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1.
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.  相似文献   

2.
A one-month male Greyhound dog presented with a swinging gait of the hindlimbs, and later developed muscular atrophy of the femoral region and hyperextension of hindlimbs. The dog had positive serum IFAT titers to Neospora caninum, but a negative titer in the cerebrospinal fluid (CSF). N. caninum-specific DNA was amplified from the CSF using a semi-nested polymerase chain reaction assay. Clusters of protozoa in biopsied muscle fibers were subsequently confirmed as N. caninum tachyzoites by immunohistochemical examination. Early recognition and treatment are necessary for effective recovery of clinical canine neosporosis, but antemortem diagnosis is difficult. We suggest that the detection of parasite deoxyribonucleic acid in the CSF is a useful antemortem diagnostic method in facilitating treatment of this disease.  相似文献   

3.
First Israeli Neospora caninum isolates were obtained from brain tissues of aborted fetuses (NcIs491 and NcIs580) from dairy farms endemic for neosporosis and maintaining cattle on zero grazing. Tissues from different parts of the fetus brains were used to infect Vero cells. Tachyzoites of N. caninum were first observed in cultures from days 30 and 32 after infection. To confirm the identity of the isolated parasites, DNA extracts from brains and cultures were tested by PCR with specific primers based on the Nc5 gene. Specific fragments were amplified by PCR from infected cultures of both fetuses on day 25. Susceptible seronegative gerbils (Meriones tristrami) were inoculated intraperitoneally with 10(3) to 10(5) tenfold dilutions of subculture tachyzoites. The inoculated gerbils developed specific antibodies to N. caninum, with end-point serum dilution of 1:4096 in the IFA assay, whereas no neurological signs or deaths were seen during 4 months of observation.  相似文献   

4.
Neospora caninum was isolated from a naturally infected sheep from Brazil by bioassay in dogs. Approximately 70g of brain from each of two 4-month-old sheep with indirect fluorescent antibodies (>or=1:50) to N. caninum was offered to a different IFAT negative dog (Sheep n. 302, IFAT 1:400-Dog 1 and Sheep n. 342, IFAT 1:50-Dog 2). Parasite DNA was detected in both sheep brains using a PCR targeting the Nc-5 gene of N. caninum. Shedding of Neospora-like oocysts was noticed only in Dog 1, from 10 days post-inoculation (PI) to 25 days PI (a total of approximately 27,600 oocysts). Seventy days after infection, Dog 1 was euthanized and brain/cerebellum and medulla were collected and submitted to molecular methods, as were the oocysts, to confirm the identity of the isolate. Serum samples collected weekly from both dogs from the infection to the end of the experimental period had no antibodies anti-N. caninum by IFAT (<1:50). Oocysts, brain/cerebellum and medulla specimens of Dog 1 proved positive by a PCR assay targeting the Nc-5 gene of N. caninum. In addition, the oocysts have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid ITS1 sequence. The PCR products of ITS1 were sequenced, confirming again the isolate as N. caninum. Oocysts were also orally inoculated in two Swiss white mice two Mongolian gerbils (Meriones ungulatus) and two large vesper mice (Calomys callosus) (10(3)oocysts/animal). The rodents were sacrificed 2 months PI, and fresh preparations of brains showed Neospora thick-walled cysts in gerbil brains, but molecular detection using the Nc-5 PCR assay revealed DNA parasite in gerbil and also C. callosus brains. This is the first report of isolation and sequencing of N. caninum from a Brazilian sheep and the first report of molecular detection of N. caninum from C. callosus.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
The role of rodents in the epidemiology of neosporosis was investigated by assaying brain tissue of feral mice (Mus musculus) and rats (Rattus norvegicus) for Neospora caninum. Both mouse and rat brain tissue were extracted for total DNA, and subjected to two different N. caninum-specific nested polymerase chain reaction (PCR) assays. A portion of brain tissue from the mice and rats were also assayed for N. caninum in gerbils or gamma-interferon gene knockout (KO) mice. Of the 105 feral mice tested, 10% were positive in the N. caninum-specific PCR assays. Of the 242 rats tested, 30% were positive in both assays. Although mice and rats had N. caninum by PCR testing, clinical signs of N. caninum infection were not observed nor were N. caninum parasites observed in gerbils or KO mice inoculated with the rodent brain tissue.  相似文献   

9.
In vitro isolation and characterization of bovine Neospora caninum in Korea   总被引:5,自引:0,他引:5  
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.  相似文献   

10.
Neospora caninum is widely distributed in the world and this parasite is one of the major causes of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum and several species of domestic and wild animals are intermediate hosts. Dogs can become infected by the ingestion of tissues containing cysts and then excrete oocysts. It is not yet known whether sporulated oocysts are able to induce a patent infection in dogs, i.e. a shedding of N. caninum oocysts in feces. The objective of this study was to experimentally examine the infection of dogs by sporulated oocysts. The oocysts used in the experiment were obtained by feeding dogs with brain of buffaloes (Bubalus bubalis) positive for anti-N. caninum antibodies by indirect fluorescent antibody test (IFAT ≥200). Oocysts shed by these dogs were confirmed to be N. caninum by molecular methods and by bioassay in gerbils, and sporulated N. caninum oocysts were used for the oral infection of four dogs. The dogs were 8 weeks old and negative for antibodies to N. caninum and Toxoplasma gondii. Dogs 1 and 4 received an inoculum of 10,000 sporulated oocysts each; dog 2 an inoculum of 5000 sporulated oocysts and dog 3 received 1000 sporulated oocysts of N. caninum. The total feces excreted by these dogs were collected and examined daily for a period of 30 days. No oocysts were found in their feces. The dogs were monitored monthly for a 6-month period to observe a possible seroconversion and when this occurred the animals were eliminated from the experiment. Dogs 1 and 4 seroconverted 1 month after the infection with titer, in the IFAT, of 1600 and 800, respectively; the other two dogs presented no seroconvertion during the 6-month period. Dogs 1 and 2 were euthanized 180 days after infection and were examined for the detection of N. caninum in tissues (brain, muscle, lymph node, liver, lung, heart and bone marrow) by immunohistochemistry and PCR with negative results in both techniques. Bioassay in gerbils with brain of these dogs was also performed and again the results were negative. In conclusion, dogs infected with sporulated oocysts of N. caninum were not able to shed oocysts in feces. However, a higher dose of infection stimulated the production of antibodies against N. caninum in the dogs.  相似文献   

11.
The aim of this study was to compare systemic humoral immune responses in pregnant heifers inoculated with Neospora caninum tachyzoites by conjunctival and intravenous routes. Twenty nine heifers separated in three experimental groups were studied: Group 1 (n=10 animals) and Group 2 (n=9 animals) were inoculated with 10(8) of N. caninum tachyzoites by conjunctival and intravenous routes at 5th month of gestation, respectively; Group 3 (n=10 animals) were non-inoculated control animals. An indirect fluorescent antibody test (IFAT) and western immunoblotting (IB) were used to analyze the humoral immune response. All animals from Group 1 developed N. caninum specific antibody responses after conjunctival inoculation recording the highest antibody titer (mean+/-SE: 160+/-49.9) at 6th month of gestation. There were statistical differences between humoral immune responses found in Group 1 and 2 being higher in the second one at 6.5th, 8.5th and 9th months of gestation (P<0.05). Interestingly, all heifers from Group 1 reverted to seronegative status at the end of gestation. No increase in antibody was detected in the uninfected control group. Same pattern of N. caninum antigens was recognized by sera from heifers inoculated by conjunctival route and heifers inoculated by intravenous route. Recognized antigens were 116, 92, 84, 77, 45, 40, 25-26 and 17-18 kDa. The conjunctival instillation of N. caninum tachyzoites in pregnant heifers induces specific systemic antibodies. Further work is needed in order to clarify the consequences of this novel experimental route of infection not only on the fetus but also on the dam.  相似文献   

12.
OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the detection of Toxoplasma gondii and Neospora caninum DNA in canine and feline biological samples. SAMPLE POPULATION; Biological samples from 7 cats with systemic (n = 4) or CNS (3) toxoplasmosis, 6 dogs with neospora- or toxoplasma-associated encephalitis, and 11 animals with nonprotozoal disease. PROCEDURE: Primers for T gondii, N caninum, and the canine ferritin gene (dogs) or feline histone 3.3 gene (cats) were combined in a single PCR assay. The DNA was extracted from paraffin-embedded brain tissue, CSF, or skeletal muscle. The PCR products with positive results were cloned, and sequence identity was confirmed. RESULTS: Of 7 cats and 4 dogs with immunohistochemical or serologic evidence of toxoplasmosis, PCR results were positive for all cats and 3 dogs for T gondii, and positive for T gondii and N caninum for 1 dog. Another dog had negative PCR results for both parasites. Of 2 dogs with immunohistochemical or serologic evidence of neosporosis, PCR results were positive for 1 for N caninum and positive for the other for T gondii. All negative-control samples yielded negative results for T gondii and N caninum on the PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: Standard tests for toxoplasmosis or neosporosis associated with the CNS rely on serologic, histologic, or immunohistochemical analysis and can be difficult to interpret. The multiplex PCR assay with built-in control reactions could be a complementary clinical tool for the antemortem diagnosis of toxoplasmosis or neosporosis associated with the CNS.  相似文献   

13.
Clinical neosporosis was diagnosed in a litter of five pups born to a Beagle bitch from Virginia, USA. Four of the pups developed limb weakness starting at 4 weeks of age. The dogs were suspected to have neosporosis based on clinical signs and empirically treated with Clindamycin (75 mg, oral, twice daily, total 150 mg) starting at 9 weeks of age and the dosage was doubled at 13 weeks of age. Antibodies to Neospora caninum were detected in sera of the dam and pups when first tested serologically at the age of 4 months. The owner donated the pup with the worst clinical signs and the dam for research; both dogs were euthanized. Viable N. caninum was isolated in gamma interferon gene knock out (KO) mice and in cell culture from the pup killed at 137 days of age. Tissue cysts, but no tachyzoites, were found in histological sections of brain and muscles. The isolate was also identified as N. caninum by PCR and sequence analysis and designated NC-9. N. caninum was neither isolated by bioassay in KO mice nor found in histological sections of tissues of the bitch. Clinical signs in the remaining three pups improved considerably after a 6-month treatment with Clindamycin; N. caninum antibody titers were still persistent in these pups at 23 months of age. Results indicate that medication with Clindamycin can improve clinical condition but not eliminate N. caninum infection.  相似文献   

14.
Experimental infections with the NC-1 strain of Neospora caninum were conducted in gerbils (Meriones unguiculatus) to determine their acute responses to experimental intraperitoneal infection. Five groups of five female gerbils were used and they were intraperitoneally infected with 1x10(6), 2x10(6), 3x10(6), 4x10(6) or 5x10(6) tachyzoites. Gerbils in all groups developed clinical signs of neosporosis which consisted of inactivity 4-5 days post-inoculation. Morbidity and mortality were observed in all groups. Grossly there was a clear fibrinous exudate in the abdominal cavity and adhesions of the spleen and pancreas to the stomach in gerbils suffering from acute neosporosis. The LD50 was calculated as 9.3x10(5) tachyzoites per gerbil. The results indicate that gerbils can be used as a suitable model of acute neosporosis. This model can be used to screen candidate treatments and to test the efficacy of vaccines for neosporosis without the need to use histology or PCR to demonstrate treatment efficacy.  相似文献   

15.
Neospora caninum was isolated from the brain of an adult dog in Brazil. Cerebral tissue from the dog was inoculated into Mongolian gerbils. Gerbils were euthanized 3-4 months later and bradyzoite-containing tissue cysts were observed in their brains. N. caninum (designated NC-Bahia) was isolated in cell culture after inoculation with tissue cysts from the gerbils. The identity of the parasite was confirmed by immunohistochemical examination and polymerase chain reaction (PCR). Gerbils may be a useful alternative to immunosuppressed mice for isolation of N. caninum and for production of encysted bradyzoites.  相似文献   

16.
The role of domestic dogs in the epidemiology of Neospora caninum as well as the relationship between N. caninum infection of farm dogs and cattle were demonstrated, however, evidence is scarce regarding the role of wild canids in domestic animal neosporosis. The present study was undertaken to evaluate the role of wild canids in the epidemiology of bovine neosporosis in Israel by analyzing the prevalence of antibodies to N. caninum in wild canids. Sera samples were collected from 114 free ranging wild golden jackals (Canis aureus), 24 red foxes (Vulpes vulpes) and nine wolves (Canis lupus), which were collected in Israel during the years 1999-2004. Of a total of 147 wild canids tested antibodies to N. caninum were only found in two golden jackals with IFAT titers of 1:50, and in one red fox and one wolf with IFAT titer of 1:400. The low seroprevalence found in this study (2.7%) indicated that wild canids probably do not have an important role in the epidemiology of N. caninum in Israel. However, since the diet of different species of wild canids and even diverse populations of the same canid species vary, it is possible that other results might be obtained from specific wild canids populations, which scavenge in the vicinity of infected bovines.  相似文献   

17.
AIMS: To investigate the seroprevalence of Neospora caninum infection in breeder bulls. In addition, the level of agreement of serological methods used was evaluated. METHODS: A total of 285 bulls of different ages and breeds raised for reproductive purposes in Spain were sampled for serum antibody activity to N. caninum. Sera were tested using three validated assays: the indirect fluorescent antibody test (IFAT) and two commercially available enzyme-linked immunosorbent assays (ELISA). RESULTS: Antibodies to N. caninum were found in 13.7% of bulls sampled by at least one of the three validated serological assays. The seroprevalence of neosporosis oscillated between 11.2 and 13.3% depending on the serological technique used. No significant associations (P > 0.05) were observed between N. caninum infection and bull breed or age. The serum antibody levels were predominantly low and close to threshold levels in all positive samples. A very good agreement was found among the serological methods used. CONCLUSIONS: This is the first seroprevalence study of bovine neosporosis in breeder bulls and results showed a moderate presence of N. caninum chronic infections.  相似文献   

18.
Little information is available regarding a delayed type hypersensitivity (DTH) reaction in neosporosis. In this study, we examined the elicitation of a DTH reaction in mice infected with Neospora caninum by inoculation of the footpad with tachyzoite antigens. The footpads of BALB/c mice infected with N. caninum and those of non-infected were injected with either the tachyzoite extract, or paraformaldehyde-fixed tachyzoites. In mice inoculated with N. caninum antigens on day 7 p.i. swelling peaked at 6h after injection of the tachyzoite extract. In mice inoculated on days 14, 28 and 56, swelling was observed between 6 and 72 h afterwards. Mice immunized with the tachyzoite extract plus adjuvant showed peak footpad swelling at 6h post injection, and the swelling had decreased at 24h or later. In contrast, mice injected before infection showed no specific swelling. In sections of footpads injected with the tachyzoite extract, exudate had accumulated at 6h post injection and clusters of infiltrated lymphocytes were observed at 48 h post injection. In mice administered anti-CD4+ cell monoclonal antibodies swelling had decreased at 24h post injection of the extract. These results indicate that mice infected with N. caninum produce a DTH reaction, which is a good indicator of the development of type 1 immune responses.  相似文献   

19.
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.  相似文献   

20.
A 16-week-old, male boxer dog developed multifocal nodular dermatitis followed by rapidly progressive and fatal neuromuscular disease. Protozoal tachyzoites were demonstrated by aspiration and biopsy of dermal lesions. Necropsy and histology revealed necrotising inflammation associated with intralesional protozoal organisms in various organs including the brain, heart, skeletal muscle and skin. Serology suggested active infection with Neospora caninum. Immunohistochemistry provided a definitive diagnosis. Dermatitis is a finding rarely associated with juvenile neosporosis. The possible role of immunosuppression is discussed.  相似文献   

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