Specificity of an immunochromatographic test for anthrax.

OBJECTIVE: To evaluate the specificity of an immunochromatographic test (ICT) for anthrax in cattle. DESIGN: A comparison of an ICT with blood smear and culture in uninfected cattle. PROCEDURE: Two hundred and forty blood samples were collected from dead cattle at two knackeries within Victoria and tested on-site with an ICT for the detection of protective antigen (PA) of Bacillus anthracis. Blood smears were prepared on-site and blood samples transported to the laboratory for aerobic and anaerobic culture. The results of the ICT were compared with blood smear and culture. Animals were regarded as not infected with B anthracis if the organism was not detected in a stained blood smear or on culture. Ten healthy yearling cattle were vaccinated with live spore anthrax vaccine and blood samples collected on days 0 to 7 and day 15 were tested in the ICT for the presence of PA. RESULTS: All blood samples from the 240 knackery cattle were ICT, smear and culture negative. All blood samples from the 10 vaccinated cattle were ICT negative. CONCLUSION: The ICT is a test with high specificity in cattle (98.5 to 100%; 95% CI) and recent vaccination of cattle does not give rise to positive reactions.

Molecular characterisation of Victorian Newcastle disease virus isolates from 1976 to 1999.

OBJECTIVE: To characterise Newcastle disease virus isolates obtained in Victoria from 1976 to 1999 and identify the diversity of FO cleavage signal. DESIGN: RT-PCR using viral RNA extracted from positive NDV allantoic fluid was performed to amplify a segment of the NDV F and HN genes. Molecular characterisation of the nucleotide and amino acid sequences within the FO cleavage site was undertaken. RESULTS: All isolates contained 'avirulent FO cleavage signal sequence of varied amino acid composition. CONCLUSIONS: Molecular characterisation of past and present NDV FO cleavage signal sequences will provide valuable epidemiological information and assist in understanding the genetic origins and relationships of outbreaks.

An estimate of specificity for a Johne's disease absorbed ELISA in northern Australian cattle.

OBJECTIVE: To estimate the specificity of an absorbed enzyme-linked immunosorbent assay kit for Johne's disease (JD) when used in mature cattle populations resident in northern Australia. DESIGN: Blood samples were collected from beef cattle in northern Queensland, the Northern Territory and northern Western Australia, and from dairy cattle in northern Queensland. The specificity of a serological test for JD was estimated by testing the blood samples with an absorbed ELISA kit. Further samples were collected from cattle with positive ELISA results to determine the presence or absence of infection with Mycobacterium avium subsp paratuberculosis. PROCEDURE: During 1995 and 1996, blood, tissue and gut contents were collected from beef cattle at abattoirs in Queensland and the Northern Territory; and blood and faecal samples were collected from dairy cattle in herds assessed to be most at risk for JD in northern Queensland. The blood samples were tested using an absorbed ELISA kit. Tissues and gut contents from beef cattle that had positive ELISA results were cultured for M. avium subsp paratuberculosis, and tissues were examined histologically. Faecal samples from dairy cattle with positive ELISA results were cultured for M. avium subsp paratuberculosis. RESULTS: Estimates of specificity for this absorbed ELISA in mature northern Australian cattle were 98.0% (97.0 to 98.8%, 95% CI) in beef cattle, and 98.3% (96.7 to 99.3%, 95% CI) in dairy cattle. CONCLUSION: Estimates of specificity in this study were lower for beef cattle from the Northern Territory and northern Western Australia and for dairy cattle from northern Queensland than those quoted from studies on cattle in southern Western Australia. This should be considered when serological testing using the JD ELISA is carried out on northern Australian cattle.

Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis.

OBJECTIVE: To determine the sensitivity and specificity of an absorbed ELISA and an AGID test for the detection of clinical and subclinical paratuberculosis in sheep. DESIGN: By testing a panel of sera from 1257 Australian Merino and crossbred sheep greater than 1 year of age, of which 1137 sheep were not infected with Mycobacterium avium subsp paratuberculosis and 120 sheep had paratuberculosis. PROCEDURE: Sera were collected from 457 sheep in Victoria and 800 sheep in Western Australia. Presence of M a paratuberculosis infection in Victorian sheep was determined by histological examination of intestinal tissues, whereas sheep from Western Australia were presumed to be free of Johne's disease. The ability of an absorbed ELISA to discriminate between infected and uninfected sheep was described by test sensitivity and specificity, the distribution of ELISA OD, and the area under a receiver operating characteristic curve. RESULTS: The absorbed ELISA had a specificity of 98.2 to 99.5% (CI) and a sensitivity of 35 to 54% (CI). In sheep from infected flocks in Victoria, the AGID test had a specificity of 99 to 100% (CI) and a sensitivity of 38 to 56% (CI). The sensitivity of serological tests was higher in sheep with a body condition representative of the lower quintile of their flock of origin. CONCLUSION: The AGID test and absorbed ELISA are useful tests for the detection of ovine paratuberculosis. Although the tests had a similar accuracy, they detected different subpopulations of infected sheep with only moderate overlap. The AGID test had a higher specificity than the absorbed ELISA.

Experiences with vaccination and epidemiological investigations on an anthrax outbreak in Australia in 1997.

Between January and February 1997, there was a severe outbreak of anthrax on 83 properties in north-central Victoria, Australia. Vaccination was used as a major tool to control the outbreak by establishing a vaccination buffer zone 30 km by 20 km. In all, 78, 649 cattle in 457 herds were vaccinated in a three week program. In the face of the outbreak, there was a delay before vaccination was able to stop deaths. In the 10 days following vaccination 144 cases of confirmed anthrax occurred and 38 cases occurred more than 10 days after vaccination. When all cattle on at-risk properties were revaccinated in October and early November 1997, there were only two confirmed cases of anthrax in vaccinated seven and nine month old calves in the following anthrax season. Investigations into the epidemiology of the outbreak were unable to establish a single major association for the spread of the disease by flies, biting insects, carrion scavengers, wind, manufactured feed, milk factory tanker routes, veterinary visits, animal treatments, movements of personnel between farms or burning of carcases. The weather conditions in the outbreak area were part of a long dry spell with periods of high daily and night temperatures, continuing high humidity over the period and higher than normal soil temperatures. It is possible that extensive earth works in the district involving irrigated pasture renovation and water channel and drainage renovation could have disturbed old anthrax graves. It is postulated that these works released spores that were dispersed in the preceding wet winter across poorly drained areas that formed the axis for the outbreak. The earth moving renovations establishing irrigation in the area were conducted in the late 1890s, and before the occurrence of anthrax outbreaks were recorded. The axis of the outbreak was the major stock route for cattle and sheep moving from southern Victoria to northern Victoria and southern New South Wales, and undoubtedly there would have been extensive anthrax outbreaks before vaccine became available in the 1890s. In respect of other outbreaks, the events in Victoria most resembled outbreaks of anthrax recorded in the United States of America in the 1950s, 1960s and 1970s.

Evaluation of diagnostic tests for Johne's disease in young cattle.

OBJECTIVE: To investigate the development of immune responses in calves experimentally and naturally infected with Mycobacterium paratuberculosis and to evaluate the potential for diagnostic tests to detect infected calves. DESIGN: Sequential testing of four treatment groups of calves over a 2 year period. PROCEDURE: Twenty-nine calves were allocated to four groups. Group D calves were orally dosed with M paratuberculosis, group N calves naturally exposed to M paratuberculosis, group V calves vaccinated for M paratuberculosis, and group C were control calves (not infected or vaccinated). Blood and faecal specimens were collected from each calf at monthly intervals to 18 months of age and then every 2 months until they were slaughtered between the ages of 21 and 29 months. Specimens were tested using absorbed EIA, IFN-gamma EIA and faecal culture. The infection status of the calves was confirmed by extensive histopathological examination and tissue culture. RESULTS: M paratuberculosis infection was confirmed in 10 calves, comprising six of eight orally dosed calves, three of five naturally exposed calves and one of nine vaccinated calves. The six artificially infected calves and one naturally infected calf were detected shedding M paratuberculosis in their faeces. Results with positive absorbed EIA were obtained from one artificially infected calf, one naturally infected calf and three vaccinated calves. All calves including controls had positive results on at least one occasion using the IFN-gamma EIA. In addition, seven calves had positive bovine tuberculosis results using the IFN-gamma EIA, even though bovine tuberculosis has been eradicated from Australia. CONCLUSION: Detection of M paratuberculosis infection in young cattle continues to be difficult using current tests.

Morphometric analysis of renal proximal tubules in cadmium-treated rats.

The morphological changes in renal proximal tubules of Sprague Dawley rats given acute or chronic exposure to cadmium were analysed. Cadmium chloride was administered either by five subcutaneous injections of cadmium at 2 mg kg-1 body weight or in drinking water at 100 micrograms ml-1 for 39 weeks. The mean cadmium concentration in the kidneys of these rats was 45 and 102 micrograms g-1 wet weight respectively. The rats were anaesthetized and the kidneys were fixed by perfusion and processed for electron microscopy. Proximal tubule profiles were larger in the acute exposure rats. The brush border in both groups of treated rats was shorter and there were focal areas of loss of microvilli. The surface density of microvillus membrane per unit cell volume was reduced by 25% and 19% for chronic and acute dosed rats respectively. There were few significant changes in organelles detected by morphometric analysis of the entire kidney tissue, however there was a reduction in volume density of lysosomes following chronic exposure and individual necrotic cells and distorted nuclei were observed. The morphological changes observed in chronic and acute dosed rats were consistent with a primary site of toxic insult on the apical plasma membrane. There appeared to be no evidence for change in fluid and electrolyte homeostasis in the epithelium. The results suggest that the cadmium may produce a selective deficit of transport mechanisms for macromolecules.

X-ray microanalysis of renal proximal tubules in cadmium-treated rats.

Cadmium chloride was administered to Sprague-Dawley rats in drinking water at 100 ppm for 39 weeks. The mean cadmium concentration in kidneys from 5 rats at the end of this period was 102 +/- 17 micrograms g-1 wet weight (mean +/- SE) compared with 0.22 +/- 0.15 microgram g-1 wet weight in a similar group of control rats. Right kidneys were rapidly frozen in propane and small pieces of cortex were prepared for X-ray microanalysis by freeze-substitution in ether/acrolein for 21 days. Analysis of freeze-substituted sections showed that Cd concentrations rose to 3.9 mmol kg-1 of resin embedded tissue (approximately wet weight) in cytoplasm and 6.5 mmol kg-1 in nuclei. At the same time S concentrations rose by 31 percent in nuclei and 23 percent in the cytoplasm. Cd was also present in lysosomes. Cytoplasmic and nuclear ionic (Na, K, Cl, Ca, Mg) concentrations did not change in spite of a considerable decrease in microvillus membrane surface area. The concentration of P also did not change suggesting that nucleoside phosphate levels remained stable. The distribution of Cd and S supports current concepts of cadmium toxicity. Although total kidney Cd was at the critical concentration and extensive damage to microvilli had occurred, there appeared to be no effect on ionic permeability and cell electrolyte balance and, by inference, NaCl reabsorption.

Comparison of the Johne's absorbed EIA and the complement-fixation test for the diagnosis of Johne's disease in cattle.

A commercially available absorbed ELISA for the diagnosis of Johne's disease (JD) (paratuberculosis) in cattle, the Johne's Absorbed EIA, was compared with the conventional complement-fixation test (CFT) used in Australia. Stored plasma from 3 Victorian dairy herds with a history of JD, sera from specimens submitted from animals showing clinical signs of JD and sera from the US National Repository for Paratuberculosis Specimens were used to determine the sensitivity of each test. The EIA detected 48.8% of 43 Australian animals with subclinical JD, while the CFT detected only 12 (21.4%) of 56 subclinically affected cattle. Of 150 subclinically infected US cattle, the EIA detected 47.3% and the CFT detected 52.0%. The EIA detected 59.7% of animals which at the time of sampling were shedding Mycobacterium paratuberculosis in their faeces, but showed no clinical signs of JD, while the CFT detected 57.3%. The EIA correctly identified 88.2% of 136 histologically confirmed clinical cases, and the CFT detected 83.4%. The specificity of each test was determined by testing sera collected at slaughter from animals residing in a known JD-free area of Australia, and from samples from the US National Repository of Paratuberculosis Specimens collected from certified-free herds in Wisconsin. The EIA was found to have a specificity of 99.8% when 998 Australian animals were used as the test population, and 99.0% when 196 US animals were used. The specificity of the CFT using Australian samples was 96.9% and 95.2% using American samples.

X-ray microanalytical investigation of the response of chicken proximal tubule cells to infection with avian infectious bronchitis virus.

The technique for X-ray microanalysis of frozen-hydrated bulk specimens was used to determine the intracellular and luminal fluid electrolyte concentrations in the proximal tubules of kidneys from chickens infected with infectious bronchitis virus. Eight days post-infection with this virus there were significant changes in the electrolyte composition when compared with values from normal control chickens. The intracellular sodium decreased from 43 to 36 mmol/l, the chloride fell from 41 to 31 mmol/l and the potassium went from 125 to 115 mmol/l. Sodium counts in the luminal fluid rose from .73 to 1.03 cps. These disturbances in electrolyte composition are consistent with alterations in sodium reabsorption in the proximal tubule due to decreased transport of sodium into the cells across the microvillus membrane. It appears that the Na-K-ATPase pump is unaffected. The results demonstrate the value of X-ray microanalysis methods for the study of electrolyte transport in pathologically affected cells and provide further information for the definition of viral-host cell interactions in the pathogenesis of viral disease. As a check on methodology two normal rat kidneys were analysed in the same way. Intracellular sodium and potassium concentrations were 22 and 138 mmol/l respectively.

The duct system of the avian salt gland as a transporting epithelium: structure and morphometry in the duck Anas platyrhynchos.

The duct system of the nasal salt gland of the duck comprises central central canals, secondary ducts and main ducts. The secondary and main ducts consist of a layer of columnar cells overlying a layer of small cuboidal cells. The columnar cells have complex intercellular spaces showing evidence of Na+K+-ATPase at the apical regions. Approximately 70% of surface area of the duct system is external to the gland. During adaptation to salt water the duct system increases in size as does the gland. Although the components of the gland of adapted ducks, including the duct system within the gland, increase in size compared with normal ducks, the percentage volume densities of the components remain similar in both categories of ducks, i.e. the duct system increases in size in proportion to the glandular tissue. The volume of the duct system external to the gland is six to seven times larger than the volume within the gland. Thus, if ductal modification of secreted fluid occurs, it will be most likely to take place in the ducts external to the gland. Total surface areas of the duct system were measured from serial sections of glands and ducts from one normal and one adapted duck. These were used to calculate possible flux rates of water and sodium across the duct epithelium, assuming the occurrence of either water reabsorption of sodium secretion. Although these flux rates are high it is shown that they are similar to calculated flux rates across the luminal surface of the secretory tubules.

Inhomogeneity in epoxy resin standards for quantitative biological X-ray microanalysis.

It is shown by X-ray point analyses and line scans that the concentrations of sodium and potassium, as crown ether complexes, in epoxy resin may not be of uniform distribution. The concentrations may be substantially higher in a thin layer at the base of the block. It is recommended that chemical analysis of a selected central region of a block, not the intact block, be carried out to establish the true concentration. This may be substantially lower than the nominal concentration. This problem appears to be less acute with cryptate complexes of sodium and potassium but a similar trend is nevertheless apparent.

Pathogenesis of infectious bronchitis nephritis. 1. Morphometric analysis of kidney proximal tubular epithelium in chickens.

The ultrastructure of the proximal tubular epithelial cells in chicken kidneys was examined throughout the course of an experimental infection with infectious bronchitis virus. A quantitative assessment of the structural changes in the cells was related to these in normal cells. Significant alterations were detected in the membrane structures and in the mitochondria. There was a reduction in surface area of the microvillus membrane, the basolateral membrane and the apical tubular membrane. There were alterations in the shape of mitochondrial profiles and a decrease in the volume density of mitochondria. Vesicular structures, which are a possible site of viral release, were observed in the lateral surface of cells. These changes in the functional components of the cells indicate impaired transport of ions and water. The results demonstrate the value of stereological methods for the study of viral-host cell interactions in the pathogenesis of viral disease.

Pathogenesis of infectious bronchitis nephritis. 2. Studies of water and electrolyte balance in colostomised chickens.

Water and electrolyte balance was examined throughout the course of an experimental infection with T strain infectious bronchitis virus in colostomised chickens. Significant losses of water and negative sodium and potassium balances were observed. The major change in the electrolyte balance was the increased output of sodium in urine and this was associated with a diuresis. A decrease in food intake was the most important contribution to the negative potassium balance. Death resulted from acute renal failure. The implication of the results for electrolyte replacement therapy is discussed and a method for colostomies in birds weighing less than 0.5 kg is described.

Experimental infection of mares with Haemophilus equigenitalis.

Inoculation of Haemophilus equigenitalis into the uterus of 7 mares caused a disease clinically indistinguishable from contagious equine metritis. The duration of clinical signs varied from 4 to 11 days. The causative organism persisted for a relatively short time (2 to 10 weeks) in 5 mares, but in 2 others it established a carrier status and persisted until they were killed 6 and 10 months after infection. H. equigenitalis was recovered from the vestibule of the vagina and from a combined swab of the clitoral fossa and sinuses throughout the course of the infection. In some mares there were extended periods (2 weeks) when it could not be reisolated. All mares experienced a transitory serological response to infection. The complement fixation test was generally negative 12 weeks after infection whereas antibodies detected by the passive haemagglutination test and serum agglutination test were more persistent. In some animals the PHT and SAT titres increased during the breeding season following infection. The serological response did not appear to be related to the duration of infection.