Proteins and antigens of Pasteurella multocida serotype 1 from fowl cholera.

The protein profiles of Pasteurella multocida serotype 1 isolates and the response of chickens to serotype 1 antigens were investigated using SDS-PAGE. Patterns obtained with Coomassie blue staining of soluble protein extracts were similar. The major difference between isolates was the position of one of the major proteins in the 34-38 kDa region. When chickens were experimentally infected with a clinical isolate of P. multocida serotype 1 various proteins were recognised by immunoblotting, including one with a relative molecular weight of 34 kDa; however, no reactions were observed in the region where LPS is known to migrate. When these infection sera were used in an EIA with purified LPS obtained from Heddleston serotype 1 type strain (X-73) they reacted strongly. Serum used for serotyping isolates in the gel diffusion precipitin test recognised many antigens in common with sera from infected birds, but some antigens were specific to typing sera.

The sensitivity and specificity of a modified ELISA for the diagnosis of Johne's disease from a field trial in cattle.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Mycobacterium paratuberculosis in cattle was evaluated in three herds known to have Johne's disease. Prior to testing, the plasma was absorbed with dried Mycobacterium phlei in order to remove cross-reacting antibody specificities. The sensitivity and specificity of the ELISA were calculated after repeatedly testing 327 cattle in the infected herds. Of these, 53 animals had one or more positive faecal cultures or had post-mortem histopathological evidence of infection. The other 274 had three or more negative faecal culture results, and were regarded as non-infected for the purpose of evaluation of the ELISA. Using these criteria for the presence or absence of infection, the M. phlei-absorbed ELISA under field conditions had a 57% sensitivity and a 98.9% specificity. The sensitivity of the absorbed ELISA depended on the stage of disease of the animal under test. In general, it appeared that animals in the more advanced stages of disease were absorbed ELISA positive, whereas those in the early stages of infection were not detected. These results indicate that the M. phlei-absorbed ELISA has an important role as a test for the diagnosis and control of Johne's disease in cattle.

In vitro responses of lymphocytes from cattle with advanced Mycobacterium paratuberculosis infection to homologous and heterologous antigens.

The accuracy with which the purified lymphocyte and whole blood modifications of the lymphocyte stimulation test were able to detect animals with clinical Johne's disease was compared with that of the complement fixation test and microscopic faecal examination. Confirmatory diagnosis of Johne's disease was based upon histopathological examination of intestinal tissue. False positive results were obtained only with the complement fixation test. The only test not giving rise to false negative results was the purified lymphocyte modification of the lymphocyte stimulation test using johnin at 20 micrograms/ml and avian tuberculin at 20 micrograms/ml or 2 micrograms/ml. The whole blood technique was less accurate than the purified lymphocyte technique for the in vitro detection of cell-mediated immune responses to antigens. The purified lymphocyte technique appears to have potential as an diagnostic test for Johne's disease in cattle and merits further evaluation.

Analysis by ELISA and Western blotting of antibody reactivities in cattle infected with Mycobacterium paratuberculosis after absorption of serum with M phlei.

Preabsorption of cattle serum with Mycobacterium phlei was of value in eliminating falsely positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against M paratuberculosis. Specific antibody titres from 16 animals naturally infected with M paratuberculosis were unaffected by absorption. Analysis by Western blotting indicated that a different set of antigens of M paratuberculosis were recognised by serum from falsely positive reactors compared with that from animals with established infection. After experimental infection the time required for seroconversion in the ELISA in nine calves lay between 10 and 28 months, although one animal had not seroconverted after 30 months when the experiment ended. All animals shed M paratuberculosis in their faeces before seroconversion.

The prevalence of anti-leptospiral agglutinins in sera of wildlife in southeastern Australia.

Anti-leptospiral agglutinins were found in the serum from 18 (7 species) of 419 (25 species) animals sampled from various areas of southeastern Australia. Positive serologic reactions were observed in 5 of 25 (20%) brush-tailed possum (Trichosurus vulpecula), 1 of 26 (3.8%) tammar wallaby (Macropus eugenii), 2 of 12 (16.7%) swamp wallaby (Wallabia bicolor), 1 of 3 (33.3%) koala (Phascolarctos cinereus), 3 of 41 (7.3%) common wombat (Vombatus ursinus), 2 of 100 (2%) bush rat (Rattus fuscipes) and 4 of 12 (25%) rusa deer (Cervus timorensis). The majority (55.5%) of serologic reactions were to serovar hardjo. No serologic reactions were observed in samples from echidna (Tachyglossus aculeatus), brown antechinus (Antechinus stuartii), swainson's antechinus (Antechinus swaisonsii), long-nosed bandicoot (Perameles nasuta), brown bandicoot(Isoodon obesulus), common ringtail (Pseudocheirus peregrinus), greater glider (Schoinobates volans), eastern grey kangaroo (Macropus giganteus), red-necked wallaby (Macropus rufogriseus), rabbit (Oryctolagus cuniculus), water rat (Hydromys chrysogaster), black rat (Rattus rattus), eastern swamp rat (Rattus lutreolus), broad-toothed rat (Mastacomys fuscus), fox (Vulpes vulpes), sambar deer (Cervus unicolor), hog deer (Axis porcinus) and fallow deer (Dama dama).

The prevalence of antibodies to members of Leptospira interrogans in cattle.

A total of 1,355 random samples taken from bovine serums submitted for brucellosis testing in Victoria were submitted to the microscopic agglutination test for the presence of antibody to 12 serovars of Leptospira interrogans. The most common reaction obtained was to serovar hardjo, although the percentage of reactors varied from 24.8% in the metropolitan area to 56.3% in north-eastern Victoria (mean 40.6%). A total of 86.3% of farms from which 3 or more samples were taken had at least one reactor to serovar hardjo. The prevalence of antibody to other serovars was tarassovi (7.8% of reactors), ballum (3.7%), pomona (2.4%), autumnalis (1.8%) and bataviae (1.2%). Reactions to other serovars were observed in serums of less than 1% of cattle tested; serums from 50.8% of cattle did not react to any antigen.

Development and evaluation of a rapid absorbed enzyme immunoassay test for the diagnosis of Johne's disease in cattle.

An absorbed enzyme immunoassay (EIA) test for Johne's disease in cattle was developed in which absorption of cross-reacting antibodies occurred as a rapid reaction in solution rather than overnight with whole organisms and a subsequent centrifugation step. Total test time was reduced to less than 2 h with a minimum of manipulations. The test was evaluated in cattle herds from Johne's disease-endemic and Johne's disease-free regions of Australia. Specificity was 99.8%. Calculations of sensitivity were affected by the history of the herd under test. However, the EIA detected in excess of 80% of animals before onset of clinical disease and 65% of faecal shedders were EIA positive on, or before, first detection of Mycobacterium paratuberculosis in their faeces. The test should aid epidemiological studies and be a useful tool in the management and control of Johne's disease.

Leptospira serogroup Hebdomadis infection as an Australian zoonosis.

Two cases of leptospirosis due to infection with a member of the Hebdomadis serogroup are described in farm workers on a Victorian dairy farm. The source of infection appeared to be the milking herd which had elevated serum antibody titres against a member of the Hebdomadis serogroup. Agglutinin-absorption testing of one patient's serum indicated that the infecting serovar was hardjo. A survey of 1,144 cattle entering abattoirs throughout the State indicated that 44.3% of these animals showed serological evidence of past or present infection with leptospira of the Hebdomadis serogroup and the potential risk for people with occupational contact with cattle is emphasised. The importance of considering leptospirosis in the differential diagnosis of all patients with influenza-like symptoms or pyrexia of unknown origin and having occupational contact with animals, especially cattle, is discussed.

A comparison of the interferon gamma assay with the absorbed ELISA for the diagnosis of Johne's disease in cattle.

Two interferon gamma (IFN-gamma) assays, the IFN-gamma enzyme immunoassay (EIA) and the IFN-gamma bioassay and an absorbed ELISA were used to screen 6 cattle herds for Johne's disease. Each herd had a history of Johne's disease but the majority of infected animals did not show clinical signs. The disease status of the cattle, which were removed from the herds, was confirmed by bacteriological culture of faeces or histopathological examination and culture of tissues collected at necropsy. The sensitivities of the IFN-gamma assays and the absorbed ELISA were determined using test results from infected animals. The sensitivity of the IFN-gamma EIA in detecting subclinical (71.8 to 93.3%) and clinical animals (100%) was not significantly different. However, the IFN-gamma bioassay and the absorbed ELISA were more sensitive in detecting cattle with advanced infections (80%) than those that were subclinically affected (16.7 to 33.3%).

Trichostrongylus colubriformis: analysis of monoclonal antibody and lectin binding to the larval cuticle.

A monoclonal antibody (VRI 86-1) raised against third stage Trichostrongylus colubriformis preferentially bound to the excretory pore of living exsheathed larvae, with little or no binding to other sites on the parasite surface. A similar binding pattern was observed with fluorescein-labelled wheat germ agglutinin (WGA), although the anterior end of the parasite was also stained. To gain information at the molecular level regarding the parasite components at these sites, the residues recognized by WGA (i.e. N-acetylglucosamine and N-acetylneuraminic acid) were radiolabelled on the surface of living larvae. After homogenization and detergent extraction of the larvae, four dominant bands and a number of minor bands were revealed by SDS-PAGE and fluorography. None of these bands was specifically immunoprecipitated or recognized on a Western blot by VRI 86-1, suggesting that the epitope recognized by this antibody either resides on a different molecule or is destroyed or changed during the radiolabelling procedures. These results provide further evidence that the nematode cuticle is not uniform at the molecular level, and that the excretory pore contains molecules and antigens that may be unique to that site.

The role of colostrum and milk in protection of the neonatal mouse against peripheral infection with Ross River virus. Brief report.

Neonatal mice can be protected against peripheral infection with Ross River virus by colostral and milk IgG after suckling on an immune mother. This protection supplements the transplacentally acquired protection against in utero infection.

Pathogenesis of in utero infections with abortogenic and non-abortogenic alphaviruses in mice.

The initial stages of infection of pregnant mice at gestation day 11 with either the T48 strain of Ross River virus or avirulent Semliki Forest virus are similar. With both infections, a hematogenous spread of virus to the placenta occurs. The viruses subsequently replicate to high titer in all placentas and are able to persist in the presence of specific maternal antiviral antibodies. There is a delay of at least 1 to 2 days between the initial detection of virus in the placenta and the onset of fetal infection. With Semliki Forest virus, abortion occurred in all mothers and appeared to be preceded by infection of all fetuses. However, when Semliki Forest virus was given at other stages of pregnancy, abortion was less common, and in all non-aborted pregnancies at least one uninfected fetus was observed. This situation was similar to that with Ross River virus, in which abortion was not observed and fetal infection and death were only seen in a proportion of fetuses. Within each pregnancy, the outcome of the two in utero infections appeared to result from similar mechanisms, with the fate of an individual fetus depending upon the timing of the passive transfer of anti-viral immunoglobulin G from the mother relative to the timing of fetal infection by virus from the placenta. Although the passive maternal immunoglobulin G protected susceptible fetuses against infection, antibody did not mediate in utero recovery of infected fetuses or clear placental infection.

Two-dimensional electrophoretic comparison of the antigens and biosynthetically labelled proteins of Trichostrongylus colubriformis and Ostertagia circumcincta.

The proteins and antigens of different life stages of Trichostrongylus colubriformis were compared with those of Ostertagia circumcincta in an attempt to identify the subset of parasite molecules that is genus-specific and that may therefore be involved in the induction of genus-specific, host-protective immunity. Novel short-term culture techniques were instituted to label biosynthetically the proteins of the infective larval and adult stages of the parasites using 35S-methionine. High resolution, two-dimensional electrophoretic profiles of the labelled proteins indicated that the majority of proteins synthesized by adults were also present in the larval stages. Qualitative differences in the levels of these common proteins were observed, indicating differences in protein expression or turnover. There was extensive homology between larvae from the different species, with only eight major differences apparent in their profiles of biosynthetically-labelled proteins. Western blot analysis using immune sheep sera indicated that extensive homology also existed between the antigens of T. colubriformis and O. circumcincta larvae.

Intrathecal administration of morphine for elective Caesarean section. A comparison between 0.1 mg and 0.2 mg.

This study compared the quality of analgesia and incidence of adverse effects with two doses of intrathecal morphine in patients undergoing elective Caesarean section. Fifty patients were randomly allocated to receive either morphine 0.1 mg or 0.2 mg in addition to a standard intrathecal dose of 2.5 ml bupivacaine 0.5% in 8% dextrose. The quality of analgesia was assessed using visual analogue scores and the incidence of nausea, vomiting and itching were recorded during the first 24 h postoperatively. There was no statistically significant difference in the quality of analgesia nor in the incidence and severity of itching between the two groups. Fewer patients in the 0.1 mg morphine group experienced postoperative nausea and vomiting (7 versus 14, p < 0.05). We conclude that the use of 0.1 mg morphine intrathecally produces comparable analgesia to 0.2 mg after Caesarean section with significantly less nausea and vomiting.

Effect of pregnancy on stimulation of alphavirus immunity in mice.

The state of pregnancy was associated with a marked enhancement of both proliferative and cytotoxic T cell antiviral immune responses after infection of mice with avirulent Semliki Forest virus. Strong responses were obtained from the spleen and the para-aortic lymph nodes that drain the uterus. This immune enhancement seemed to be in response to the increased antigenic challenge that originated from infected placental and fetal tissue and was released during the process of abortion. Immune enhancement was not observed in pregnant mice similarly infected with Ross River virus, which also established an intrauterine infection but does not cause abortion.

Fasciola hepatica: immunoprecipitation analysis of biosynthetically labelled antigens using sera from infected sheep.

The antigenicity of the biosynthetically labelled somatic and excretory/secretory proteins of adult Fasciola hepatica was investigated over 20 weeks of an infection with F. hepatica in sheep. The antibody response was initially detected by ELISA 2 weeks after infection, and was sustained at this level for the remainder of the infection. Immunoprecipitation analysis indicated that a large number of proteins were recognized by the sheep, with several dominant antigens occurring in the 29-31 kilodalton molecular weight range. No differences were found between the antigens recognized by sheep in the early or late stages of infection suggesting that there are similarities between the antigens of the immature and mature forms of the parasite.

Effect of demand frequency on lockout period. The Abbott Provider 5500 Patient-Controlled Analgesia machine.

An Abbott Provider 5500 Patient Controlled Analgesia machine was noticed to have developed a prolonged lockout period when frequent analgesic demands were being made. This study tested five other machines and demonstrated that the duration of the lockout period was influenced by the frequency of analgesic demands. This finding could have clinical implications and we recommend that the duration of the lockout period should be specifically examined during the testing of patient-controlled machines.