New generation vaccines and delivery systems for control of bovine tuberculosis in cattle and wildlife

Advances in the understanding of protective immune responses to tuberculosis are providing opportunities for the rational development of improved vaccines for bovine tuberculosis. Protection requires activation of macrophages through stimulation of a Th 1 type immune response. Ideally, a vaccine for cattle should induce protection without causing animals to react in a tuberculin test when exposed to Mycobacterium bovis. A number of new tuberculosis vaccines including attenuated M. bovis strains, killed mycobacteria, protein and DNA vaccines have been developed and many of these are being assessed in cattle. The requirements for a tuberculosis vaccine for wildlife differ from those for cattle. The major goal of a wildlife vaccine is to prevent the transmission of M. bovis to cattle and other wildlife. Although there are a number of technical problems associated with the development of a vaccine delivery system for wildlife, attenuated M. bovis vaccines administered via oral baits or aerosol spray to possums have already been shown to reduce the severity of a subsequent M. bovis infection.     

Effects of time and concentration on mortality of phosphine-resistant Sitophilus oryzae (L) fumigated with phosphine

The effects of exposure period and phosphine concentration on mortality of susceptible and resistant Sitophilus oryzae (L) were investigated. Although S oryzae is one of the world's most serious pests of stored grain there are few data on the practical significance of phosphine resistance in this species. The strains investigated were an Australian susceptible strain, a homozygous resistant strain exhibiting a level of resistance common in Australia and an unselected field strain from China with a much stronger resistance. Fumigations were carried out at 25 degrees C on adults and mixed-age cultures. For adults of all three strains and mixed-age cultures of the susceptible and resistant Australian strains, the relationship between concentration and time could be described by equations of the form Cnt = k. In all cases n < 1, indicating that time was a more important variable than concentration. In all fumigations of adults the resistant strains were harder to kill than the susceptible strain. However, in fumigations of mixed-age cultures, which contained the tolerant pupal stage, the difference between susceptible and resistant strains was more pronounced at lower concentrations than higher concentrations. For example, at 0.02 mg litre-1 the estimated LT99.9 for mixed-age cultures of the Australian resistant strain (27 days) is 3.4 times that of the susceptible strain (8 days), but at 1 mg litre-1 there is no difference between the two strains (4 days). Limited data on the Chinese resistant strain supported this finding. Twenty-three days exposure at 0.02 mg litre-1 had no effect on mixed-age cultures of this strain, but there were no survivors after 5 days exposure to 1 mg litre-1.     

Use of a circular external skeletal fixator for stifle arthrodesis in a dog

Arthrodesis of the right stifle was performed in a 21 kg German shepherd dog that had sustained a comminuted fracture of the lateral femoral condyle as a result of a gunshot wound. The arthrodesis was stabilised with a circular external skeletal fixator. A simplified technique to facilitate creation of the osteotomies of the femur and tibia, and positioning of the stifle at the desired angle, is described. Compression of the subchondral bone surfaces of the distal femur and proximal tibia was achieved using tensioned coiled transarticular Kirschner wires. Arthrodesis of the stifle was confirmed radiographically 21 weeks following surgery, and the fixator was removed. After union of the arthrodesis and removal of the fixator, the dog was fully weightbearing when standing or walking, and intermittently weightbearing or non-weightbearing at faster gaits.     

Characterisation of monoclonal antibodies to ovine interleukin-6 and the development of a sensitive capture ELISA

A purified recombinant ovine (rOv) interleukin-6 (IL-6) was used to generate specific murine monoclonal antibodies (mAbs) and a polyclonal rabbit antisera to this cytokine. From the 31 initial hybridoma cell lines generated, three stable clones were established which secreted mAbs to rOvIL-6, as judged by a direct enzyme-linked immunosorbent assay (ELISA) and Western blotting. Their specificity was further confirmed by demonstrating that none of the mAbs recognised any of the six other irrelevant recombinant ovine cytokines tested by direct ELISA. All three mAbs displayed cross-reactivity with human and African green monkey IL-6 as demonstrated by direct ELISA and Western blotting. In contrast, the polyclonal antibodies only cross-reacted with bovine IL-6 and not with either of the human or monkey homologues. By combining a mAb with the polyclonal antisera a sensitive, IL-6-specific, capture ELISA was developed that had a sensitivity of 150 pg/ml. This detection system was unequivocally validated by demonstrating that native OvIL-6 could be detected in efferent lymph draining from a stimulated popliteal lymph node. In addition, one of the mAbs was shown to allow the detection of OvIL-6 by intracellular cytokine staining and flow cytometry.     

The pathology of spontaneous paratuberculosis in the North American bison (Bison bison)

Gross and histopathologic examinations were performed on 70 North American bison (Bison bison) from a Mycobacterium avium paratuberculosis culture-positive herd. The bison examined were part of a breeding herd totaling 2,800 animals. Eight of 70 (11%) animals had gross findings of intestinal mucosal thickening, and 16 of 70 (23%) of the animals had enlarged mesenteric lymph nodes. Histologic lesions compatible with Johne's disease were diagnosed in 30 of 70 (43%) bison on the basis of the demonstration of noncaseating granulomatous inflammatory infiltrates and of one or more acid-fast bacilli characteristic of Mycobacterium avium paratuberculosis. A suspicious diagnosis of Johne's disease was obtained in 11 of 70 (16%) bison on the basis of the observation of noncaseating granulomatous inflammatory infiltrates without demonstrable acid-fast bacteria. Twenty-nine of 70 (41%) animals were assessed as histologically paratuberculosis free. Histologic results were compared to Johne's disease tests such as culture, serology, and polymerase chain reaction, which were performed on some of the cohort animals.     

Equine conjunctival pseudotumors

Five horses presented with unilateral pink, smooth, nonulcerated conjunctival masses with histologic features characteristic of inflammatory pseudotumors, i.e. proliferative inflammatory lesions clinically resembling true neoplasia. Although causes for the inflammatory lesions were not determined, based on the presence histologically of mononuclear (predominantly lymphocytic) inflammatory cell infiltrates and the absence of infectious agents, parasites or foreign bodies, an immune-mediated pathogenesis was suspected. Affected horses ranged from 5 to 8 years of age with no apparent breed or sex predilection. Conjunctival lesions were nodular in two cases and relatively flat and more diffuse in three cases. Third eyelid lesions were present in three cases and two affected eyes had corneal involvement. Based on findings from these five cases, the prognosis for equine conjunctival pseudotumors appears to be good when lesions are treated by partial or complete surgical excision, local administration of anti-inflammatory agents, or a combination of surgery and anti-inflammatory therapy.     

Goat paratuberculosis in Chile: first isolation and confirmation of Mycobacterium avium subspecies paratuberculosis infection in a dairy goat.

In October 2004, 41 goats > 2 years old from a Saanen dairy goat herd located in Purranque County, 10th Region, Chile, were sampled and tested for paratuberculosis. While collecting samples it was observed that several goats were thin and emaciated. One goat was sufficiently debilitated to warrant humane euthanasia. This animal was brought to the Veterinary School at the Universidad Austral de Chile for necropsy. The goat selected for necropsy was a 12-year-old doe. The animal showed classical clinical signs of caprine paratuberculosis: emaciation despite willingness to eat, dry and rough hair coat, and no evidence of diarrhea. Gross pathology and histopathology of the necropsied goat were consistent with paucibacillary paratuberculosis. Bacteriology, serology, and PCR confirmed the diagnosis. This is the first published report of goat paratuberculosis in Chile confirming a case of caprine paucibacillary paratuberculosis.     

Diagnosis of paratuberculosis by fecal culture and ELISA on milk and serum samples in two types of Chilean dairy goat herds.

Fecal culture has been the primary method used to diagnose paratuberculosis in goats. It is laborious, slow, and expensive. Validation of enzyme-linked immunosorbent assays (ELISAs) on milk samples could make paratuberculosis testing more widely available for goat farmers. The aim of this study was to determine the accuracy of serum and milk ELISAs for paratuberculosis, relative to fecal culture, in Chilean dairy goats. Eight dairy goat herds were selected. Feces, blood, and milk samples were collected from all female goats >2 years old. Fecal samples were cultured using Herrold egg yolk medium with mycobactin J and antibiotics. Serum and milk samples were tested using a commercial ELISA kit for Mycobacterium avium subsp. paratuberculosis antibody detection. A total of 383 goats were tested by ELISA and fecal culture. The sensitivity of ELISA on serum and milk relative to fecal culture was 74.3% (95% CI: 59.8-88.8) and 60% (95% CI: 43.8-76.2), respectively. The corresponding values for ELISA specificity based on the percentage of non- M. avium subsp. paratuberculosis-infected goats testing ELISA-negative were 98.6% (95% CI: 96.6-100) and 99.3% (95% CI: 97.9-100) on serum and milk, respectively. Proportions of positive results for serum and fecal samples were significantly different, whereas the proportions of positive results for milk and fecal samples were not significantly different. The milk ELISA had a moderate level of agreement with fecal culture results (Kappa = 0.57). The paratuberculosis ELISA on goat milk samples may be a cost-effective, accurate alternative to fecal culture.