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C57BL/10ScCr mice, lack Toll-like receptor 4 and a functional Interleukin-12 receptor. Taking this into account, susceptibility of these mice to Neospora caninum infection was assessed comparatively to that of immunocompetent C57BL/10ScSn mice. C57BL/10ScCr mice inoculated intraperitoneally with 5x10(5)N. caninum tachyzoites showed a high susceptibility to this parasite. All infected C57BL/10ScCr mice were dead by day 8 post-infection whereas all control C57BL/10ScSn mice survived this parasitic challenge. Immunohistochemical analysis of infected C57BL/10ScCr mice showed N. caninum tachyzoites spread in the pancreas, liver, lung, intestine, heart and brain whereas no parasites were detected in similarly infected C57BL/10ScSn controls. The higher susceptibility of C57BL/10ScCr mice to neosporosis correlates with reduced interferon-gamma mRNA expression and increased IL-4 mRNA expression, comparatively to C57BL/10ScSn controls, detected in the spleen after the parasitic challenge. C57BL/10ScCr mice could thus be used as a new experimental model where to study immunobiological mechanisms associated with host susceptibility to neosporosis.

To study experimental Neospora caninum infection initiated at the gastrointestinal tract, Toll-like Receptor 4- and functional IL-12Rbeta2 chain-deficient C57BL/10 ScCr mice were challenged intragastrically with 5 x 10(6) N. caninum tachyzoites. All parasite-inoculated mice eventually died with disseminated infection. In contrast, immunocompetent BALB/c mice challenged with 1 x 10(7) N. caninum tachyzoites by the intragastric (i.g.) or the intraperitoneal (i.p.) route remained alive for at least 6 months. Expansion of splenic B- and T-cells, the latter displaying both activated and regulatory phenotypes, and increased levels of IFN-gamma and IL-10 mRNA were detected in both groups of infected BALB/c mice compared with non-infected controls, whereas in the Peyer's patches only IFN-gamma mRNA levels were found to be increased. Parasite-specific IgG1, IgG2a and IgA antibody levels were elevated in the sera of all infected mice, whereas increased N. caninum-specific IgA levels were detected in intestinal lavage fluids of i.g. challenged mice only. These results show that N. caninum infection can be successfully established in mice by i.g. administration of tachyzoites. They also show that the immune response elicited in i.g. or i.p. infected BALB/c mice, although conferring some degree of protection, was not sufficient for complete parasite clearance.

Neosporosis is a major cause of abortion in cattle all over the world. Congenital transmission as well as horizontal transmission by ingestion of oocysts has been described. The detection of Neospora caninum DNA in bull semen warrants the investigation of possible transmission through the use of contaminated semen. In this experiment four cows were artificially inseminated with frozen-thawed semen contaminated in vitro with viable N. caninum tachyzoites (group A) and four control cows were inseminated with tachyzoites-free frozen-thawed semen, from the same bull (group B). Serum samples were collected 15 days before the artificial insemination (AI) and at days 10, 14, 21, 28, 45, 60 and 75 post-insemination. All sera samples were tested for neosporosis by direct agglutination test (DAT). Three of the cows from group A had negative DAT titers (< or =1:20) in all of the samples, while the fourth cow from this group had a low titer of antibodies (1:80) at day 10, and became negative at day 45, suggesting a stimulation of the immune system by the tachyzoites placed in uterus, rather than the induction of an infection. All of the cows from group B had negative DAT titers (< or =1:20) in all of the samples. These results suggest that transmission of neosporosis by artificial insemination with frozen-thawed semen is an unlikely event.

Activation of B cells occurring in hosts infected with protozoan parasites has been implicated either in protective or parasite-evasion immune-mediated mechanisms. Intraperitoneal inoculation of Neospora caninum tachyzoites into BALB/c mice induces an acute response characterized by a rapid increase in the numbers of CD69-expressing peritoneal and splenic B cells. This early B-cell stimulatory effect preceded an increase in the numbers of total and immunoglobulin-secreting splenic B cells and a rise in serum levels of N. caninum-specific immunoglobulins, predominantly of the immunoglobulin G2a (IgG2a) and IgM isotypes. Increased numbers of B cells expressing the costimulatory molecules CD80 and CD86 were also observed in the N. caninum-infected mice. The B-cell stimulatory effect observed in mice challenged with N. caninum tachyzoites was reduced in mice challenged with gamma-irradiated parasites. Contrasting with the peripheral B-cell expansion, a depletion of B-lineage cells was observed in the bone-marrow of the N. caninum-infected mice. Intradermal immunization of BALB/c mice with diverse N. caninum antigenic preparations although inducing the production of parasite-specific antibodies nevertheless impaired interferon-gamma (IFN-gamma) mRNA expression and caused lethal susceptibility to infection in mice inoculated with a non-lethal parasitic inoculum. This increased susceptibility to N. caninum was not observed in naïve mice passively transferred with anti-N. caninum antibodies. Taken together, these results show that N. caninum induces in BALB/c mice a parasite-specific, non-polyclonal, B-cell response, reinforce previous observations made by others showing that immunization with N. caninum whole structural antigens increases susceptibility to murine neosporosis and further stress the role of IFN-gamma in the host protective immune mechanisms against this parasite.

This study was carried out to determine the prevalence of neosporosis in an area of intensive dairy production, in Portugal. Sera samples were obtained in a random basis from 114 cows in 49 herds (group A), and from 1237 cows in 36 herds with a history of abortion outbreaks (group B). All sera samples were tested for neosporosis by direct agglutination test (DAT). Additionally, attempts to isolate Neospora caninum in 42 aborted bovine fetuses from 38 dairy herds (group C) were carried out, utilizing a bioassay with immuno-depressed Swiss Webster mice. Parasitological confirmation was done by indirect fluorescent antibody test (IFAT). The prevalence of neosporosis in the group A was 28%. Group B had a significantly (P < 0.001) higher prevalence (46%) and Neospora caninum was isolated in 36% of the aborted fetuses (group C). These results indicate that neosporosis, a disease only recently (2001) diagnosed in Portugal, has a high prevalence in the country, particularly in populations with a story of abortion. Thus, neosporosis should systematically be considered in the differential diagnosis of abortion. In the context of embryo transfers, the importance of selecting Neospora-free embryo recipients is discussed, as well as the pertinence of assessing the Neospora status of traded and imported cattle.

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.

A definitive diagnosis of neosporosis in cattle implies the examination of the aborted fetus. However, in many instances fetal material is not available. Therefore, most diagnosis are based on serological tests. At the moment, there are no consensuses about the cut-off for serodiagnosis of neosporosis in cattle, for any test. The objective of the present study was to estimate the best cut-off for the Neospora agglutination test (NAT) for serodiagnosis in cattle. For that purpose, 246 serum samples from 4 groups of dairy cows (aborted Neospora positive; not aborted healthy; aborted other diseases and herds endemic neosporosis) were collected and antibodies anti-N. caninum were determined by NAT. Additionally, immunoblot (IB) was performed with sera from all cows of the endemic neosporosis group and the patterns of seroreactivity were contrasted with the NAT titers for this group of cows. Evaluation of the optimized sensitivity and specificity was calculated using Youden's J-statistics. The best Youden's J-statistic was obtained at 1:40 titer, presenting 100% of sensitivity and 90.4% of specificity with negative and positive predictive values of 100 and 75.0%, respectively. The comparison between NAT titers and the IB banding pattern support the results of the statistical analysis, i.e. titers of 1:40 and higher showed a complex pattern of bands, while titers lower than 1:40 did not precipitate any bands. These results indicate that 1:40 was an optimized NAT cut-off for serodiagnosis in cattle.

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal., (Copyright 2002 Elsevier Science B.V.)