The Chinese hamster FANCG/XRCC9 mutant NM3 fails to express the monoubiquitinated form of the FANCD2 protein, is hypersensitive to a range of DNA damaging agents and exhibits a normal level of spontaneous sister chromatid exchange.

Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustard-hypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein's subsequent interaction with BRCA1, is involved in maintaining genomic stability in response not only to DNA interstrand crosslinks but also a range of other DNA damages including DNA strand breaks. NM3 and other "FA-like" Chinese hamster mutants should provide an important resource for the study of these processes in mammalian cells.

DNA analysis in disputed parentage: the occurrence of two apparently false exclusions of paternity, both at short tandem repeat (STR) loci, in the one child.

In cases of disputed paternity investigated by DNA analysis, it is generally accepted that at least two independent exclusions be observed before concluding that a putative father is not the biological father of the child. We report here a case in which two apparent exclusions of paternity were obtained (at the loci FESFPS and TPOX), from a total of nineteen loci examined (short tandem repeats, AMFLPs, DQA1, Polymarkers). The combined paternity index of the other seventeen loci, and of two multilocus probes (33.15 and 33.6) would exceed twenty-eight trillion to one. In the absence of any alternative putative father (including close relatives of the man tested), we concluded that the most likely explanation for these results was that the putative father was indeed the biological father, but that two independent mutations had occurred in either the ovum and/or spermatozoon. We have altered our internal laboratory criterion for exclusion of paternity to require exclusions in three independent loci.

Salmonella Dublin infection in Queensland dairy cattle.

OBJECTIVE: To investigate the presence of Salmonella Dublin in Queensland cattle. DESIGN: An epidemiological study using diagnostic laboratory information and farm records. PROCEDURE: Outbreaks of gastroenteritis or pneumonia in calves, and abortions and enteritis in cows were routinely investigated for the presence of salmonellae. Where S Dublin was isolated, attempts were made to gather further epidemiological information. RESULTS: Prior to 1983 only two outbreaks of S Dublin have been recorded in Queensland dairy cattle. In 1983 S Dublin abortions were diagnosed in dairy heifers introduced from southern Australia to south-east Queensland. Sampling indicated that at least 10% of the 500 introduced heifers were faecal excretors of S Dublin. On 3 of the 7 farms from which S Dublin was recorded, infection spread to other cattle that were in contact. From February 1985 to February 1996, 29 outbreaks of S Dublin in cattle occurred on 29 farms (28 in south east Queensland and 1 in north Queensland). Calves were primarily affected. Continuing outbreaks were confirmed on only 4 of these 29 farms. On 15 farms S Dublin infections were associated with the purchase of infected calves or cows, while another farm adjoined 2 previously infected farms. No source of S Dublin was evident for the other 13 farms, where histories were often inadequate. CONCLUSION: There has been a marked increase in S Dublin outbreaks in Queensland dairy cattle since 1983. Introduction of S Dublin carrier and aborting dairy heifers from southern Australia, where S Dublin is not uncommon, was associated with the initial outbreaks.

Suspected botulism in three intensively managed Australian cattle herds.

Serious outbreaks of a paralytic disease in cattle occurring in the spring and summer of 1988 were investigated on three farms in south eastern Queensland, Australia. On one farm 237 (31 per cent) of 770 cattle died, on the second 109 (40 per cent) of 271 cattle died and on the third 30 (8 per cent) of 380 cows died. Botulism was suspected on the basis of the clinical signs, the lack of significant pathology, a failure to incriminate other agents and a positive feeding trial in one sheep. Laboratory tests for the presence of botulinum toxin failed to confirm this diagnosis, and further feeding trials using ingredients of two rations were also negative.

Red cells suspended in a phosphate-fortified additive: biochemical enhancement and longer storage.

When compared to our current storage regime, an additive solution, with 75 mM inorganic phosphate, improved certain biochemical and morphological parameters measured in suspensions of packed red cells throughout 49 days of liquid storage. In particular, the mean ATP level of 20 donations stored with the high phosphate additive was consistently and significantly higher than in standard (n = 6) suspensions. The increased amount of inorganic phosphate and the higher pH of the new additive stimulated ATP production and provided better pH buffering than the standard solution currently in use. Although survival study results indicated adequate 24h survivals for erythrocytes stored for 42 days with the new solution, after 49 days storage, the mean 24h survival of autologous erythrocytes was only 58 +/- 7% (n = 6).

Lymphocyte subpopulations in sheep with lymphosarcoma resulting from experimental infection with bovine leukaemia virus.

Sheep were experimentally infected with bovine leukaemia virus (BLV) and developed leukaemia and lymphosarcoma 30-88 weeks later. Ten sheep with lymphosarcoma were necropsied and lymphocyte subpopulations were evaluated in peripheral blood lymphocytes (PBL) and lymphocyte suspensions prepared from a range of lymph nodes, tumours and spleen. The leukaemic phase of BLV infection was characterized by an increase in the number of circulating B lymphocytes. The number of T lymphocytes was also increased with the CD8+ subpopulation proliferating at a much greater rate than the CD4+ subpopulation. In PBL the CD4:CD8 ratio fell rapidly as leukaemia developed, being 1.15 (+/- 0.18) 5-8 weeks before necropsy and 0.38 (+/- 0.09) at necropsy. During this period the number of B lymphocytes increased from 11.2 (+/- 0.7) to 379.4 (+/- 85.8) x 10(9)/L. CD4:CD8 ratios were also low in all lymph nodes and spleens of leukaemic sheep at necropsy. Most of the cells in solid tumours were B lymphocytes but a small population of T lymphocytes with a low CD4:CD8 ratio was identified.

Lymphocyte subpopulations in sheep during the early stage of experimental infection with bovine leukaemia virus.

Sheep were experimentally infected with bovine leukaemia virus (BLV) by the inoculation of PBL from leukaemic sheep. Antibodies to viral structural proteins were detected at from 2 to 6 weeks after inoculation. At seroconversion, all sheep had a marked increase in the number of circulating lymphocytes, due essentially to an increase in the number of B cells. The number of circulating B cells then decreased but remained higher than pre-infection levels. A second increase in this population preceded the development of a B cell lymphoblastic leukaemia. Generalized lymphosarcoma was diagnosed at necropsy of all sheep. Variation between individual sheep in the time from infection to the development of tumours allowed two clearly delineated groups of nine sheep to be compared. A study of changes in the B cell and T cell populations during the first 16 weeks of infection suggested that the initial response to infection influences the subsequent rate of leukaemogenesis. At seroconversion the number of circulating B cells was significantly higher in group 1 (10.16 +/- 1.51 X 10(9)/l) than in group 2 (6.47 +/- 2.76 X 10(9)/l). Group 1 sheep became leukaemic at 20-50 weeks after infection, whereas group 2 sheep did not do so until 70-95 weeks after infection.

Evaluation of four serological tests for the diagnosis of caprine melioidosis.

A complement fixation (CF) test, 2 indirect haemagglutination (IHA-A; IHA-L) tests which differed in antigen preparation and technique, and a microtitre agglutination (MA) test were compared in the serodiagnosis of melioidosis in goats. One hundred and eighteen experimental serums and 3143 field serums from goats in endemic and non-endemic areas of north Queensland were used in the evaluation. Culture of samples for Pseudomonas pseudomallei from 112 goats provided substantiating evidence of infection. The IHA-A test was the most sensitive, and the CF test the most specific. We advocate the use of the IHA-A as a screening test followed by the CF test for confirmation of active melioidosis. The IHA-A test is the better indicator of past infection.

Clinical and pathological observations on goats experimentally infected with Pseudomonas pseudomallei.

The effects in goats of the subcutaneous injection of varying doses of Pseudomonas pseudomallei (90 to 500,000 bacilli) suspended in normal saline are described. High doses (greater than or equal to 500 bacilli) caused acute, fatal infections. Lower doses (90 to 225 bacilli) caused acute or chronic disease when infection became established. However, 11 of 18 goats injected with the lower doses of bacilli showed no sign of infection on clinical or bacteriological examination. Response to antibiotic therapy with long acting tetracycline and chloramphenicol was minimal. Goats surviving the initial phase of infection tended to overcome the disease with a corresponding increase in the number of abscesses that were sterile at necropsy. In infected goats, clinical signs included undulating fever, wasting, anorexia, paresis of the hind legs, severe mastitis and abortion. At necropsy, abscesses were found predominantly in the spleen, lungs, subcutaneous injection site and its draining lymph node.

Susceptibility of Bos indicus and Bos taurus to Anaplasma marginale and Babesia bigemina infections.

The reaction of Bos taurus and pure-bred Bos indicus heifers to infection with the intraerythrocytic parasites Anaplasma marginale and Babesia bigemina was studied. B. bigemina infection at 18 months and A. marginale infection at 13 or 24 months resulted in slightly less severe reactions in pure-bred Bos indicus cattle than in Bos taurus. In both breeds, the reaction to A. marginale infection was more severe in older cattle. The severity of B. bigemina infection was not affected by a previous infection with A. marginale.

Examination of the origin of increased equine serum alkaline phosphatase concentrations.

Serum alkaline phosphatase activity was found to be increased in 32.6% of equine samples analyzed at the Ontario Veterinary College over an 18 month period. An attempt was made using sensitivity to L-phenylalanine and heat to identify the origin of increased serum alkaline phosphatase isoenzymes present in 44 clinical cases. No difference in sensitivity to either procedure was observed for serum alkaline phosphatase from groups of foals and horses representing different clinical problems. Alkaline phosphatase of osseous tissue origin appeared to be the major source of activity for each group of animals reported.

An evaluation of the anamnestic test for brucellosis in cattle of the northern pastoral area.

An investigation of the anamnestic test for brucellosis using Brucella abortus 45/20 vaccine was carried out in 3 groups of weaner cattle on 2 farms in western Queensland. Each group originally consisted of about 500 cattle. They were bled before and at 6 or 10 weeks after vaccination and again in the following year. The serums were tested by the complement fixation (CFT), Rose Bengal (RBT) and indirect haemolysis tests (IHLT). Most of the cattle reacting to one or more of the tests were killed and selected tissues were subjected to bacteriological examination for B. abortus. B. abortus was isolated from 19 of 30 (63%) pre-vaccinal reactors, 23 (24%) of 96 cattle reacting at 6 or 10 weeks after vaccination (the anamnestic test) and 1 (2%) of 50 cattle reacting one year after vaccination. The reactor found to be infected the year after vaccination had high serological titres in each of the 3 serological tests: RBT of 3, CFT of 128 and IHLT of 256. A subsequent test showed the group to be brucellosis-free. The CFT was the most efficient test. In the pre-vaccination tests 17 of 19 infected animals were positive in the CFT compared with 11 positive in the IHLT and 17 in the RBT. In post vaccination tests 22 of 23 infected animals were positive in the CFT compared with 18 in the IHLT and 19 in the RBT. At the pre-vaccinal and anamnestic tests (6 or 10 weeks after vaccination) 19 of 57 (33%) cattle with CF titre of 4 or 8 yielded B. abortus on culture compared with none of 26 cattle with similar titres in the year after vaccination. The interpretation of CF titres in cattle following 45/20 vaccination needs to be re-examined.

Bluetongue virus serotype 20 infection in pregnant Merino sheep.

Eleven maiden Merino ewes, free of antibody to bluetongue virus serotype 20 (BTV-20) in agar gel immunodiffusion and serum neutralisation tests, were mated once with a ram. Ten ewes were inoculated with BTV-20 35 to 42 days after service, and one ewe was left as an uninoculated control. One of the inoculated ewes and the control ewe remained uninfected throughout the experiment. Eight of the remaining 9 ewes showed clinical signs ranging from mild to moderate, and the other showed no clinical signs of infection. BTV-20 viraemia was detected in ewes between days 3 and 11 post inoculation, and the serum antibody response was followed. The control ewe and 5 of the 9 infected ewes were pregnant when examined 90 to 97 days after service. Each of these animals produced a normal lamb. There was no evidence of abortion in the remaining 5 ewes, and no transplacental transfer of virus was detected in the lambs of the 5 infected ewes. At necropsy, 46 days after the birth of the last lamb, no gross or microscopic lesions were observed in either the ewes or lambs.

Susceptibility of Bos indicus crossbred and Bos taurus cattle to Anaplasma marginale infection.

The effects of infection with Anaplasma marginale in groups of Bos indicus-Brahman crossbred and Bos taurus cattle aged 27 and 48 months were studied using the parameters of body weight change, packed cell volume, parasitaemia and humoral antibody. Twenty-six animals (8 Bos taurus and 18 Bos indicus crosses) of each age group were infected. No significant differences between any of the groups were observed.

A comparison of direct fluorescent antibody and Giemsa staining for the post-mortem diagnosis of anaplasmosis.

Direct fluorescent antibody (DFA) and Giemsa staining of Anaplasma marginale were compared in smears collected serially at post-mortem (PM) from 11 experimentally infected calves. Once smears had been prepared and air-dried they could be held for at least 5 days before staining with either technique with no noticeable change in staining quality. DFA staining was more sensitive in detecting anaplasms in smears than Giemsa staining. Anaplasma spp could be differentiated from Babesia bovis and B. bigemina by DFA staining but there were cross reactions between A. marginale and A. centrale. Blood smears prepared from subcutaneous vessels in the legs provided better diagnostic material than kidney, heart and lung smears. Brain smears were not suitable for PM diagnosis using either staining technique.