Detection of chicken antibody to the protozoan parasite Eimeria tenella by antigen-coated microspheres.

A sensitive immunoassay for detecting antibodies to the protozoan parasite of poultry Eimeria tenella, has been developed. Microspheres coated with parasite antigen are reacted with antibody-containing chicken serum followed by a fluorescent labeled second antibody and analyzed by flow cytometry. The assay was used to demonstrate that chickens, vaccinated with E. tenella sporozoite antigen, showed significant elevations in serum antibody levels after exposure to infection compared to non-vaccinates. The assay is reproducible and because antigen-coated microspheres are stable for up to 4 weeks at 4 degrees C, a single batch can be used to analyze multiple serum samples.

Trypanosoma cruzi: efficacy of the 2-substituted, 5-nitroimidazoles, MK-436 and L634,549, in tissue culture and mice.

Experimental studies were undertaken in tissue culture and mice infected with a cloned derivative of Trypanosoma cruzi, Y strain to determine the efficacy of two 2-substituted 5-nitroimidazole compounds, MK-436 and L634,549. The use of an X-irradiated myoblast culture system proved better than a conventional fibroblast culture for assaying the activity of compounds against intracellular parasite stages. MK-436 showed activity against amastigotes at a level of 25 micrograms/ml and L634,549 a dihydroxy metabolite of MK-436 showed activity 2 micrograms/ml. Neither compound caused morphological damage to the host cells at levels tested (250 micrograms/ml). By contrast, nifurtimox, which was active at 2 micrograms/ml, caused significant host cell damage at 100 micrograms/ml. In mice, studies in the chronic infection showed that MK-436 was curative at a level of 30 mg/kg if given daily for 20 days. Neither nifurtimox nor benznidazole were fully curative when given at a level of 100 mg/kg daily for 20 days. These studies showed that administration of MK-436 with a suitable solvent, PEG 400, enhanced its efficacy fourfold, and that efficacy was also enhanced by increasing the treatment interval. Since MK-436 showed better efficacy in chronic rodent infections than either nifurtimox or benznidazole, such compounds should be evaluated for efficacy in human Chagas' disease.

An improved parasitological technique for the diagnosis of African trypanosomiasis.

Light microscopic examination of the buffy coat zone of a microhaematocrit capillary tube expressed on to a slide was found to be consistently more reliable than other standard techniques in detecting trypanosomes in the circulation of cattle. This method alaos allowed identification of different trypanosome species. Optimal results were obtained using darkground illumination.

Treatment with suramin and 2-substituted 5-nitroimidazoles of chronic murine Trypanosoma brucei infections with central nervous system involvement.

Mice infected with either of two isolates of Trypanosoma brucei, GVR 23/1 or GVR 35/1, develop a chronic infection in which trypanosomes are localized in the central nervous system. These infected mice were used to evaluate the efficacy of a combination drug treatment comprising suramin and one of three 2-substituted 5-nitroimidazoles. None of the three 5-nitroimidazoles tested alone, cured mice when administered 21 days after infection. However, it was found that T. brucei GVR 23/1 infections could be cured by a single dose of 20 mg/kg suramin followed by a single dose of 80 mg/kg L611,744 [3a,4,5,6,7,8,9,9a-octahydro-3-(1-methyl-5-nitroimidazol-2yl)cycloocta(D) isoxazole]. The single dose of 20 mg/kg suramin had to be followed by four doses of 80 mg/kg L611,744 to cure mice infected with another stabilate, T. brucei GVR 35/1. A single dose of 20 mg/kg suramin followed either by four doses of 250 mg/kg MK 436 [3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5nitro-1H-imidazol-2-yl)-1, 2-benzisoxazole] or four doses of 70 mg/kg of a dihydroxy analogue of MK 436 [cis-3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1, 2-benzisoxazole-6,7-diol] also permanently cured all T. brucei GVR 35/1.

Prospects for molecular vaccines in veterinary parasitology.

Despite the profound developments in recombinant DNA technology there is only one marketed recombinant vaccine (for human viral hepatitis B). The development of others proceeds with great difficulty. Molecular vaccines against veterinary parasites are at the utmost pole of complexity in the spectrum of potential vaccines since these parasites are complex eukaryotic organisms, often dwelling at mucosal surfaces where anamnestic responses are problematic, where the immunogenicity of the parasite components is poorly understood and where the effector mechanisms of immunity are unresolved. Cloning a "protective" gene is only the first step, and perhaps the easiest, in a long process which will be necessary to develop vaccines against parasites. Additional steps will involve comprehensive analyses of the immunological responses to ensure that vaccine antigens contain the correct epitopes to induce appropriate immune effector mechanisms for parasite elimination and immunological memory and that these responses are not genetically restricted. The great expectations for recombinant vaccinia-based vaccines must be modified substantially in the light of recent evidence indicating immunological and other constraints on this approach. The use of anti-idiotype vaccines is an underexplored opportunity for practical parasite vaccines since they have several potentially important advantages. The need to include T cell antigenic peptides in peptide vaccines to extend the range of genetic responsiveness and to induce anamnestic responses is now clear. New algorithms for the prediction of such sites exist and these can be tested experimentally with synthetic peptides. There are no major technical obstacles to the development of vaccines for parasites which cannot be overcome. However substantial long term basic research is needed over a range of disciplines to achieve this worthwhile objective.

Trypanosoma cruzi: selective isolation of pure trypomastigotes from cultured muscle cells.

Trypomastigote or trypomastigote-amastigote populations of Trypanosoma cruzi (Y strain, MERC 2C) entirely free of epimastigotes were obtained from infected muscle cell cultures, and separated from host-cell debris by passage through a DEAE-cellulose column. Approximately 75% of the parasites were recovered and mouse infectivity titrations with postcolumn trypomastigotes showed only a slight reduction in infectivity compared to starting material. Light and electron microscopic examination of material showed a high degree of purity had been achieved by the column procedure. No host-cell debris could be identified in the eluate and parasites were morphologically intact. Enumeration of trypomastigotes and amastigotes in mixed populations before and after column purification showed that there was no preferential loss of either stage and both had the same residence time. This procedure may be used to obtain clean, minimally altered, parasite material free of the invertebrate epimastigote stage and host-cell debris for studies which are sensitive to these contaminants.

Parasite specific antibodies in three strains of mice after infection with Trypanosoma cruzi.

Three inbred strains of mice (BALB/cJ, C3H/HeJ and NZB/BInJ) were infected with trypomastigotes of Trypanosoma cruzi. Sera were taken at different times after infection and radioimmunoprecipitation assays were used to detect antibodies against individual T. cruzi epimastigote and trypomastigote antigens. The mouse strains differed in regard to the spectrum of antibodies and the time after infection when the various epimastigote specific antibody species appeared. NZB mice had antibodies against at least 25 polypeptides ranging in molecular weight from 20,000 to 90,000 D at 3 wk after infection, and these persisted until at least 10 wk post-infection. C3H and BALB/c had antibodies against fewer than 5 antigens at 3 wk after infection; whereas by week 10, antibodies against at least 25 polypeptides were detected. C3H mice that were most susceptible to infection (but not NZB or BALB/c mice) had antibodies against a 25,000 D molecular weight epimastigote antigen. The antibody response against trypomastigote polypeptides was more uniform. Sera from all mouse strains at 3 wk after infection precipitated the same polypeptides and the radioimmunoprecipitation patterns did not change as a function of time after infection.

Clinical signs and humoral immune response in horses following equine herpesvirus type-1 infection and their susceptibility to equine herpesvirus type-4 challenge.

A group of three horses was experimentally infected with equine herpesvirus type 1 (EHV-1) and showed clinical signs characterised by a biphasic febrile response, leucopenia and cell associated viraemia accompanied by virus shedding from the nasopharynx. A second exposure to the virus 18 days later resulted in the isolation of virus from the nasopharynx of one horse. This and a further group of three EHV-1 seropositive horses were subsequently infected with equine herpesvirus type 4 (EHV-4) 147 days after the initial EHV-1 infection and virus was shed from the nasopharynx in the absence of clinical disease. Following the first EHV-1 infection, virus specific immunoglobulin M (IgM) was present by day 5 and remained high until the second exposure at day 18 at which point levels decreased. In contrast, EHV-1 specific IgG, detected at day 6 peaked at day 18, after which time levels remained high. Virus neutralising antibodies and antibodies able to mediate antibody-dependent cellular cytotoxicity were present by day 10. The immune response to EHV-1 is discussed with reference to the disease.

The blood volumes and erythrokinetics of Ndama and Zebu cattle experimentally infected with Trypanosoma brucei.

The responses of susceptible Ndama and Zebu cattle to experimental infection with Trypanosoma brucei were compared using haematological, parasitological and radioisotopic methods. Animals of both breeds became anaemic, but this was more severe in the Zebu cattle, one of which died. Although the prepatent period was the same in animals of both breeds, the levels of the first and subsequent peaks of parasitaemia were higher in the Zebu. The anaemia was due to an accelerated rate of red cell break-down which was more marked in the Zebu cattle. Haemodilution was not a feature. There was no evidence of dyshaemopoiesis but iron reutilisation from degraded erythrocytes was impaired. The greater resistance of the Ndama to T brucei infection could not be attributed to the capacity of this breed to mount a more effective erythropoietic response than the Zebu.

An overview of the roles and structure of international high-security veterinary laboratories for infectious animal diseases.

The unique structure, role and operations of government high-security (HS) laboratories which work on animal diseases are described, with particular reference to the laboratories of nine countries. High-security laboratories provide cost-effective insurance against catastrophic losses which could occur following exotic disease outbreaks. The importance of these laboratories is reflected in the fact that several new laboratories have recently been constructed at considerable expense and older facilities have undergone major renovations. Biosecurity is fundamental to the operation of high-security laboratories, so good facility design and microbiological security practices are very important. High-security laboratories conduct exotic disease diagnosis, certification and surveillance, and also perform research into virology, disease pathogenesis and improvements to diagnostic tests and vaccines. The mandate of these laboratories includes the training of veterinarians in the recognition of exotic diseases. One extremely important role is the provision of expert advice on exotic diseases and participation (both nationally and internationally) in policy decisions regarding animal disease issues.

Immune response and protection of cattle and pigs generated by a vaccinia virus recombinant expressing the F protein of rinderpest virus.

The immune response of cattle and pigs to a vaccinia recombinant virus containing the fusion (F) protein gene of rinderpest virus was examined. Half the cattle and all the pigs gave humoral response to primary vaccination and all the cattle gave an anamnestic response to a second vaccination 28 days after the primary vaccination. All the cattle after a single or secondary vaccination were completely protected clinically after exposure to a lethal dose of the Saudi 1/81 strain of virus. Prior vaccination with another TK- vaccinia recombinant (VVCAT) suppressed, but did not abrogate, the immune response to the rinderpest F recombinant. The pigs gave a humoral immune response in the absence of any local reaction at the site of vaccination.

Vaccines for bluetongue.

Isolation of 8 serotypes of bluetongue virus (BTV) in Australia has led to widespread debate on how to prepare for an outbreak of bluetongue disease and the type of vaccine best suited to control bluetongue in Australia. This article describes the vaccine options under consideration by research workers and animal health administrators. The most widely discussed options are live attenuated virus, killed virus and virus-like particles (VLP) generated by recombinant baculoviruses. Attenuated virus vaccines are cheap and easy to produce and are administered in a single dose. They replicate in sheep without causing significant clinical effects and provide protection against challenge with virulent virus of the same serotype. The possibility that insects could acquire vaccine virus by feeding on vaccinated animals and transmit it to sheep or cattle cannot be eliminated. This poses a risk because attenuated viruses are teratogenic if ewes are infected in the first half of pregnancy. In addition, vaccine virus replication in insects and ruminants may lead to a reversion to virulence. Killed virus vaccines have been shown to be efficacious in small laboratory trials and cannot be transmitted to other animals in the field, but are significantly more expensive to produce than attenuated viruses and require at least 2 doses with adjuvant to elicit an immune response. More work is needed to properly assess their effectiveness and determine their cost of production. Recombinant VLP contain the 4 major structural proteins of BTV but no nucleic acid. VLP are relatively easy to isolate, but it is unlikely that the purification methods currently used in laboratories will be adapted for use commercially. Despite the enthusiasm of recent years, little commercial progress appears to have been made. Although scientific research in Australia and overseas has provided a number of options for development of bluetongue vaccines, the decisions on which to use in an outbreak are complex and will require, not only consideration of factors discussed here, but also agreement from industry and government.

Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats.

OBJECTIVE: To determine the infectivity and transmissibility of Hendra virus (HeV). DESIGN: A disease transmission study using fruit bats, horses and cats. PROCEDURE: Eight grey-headed fruit bats (Pteropus poliocephalus) were inoculated and housed in contact with three uninfected bats and two uninfected horses. In a second experiment, four horses were inoculated by subcutaneous injection and intranasal inoculation and housed in contact with three uninfected horses and six uninfected cats. In a third experiment, 12 cats were inoculated and housed in contact with three uninfected horses. Two surviving horses were inoculated at the conclusion of the third experiment: the first orally and the second by nasal swabbing. All animals were necropsied and examined by gross and microscopic pathological methods, immunoperoxidase to detect viral antigen in formalin-fixed tissues, virus isolation was attempted on tissues and SNT and ELISA methods were used to detect HeV-specific antibody. RESULTS: Clinical disease was not observed in the fruit bats, although six of eight inoculated bats developed antibody against HeV, and two of six developed vascular lesions which contained viral antigen. The in-contact bats and horses did not seroconvert. Three of four horses that were inoculated developed acute disease, but in-contact horses and cats were not infected. In the third experiment, one of three in-contact horses contracted disease. At the time of necropsy, high titres of HeV were detected in the kidneys of six acutely infected horses, in the urine of four horses and the mouth of two, but not in the nasal cavities or tracheas. CONCLUSIONS: Grey-headed fruit bats seroconvert and develop subclinical disease when inoculated with HeV. Horses can be infected by oronasal routes and can excrete HeV in urine and saliva. It is possible to transmit HeV from cats to horses. Transmission from P poliocephalus to horses could not be proven and neither could transmission from horses to horses or horses to cats. Under the experimental conditions of the study the virus is not highly contagious.