Susan Liddell

Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses.

Neospora caninum has been identified as a major cause of abortion in cattle in a number of countries throughout the world. Until the recent demonstration that dogs can serve as a definitive host of this parasite, it was not possible to study the infection in cattle orally exposed to oocysts. The aim of this study was to investigate the potential of N. caninum oocysts to infect calves, and to define initial immune responses that arise after oral infection. Seven calves were fed approximately 10(4)-10(5) N. caninum oocysts, three calves served as uninfected controls. Before infection, all calves were serologically negative for anti-Neospora antibodies and the calves were non-reactive to Neospora antigen in an in vitro lymphocyte proliferation assay. Peripheral blood lymphocytes from inoculated calves were able to mount in vitro proliferative responses to crude N. caninum antigen extract as early as 1 week p.i. Within 2 and 4 weeks p.i., Neospora-specific IgG1 and IgG2 antibodies were detected by IFAT and ELISA in serum from infected calves but not from sham-infected calves. The continued presence of reactive cells in the blood, spleen and mesenteric, inguinal, bronchial lymph nodes was seen as late as 2.5 months p.i., and parasite DNA was detected in the brain and spinal cord of the infected animals by PCR, indicating that the cattle were infected by oral inoculation of N. caninum oocysts collected from dogs, and that the animals were systematically sensitised by parasite antigen.

Molecular cloning and characterisation of a putative aspartate proteinase associated with a gut membrane protein complex from adult Haemonchus contortus.

A cDNA was isolated from an adult Haemonchus contortus cDNA expression library the deduced amino acid sequence of which showed significant homology to mammalian pepsinogen sequences. The library was screened with antisera raised against Haemonchus galactose-containing glycoprotein complex, a gut membrane protein complex with aspartyl proteinase activity which has shown considerable potential as a protective antigen. The amino acid sequence obtained corresponded very closely in part to the N-terminal amino acid sequences of two polypetides within the complex. The enzyme was shown to be almost exclusively expressed by the blood-feeding parasite stages. The cDNA was expressed in E. coli, and antibody produced to the recombinant protein bound to the luminal surface of the gut in the adult parasite. The proteinase may play a central role in digesting the blood meal and is considered a potential sub-unit vaccine candidate.

Canine neosporosis: clinical signs, diagnosis, treatment and isolation of Neospora caninum in mice and cell culture

Clinical signs, diagnosis, treatment and isolation of Neospora caninum from two littermate dogs are described. Three of six pups from a Labrador bitch developed paralysis. Neosporosis was diagnosed ante mortem by serological examination in two of the affected pups. At necropsy, tissue cysts were seen in unstained smears and in histologic sections of their brains. Tissue cysts were often thin-walled (approximately 1 micron) but antigenically and ultrastructurally identified as N. caninum. Furthermore, N. caninum (isolates NC-4, NC-5) was isolated in mice and in cell cultures inoculated with neural tissues of these two dogs. Serological diagnosis of neosporosis using a variety of tests is discussed.

Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination.

Neosporosis is an important cause of abortion and neonatal morbidity in dairy cattle. The disease is caused by Neospora caninum, an intracellular protozoan parasite. In this report, we describe the use of a mouse model in the preliminary evaluation of vaccination as a means to prevent vertical transfer of N. caninum. Parasites present in the tissues of the offspring were detected using an N. caninum-specific polymerase chain reaction assay. Immunization of dams with a single inoculation of a crude lysate of N. caninum tachyzoites appeared to induce complete protection against infection of the offspring.

A competitive PCR assay for quantitative detection of Neospora caninum.

A quantitative-competitive PCR (QC-PCR) assay was developed for measurement of Neospora caninum levels in the tissues of infected animals. A molecule was synthesised for use in PCR as a competitor to the target Neospora-specific Nc5 genomic sequence. The assay was used to evaluate the relative level of parasites in the brain and lungs of mouse pups in a model of vertical transmission of N. caninum. Infection on day 11 of gestation resulted in similar levels of parasites in all offspring. The assay should be useful in evaluation of vaccines against Neospora infection. Incorporation of the competitor molecule in the detection assay also provides a control for PCR failure and facilitates identification of truly negative samples.

CHARACTERIZATION OF THE OREGON ISOLATE OF NEOSPORA HUGHESI FROM A HORSE

Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (≥1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical signs, and Neospora organisms were not demonstrable in their tissues. Tissue cysts were not found in gerbils, nude mice, KO mice, immunosuppressed outbred Swiss Webster mice, or BALB/c mice injected with the Oregon isolate of N. hughesi. Ultrastructurally, tachyzoites of the Oregon isolate from the myocardium of infected KO mice and from cell culture were similar to N. caninum tachyzoites. Western blot analysis using NcSAG1 and NcSRS2 polyclonal and monoclonal antibodies and characterization of the internal transcribed spacer 1 sequences from the equine isolates and different isolates of N. caninum from dogs and cattle indicated that the Oregon isolate of N. hughesi is distinct from N. caninum isolates from cattle and dogs.

Immunization of mice with plasmid DNA coding for NcGRA7 or NcsHSP33 confers partial protection against vertical transmission of Neospora caninum

The purpose of the present study was to use direct plasmid deoxyribonucleic acid (DNA) injection to identify specific antigens that confer protection against congenital transfer of Neospora caninum. Inbred BALB/c mice were vaccinated before pregnancy with a recombinant plasmid containing sequences encoding N. caninum antigen NcGRA7 or NcsHSP33. The mice were challenged with N. caninum tachyzoites at 10–12 days of gestation. Whereas 100% of pups born from dams immunized with control plasmid contained detectable levels of N. caninum DNA in a Neospora-specific polymerase chain reaction assay, only 46% of pups from pCMVi-NcGRA7–immunized mice and 53% of pCMVi-NcsHSP33–immunized mice were N. caninum positive, and none of the mice immunized with tachyzoite extract contained N. caninum DNA. Thus, immunization of mice with plasmid DNA expressing N. caninum antigens conferred partial protection against congenital neosporosis.

Vertical transmission of Neospora caninum in BALB/c mice determined by polymerase chain reaction detection.

Vertical transmission of Neospora caninum was evaluated in BALB/c mice using an N. caninum-specific polymerase chain reaction (PCR) assay as a means of detecting parasite transmission to offspring. BALB/c mice were infected with the NC-1 isolate of N. caninum during pregnancy (days 8-15 gestation). Transmission of parasite, detected by PCR, was determined in 2- to 23-day-old offspring. When dams were infected on days 13-15 of gestation, transfer of parasites was detected in only a proportion of the litter. Infection between days 8 and 12 of gestation resulted in a high frequency of parasite transmission; every offspring from all litters was infected. The tissue locations of parasites in pups of different ages were determined. In young pups (2- to 4-days-old), the predominant sites of infection were the lungs and the brain. In older pups (7- and 23-days-old) the predominant site of infection was the brain. This study shows that PCR may be useful for evaluation of candidate vaccines against horizontal N. caninum infection, vertical transmission, or both.

Specific detection of Neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction.

Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated, and genomic DNA was extracted. A polymerase cahin reaction (PCR) targeting the N. caninum-specific Nc 5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of PCR-amplified competitor to that of oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts and to assess the sensitivity of the assay. The specificity of the assay was determined using the Nc 5–specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidians Hammondia heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces, such as Hammondia hammondi, Toxoplasma gondii, or Eimeria tenella, were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.

EXPERIMENTAL INFECTION OF SHEEP WITH NEOSPORA CANINUM OOCYSTS

The purpose of the present study was to investigate the potential of Neospora caninum oocysts to infect sheep and determine whether N. caninum DNA could be detected by polymerase chain reaction (PCR) assay in blood and brain of sheep after oocyst inoculation. Six ewes were inoculated per os with 104 N. caninum oocysts, whereas 2 ewes served as uninoculated controls. All sheep were bled weekly for 7 wk after inoculation. Blood was analyzed for the presence of N. caninum DNA by 2 different PCR assays, as well as for the presence of antibodies to recombinant and native N. caninum antigens. Neospora caninum DNA was detected in 2 sheep as early as 7 days after oocyst inoculation (DAOI). All 6 sheep were PCR positive by 32 days and remained positive until the end of the study at 49 DAOI. Aside from 1 ewe, all sheep inoculated with N. caninum oocysts contained detectable N. caninum DNA in the brain tissue collected at 49 DAOI. Unlike with PCR, no lesion or parasite was detected by immunohistochemistry. Antibodies were detected by enzyme-linked immunosorbent assay, Neospora agglutination test, or immunoblotting to either native or recombinant N. caninum antigens in sheep inoculated with oocysts.