Poisoning of livestock in oregon in the 1940s to 1960s attributed to corynetoxins produced by Rathayibacter in nematode galls in chewings fescue (Festuca nigrescens).

Tunicaminyluracil antibiotics, similar to the corynetoxins produced by Rathayibacter toxicus in Australia and South Africa, were found in old nematode seed-galls from Festuca nigrescens from New Jersey (USA) and New Zealand (NZ). The toxin profiles from the NZ and USA galls were similar to each other, but differed from those produced by R toxicus from Australia and South Africa, suggesting that a geographical variant of R toxicus or closely related species may be involved. The NZ galls gave a positive response to a R toxicus-specific monoclonal antibody assay, albeit a considerably weaker response than that seen with Australian R toxicus galls, but the older USA galls were negative, possibly due to deterioration of the antigen. From these findings, it is postulated that livestock deaths associated with the feeding of nematode and bacterial infected screenings of F nigrescens in Oregon, USA, in the 1940s to 1960s were caused by corynetoxin-like toxins produced by the bacterium.

Sequence data suggests big liver and spleen disease virus (BLSV) is genetically related to hepatitis E virus.

A monoclonal antibody (mAb) that reacted specifically with a 16 kDa big liver and spleen disease virus (BLSV) protein was used to identify the protein in western immunoblots of infected liver extracts and enable partial amino acid sequence analysis of the protein. Based on this sequence, a degenerate primer was designed that was used in conjunction with random hexamers in a reverse transcriptase-POR (RT PCR), to amplify a 523 bp product from RNA extracted from homogenates of BLSV-infected livers. There was 62% nucleotide sequence identity between this sequence and the sequence of the helicase gene of human hepatitis E virus (HEV). POR primers designed from this 523 bp fragment were able to amplify a 490 bp product from livers of virus-infected chickens but not chickens from virus-free flocks.

Comparative immunogenicity of two bivalent botulinum vaccines.

OBJECTIVE: To compare the ability of a new single-dose botulinum vaccine containing a non-mineral oil adjuvant with a single dose of a conventional botulinum vaccine product to produce antibody to Clostridium botulinum types C and D in cattle in Northern Australia. DESIGN AND PROCEDURE: One hundred and fifty Brahman steer weaners were randomly divided into two groups receiving either a single dose of CSL Bivalent Botulinum vaccine or Websters Singvac. Blood samples were collected at 0, 8 and 24 weeks and tested by antibody ELISA. The final samples were also tested by the toxin neutralisation test, to test titres of neutralising antibody. RESULTS: Six months after inoculation, cattle vaccinated with Websters Singvac had ELISA antibody response twice that of CSL conventional product. However, this difference was only evident for neutralising antibody to type C botulinum toxin. Both products produced similar titres of type D neutralising antibody after a single dose. CONCLUSION: Websters' Singvac produces a greater neutralising antibody response to type C botulism upon single inoculation than a conventional vaccine. The product produces an equivalent neutralising antibody response to type D.

Human papillomavirus DNA and virus-encoded antigens in cervical carcinoma.

The present study was undertaken to evaluate the prevalence of HPV in formalin-fixed, paraffin-embedded cervical carcinoma tissues using PCR followed by non-radioactive Southern hybridization with type-specific oligonucleotides for HPV 16 and 18. In addition, the tissue sections were immunohistochemically screened with two monoclonal antibodies, for expression of HPV 16 L1 and HPV 18 E6 proteins. A total of 57 of 60 cervical carcinomas (95.0%) were found with HPV using both techniques. HPV 16 and HPV 18 were present in equal proportions. Results of both DNA hybridization and immunohistochemistry were in agreement for the majority of the cases. HPV 16 and 18 DNA and virus-encoded antigens, L1 and E6 were found highly prevalent in these cervical carcinomas. Due to the high prevalence of HPV with cervical carcinoma in Malaysia, the implementation of routine diagnosis for the virus in cervical biopsies would be clinically useful.

Use of enzyme-linked immunoassays for antibody to types C and D botulinum toxins for investigations of botulism in cattle.

The development of specific enzyme-linked immunosorbent assays (ELISA) for antibody to types C and D Clostridium botulinum toxins for investigation of botulism in cattle is described. Partially purified type C and D toxins were used as antigens to develop these ELISAs. Specificity of the ELISAs was evaluated on sera from 333 adult beef and dairy cattle from areas with no history or evidence of botulism in animals or water birds. The test was also evaluated on sera from 41 herds that included herds vaccinated against botulism, confirmed botulism cases and herds from areas where the disease is considered endemic. The ELISAs detected the presence of antibody to botulinum toxins in samples from vaccinated cattle and both convalescent and clinically normal animals from unvaccinated herds with outbreaks of botulism. Antibody was also found in unvaccinated animals from herds in which there had been no diagnosed botulism cases in areas where botulism was considered endemic. Sera from some unvaccinated cattle with high ELISA reactivity was shown to be protective for mice in botulinum toxin neutralisation tests. The use of these tests in investigations of botulism in cattle is discussed.

An antigen detection immunoassay for big liver and spleen disease agent.

An enzyme-linked immunosorbent assay (ELISA) able to detect an antigen associated with infection with big liver and spleen (BLS) agent was developed. The assay is a capture ELISA (C-ELISA) procedure using ELISA plates coated with mouse monoclonal antibody (mAb 1H4) for antigen capture and a polyclonal chicken Ig preparation, extracted from yolk of BLS antibody positive birds, for detection of captured BLS antigen. mAb 1H4 was produced to a partially purified antigen preparation from liver suspensions of naturally BLS infected birds. The C-ELISA was evaluated for specificity and sensitivity in naturally and experimentally BLS infected and uninfected broiler and layer breeds of domestic fowl.

Dermatophilus chelonae sp. nov., isolated from chelonids in Australia.

Three isolates of a previously undescribed Dermatophilus sp. obtained from chelonids (two strains obtained from turtles and one strain obtained from a tortoise) were compared with 30 Dermatophilus congolensis isolates obtained from Australian mammals. The microscopic appearance, the colony morphology, and most biochemical test results for the chelonid isolates were characteristic of the genus Dermatophilus. Our isolates differed from the mammalian D. congolensis isolates in a number of cultural characteristics, including faster growth at 27 degrees C than at 37 degrees C, formation of two hemolysis zones around colonies on blood agar at 37 degrees C in the presence of 10% CO2, poor motility, and production of a distinctive odor. The DNA restriction enzyme digestion and protein electrophoresis patterns of our strains were distinct. The electrophoretic mobilities of 11 enzymes differed from the mobilities observed with D. congolensis strains. A monoclonal antibody to a surface antigen of an ovine isolate did not react with zoospores or filaments of the chelonid isolates. Biochemical differences between our isolates and D. congolensis included the ability of the chelonid isolates to reduce nitrate to nitrate and the fact that the chelonid isolates exhibit collagenase activity in vitro. We propose that the chelonid isolates should be placed in a new species, Dermatophilus chelonae. Strain W16, which was isolated from a nose scab on a snapping turtle, is the type strain; a culture of this strain has been deposited in the American Type Culture Collection as strain ATCC 51576.

Cytopathologic changes associated with intrauterine contraceptive devices. A review of cervico-vaginal smears in 350 women.

Cervico-vaginal smears from 350 IUCD users were analysed to ascertain the range of abnormalities induced in the genital tract of these women. Alteration of the microbial environment, inflammatory, degenerative, reparative and proplastic epithelial changes were the salient cytological findings. The clinical implications of these are briefly discussed.

PIP: Researchers examined results of cervico-vaginal smears of 350 women aged 23-45 years fitted with IUDs at various family planning clinics in Kuala Lumpur, Malaysia, to examine abnormalities in their genital tract. All the women had undergone preinsertion cervico-vaginal smears. They used the IUD for 1-8 years. Around 66% exhibited symptoms after IUD insertion. 40% had vaginal discharge, especially mucous. 3% had pelvic pain and intermittent low grade fever, suggesting pelvic inflammatory disease. 80% had an increase in the number of leukocytes in their blood. 42% had an increase in the number of histiocytes with multinucleate giant forms. The following microorganisms were present: Gardnerella vaginalis (42%), Trichomonas vaginalis (32%), Candida (28%), Actinomyces-like organisms (2%), and non-pathogenic Amoeba (0.6%). Both endocervical and squamous columnar cells exhibited morphological atypias (inflammatory, degenerative, or reparative changes). 70% of atypias were benign and varied from mild to severe. 14 women (4%) had cervical intra-epithelial neoplasia (CIN). 3% of the women had atypical single cells. The IUDs were removed from all of these women. 6 months after IUD removal, the cervixes with mild dysplasia had reverted to normal. Two women with severe dysplasia underwent cervical biopsy, which revealed a CIN III lesion. 28% of smears had abnormal or irritated glandular epithelial endocervical and endometrial cells with hyperchromatic nuclei, an increased nucleo-cytoplasmic ratio, and bubble-gum vacuolation of the cytoplasm. 31% of the women had normal or inflamed out-of-phase (beyond day 11 of the menstrual cycle) endometrial cells. 80% of these 109 women had menorrhagia or intermenstrual bleeding. The researchers recommend that serious epithelial atypias be followed up and the IUD be removed. IUD removal allows clinicians to determine whether atypias will regress in the absence of an IUD or are truly neoplastic.

Variation in cultural, morphological, biochemical properties and infectivity of Australian isolates of Dermatophilus congolensis.

Recent vaccination studies with Dermatophilus congolensis showed that variation of challenge strains had a considerable influence on protection afforded by the vaccines. In this study cultural, morphological and biochemical properties of 30 D. congolensis isolates from throughout Australian were investigated. The infective dose required to produce lesions of equivalent severity by these isolates for sheep, rabbits and guinea pigs was also examined and the isolates were grouped into four clusters of similar infectivity ranking. Analysis of the relationship between cultural, morphological and biochemical characteristics and infectivity rankings of clusters was undertaken to determine if certain properties were linked to infectivity. Considerable variability was found in haemolytic activity on blood agar, mucoid nature of colonies, motility, flagella density and polarity, capsule width, restriction enzyme profiles of bacterial DNA, protein electropherotype, carbohydrate content, and enzymic activity against proteins, maltose, chondroitin-4-sulphate, phospholipids and lipids. Of these properties haemolytic activity and enzyme activity against casein, chondroitin-4-sulphate and lipids showed some link with infectivity ranking for these isolates.

Cancer risk--does anyone really care?

Congress has enacted numerous laws in an attempt to protect people from carcinogens. But neither regulators nor oncologists can hope to accomplish this protection via studies in epidemiology. Nor can they hope to accomplish this using unverified models in carcinogenesis bioassays. The only viable hope is to test and utilize models of carcinogenicity that will tell us what we really need to know; i.e., does this substance pose a reasonable risk of carcinogenesis in man? And if so, what is a quantitative estimate of that risk? There is a substantial number of known human carcinogens. Yet, the most elaborate cancer-testing facility in the world continues to operate without the benefit of validating its results using positive controls (known human carcinogens). Without such controls to gauge the potency of the response in the test animal, Congress can pass 10,000 more laws and still the public will remain unprotected.

Evaluation of a toxoid for protection of rabbits against enterotoxaemia experimentally induced by trypsin-activated supernatant of Clostridium spiroforme.

Investigations were conducted into an enterotoxaemia caused by Clostridium spiroforme responsible for significant losses in commercial rabbit farms in Western Australia. Two trials using laboratory and farm bred rabbits were performed to evaluate the protective value of a toxoid prepared from the supernatant of C. spiroforme cultures against intraperitoneal challenge with the trypsin-activated toxin of C. spiroforme. The trials showed clearly that a single vaccination at weaning (four weeks) was protective against toxin but more complete and lasting protection was conferred following a second vaccination administered 14 days after the first. Adults likewise showed similar levels of protective antibodies but did not appear to pass on this protection to their kits although ELISA results indicated levels of antibody in kits from unvaccinated mother to be lower than progeny from vaccinated mothers. However antibody levels in kits from vaccinated mothers were very low and did not protect against challenge with toxin.

Uncertainty in health risk assessments.

Considerable scientific evidence has accumulated in the area of risk assessment. Using physiologically based pharmacokinetic models and biologically based dose-response models, more precise estimates of risk are becoming available. Uncertainty analysis performed at three steps of the process will enhance a clearer understanding of the assumptions being made regarding a public health decision. Recommendations are made on how uncertainty in risk assessments can be addressed and the types of future research that are required to this end.

Inflammatory cell and immune function in Merino sheep with chronic dermatophilosis.

Components of inflammatory and immunological responses were compared in 17 Merino sheep with chronic dermatophilosis (Group 1) and 15 Merino sheep that had recovered from the disease (Group 2). The functions studied included: (i) total and differential white cell counts; (ii) phagocytic function and intracellular killing by neutrophils; (iii) humoral immune response to T-dependent and T-independent antigens and to Dermatophilus congolensis. (iv) lymphocyte blastogenic responses to phytohaemagglutinin; (v) bovine serum albumen and D. congolensis antigens; (vi) quantification of T-lymphocyte subsets in skin lesions resulting after re-infection with D. congolensis zoospores. After all lesions were treated and the sheep were shorn, both groups of sheep were re-infected with D. congolensis. Both groups had similar infection rate, severity of lesions and rate of resolution after re-infection. The Group 2 sheep had significantly higher primary and secondary antibody responses to killed Brucella abortus cells than Group 1 sheep, but Group 1 sheep had higher levels of specific D. congolensis antibody throughout the trial. Neutrophils from Group 1 sheep showed a higher phagocytic rate for D. congolensis zoospores than Group 2 sheep when the zoospores were opsonised by sera from the Group 1 sheep, but there was no difference in their ability to kill ingested zoospores. Although there were some differences between the groups in the proportion of lymphocytes in lesions that reacted with monoclonal antibodies to T4, T8 and T19-19 lymphocyte markers at various times after re-infection, the sheep in Group 2 consistently had higher levels of lymphocytes reacting to a monoclonal antibody for the T6 lymphocyte antigen in skin biopsies collected 9, 15 and 21 days post-inoculation (p.i.) than did sheep in Group 1. Group 2 sheep also had higher levels of epidermal cells with immunohistochemical properties of Langerhans cells at lesion sites 15 and 21 days p.i.

Species comparisons in evaluating carcinogenicity in humans.

Some species and strains of experimental animals have such unique mechanisms of developing cancer that the extrapolation of such bioassay results to the human situation would be fraudulent. This fraudulent extrapolation could occur both qualitatively and quantitatively. Although it will be expensive, species other than the rat, mouse, and hamster should be tested, and tested at wider dose ranges than presently used, before risk assessors will have sufficient data to make legitimate risk estimates. Both species- and strain-unique mechanisms and pharmacokinetic information must be made available to the risk assessors before their estimates can be any better than "guesstimates." As more and more data become available, it will become essential that newer techniques of visualization of the data be used in order to evaluate the weight of evidence that an animal carcinogen is or is not a human carcinogen.

Serum and skin surface antibody responses in merino sheep given three successive inoculations with Dermatophilus congolensis.

Three antigens prepared from different phases of the life cycle of Dermatophilus congolensis were used in an enzyme-linked immunosorbent assay to measure serum and skin surface antibody responses in sheep after a first, second and third inoculation with D. congolensis. After the first inoculation, a strong antibody response to the flagella, filament and soluble antigens was detected after 7-21 days in the sera from sheep that were regularly biopsied; the antibody response at the skin surface was detected 28-42 days after inoculation, when the lesions were resolving. Strong anamnestic responses were detected in the serum of sheep that were biopsied and some of the nonbiopsied sheep after the second and third inoculations, but the skin surface antibody response at these times was variable.

Cellular responses in the skin of merino sheep to repeated inoculation with Dermatophilus congolensis.

The cellular response in the skin of Merino sheep was examined after three successive inoculations with Dermatophilus congolensis. There was a massive neutrophil influx into the infected epidermis and underlying dermis at 4-10 days after the first inoculation. A lymphocyte-macrophage response occurred at 10-12 days, followed by a plasma cell response at 14-38 days. Resolution of skin lesions after the first inoculation corresponded to the time when the plasma cell response in the skin was most intense. A second inoculation with D. congolensis, 70 days after the first, failed to produce skin lesions typical of dermatophilosis. Typical lesions of dermatophilosis did develop after a third inoculation of the same sheep 140 days after the first inoculation, but the lesions resolved in most sheep within 13 days. Dermatophilosis did not develop in some of these sheep at sites inoculated with 100-1000-fold lower infective doses of D. congolensis, whereas control sheep did develop lesions.