Estimation of clinical and economic effects of prophylaxis against venous thromboembolism in medical patients, including the effect of targeting patients at high-risk.

BACKGROUND: The clinical and economic effects of medical thromboprophylaxis (MT) using low molecular weight heparin in Australia are unknown. AIM: To estimate the effects of MT in Australia. METHODS: A decision tree model of MT was populated with national data for medical admissions. The Prevention of Recurrent Venous Thromboembolism (PREVENT) trial was chosen as the primary data source because its design uniquely avoided bias caused by treatment of sub-clinical events. Clinical efficacy and costs were estimated compared with no prophylaxis, assuming full compliance and according to three definitions of eligibility. Effectiveness was estimated as thrombotic events saved, mortality from bleeding or pulmonary embolus (PE), cost and $/year of life saved. Model outputs were subjected to sensitivity analysis. RESULTS: MT decreased thrombotic events, and the numbers avoided increased as eligibility broadened (deep vein thrombosis (DVT): 2597, 2771 and 3232 at restricted, intermediate and broad eligibility; PE: 454, 484 and 565 respectively). The annual cost of no prophylaxis was $88.7 m. Costs were reduced at most restricted eligibility (-$7.9 m), but increased by $3.0 and $32.1 m at broader eligibility. PE deaths declined, but this was offset by deaths from haemorrhage, causing a net increase (158, 299 and 672 respectively). Estimates were sensitive to the incidence of venous thromboembolic event (VTE), case-fatality rates for PE and bleeds and the relative risk reduction for PE with prophylaxis. CONCLUSIONS: Under PREVENT trial conditions, MT avoids up to 3200 DVT and 565 PE events annually, but may increase mortality.

A study on the severity and relevance of porcine circovirus type 2 infections in Dutch fattening pigs with respiratory diseases.

This study was set up to get more insights in the severity and relevance of porcine circovirus type 2 (PCV2) infections in Dutch fattening farms in an endemic PCV2-situation with no clinical signs of post-weaning multisystemic wasting syndrome (PMWS). In part A of the study, in total 29 commercial fattening farms with varying percentages of pneumonia and pleurisy at slaughter were examined. Blood samples were collected at random by cross-sectional sampling; 10 in the age of 10-12 weeks, 10 at the age of 16 weeks and 10 blood samples at the end of the finishing period (20-22 weeks of age). Serum samples were examined for the presence of PCV2 IgM and IgG antibodies and for antibodies against other porcine lung pathogens. In part B, 8 "high" and 8 "low" herds were selected. The 8 "high" herds were defined as herds having high percentages of lung lesions (pneumonia) at slaughter, and the 8 "low" herds had low percentages of pneumonia at slaughter. For both the "high" and "low" herds, 3 pigs showing signs of respiratory distress were selected for necropsy (n=48). Lung tissue samples were examined post-mortem for macroscopic and histopathological lesions, and for the presence of bacteria and viruses. The results of part A showed that, pigs at 16 weeks of age with IgM antibodies against PCV2 had a lower probability of having pleuritis at slaughter (OR 0.34, P<0.000). Pigs in the age category of 20-22 weeks, and with IgM antibodies against PCV2, also had a lower probability of having pneumonia at slaughter (OR 0.29, P=0.032). In part B lobus apicalis pneumonia, PCV2 in macroscopically unaffected lungs, Pasteurella multocida, Mycoplasma hyopneumoniae, and swine influenza viruses were all found significantly more often in "high" than in "low" pigs at autopsy. High PCV2 DNA loads (>10(4) PCV2 DNA copies/mg) were found in lungs of 14 (58%) "high", and in 7 (29%) of the "low" pigs (P=0.13). In 11 of the 19 affected lungs from "high" pigs, high PCV2 DNA loads were found in combination with one or more other lung pathogens, while this was found only in 5 of the 17 affected lungs from "low" pigs (P=0.02). This study confirms the hypothesis that PCV2 plays a role in pneumonia and pleurisy in 10-24 weeks old fattening pigs, not only in herds with a high prevalence of PMWS, but also in herds with no clinical signs of PMWS.

Expression of cellular adhesion molecule 'OPCML' is down-regulated in gliomas and other brain tumours.

The four GPI-anchored cell adhesion molecules that exemplify the IgLON family are most highly expressed in the nervous system and associate to form up to six different heterodimeric 'Diglons' that can modify cell adhesion and inhibit axon migration. Recently, two members, OPCML and LSAMP, were identified as putative tumour suppressor genes in ovarian and renal carcinomas respectively. In this study, we investigated OPCML expression in nonneoplastic brain tissue and 35 brain tumours (18 glioblastoma multiformes, five anaplastic gliomas, five meningiomas, six metastases and one medulloblastoma) and four glioma cell lines using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). OPCML was highly expressed in cerebellum, less so in cerebral cortex, frontal lobe and meninges and was significantly reduced or absent in 83% of brain tumours and all cell lines compared with nonneoplastic whole brain. Two OPCML splice variants have been identified in humans, termed alpha1 and alpha2, but the latter has not been demonstrated in human neural tissues. Using PCR with specific primers, nonneoplastic brain and 3/6 of tested brain tumours expressed both splice variants, whereas the remaining brain tumours only expressed the alpha2 variant. Hypermethylation of the alpha1 OPCML promoter, associated with down-regulation of expression in ovarian tumours, did not correlate with expression levels in the subset of brain tumours tested, implying transcription of OPCML from an alternative promoter or a different mechanism of down-regulation. This study demonstrates that OPCML down-regulation occurs in the majority of brain tumours tested, warranting further investigation of OPCML and other IgLONs in the development and progression of brain tumours.

Expression of ADAMTS-8, a secreted protease with antiangiogenic properties, is downregulated in brain tumours.

Angiogenesis and extracellular matrix degradation are key events in tumour progression, and factors regulating stromal-epithelial interactions and matrix composition are potential targets for the development of novel anti-invasive/antiangiogenic therapies. Here, we examine the expression of ADAMTS-8, a secreted protease with antiangiogenic properties, in brain tissues. Using quantitative RT-polymerase chain reaction (PCR), high, equivalent expression of ADAMTS-8 was found in normal whole brain, cerebral cortex, frontal lobe, cerebellum and meninges. ADAMTS-8 expression in 34 brain tumours (including 22 high-grade gliomas) and four glioma cell lines indicated at least two-fold reduction in mRNA compared to normal whole brain in all neoplastic tissues, and no detectable expression in 14 out of 34 (41%) tumours or four out of four (100%) cell lines. In contrast, differential expression of TSP1 and VEGF was seen in nine out of 15 (60%) and seven out of 13 (54%) tumours, with no relationship in the expression of these genes. Immunohistochemistry and Western analysis indicated downregulation of ADAMTS-8 protein in >77% tumours. Methylation-specific PCR analysis of ADAMTS-8 indicated promoter hypermethylation in one out of 24 brain tumours (a metastasis) and three out of four glioma cell lines suggesting an alternative mechanism of downregulation. These data suggest a role for ADAMTS-8 in brain tumorigenesis, warranting further investigation into its role in regulation of tumour angiogenesis and local invasion.

Comparison of three polymerase chain reaction methods for routine detection of bovine herpesvirus 1 DNA in fresh bull semen.

Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection.

Serodiagnosis of neosporosis in individual cows and dairy herds: A comparative study of three enzyme-linked immunosorbent assays.

The performance of three enzyme-linked immunosorbent assays (ELISA) for detection of antibodies to Neospora caninum in bovine sera was evaluated by using various categories of sera. Two commercial ELISA methods, one based on chemically fixed intact tachyzoites and one based on a sonicate lysate of whole tachyzoites, were compared with an in-house ELISA based on a detergent lysate of whole tachyzoites. A brief description of the development of the latter ELISA is also given. There was good agreement among all three tests with regard to postabortion sera. By using acute-phase abortion sera from cows with confirmed N. caninum-induced and non-N. caninum-induced abortions, satisfactory levels of sensitivity and specificity were calculated for all tests. In addition, similar test results were obtained with postpartum samples from dams and calves. However, considerable differences were found between test results of sequential samples and cross-sectional and total-herd samples. Apparently, these discrepancies were due to different sensitivities of the tests for detection of low antibody levels in chronically infected animals. It is suggested that these differences were primarily due to the use of different antigens and different test sample dilutions. It is concluded that all tests are applicable as an additional diagnostic tool in cases of abortion in cattle and for monitoring of congenitally infected calves. For herd screening, the lysate-based ELISAs appear to be more adequate because of their higher sensitivities.

Effect of freezing on somatic cell count of quarter milk samples as determined by a Fossomatic electronic cell counter.

The objective of this study was to determine the effect of freezing and the duration of freezing on somatic cell count (SCC) in milk samples from quarters with no signs of clinical mastitis. Effect was determined by a fluoro-opto-electronic cell counter. Samples were frozen in a commercial freezer at approximately -20 degrees C. The milk samples were thawed at 1, 2, 3, 4, 7, 14, or 28 d. Another batch of samples was frozen 1 d, thawed, frozen again for 1 d, and then thawed. The SCC was determined on the fresh samples and after any one of the freezing periods. On average, the SCC were lower after all freezing periods than they were before freezing. However, the difference in SCC before and after freezing was small and relatively constant on the natural logarithm scale. The decrease in SCC was larger with longer freezing periods. When SCC thresholds of 200,000, 250,000, and 500,000 cells/ml were used to predict infection, freezing appeared to have little impact on the sensitivity and specificity of diagnostic parameters. Therefore, SCC, as determined by a fluoro-opto-electronic cell counter, determined for frozen quarter milk samples can be used in mastitis control and research programs.

The use of a polymerase chain reaction assay for the detection of bovine herpesvirus 1 in semen during a natural outbreak of infectious bovine rhinotracheitis.

Nasal swabs from two bulls at an artificial insemination (AI) station were submitted to our laboratory. The animals showed clinical signs of Infectious bovine rhinotracheitis (IBR), although the station was supposedly free of bovine herpesvirus 1 (BHV1). DNA of BHV1 was detected using a polymerase chain reaction (PCR). Subsequently nasal swabs from 100 animals that could have been in contact were submitted. BHV1 DNA was detected in swabs from 23 animals. Using the PCR, BHV1 could only be detected in a limited number of semen samples over a period of two months prior to the outbreak or two months after the outbreak. Also, not all animals that shed BHV1 from the nose harboured detectable BHV1 in the semen. Finally BHV1 was detected in the semen of one bull, approximately six weeks before seroconversion. Presently the PCR is being used as a means of quality control of fresh semen from bulls that are seropositive for BHV1. We are able to produce a result within 6 h after the semen samples have been submitted, allowing the AI-station manager to take measures before semen distribution in the event of a positive reaction. So far 11 out of 318 samples were shown to contain BHV1 DNA. In order to be able to interpret these results an interlaboratory comparative study is proposed. In countries endemically infected with BHV1 the PCR can be a cost-effective method to minimize the risk of transmitting virus by semen.

Behaviorally conditioned modulation of natural killer cell activity: enhancement of baseline and activated natural killer cell activity.

Natural killer (NK) cells comprise a sub-population of lymphocytes functionally defined by their ability to spontaneously lyse select tumor and virally infected cells. NK cell lytic function is sensitive to modulation by cytokines and neuroendocrine mediators. Behavioral conditioning trains an animal to cognitively associate a novel environmental cue (i.e. odor) with a physiologically active agent (i.e. Polylnosinic:PolyCytidilic acid, Poly I:C). Poly I:C induces interferon production resulting in activation of NK cells and enhanced NK cell lytic activity. Subsequent to behavioral conditioning, independent presentation of the odor should elicit similar responses (enhanced NK cell activity). We have shown that animals pre-exposed to the conditioning apparatus or manual restraint prior to behavioral conditioning demonstrate enhanced baseline NK cell activity or no effects respectively. To assess the influence of NK cell activation in conjunction with behavioral conditioning, we have re-presented the odor to animals with activated or baseline NK activity. Lymphocytes were then incubated for five days with IL-2 and cellular cytotoxic activity re-assessed. Behavioral conditioning significantly enhanced baseline and activated NK cell activity in animals previously exposed to the conditioning apparatus. No differences in lytic activity versus NK sensitive targets were observed following IL-2 activation. In contrast, animals manually restrained prior to conditioning showed no differences in baseline or in vivo activated NK activity, but previously non-activated lymphocytes stimulated with IL-2 demonstrated significantly higher lytic activity in conditioned animals re-presented with the odor. These results demonstrate central nervous system (CNS) modulation of NK cell activity and the presence of an interplay between cytokine activation and responsiveness to CNS immunoregulatory signals.

Behaviorally conditioned modulation of natural killer cell activity: effects of pre-conditioning manual restraint and apparatus exposure.

This study investigated behaviorally conditioned modulation of natural killer cell (NK) activity. One and three trial behavioral conditioning training trial designs were examined. In addition, the influence of pre-conditioning exposure to manual restraint and the conditioning apparatus upon behaviorally conditioned natural killer cell responses was assessed. Prior to one trial training, animals were exposed to either the conditioning apparatus, manual restraint, or were undisturbed. The animals were then trained to associate the conditioned stimulus (CS) and the unconditioned stimulus (US), and later re-presented with the CS. Behaviorally conditioned animals demonstrated significantly enhanced NK cell activity, no difference in NK activity, or significantly decreased NK activity, respectively. There was also a significant interaction between the manual restraint and behavioral conditioning interventions. Animals in a three training trial design were either manually restrained daily prior to and during behavioral conditioning or were left undisturbed. In this design behavioral conditioning significantly decreased NK cell activity and manual restraint significantly increased NK cell lytic activity. These data illustrate behaviorally conditioned decreases and increases in NK cell lytic activity dependant upon the experimental design. Behaviorally conditioned NK cell activity is influenced by additional behavioral interventions (i.e. manual restraint and novelty of conditioning environment) inherent within the behavioral conditioning procedures.

Production of alpha interferon in Cowdria ruminantium-infected cattle and its effect on infected endothelial cell cultures.

Cattle that resisted experimental heartwater infection caused by the rickettsia Cowdria ruminantium produced significant levels of circulating alpha interferon (IFN-alpha), whereas animals that died from heartwater did not. In vitro, recombinant bovine IFN-alpha was found to significantly reduce the yield of Cowdria organisms in bovine endothelial cells, but even at a high concentration (1,000 U/ml), IFN-alpha did not completely prevent the growth of Cowdria organisms in these cells. This limited inhibitory effect of IFN-alpha is in agreement with the in vivo situation where an infectious process has to take place to induce a protective immune response. Our results suggest that IFN-alpha produced in vivo in response to Cowdria infection may represent an efficient way to slow down the infection and allow the animal to mount a protective immune response. IFN-alpha is the first endogenously produced factor shown to have anti-Cowdria activity.

Progress in research on tick-borne diseases: theileriosis and heartwater.

The rapid population growth in subsaharan Africa necessitates a great increase in animal production in the more humid zones. Vector-borne diseases occurring in these zones will assume more importance, but are difficult to control. They include theileriosis and heartwater. Recent developments in research on these diseases are presented. Indigenous animal populations in endemic areas, subjected to natural selection, are far less susceptible than exotic stock. Heartwater, caused by the rickettsia Cowdria ruminantium, transmitted by Amblyomma ticks, causes high mortality in exotic ruminants. It has received much attention in recent years, partly because the disease has been introduced from Africa into the Caribbean and threatens the American mainland. Since the recent success of in vitro culture, much progress in research has been made, but so far prevention still relies mainly on acaricidal tick control; an infection and treatment method is used on a limited scale. Antigenic diversity is a complication for immunization procedures. Theileria parva (East Coast fever, Corridor disease and January disease) and T.annulata (Mediterranean or tropical theileriosis) are the most pathogenic of the 6 species of this protozoan genus that infect cattle. Great progress has been made in recent years in knowledge on the immunology, the epidemiology, the taxonomy and the chemotherapy of theileriosis. Intensive acaricidal tick control can now be supplemented by an attenuated schizont vaccine against T.annulata, while immunization against East Coast fever is carried out on a limited scale using virulent sporozoite infection and treatment. Research on recombinant vaccines is promising. Antigenic diversity in T.parva is a serious complication.

Role of interferons in infectious diseases in the bovine species: effect on viruses and rickettsias.

Successful protection was obtained with interferon treatment in experimental viral infections in the bovine species in a number of cases. The efficacy of the treatment against vaccinia virus infection and against rotavirus infection have been demonstrated. On the contrary, bovine herpes virus 1 (BHV 1-causing rhinotracheitis and part of the shipping fever complex) infections were not inhibited by interferon (IFN). The authors have undertaken a study in cattle in Zimbabwe to assess the role of interferon in the resistance of the animals to Cowdria ruminantium. A good correlation between production of interferon by the animal following the infection, and the resistance of the animals against the rickettsia was demonstrated. This pointed out the possible "adjuvant" role of interferons and other cytokines.

[The presence of antibodies against glycoprotein I (gI) of the virus of Aujeszky's disease in colostrum and milk of sows]. / De aanwezigheid van antistoffen tegen glycoproteïne I (gI) van het virus van de ziekte van Aujeszky in biest en melk van zeugen.

The possibility of using sow's milk to detect antibodies to the gI protein of Aujeszky's virus was studied by the present authors. Antibody titres in serum (collected less than 30 days ante partum) and colostrum from sixty-three sows were determined using a commercially available ELISA; twenty-four of thirty-seven animals vaccinated with gI-positive vaccines showed an antibody response in both colostrum and serum. The titre in colostrum was significantly higher than it was in serum. Antibodies could not be detected in serum or in colostrum of the twenty-six animals which were only vaccinated with gI-negative vaccines. Subsequently, anti-gI titres were determined in milk collected during lactation. Antibodies were also detectable in milk of animals showing high colostrum titres (greater than 128) for at least seventeen days. This period decreased as the initial colostrum titre became lower. Antibodies could still be detected in milk of animals vaccinated with gI-positive vaccine, which were sero-negative at the time of the experiment. Milk therefore seems to be a useful alternative to serum in detecting anti-gI antibodies in sows.

Genetics of resistance to the African trypanosomes. VI. Heredity of resistance and variable surface glycoprotein-specific immune responses.

The question of genetic linkage of parasite-specific immune responses to resistance to infection in experimental African trypanosomiasis was addressed. For this purpose, major histocompatibility complex-compatible resistant and susceptible inbred mouse strains and their F1 hybrid, F2 hybrid, and backcross offspring were infected with Trypanosoma brucei rhodesiense LouTat 1. Immunologic control of the first peak of parasitemia and survival times were the parameters measured. As we have reported previously (R. F. Levine and J. M. Mansfield, J. Immunol. 133:1564, 1984), B10.BR/SgSnJ mice are relatively resistant and controlled the growth of the infecting variant antigenic type (VAT) by mounting an antibody response to exposed epitopes of the variable surface glycoprotein (VSG). Fluctuating parasitemias resulting from sequential growth of different variable antigenic types occurred subsequently, and these mice died with a median survival time of 48 days. C3HeB/FeJ mice, relatively susceptible, did not control the infecting VAT and did not exhibit VSG-specific antibodies. These mice died with a median survival time of 22 days. The (B10.BR X C3H)F1 hybrids derived from crosses between resistant and susceptible mice all exhibited VSG-specific antibody responses and controlled the infecting VAT population. However, the median survival time of the F1 hybrids (24 days) was not significantly different from the survival time of the susceptible C3H parent. These findings demonstrate for the first time that antibody-mediated control of parasitemia is inherited as a dominant trait; that overall resistance, as measured by survival time, is inherited as a recessive trait (e.g., susceptibility is dominant); and that the two events segregate independently of one another. Further analyses of the inheritance of immunity and resistance (survival time) were made in which the F2 hybrid and backcross studies revealed that there are multiple genes controlling the VSG-specific antibody response as well as determining susceptibility. An extension of the present studies to a similar but non-major histocompatibility complex-mouse model system of resistance and susceptibility (C57BL/6J and C3H/HeJ mice, F1 hybrids, and 11 recombinant inbred B X H strains derived from them) was made in order to link the strain distribution patterns of known genetic markers with control of VSG-specific antibody responses or with control of susceptibility. Results of this study showed that resistance varied independently of the ability to control parasitemia with VSG-specific B cell responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetics of resistance to the African trypanosomes. VII. Trypanosome virulence is not linked to variable surface glycoprotein expression.

The question of linkage of virulence traits to variable surface glycoprotein (VSG) expression in African trypanosomiasis was addressed. Previously we demonstrated that daughter cells arising in mice infected with a genetically homogeneous trypanosome population of Trypanosoma brucei rhodesiense were more virulent than the infecting population (J. A. Inverso and J. M. Mansfield, J. Immunol. 130:412, 1983). These virulent trypanosomes expressed differences in surface phenotype compared with the infecting variant types, and we proposed that virulence may be "linked" to VSG expression. In the present study, however, we have shown that expression of virulence is independent of the VSG phenotype displayed by trypanosome populations. A VSG-identical but highly virulent subpopulation of T. b. rhodesiense LouTat 1 was derived by rapid subpassage and subcloning in immunosuppressed mice. The virulent LouTat 1A subclone derived in this manner killed B10.BR/SgSnJ mice in 3 to 4 days postinfection compared with approximately 60 days for the parent clone, LouTat 1. The virulent subclone LouTat 1A appears to express the same VSG as the less virulent LouTat 1 population, as determined by polyspecific and monoclonal antibody-binding assays, cross-protection tests, and amino acid sequence analyses of the N-terminal portion of the VSG molecules. When LouTat 1 and subclone LouTat 1A were injected into a heterologous host species, multiple variant antigenic types (VATs) arising from each inoculum were isolated and characterized. VATs derived from the virulent subclone were as uniformly virulent for B10.BR mice as LouTat 1A. In summary, these results demonstrate that trypanosome virulence, once expressed, is a stable phenotype that does not seem to be associated with a particular VSG phenotype, nor does virulence change with the expression of different VSG genes.

Trypanosoma brucei rhodesiense infection in mice prevents virus-induced diabetes: possible role of interferon and immunological mechanisms.

B10.BR, DBA/2, and BALB/c by J mice were infected with Trypanosoma brucei rhodesiense (Lou Tat clone 1). Subsequent infection with the D variant of encephalomyocarditis virus (EMC-D) resulted in no diabetes or encephalitis, even in the susceptible DBA/2 and BALB/c by J strains. Low levels of circulating interferon (IFN) were detected in trypanosome-infected mice at the time of EMC-D infection. All strains were severely immunosuppressed as a result of trypanosome infection, as evidenced by decreased virus-specific neutralizing antibody titers, compared to virus-infected controls. We attempted to simulate some aspects of T.b. rhodesiense infection in B10.BR mice by pretreating mice with cyclophosphamide and IFN prior to EMC-D infection. Immunosuppression by cyclophosphamide greatly enhanced the pathogenesis of EMC-D, while IFN protected against the diabetogenic effect of this virus. Our results indicate that: (i) T.b. rhodesiense infection inhibited EMC-D-induced diabetes, (ii) this inhibition was not due solely to the immunosuppression generated by the trypanosome infection, and (iii) IFN generated by the trypanosome infection could play some protective role in the inhibition of EMC-D-induced diabetes by trypanosome infection.