Caninum Antigen Research Articles

Immunoassay for detection of Dipylidium caninum coproantigen in dogs and cats.

Dipylidium caninum infections in dogs and cats are underestimated because of a lack of proglottid observations and poor recovery of parasite elements by centrifugal flotation. We developed an immunoassay that employs a pair of monoclonal antibodies to capture D. caninum-specific coproantigen in fecal extracts from dogs and cats. Real-time PCR for D. caninum DNA in perianal swabs and observation of proglottids were used as reference methods. In 6 experimentally infected dogs, parasite DNA, coproantigen, and proglottid segments were first detected at 22, 23, and 26 d post-infection, respectively. Praziquantel treatment of 3 experimentally infected dogs resulted in the elimination of both coproantigen and proglottid shedding within 1-5 d post-treatment; however, parasite DNA persisted for 14 d. Immunohistochemistry on immature and mature tapeworm segments using an antibody against the coproantigen supports the premise that the antigen is produced in mature segments. We assessed the performance of our coproantigen test in natural infections in 78 dogs from a flea-endemic area. Of the 12 antigen-positive samples, 11 were confirmed with a positive PCR test and/or proglottid observation. Finally, we evaluated a convenience sample set of 730 canine and 163 feline fecal samples obtained from a commercial diagnostic laboratory; D. caninum antigen was detected in 4.1% of the canine and 12.9% of the feline samples, whereas parasite elements were observed in only 0.028% of samples. Our coproantigen immunoassay provides a sensitive method for the detection of D. caninum infection in dogs and cats.

Role of TLR2/MyD88 in the production of specific IgM and IgG antibodies during the immunization of mice against Neospora caninum

Neospora caninum is a parasite relevant to the veterinary field. Innate and adaptive responses against N. caninum induce effector mechanisms that limit parasite replication, but little is known about their role in humoral response. Our work aimed to verify whether key molecules in the TLR2/MyD88-mediated response would impact the production of specific IgM and IgG antibodies in mice during immunization with soluble antigens of N. caninum. We observed that lack of IFN-gamma did not negatively affect the production of specific antibodies. However, mice genetically deficient in Toll-like receptor 2, Myeloid differentiation factor 88, Interleukin 12 and inducible nitric oxide synthase presented significant decrease in antibody levels against N. caninum antigens, which also reflected in the diversity of the antigen recognized by their serum. In that sense, we show here that molecules within this innate recognition pathway may present a direct impact in the induction of an antibody response against N. caninum.

Seroprevalence and risk factors of Neospora caninum and Bovine Viral Diarrhoea Virus in smallholder dairy cattle in Kenya

Little is known of the risk factors associated with occurrence of Neospora caninum and Bovine Viral Diarrhoea Virus (BVDV) infection in Kenya. This cross-sectional study hypothesized that there are significant biosecurity measures associated with N. caninum and BVDV infections on smallholder dairy farms in Kenya that could be adopted to reduce seroprevalence and impacts. From 158 randomly selected farms in Meru County, Kenya, 470 serum samples were collected from dairy cattle (over six months of age and unvaccinated for these two pathogens). Sera were analyzed for antibodies to N. caninum and antibodies and antigens to BVDV. Data on risk factors were obtained through face-to-face interviews with the farmers. Multivariable logistic regression models were used to identify significant risk factors associated with seropositivity for the pathogens. The apparent seroprevalence of N. caninum, BVDV antibody, BVDV antigen, and co-infection with N. caninum and BVDV antibody and/or antigen were 35.1%, 47.1%, 36.2% and 18.5%, respectively. Risk factors associated with N. caninum antibody included: introducing milking cows into the farm, lending of cattle between farms, farm dogs having access to bovine aborted fetuses, and dogs whelping in the farm compound, with an interaction between the last two variables. BVDV antigen was associated with cattle having contact with pigs, and an interaction between cattle age and whether farms introduced new calves onto farms, and cattle age and whether visiting dairy farmers have access to the cow shed. Cows had higher odds of having BVDV antibodies compared to heifers. Factors associated with co-infection included cow parity, direct contact between dairy cattle, dogs and goats, and introducing new milking cows into the farms. Antibody and antigen results may be partly a function of classical swine fever virus or border disease virus interactions. Farmer education on these biosecurity measures is recommended, along with introduction of BVDV vaccination.

Neospora caninum truncated recombinant proteins formulated with liposomes and CpG-ODNs triggered a humoral immune response in cattle after immunisation and challenge

Abortions caused by Neospora caninum are a serious problem in cattle production and require effective immunoprophylaxis. The objective of this work was to assess the humoral immune response to four recombinant (r) N. caninum antigens in cattle after immunisation and challenge. MIC1 and MIC3 proteins from the micronemes, SRS2 from the surface of tachyzoites, and GRA7 from the dense granules were expressed as truncated recombinant proteins in Escherichia coli. Cationic liposomes (Lip) and CpG oligodeoxynucleotides (CpG-ODNs) were used as adjuvant. Steers were assigned to three groups of six steers each and were inoculated twice subcutaneously, 21 days apart. The rP + Lip + CpG-ODN group received the truncated recombinant proteins rMIC1, rMIC3, rSRS2 and rGRA7 formulated with the adjuvant; the Lip + CpG-ODN group received the adjuvant alone; and the PBS group received sterile phosphate-buffered saline. All steers were subcutaneously challenged with the NC-1 strain of N. caninum 35 days after the second dose of immunisation. Steers from the rP + Lip + CpG-ODN group developed specific IgG, IgG1 and IgG2 against the four recombinant proteins after immunisation. After challenge, IgG against rMIC1 and rMIC3 was detected in rP + Lip + CpG-ODN group and against rSRS2 and rGRA7 in all groups. IgG1 and IgG2 against the four recombinant proteins remained high after challenge in the rP + Lip + CpG-ODN group. Indirect ELISA detected anti-N. caninum antibodies after challenge in all groups, with the highest level of antibodies being detected in the rP + Lip + CpG-ODN group. The recombinant vaccine formulated with rMIC1, rMIC3, rSRS2 and rGRA7 using Lip + CpG-ODN as adjuvant was immunogenic in cattle and the humoral immune response after challenge was enhanced in vaccinated cattle.

Evaluation of Neospora caninum serodiagnostic antigens for bovine neosporosis

Abortion and reproductive failure caused by Neospora caninum infection has a dramatic negative economic impact on the cattle industry. To date, no definitive serodiagnostic tool for assessing N. caninum abortion has been reported. In this study, we evaluated the diagnostic performance of numerous N. caninum antigens in relation to abortion in cattle. Five recombinant proteins with potential as diagnostic antigens (NcGRA6, NcGRA7, NcGRA14, NcCyP, and NcSAG1) were compared by indirect enzyme-linked immunosorbent assay (iELISA) using sera from mice and cattle experimentally infected with N. caninum. The best-performing three antigens (NcSAG1, NcGRA7, and NcGRA6) were evaluated by IgG-iELISAs to assess their utility in diagnosing Neospora abortion using sera from confirmed N. caninum-aborted dams based on immunohistochemical assays (IHC). Additionally, all samples were tested using a commercial N. caninum antibody competitive ELISA (cELISA). The iELISAs against both NcSAG1 and NcGRA7 could efficiently distinguish IHC positive and negative samples compared with iELISAs against NcGRA6 and the cELISA. Furthermore, antibody levels against NcSAG1 and NcGRA7 were significantly higher in aborting cows comparing with infected but non-aborted dams in a herd experiencing a Neospora abortion outbreak. Tracking the dynamics of antibody levels during pregnancy revealed a marked increase in NcSAG1- and NcGRA7-specific antibodies at the last trimester of pregnancy. In contrast, no marked differences in antibody levels against either antigen were noted in neurologically symptomatic calves compared with non-symptomatic infected calves. Our data suggests NcSAG1 and NcGRA7 as indicators for Neospora abortion.

Abortion outbreak caused by Campylobacter fetus subspecies venerealis and Neospora caninum in a bovine dairy herd

In November 2015, an abortion outbreak occurred in a commercial dairy herd of 650 Holstein cows in Florida department, Uruguay. Forty-five cows aborted within three weeks. Five fetuses were subjected to gross and microscopic pathologic examination, and microbiological testing. One fetus had fibrinous epicarditis and peritonitis, and neutrophilic bronchopneumonia. Campylobacter fetus subsp. venerealis was detected by direct immunofluorescence, isolated and identified by PCR and sequencing of the 16S rDNA in the abomasal fluid and/or lung. Histologic examination of two other fetuses revealed non-suppurative necrotizing encephalitis, lymphohistiocytic myositis and myocarditis, and lymphocytic interstitial nephritis. In these fetuses, N. caninum antigen was detected intralesionally by immunohistochemistry, and N. caninum DNA was amplified by PCR on formalin-fixed paraffin-embedded brain. Antibodies against N. caninum were detected by indirect immunofluorescence in 10 of 27 cows, with titers ranging from 1/200 to 1/3200. The results indicate that two abortigenic microorganisms may coexist and cause contemporaneous abortion in a herd. We highlight the importance of performing multiple diagnostic tests in various aborted dams and fetuses from the same herd for the etiologic confirmation of bovine abortion syndrome.

Neospora caninum antigens displaying virus-like particles as a bivalent vaccine candidate against neosporosis

Neospora caninum is a causative and transmissible agent of dog and bovine neosporosis. The resulting reproductive failures in infected cattle lead to significant economic losses worldwide. However, there is no satisfactory treatment or vaccine currently available to combat this pathogen. Thus, the development of appropriate vaccines to manage its infection and transmission is urgently needed. In this study, we expressed Rous sarcoma virus-like particles (RSV-LP) that displayed dual N. caninum antigens in silkworms. The antigen candidates are modified by adding a transmembrane domain of GP64 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) to the C-terminus of surface antigen 1 (NcSAG1) and SAG1-related sequence 2 (NcSRS2). The NcSRS2 alone or the NcSAG1/NcSRS2 bivalent form displaying RSV-LPs were purified using sucrose density gradient centrifugation. These purified VLPs were then used for immunizations in gerbils, Meriones unguiculatus, to evaluate the anti-N. caninum effects in vivo. The results demonstrated that antigens displaying RSV-LPs in immunized gerbils produced the antigen-specific antibody, leading to a relatively lower parasite load after infections of N. caninum. To the best of our knowledge, this is the first study to present an RSV-LP vaccine displaying bivalent antigens from neosporosis. Taken together, our strategy suggests that silkworm-expressed virus-like particles (VLPs) are promising bivalent vaccine candidates against N. caninum infections.

Immunization with a cocktail of antigens fused with OprI reduces Neospora caninum vertical transmission and postnatal mortality in mice

OprI is an outer membrane lipoprotein from Pseudomonas aeruginosa, and when fused to a recombinant antigen, will exert adjuvant properties by engaging Toll-like receptor 2, leading to dendritic cell activation. Previous studies have shown that the Neospora caninum (Nc) antigens NcPDI, NcROP2 and NcROP40 are implicated in host cell interactions and are promising vaccine candidates. In two independent experiments, the efficacy of a polyvalent vaccine formulation composed of OprI-NcPDI, OprI-NcROP2 and OprI-NcROP40 (collectively named O-Ags) was assessed in non-pregnant and pregnant Balb/c mouse models challenged with tachyzoites of the high-virulence isolate Nc-Spain7. Parameters that were investigated were clinical signs, fertility, parasite burden in adult mice, humoral and cellular immune responses at different time-points prior to and after challenge infection, vertical transmission and post-natal survival of offspring mice, all to explore potential correlations with efficacy. Vaccination of mice with O-Ags induced a mixed Th1/Th2 immune response in adult mice and led to significantly increased protection against cerebral infection. Vaccination with O-Ags also resulted in reduced vertical transmission, and postnatal disease in offspring was significantly inhibited at a rate not observed in mice infected with a high-virulence isolate to date. However, O-Ags mixed with TLR ligands targeting TLR3 and TLR7, which are known to induce clear Th1-biased responses, or vaccination with OprI fused to the non-N. caninum antigen ovalbumin (OprI-OVA) did not confer protection.

Delayed type hypersensitivity induced by intradermal inoculation of a Neospora caninum tachyzoite antigen in previously exposed cattle

The aim of this study was to evaluate delayed type hypersensitivity (DTH) induced by the intradermal inoculation of a Neospora caninum tachyzoite soluble lysate in cattle previously exposed with the protozoa. Four experimental groups were selected according to the prior exposure to N. caninum antigen. All cows were intradermally injected with a N. caninum tachyzoite soluble lysate and skinfold thickness growth at the inoculation sites was measured at 0, 24, 48, 72 and 96 h post inoculation (hpi). Additionally, specific antibodies and IFN-γ production were assessed. Cows experimentally infected with live N. caninum tachyzoites and cows naturally exposed to N. caninum developed skin reactions compatible with DTH between 24 and 96 hpi (p < 0.05). Moreover, cows inoculated with an experimental N. caninum vaccine and cows without evidence of exposure to N. caninum did not show a significant increase in skin thickness (p > 0.05). Furthermore, serological status of the animals was not modified due to the intradermal inoculation. The highest IFN-γ production was observed at 15 days after intradermal inoculation (p < 0.05). Therefore, these results suggest that cattle previously exposed to N. caninum develop a reaction compatible with DTH which could be useful as in vivo cell mediated immunity parameter for assessed bovine neosporosis.

Identification of suitable adjuvant for vaccine formulation with the Neospora caninum antigen NcSRS2

The parasite Neospora caninum is the main cause of abortion in cattle in many countries around the world, so a vaccine is a rational approach method for the control of the disease. An effective vaccine should be able to prevent both, the horizontal and vertical transmission of N. caninum. In this study, the immune vaccinal response of the recombinant protein rNcSRS2 of N. caninum expressed in Pichia pastoris and formulated with water-in-oil emulsion, xanthan gum, and alum hydroxide was assessed in an experimental murine model. Groups of 10 Balb/c mice were subcutaneously inoculated with two doses of prNcSRS2 twenty-one days apart. After the second immunization, four mice from each group were euthanized, and splenocytes were stimulated ex vivo with recombinant protein. The IgG dynamics were evaluated by indirect ELISA, and the splenocytes cytokines transcription by qPCR. All groups elicited specific antibodies against prNcSRS2, with the water-in-oil group showing significantly (p ≤ .05) elevated titers compared to the other groups. The prNcSRS2 protein alone did not induce a significant ex vivo splenic transcription level of IFN-γ, TNF-α, IL-4, IL-10, and IL-12 cytokines, except for IL-17A, and the adjuvant associations with the prNcSRS2 protein induced different cytokine transcription profiles. The water-in-oil emulsion modulated the expression of TNF-α; the xanthan gum modulated IL-4, IL-10, and IL-12; and alum hydroxide modulated IFN-γ, TNF-α, IL-4, IL-10, and IL-12. In conclusion, it was found that the association of the recombinant prNcSRS2 protein with different adjuvants induced different levels of specific antibody, and a distinct splenic cytokine profile in an adjuvant-dependent manner. The mechanisms of adjuvancity activity is complex, so adjuvant formulation may help in the design of efficient vaccine to control Neosporosis.

Experimental Neospora caninum infection in chickens (Gallus gallus domesticus) with oocysts and tachyzoites of two recent isolates reveals resistance to infection

The importance of birds in the biological cycle of Neospora caninum is not clear. We report unsuccessful Neospora infection in chickens (Gallus gallus domesticus) using two isolates of N. caninum. In experiment #1, 30 White Leghorn chickens were orally inoculated with viable N. caninum oocysts (NC-SP1 isolate, 200 oocysts per bird) via the crop at 21days of age. Groups of three birds were euthanised at intervals of 7days (a total of 9weeks) and one group was challenged with the same oocyst dose at 37daysp.i. and observed for 11weeks. Blood samples were collected weekly, and sera were tested using IFAT. Chicken tissues were collected for PCR, quantitative PCR and immunohistochemistry. Two dogs approximately 45days of age were fed with tissues from chickens euthanised at 138 and 159daysp.i. The results indicated that the chickens were resistant to neosporosis as revealed by failure to seroconvert, to detect parasite DNA or N. caninum antigen by immunohistochemistry in inoculated bird tissues, and by no oocyst excretion by the dogs fed avian tissues. Similar results were obtained in experiment #2, in which 34 1-week-old chickens were each s.c. inoculated with 100,000 tachyzoites of the NcWTDMn1 isolate of N. caninum. The chickens were euthanised on days 7, 15, 22, 28, 36 and 60p.i. At necropsy, all tissues and serum from each bird were collected. All chickens remained asymptomatic, and N. caninum antigen was not detected by immunohistochemistry. Seven chickens euthanised at day 60p.i. demonstrated low (1:25 dilution) levels of antibodies by using the Neospora agglutination test. Two 12-week-old dogs fed tissues pooled from 10 inoculated chickens euthanised at day 60p.i. did not excrete N. caninum oocysts. This investigation indicates that chickens are resistant to experimental infection by N. caninum.

In vitro screening of the open source Pathogen Box identifies novel compounds with profound activities against Neospora caninum

Neospora caninum is a major cause of abortion in cattle and represents an important veterinary health problem of great economic significance. The Medicines for Malaria Venture (MMV) Pathogen Box, an open-source collection of 400 compounds with proven anti-infective properties against a wide range of pathogens, was screened against a N. caninum beta-galactosidase reporter strain grown in human foreskin fibroblasts. A primary screening carried out at 1µM yielded 40 compounds that were effective against N. caninum tachyzoites. However, 30 of these compounds also affected the viability of the host cells. The 10 remaining compounds exhibited IC50 values between 4 and 43nM. Three compounds with IC50 values below 10nM, namely MMV676602, MMV688762 and MMV671636, were further characterized in vitro in more detail with respect to inhibition of invasion versus intracellular proliferation, and only MMV671636 had an impact on intracellular proliferation of tachyzoites. This was confirmed by transmission electron microscopy, showing that the primary target of MMV671636 was the mitochondrion. MMV671636 treatment of experimentally infected mice significantly reduced the number of animals with lung and brain infection, and these mice also exhibited a significantly reduced titer of antibodies directed against N. caninum antigens. Thus, MMV671636 is a promising starting point for the development of a future neosporosis therapy.

Transduction of a Neospora caninum antigen gene into mammalian cells using a modified Bombyx mori nucleopolyhedrovirus for antibody production

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can easily enter and transduce foreign genes into mammalian cells, but these functions are difficult for Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, we investigated the induction of antibody production in mice immunized with an engineered BmNPV. The GP64 of BmNPV (BmGP64) was replaced with the GP64 of AcMNPV (AcGP64); this construct, designated BmNPVΔbgp/AcGP64, displays AcGP64 on the surface of BmNPV. The Neospora caninum antigen (NcSRS2) expression cassette, consisting of the cytomegalovirus immediate-early promoter and NcSRS2 from N. caninum, was inserted into BmNPVΔbgp/AcGP64; this construct was designated BmNPVΔbgp/AcGP64/SRS2. For comparison, AcMNPV/SRS2, which contains the same NcSRS2 expression cassette as for BmNPVΔbgp/AcGP64, was also constructed. NcSRS2 was expressed in HEK293T cells when the engineered BmNPVs were transduced at a multiplicity of infection of 150. BmNPVΔbgp/AcGP64/SRS2 induced the production of NcSRS2-specific antibodies in mice, whereas AcMNPV/SRS2 and the control BmNPV did not. These results suggest that BmNPV prepared from silkworm hemolymph induces the production of antigen-specific antibodies in immunized mice and can be used for antibody production and vaccine development.

Neospora caninum abortion in a Malayan tapir (Tapirus indicus)

A captive 17-year old female Malayan tapir (Tapirus indicus) aborted a fetus with a crown rump length of 19cm in early pregnancy. The fetus showed an early state of mummification. Histologically, a multifocal mononuclear encephalitis, myocarditis and periportal hepatitis was present indicating a possible protozoal cause of abortion. Although immunohistologically, Neospora (N.) caninum antigen could not be demonstrated, N. caninum DNA was detected by Polymerase Chain Reaction (PCR) in brain, heart, liver and lung of the fetus. N. caninum DNA was extracted from the aborted fetus and the microsatellite marker MS10 was amplified by PCR and sequenced. The obtained MS10 microsatellite pattern has not been described in Germany yet. Nevertheless, the MS10 pattern was very similar to those reported for N. caninum isolated from dogs and cattle in Germany. Because of the histological pattern and extent of the lesions, neosporosis was suspected as the cause of fetal death and abortion. This case report describes for the first time transplacental transmission of N. caninum and abortion due to neosporosis in a tapir.

Mucosal immunization confers long-term protection against intragastrically established Neospora caninum infection

Neospora caninum is an obligate intracellular protozoan parasite responsible for heavy economic losses in dairy and beef cattle farms worldwide. Although vaccination is widely regarded as the preferable strategy to prevent neosporosis no commercial vaccine is currently available. We have previously shown that intranasal immunization with an N. caninum antigen extract enriched in hydrophobic proteins plus CpG adjuvant protected mice against intragastrically established neosporosis. Nevertheless, the antigen specificity as well as the long-term protective effect of this immunization strategy were not determined. Here, we show that the protective effect of this intranasal immunization procedure lasted for at least 20weeks. Protection was accompanied by long-lasting elevated levels of parasite-specific serum IgG and intestinal IgA. Moreover, spleen and mesenteric lymph node cells obtained from non-infected long-term immunized mice responded by producing interferon-γ following in vitro parasite-antigen recall. Analysis of serum IgG and intestinal IgA antibody reactivity in immunized mice identified dense granule antigen 7 (NcGRA7) and microneme associated protein 1 (NcMIC1) as immunodominant antigens respectively recognized by those antibody fractions. In summary, this work shows that a previously reported mucosal immunization strategy against N. caninum infection established through the gastrointestinal tract is effective in the long term.

Maternal and foetal cytokine production in dams naturally and experimentally infected with Neospora caninum on gestation day 110

In the present study, IFN-γ (Th1), IL-17A (Th17) and IL-4 (Th2) concentrations in response to concanavalin (ConA) and Neospora caninum antigen (Nc-1) stimulation were determined in cultures of cells from control uninfected (n=4), naturally N. caninum-infected (n=3) and experimentally N. caninum-infected (n=6) pregnant dams and their foetuses. Experimental animals were infected at 110days of gestation and euthanized 6weeks post-infection. In culture supernatants from the dams, significantly higher IFN-γ and IL-4 levels were found in the experimentally-infected animals compared to the control or naturally-infected dams. However, among the experimentally-infected dams no significant differences in IFN-γ production were observed regardless of the incidence of live or aborted/dead foetuses, though spleen cultures of dams carrying live foetuses showed the highest levels of IFN-γ. IL-17A production was very low and occasional in the dams infected with N. caninum and did not seem to be a major regulator of IFN-γ production in this model. Experimentally infected dams with live foetuses showed higher IL-4 levels and accordingly IFN-γ/IL-4 ratios were significantly lower than ratios recorded for cows with aborted/dead foetuses. In the infected foetuses of these dams, only spleen cultures showed high levels of IFN-γ and IL-4 after Nc-1 antigen and ConA stimulation, respectively. No IL-17A was detected in the foetuses. As conclusion, although we could not clearly relate a protective immune response against N. caninum abortion only to IFN-γ levels in cell cultures, our results highlight the important role of an inverse IFN-γ/IL-4 balance in conferring protection against abortion induced by this parasite.

Prevalence of Antibodies against Neospora caninum in Père David's Deer ( Elaphurus davidianus ) in Beijing, China.

Neospora caninum is an apicomplexan protozoan and causes neuromuscular disorders in canids and abortions in cattle worldwide. We screened sera samples from 49 free-ranging Père David's deer ( Elaphurus davidianus ) in a nature reserve in Beijing, China, for antibodies against N. caninum using indirect fluorescence antibody tests and western blot tests. Antibodies were found in 27% of the deer. Western blot analysis revealed antibody reactivity against immunodominant N. caninum antigens of 16, 25, and 37 kDa in size together with other visible bands.

İran’ın Batısındaki Mandra Büyükbaş Hayvanlarında Neospora caninum Enfeksiyonunun Seroprevalansı

The Neospora caninum parasite causes abortion in cattle in virtually all parts of the world with enormous economic consequences. The purpose of this study was to determine the seroprevalence of antibodies of Neospora caninum in dairy cattle in Lorestan Province, west of Iran. A total of 347 dairy cows were randomly selected. The serum of each case was analyzed for the possibility of the presence of antibody against N. caninum antigen, using the commercial kit: ELISA. The results of the ELISA test indicated that from 347 dairy cattle examined, the antibodies to N. caninum were found in 34 (9.8%). The percentage of seropositive aborted cattle was 13.33%. This study also indicated that there was no significant relationship between seropositivity and such factors as the age, breed, and abortion history of the cattle. Moreover, no significant relationship between seroprevalence of infection among rural and industrial cows was found. The neosporosis could be one of the possible causes of abortion in cattle. Further studies are recommended to determine the relationship between this parasite and the occurrence of abortion in cattle in the province of Lorestan.

Neospora caninum: the First Demonstration of the Enteroepithelial Stages in the Intestines of a Naturally Infected Dog

SummaryA 1.5-month-old Kangal breed puppy from a dairy cattle farm died after showing severe diarrhoea and incoordination. Necropsy examination revealed multifocal pulmonary consolidation and necrosis and fibrinohaemorrhagic enteritis. Microscopically, there was necrotic and purulent bronchopneumonia, myocarditis and non-purulent encephalitis. In the jejunum and ileum there was villous atrophy and crypt hyperplasia with oocyst-like and schizont-like structures in the epithelia. Immunohistochemically, Neospora caninum antigen was detected in association with the intestinal protozoal structures, degenerative neurons and areas of necrosis in the lungs and heart. Polymerase chain reaction confirmed that the organism was N. caninum and not Toxoplasma gondii. The seroprevalence for N. caninum was 74.2% (49/66 animals) for the cattle and 57.1% (4/7 animals) for dogs on this farm. This report documents fatal systemic neosporosis and enteroepithelial stages of N. caninum in a naturally infected puppy. To the authors' knowledge, this is the first definition of intestinal neosporosis in a naturally infected dog as well as the first evidence of fatal canine neosporosis in Turkey.

Evaluation of recombinant Neospora caninum antigens purified from silkworm larvae for the protection of N. caninum infection in mice

Three antigens (NcSAG1, NcSRS2 and NcMIC3) from Neospora caninum were expressed using the BmNPV bacmid system in silkworm larvae and purified from the hemolymph. From 20 silkworm larvae, 1.5, 1.2 and 1.4 mg of purified recombinant NcSAG1, NcSRS2 and NcMIC3 were obtained, respectively. When each purified recombinant antigen was immunized with Freund's incomplete adjuvant (FIA) to mice, recombinant NcSAG1 induced a Th2 immune response in immunized mice and produced a SAG1-specific antibody. In the experiment where NcSAG1-immunized mice were challenged with N. caninum, the cerebral N. caninum burden was significantly reduced compared with that of either the FIA- or PBS-immunized mice. Recombinant NcSRS2 or NcMIC3 induced both Th1 and Th2 immune responses, but NcMIC3-immunization did not induce significant production of NcMIC3-specific antibodies. These results suggest that the silkworm can produce recombinant antigens of N. caninum, which can be used as a recombinant vaccine against N. caninum.