CLINICAL RELEVANCE: Culling of replacement heifers seropositive to N. caninum may be a cost-effective strategy for minimising risk of abortion. Pregnancy testing heifers before 5 months gestation may overestimate the number that calve in N. caninum-infected herds, but would assist in documenting the occurrence of abortion. Reliance on a high (>1:2,000) IFAT titre to rule-in N. caninum as a cause of abortion is likely to produce false-positive results.     

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.     

Multimammate rat (Mastomys natalensis), Tristram's jird (Meriones tristrami) and Wagner's gerbil (Gerbillus dasyurus) as laboratory models of acute neosporosis

To test the different sensitivity of rodents of the subfamily Murinae and Gerbillinae, Wagner's gerbils (Gerbillus dasyurus), Tristram's jirds (Meriones tristrami) and multimammate rats (Mastomys natalensis) were inoculated with Neospora caninum tachyzoites. Clinical signs of neosporosis appeared in all inoculated animals. Histopathological examination confirmed the presence of tachyzoites in brains, lungs, skeletal muscle, myocardium, liver, in serosa of stomach and intestines, and in vesicular accessory genital glands. An examination of brains by PCR revealed presence of N. caninum DNA in all experimentally N. caninum infected rodents. The susceptibility of Wagner's gerbils and Tristram's jirds further proved the high sensitivity of gerbiline rodents to the N. caninum infection. The finding of N. caninum tachyzoites in the vesicular accessory genital glands of the infected gerbils suggests the usefulness of the rodent model for demonstration of N. caninum in the male reproductive system. Moreover, the multimammate rat was a susceptible experimental host to be the first immunocompetent rodent of the subfamily Murinae.     

Experimental studies on the transmission of Neospora caninum between cattle

Three studies were conducted to investigate the transmission of Neospora caninum between cattle by the oral route. In the first study, six calves were dosed with 10(7)N caninum tachyzoites (NC LivB1) in colostrum and/or milk replacer on four occasions. In the second study, two calves and two cows were fed placental tissues from N caninum -infected cows, and, in the third study, seven uninfected calves were fostered onto N caninum -infected dams. In the first study, all six calves developed antibody responses and five calves developed antigen-specific lymphoproliferation responses, including two calves initially challenged at 1 week of age. No evidence of N caninum infection was found in the brain or heart of these calves by histology or polymerase chain reaction (PCR). In the second and third studies, there was no evidence of N caninum infection in any of the calves and cows. The results confirm that calves up to 1 week of age can be experimentally infected via the oral route, but suggest that this is not an important natural route of transmission for N caninum between cattle.     

Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow

Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum.     

Confirmed clinical Neospora caninum infection in a boxer puppy from Argentina

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

Dynamics of anti-Neospora caninum antibodies during gestation in chronically infected dairy cows

The dynamics of antibody production against Neospora caninum during the gestation period was examined in chronically infected dairy cows. Data were obtained from 86 pregnant parous dairy cows, 21 of which had suffered abortion. The cows belonged to two herds in which a diagnosis of N. caninum infection had been previously confirmed in aborted foetuses. Pregnancy diagnosis and blood collection were performed on post-insemination Days 40, 90, 120, 150, 180, 210, and at parturition or until the time of abortion detection. Blood plasma was tested for antibodies against N. caninum using ELISA. The non-aborting cows were divided into two groups according to whether their antibody values in the second half of gestation had increased or not, while aborting cows were classified as those showing an antibody peak before abortion or those not showing a pre-abortion peak. Differences in antibody values throughout pregnancy in each group of non-aborting and aborting cows were analysed by GLM repeated measures of analysis of variance. While 32 non-aborting cows (49%) showed a significant and consistent increase in anti-Neospora antibody values during the second half of gestation, antibody values in the remaining 33 non-aborting cows were practically constant throughout gestation. An antibody peak around abortion was observed in 11 aborting cows (52%), while antibody values in the remaining 10 aborting cows were similar before and at abortion. Seroprevalence fluctuations, defined as seronegative blood samples at some point during the gestation period, were, furthermore, observed in 2 aborting and 11 non-aborting cows. Our results indicate two clearly distinguishable types of humoral immune dynamics throughout gestation: an increased or flat production of antibodies during the second half of gestation in non-aborting animals and before abortion in aborting cows. The observation that some Neospora-infected dams can exhibit negative antibody values at any time during gestation, particularly at parturition or abortion, prompts future studies designed to explore the use of new ELISA strategies at the farm level.     

Experimental inoculation of Neospora caninum in pregnant water buffalo

The aim of this study was to characterize the pathogenesis of Neospora caninum in experimentally inoculated pregnant water buffalo (Bubalus bubalis). Twelve Mediterranean female water buffaloes ranging in age from 4 to 14 years old and seronegative to N. caninum by indirect fluorescent antibody test (IFAT) were involved. Ten females were intravenously inoculated with 10(8) tachyzoites of NC-1 strain at 70 (n=3) or 90 (n=7) days of pregnancy (dp). Two control animals were inoculated with placebo at 70 and 90 dp, respectively. Serum samples were obtained weekly following inoculation to the end of the experiment. Three animals inoculated at 70 dp were slaughtered at 28 days post inoculation (dpi), three animals inoculated at 90 dp were slaughtered at 28 dpi and the remaining four animals inoculated at 90 dp were slaughtered at 42 dpi. Fetal fluids from cavities and tissue samples were recovered for IFAT and histopathology, immunohistochemistry and PCR, respectively. Genomic DNA from fetal tissues was used for parasite DNA detection and microsatellite genotyping in order to confirm the NC-1 specific-infection. Dams developed specific antibodies one week after the inoculation and serological titers did not decrease significantly to the end of the experiment. No abortions were recorded during the experimental time; however, one fetus from a dam inoculated at 70 dp was not viable at necropsy. Specific antibodies were detected in only two fetuses from dams inoculated at 90 dp that were slaughtered at 42 dpi. No macroscopic changes in the placentas and organs of viable fetuses were observed. Nonsuppurative placentitis was a common microscopic observation in Neospora-inoculated specimens. Microscopic fetal lesions included nonsuppurative peribronchiolar interstitial pneumonia, epicarditis and myocarditis, interstitial nephritis, myositis and periportal hepatitis. Positive IHC results were obtained in two fetuses from dams inoculated at 70 dp and slaughtered at 28 dpi. N. caninum DNA was detected in placentas and fetuses from all inoculated animals. The pattern of amplified microsatellites from placental and fetal tissues resembled the NC-1 strain. Water buffaloes, like cattle, are susceptible to experimental inoculation with N. caninum at early pregnancy.     

Serological analysis of calves experimentally infected with Neospora caninum: a 1-year study

A serological study was conducted with calves experimentally infected with the protozoan parasite Neospora caninum. The animals were inoculated with either a low or high dose of N. caninum tachyzoites and temperature responses monitored daily for the first 2 weeks after inoculation. Blood samples were collected before inoculation, and at regular intervals thereafter for 1 year. Serological analysis was achieved using an indirect fluorescent antibody test (IFAT), an enzyme-linked immunosorbent assay (ELISA) and an IgG avidity ELISA.Injection of Neospora produced a significant rise in rectal temperature in the high dose group. In addition, the lymph node draining the site of inoculation increased in size following injection in all animals, in both infected groups, before returning to normal by day 14 after injection. Both groups given N. caninum produced specific antibody that was detected by the IFAT and the ELISA, which remained elevated for the 12-month duration of the experiment. The specific Neospora antibodies produced did not cross-react in an IFAT for the detection of antibodies to Toxoplasma gondii. IgG avidity increased 2 weeks after inoculation, in both infected groups, until week 12 when infection was well established. There was a little difference between the two infected dose groups. This study demonstrates that the two different doses of N. caninum produced a similar antibody response, and that the higher dose also induced a febrile reaction. The IgG avidity ELISA was successful at distinguishing between recent and long-standing infection in this study. However, in both groups, there was fluctuation in the levels of specific antibody throughout the yearlong study, which accords with similar experiments in pregnant cattle, where it has been suggested that fluctuation may indicate periodic recrudescence of infection and a re-stimulation of antibody production by antigen.     

Susceptibility of Djungarian hamsters (Phodopus sungorus) to Neospora caninum infection

Djungarian hamsters were examined for the susceptibility to Neospora caninum infection. After 29 Djungarian hamsters were intraperitoneally inoculated with 5 x 10(6) N. caninum tachyzoites of JPA1 strain, some animals showed symptoms such as ataxia, and many tissue cysts were detected in the brain and a cyst in the muscular tunics of stomach. Especially, more than 100 cysts per head were observed after 5 weeks post inoculation. It is suggested that the Djungarian hamster is a model useful to examine neosporosis.     

In vitro isolation and characterization of bovine Neospora caninum in Korea

The brains of nine aborted bovine fetuses and two newborn calves born from dams suspected to be infected with Neospora caninum were homogenized and inoculated into Vero cells. All fetuses and calves were from cows determined as seropositive to N. caninum by an IFA test. Sera and thoracic fluids of all fetuses and calves also revealed high antibody titer to N. caninum by IFAT ranging from 1:800 to 1:3200. N. caninum was isolated from the brains of one aborted fetus and one newborn calf when the brain homogenates were grown continuously in Vero cell culture. N. caninum tachyzoites, giemsa-positive, were first observed on Days 45 and 56 postinoculation in the newborn calf and the aborted fetus, respectively. The isolates (KBA-1 and KBA-2) were morphologically and ultrastructurally similar to previously published Neospora isolates. The isolated parasites were confirmed as N. caninum by means of the antigenic reactivities, immunostaining, PCR and southern blotting, and electron microscopy.     

Serological responses to Neospora caninum in experimentally and naturally infected water buffaloes (Bubalus bubalis)

The water buffalo (Bubalus bubalis) is an important natural host for Neospora caninum. Serologic responses to N. caninum were studied in experimentally and naturally infected water buffaloes in Brazil. Antibodies were assayed by the indirect fluorescent antibody test (IFAT) using a cut off value of 1:25. Six buffaloes were each inoculated subcutaneously with 5 x 10(6) live culture-derived tachyzoites of the cattle Illinois strain of N. caninum, and two calves were kept as uninoculated controls. Post-inoculation (p.i.) blood samples were collected weekly for 8 weeks and then monthly until 1 year p.i. All inoculated buffaloes developed IFAT titers of 1:100 or more between 7 and 11 days p.i. and the titers remained elevated until 7 weeks p.i. Antibody titers peaked to 1:1600 in 1, 1:800 in 3 and 1:400 in 2, usually by 3 weeks p.i. Antibody titers declined to 1:25 or 1:50 in all the six buffaloes by 12 months p.i. IFAT titers to N. caninum remained at an undetectable level (< 1:25) in both control uninoculated buffaloes. To follow the dynamics of N. caninum antibodies, sera from 29 buffaloes and their calves were collected for 1 year and assayed for N. caninum antibodies; 23 of 29 calves were seropositive (IFAT of 1:100 or more) at 1-2 day of age. Of these 23 calves, 17 remained seropositive during the study, while six became seronegative at four (two calves), six (one calf) seven (two calves) and eight (one calf) months of age. These findings suggest a high rate of neonatal transmission of N. caninum in buffaloes.     

Influence of Neospora caninum infection in BALB/c mice during pregnancy in post-natal development

The influence of Neospora caninum infection during pregnancy on the post-natal period has been poorly investigated. In a previous study, we suggested that infection with N. caninum during pregnancy could affect the normal post-natal development of the offspring. For this reason, in the present work we evaluated the influence of N. caninum infection in pregnant BALB/c mice at days 0, 7 and 14 of gestation (groups A, B and C, respectively) on the post-natal development of the offspring from birth to day 60 post-partum (PP). Morbidity and mortality, vertical transmission, and histopathological lesions were investigated. The humoral immune response (IgG) of pups was also evaluated. Results showed that infection with N. caninum during pregnancy had fatal consequences for pups, especially during mid-gestation (day 7). Infection provoked a delay in the general development of neonates, clinical signs compatible with neosporosis and severe histopathological lesions. A high mortality rate was found in all infected groups. A 69% of mortality rate was found in group A, a 100% in group B and a 46% in group C. Necrotizing encephalitis and multifocal hepatocellular necrosis were the most severe lesions found. All neonates, except four animals from group C, had antibodies against N. caninum but the immune response was not sufficient to control parasite infection. We have demonstrated that extension of the observation period after N. caninum infection permits a more accurate study of vertical transmission, the major route of parasite transmission, and mortality rates. We propose that infection at mid-gestation (day 7) in BALB/c mice and its study during the post-natal period constitutes a valuable experimental model for testing new chemotherapeutic agents and vaccines designed to protect against congenital neosporosis, in order to select effective protocols before its use on bovine.