Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates. METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent antibody test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice. RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3-7%. CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

牛源犬新孢子虫孕鼠感染模型的建立

为建立牛源犬新孢子虫孕鼠感染模型,深入研究牛源犬新孢子虫对孕鼠的致病作用,本试验以雌性BALB/c小鼠为试验动物,分离Vero细胞中培养的牛源犬新孢子虫速殖子,分不同剂量组腹腔接种雌性BALB/c小鼠后,与雄性BALB/c小鼠合笼,每天观察小鼠临床症状和发病情况,观察主要脏器组织的病理变化,应用PCR方法检测孕鼠脑、肝脏、脾脏等脏器组织及胎盘中犬新孢子虫Nc5基因,并测定孕鼠胎盘湿重和胎盘系数。结果显示,感染模型小鼠的最佳攻虫剂量为10⁵个虫体;感染犬新孢子虫孕鼠先后出现精神不振、共济失调等临床症状,并有不同程度死亡;病理学观察模型小鼠脑、肝脏、脾脏等脏器组织出现充血、出血、肿大等病理变化;在模型小鼠脑、肝脏、脾脏等脏器组织及胎盘中检测到犬新孢子虫Nc5基因;随攻虫天数的增加,模型小鼠胎盘重量和胎鼠重量均不断增加,胎盘系数逐步降低,在第12、14、16天时,模型组与对照组相比,胎盘重量和胎盘系数均差异显著（P < 0.05）。本试验成功建立了牛源犬新孢子虫孕鼠感染模型,为犬新孢子虫致病机制研究奠定了基础。     

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.     

Induced transplacental transmission of Neospora caninum in cattle.

Three Jersey cows were inoculated SC and IM with 26 million Neospora caninum tachyzoites at 129 (cow 1), 126 (cow 2), and 81 (cow 3) days after mating. Cows remained clinically normal for at least 1 month after inoculation of N caninum. Cow 1 was euthanatized 32 days after inoculation because of gangrenous mastitis. Cow 1 had a live fetus with no gross lesions; however, microscopic lesions were seen in the fetus and consisted of severe nonsuppurative necrotizing encephalitis of the cerebral white matter. Neospora caninum was identified in lesions by staining with anti-N caninum serum in an immunohistochemical test, by bioassays in mice, and by inoculation of bovine monocyte cultures with fetal tissue homogenate. Neither N caninum nor lesions were associated with infection with the protozoon identified in tissues of cow 1. Cows 2 and 3 aborted small autolysed fetuses 101 and 74 days, respectively, after inoculation with N caninum; the fetuses and attached placenta were unsuitable for laboratory investigations. Cows 2 and 3 remained clinically normal 4 months after abortion. Results of this study indicated that N caninum can be transmitted transplacentally in cattle.     

Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates.

METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent anti-body test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice.

RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3–7%.

CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.     

Neospora caninum infected the alimentary tract of nude mice and was transmitted to other mice by intraperitoneal inoculation with the intestinal contents

Neospora caninum (BT-2 strain) that originated from the brain of a Holstein calf was serially passaged through 10 generations of BALB/c nude mice by intraperitoneal inoculation. Histological examination of the mice revealed that numerous clusters of tachyzoites appeared in the pancreas, stomach and small intestine as well as in the central nervous system (CNS) and skeletal muscles. Intestinal contents of the infected mice were inoculated intraperitoneally into uninfected nude mice and 3 of the 17 inoculated mice showed clinical signs at post inoculation days 3 to 10. The present experiments demonstrated a proliferation of N. caninum tachyzoites in the mucosa of the alimentary tract and pancreas of the nude mice and the intestinal contents of the mice were infective to other nude mice.     

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.     

Influence of adjuvant and antigen dose on protection induced by an inactivated whole vaccine against Neospora caninum infection in mice

In this study, the protection afforded by a Neospora caninum inactivated vaccine formulated with three different adjuvants (water-in-oil emulsion, aluminum hydroxide with CpG oligodeoxynucleotides and aluminum hydroxide with ginseng extract) and three different parasite doses (10(5), 5 × 10(5) or 10(6) inactivated whole tachyzoites) was evaluated using a mouse model. Mice were immunized subcutaneously twice at three-week intervals with inactivated Nc-Spain 1H tachyzoites and challenged by intraperitoneal inoculation with 10(6) live Nc-1 tachyzoites. The efficacy of the immunization was evaluated in non-pregnant BALB/c mice on days 1 and 5 (acute infection phase) and days 14 and 30 (chronic infection phase) post-challenge. The results showed the ability of water-in-oil emulsion combined with inactivated 5 × 10(5) tachyzoites to induce protection against neosporosis during the chronic stage, limiting parasite multiplication in the brain. Aluminum hydroxide-ginseng extract and inactivated tachyzoites reduced the number of parasites circulating in the blood during acute phase but failed to limit the establishment of chronic infection. On the other hand, a dose-effect was observed in groups vaccinated with aluminum hydroxide-ginseng extract in which the lesion severity increased as the inactivated tachyzoite dose. This study demonstrates that efficacy can significantly vary depending on the adjuvant, the dose of antigen and the phase of N. caninum infection in which the vaccine is tested.     

Possibility of Neospora caninum infection by venereal transmission in CB-17 scid mice

CB-17 scid and BALB/c male mice were inoculated intraperitoneally with Neospora caninum to examine the possibility of its venereal transmission. Some of these mice were killed on days 7 and 20 post-inoculation to examine the genital organs for presence of the parasite. The remaining scid male mice were housed with non-infected female mice from day 7 p.i. and kept with them for 14 days. These scid mice died between days 28 and 35 p.i. N. caninum DNA was detected in the testis of mice on days 7 and 20 p.i. by PCR and tachyzoite viability was determined by bioassay conducted by means of mouse inoculation. Microscopically, fewer tachyzoites were detected in the testis obtained on day 20 p.i., than in other organs. The inoculated BALB/c male mice survived until the end of the experiment with no clinical signs and N. caninum DNA was detected in the testis on day 7 p.i. but not on day 14 p.i. Five of eight female scid mice housed with infected males became pregnant. Tachyzoites were detected in three of these mice and their neonates (n=3, 5 and 13, respectively). In three non-pregnant mice, no parasite was detected. Two of the four female BALB/c mice housed with infected male scid mice became pregnant but the parasite was not detected in them or in the neonates (n=3 and 13, respectively). These results indicate that the tachyzoites were present in the genital organs of the immunodeficient mice from day 7 p.i. and suggest that transmission may occur through mating with male mice.     

Characterisation of the first Australian isolate of Neospora caninum from cattle

OBJECTIVE: To isolate Neospora caninum from a congenitally infected calf. PROCEDURE: A calf was obtained from a N. caninum infected dam maintained in a dairy herd of Holstein-Friesian cattle located on the south coast of NSW near Nowra. The calf was euthanased and samples collected for serology and pathology. Samples of brain and spinal cord of the calf were homogenised and injected into immunocompromised mice in an attempt to recover protozoa by in vivo culture. Sequential passage of brain homogenate through IFNgammaPKO mice was performed and tissue culture flasks were inoculated with brain homogenate. Parasites were identified by electron microscopy and DNA sequencing. The antigen profile of the isolate was analysed using Western blotting. Pathogenicity was examined in BALB/c mice and transmission of the parasite during pregnancy was examined in Qs mice. RESULTS: The calf was seropositive for N. caninum and histopathological examination of sections of cerebrum identified lesions consistent with a very mild infection with N. caninum. The parasites isolated using tissue culture were identified as N. caninum, based on the sequence of the ribosomal DNA and electron microscopy. The antigen profile of the new isolate was similar to that of the NC-Liverpool isolate, but quite different from that of Toxoplasma gondii. In BALB/c mice inoculated with the new isolate, severe clinical signs developed in only three of ten infected mice, compared with six of ten mice infected with NC-Liverpool. Mild to moderate nonsuppurative encephalitis was observed in BALB/c mice infected with the new isolate, compared with mice infected with NC-Liverpool, that developed severe nonsuppurative encephalitis. Transplacental transmission of the isolate arising from an acute infection during pregnancy occurred in about 87% of pups. CONCLUSION: This is the first isolation of bovine Neospora caninum in Australia. This isolate, called NC-Nowra, appears to be a less virulent form and may prove to be a suitable candidate for vaccine development.     

Neospora caninum: adoptive transfer of immune lymphocytes precipitates disease in BALB/c mice

Neospora caninum is a recently described apicomplexan parasite first isolated from a dog in 1988 and has subsequently been shown to infect a wide range of mammals. In mice, Neospora can cause primary pneumonia, myositis, encephalitis, radiculoneuritis, and pancreatitis. Whereas, certain aspects of the host immune response to Toxoplasma gondii have been well studied, not as much is known about the full immune response to Neospora. This paper examines whether or not immune splenocytes are able to adoptively transfer protection against N. caninum infection in BALB/c mice. Mice receiving immune enriched CD8+ cells had severe neurological signs by 19 days post infection. Mice receiving immune enriched CD4+ cells had mild neurological signs on day 22 post infection. It would appear that additional immune cells can precipitate disease in the presence of circulating lymphocytes.     

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.     

Reduced cerebral infection of Neospora caninum in BALB/c mice vaccinated with recombinant Brucella abortus RB51 strains expressing N. caninum SRS2 and GRA7 proteins

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-gamma, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.     

Footpad reaction induced by Neospora caninum tachyzoite extract in infected BALB/c mice

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.     

Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission

Neospora caninum infection was diagnosed in 5 young dogs from 2 litters with a common parentage. The pups were born healthy, but developed hind limb paresis 5 to 8 weeks after birth. The predominant lesions were polyradiculoneuritis and granulomatous polymyositis. Neospora caninum was seen microscopically in sections of naturally infected pups, and was isolated in cell cultures, mice, and dogs inoculated with infected canine tissues. Antibodies to N caninum were detected in sera of infected dogs by indirect fluorescent antibody test.     

Pathological and immunological findings of athymic nude and congenic wild type BALB/c mice experimentally infected with Neospora caninum.

Neospora is a cyst-forming coccidian parasite that causes abortions and neuromuscular disorders in a wide variety of mammals. Japanese bovine isolate JPA1 was inoculated intraperitoneally into BALB/c nu/ nu (athymic nude) and BALB/c (congenic wild type) female mice to examine the distribution of parasites and resistance mechanisms to Neospora infection. All the athymic nude mice died within 28 days after intraperitoneal injection of 2 x 10(5) JPA1 tachyzoites, whereas all the congenic wild type mice survived without exhibiting any clinical signs. Tachyzoites were identified in the uterus and pancreas and later spread to many other organs. Most tachyzoites identified in the necrotic foci were localized in the epithelium of the venules and capillaries. Nude mice developed high level of serum interferon-gamma and interleukin-6 as infection proceeded. Inflammatory response to Neospora infection might be mediated by Th1-type dependent cellular immunity.     

Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms

The coccidian parasite Neospora caninum is an intracellular protozoan, causing abortion in cattle in many countries around the world. In this study, the protective potential of the major N. caninum surface antigen NcSRS2, expressed in Escherichia coli and formulated into immunostimulating complexes (iscoms), was investigated in an experimental mouse model. The recombinant protein was specially designed for binding to iscoms via biotin-streptavidin interaction. Two groups of 10 BALB/c mice were immunised twice, on days 0 and 28 with iscoms containing either the recombinant NcSRS2 (NcSRS2 iscoms) or similar iscoms with NcSRS2 substituted by an unrelated recombinant malaria peptide (M5) as a control (M5 iscoms). A third group of 10 age-matched BALB/c mice served as an uninfected control group. Immunisation with recombinant NcSRS2 iscoms resulted in production of substantial antibody titres against N. caninum antigen, while the mice immunised with M5 iscoms produced only very low levels of antibodies reacting with N. caninum antigen. After challenge infection with N. caninum tachyzoites on day 69, mice immunised with NcSRS2 iscoms showed only mild and transient symptoms, whereas the group immunised with M5 iscoms showed clinical symptoms until the end of the experiment at 31 days post inoculation. A competitive PCR assay detecting Nc5-repeats was applied to evaluate the level of parasite DNA in the brain. The amount of Nc5-repeats in the group vaccinated with NcSRS2 iscoms was significantly lower than in the control group given M5 iscoms. In conclusion, it was found that the recombinant NcSRS2 iscoms induced specific antibodies to native NcSRS2 and immunity sufficient to reduce the proliferation of N. caninum in the brains of immunised mice.     

Occurrence of Neospora caninum antibodies in sera from dogs of the city of São Paulo,Brazil

Neospora caninum is an important cause of abortion in dairy cattle worldwide. Dogs are important in the epidemiology of this parasite because they are the only hosts known to excrete N. caninum oocysts. In order to understand the prevalence of N. caninum in dogs, sera from 500 owned dogs and from over 600 feral street dogs from the city of S?o Paulo, Brazil were assayed for antibodies to N. caninum. Sera were examined by the Neospora agglutination test (NAT) using mouse-derived tachyzoites. Antibodies (> or =1:25) to N. caninum were found in nearly 10% (49/500) of owned dogs and in 25% (151/611) of stray dogs. NAT titers for owned dogs were 1:25 in 28 (5.6%) dogs, 1:50 in 20 (4%) dogs, and > or =1:500 in 1 (0.2%) dog. NAT titers for stray dogs were 1:25 in 79 (12.9%) dogs, 1:50 in 68 (11.1%) dogs, and > or =1:500 in 4 (0.6%) dogs. These data indicate that feral dogs may be important in the epidemiology of N. caninum infection.     

Neosporosis in dogs

A bitch was inoculated subcutaneously and intramuscularly with Neospora caninum tachyzoites on Day 35 of pregnancy. Eight pups were born 28 days later. Five pups became ill and necropsies were performed before 20 days of age. Three pups and the bitch remained clinically normal for 7 weeks after parturition when they were intramuscularly injected with 40 mg kg-1 methylprednisolone acetate weekly to activate chronic N. caninum infection. Necropsies were performed 48, 17, 18, and 18 days respectively after administration of corticosteroids. Hepatic necrosis, pneumonia, encephalomyelitis, and myonecrosis were the main changes seen in these dogs.