Is moving from targeted culling to BCG-vaccination of badgers (Meles meles) associated with an unacceptable increased incidence of cattle herd tuberculosis in the Republic of Ireland? A practical non-inferiority wildlife intervention study in the Republic of Ireland (2011-2017).

Bovine tuberculosis (BTB) remains as a costly disease of cattle-herds in the Republic of Ireland (ROI). This persistence is partially attributable to the presence of M. bovis infection in a wildlife reservoir, the European badger (Meles meles). Thus, both area-wide and limited-area targeted-badger-culling have been part of the ROI-BTB control/eradication program to help reduce the future incidence of a cattle-herd BTB breakdown (i.e. a "new herd-level occurrence of BTB"). However, neither badger-culling practice can be sustained as a major component in the ongoing BTB eradication program in the ROI. Vaccination of badgers with Bacille Calmette-Guérin (BCG) has been proposed as an alternative to badger culling. Thus, in 2011, a five-year non-inferiority study was implemented in seven counties in the ROI. This study was designed to compare and contrast the cattle-herd-BTB-incidence in areas where intramuscular badger vaccination would be implemented versus the cattle-herd-BTB-incidence in the remaining area of the same county where targeted-badger-culling was maintained as the standard treatment response to probable badger-sourced BTB breakdowns. Our outcome of interest was a new cattle-herd-BTB-episode (breakdown) with a total of >2 standard skin-test (SICTT) reactors detected during the episode. Treatments (badger vaccination or targeted badger culling) were cluster allocated based on where the majority of the herd owner's land was located. To assess the impact of the two treatments, we compared the incidence-risk, of our defined outcome, for cattle herds in the area under vaccination to the outcome incidence-risk for cattle herds in the remainder of the same county after 4 and 5 years of having implemented badger vaccination. A random-effects logit model with adjustment for clustering by treatment, and statistical control of herd-type, herd-size and five-year prior-BTB-episode history was used for our analyses. Although not included in the logistic model, a relative badger density metric based on the annual number of badgers captured-per-sett-night of capturing effort was developed for each treatment area; this metric indicated that relative badger density was approximately 40 % higher in vaccination areas than in the targeted badger-culling areas during our study. Overall, our study results indicated that vaccination was not inferior to targeted badger-culling in four counties and badger vaccination was deemed to produce ambivalent results in one (County Cork North) of the seven study sites in the ROI. A post-study investigation, in County Galway, where vaccination was deemed inferior to target culling, revealed that widespread purchases of cattle from a nearby cattle mart, by herd owners in the vaccination-area, was associated with the increased herd and vaccination-area risk of BTB. No single "biasing hypothesis" was evident for the apparent vaccine inferiority in the second study site (County Monaghan) where vaccination was deemed inferior to targeted culling; hence no further investigations were conducted.

Risk of bovine tuberculosis for cattle sold out from herds during 2005 in Ireland.

A retrospective cohort study was conducted to determine the risk of bovine tuberculosis (TB) among animals sold out from herds that were free to trade animals during the year 2005 according to their bovine TB testing history during the year 2005. The present study sample comprised of 338,960 animals, of which 124,360 animals were sold out from herds that were restricted from trading at some stage during 2005 (bovine TB 'exposed') and 214,600 animals that were sold from herds which did not have their trading status withdrawn in 2005 (bovine TB 'non-exposed'). The overall risk of a diagnosis of bovine TB during the two-year period after the animals were sold out was 0.69 per cent. The odds of bovine TB were 1.91 higher for animals sold out from bovine TB 'exposed' herds compared with animals sold out from bovine TB 'non-exposed' herds (OR 95 per cent CI: 1.76 to 2.07, P<0.0001). Ten per cent of animals identified during field surveillance with bovine TB did so less than two months after being sold out in 2005, and similarly, 10 per cent of the animals classified as bovine TB positive by finding a bovine TB lesion at slaughter did so within 25 days (or less) of being sold out in 2005.

The risk of a positive test for bovine tuberculosis in cattle purchased from herds with and without a recent history of bovine tuberculosis in Ireland.

A retrospective cohort study was conducted to assess if cattle sold from Irish dairy herds within 7 months of herd de-restriction (clearance to trade) from a bovine-tuberculosis (BTB) episode had an excess risk of testing positive for BTB during the following 2 years, and to determine other risk factors associated with this outcome. If possible, a predictive metric for herds at high risk of selling future BTB-positive cattle would be generated. The unexposed cohort included all cattle sold within 7 months of the annual herd test in a random sample of dairy herds that did not test positive for BTB in 2003. The exposed cohort consisted of all cattle sold within 7 months of the date of de-restriction in all dairy herds that cleared a BTB episode in 2003. Only cattle sold from herds that were initially found to test positive for BTB using the single intradermal comparative tuberculin test (SICTT)-and not due to discovery of a BTB-positive animal at slaughter-were included as exposed cattle. To aid in the development of a predictive metric, the exposed cohort was subcategorized based on the number of reactors to the SICTT in the herd of origin during the BTB episode immediately prior to sale. The final exposure categories of 0 (unexposed), 1-7, and >or=8 total reactors were considered the unexposed, mildly exposed, and severely exposed cohorts, respectively. A multivariable logistic regression model was fit to the final BTB status of the animal using a generalized estimating equation method (GEE), assuming an exchangeable correlation structure of animals within herds, and using robust standard errors. Exposure level and the other available herd- and animal-level information were modeled. After controlling for other risk factors including the size of the herd of origin and the sex and age of the animal, the three-level exposure variable significantly improved the model (based on a change in Quasi-Akaike Information Criteria of 2.2) and demonstrated a trend of increasing risk of a future positive BTB test with increasing exposure category. The severely exposed cohort of animals had significantly higher risk of a future positive BTB test than the unexposed cohort (OR=1.78, p=0.030).

Quantification of the relative efficiency of factory surveillance in the disclosure of tuberculosis lesions in attested Irish cattle.

In Ireland, factory surveillance of cattle for gross lesions is an important supplementary method for detecting herds infected with bovine tuberculosis (tb), and in recent years between 27 and 46 per cent of all new herd breakdowns in any year have been detected by this method. The aim of this study was to determine the relative efficiency of factories in detecting lesions among attested cattle slaughtered during 2003 and 2004. National databases were available on animal slaughter, programmes of tuberculin testing for bovine tb and laboratory confirmation of suspected lesions. Factories were ranked according to their submission risk (number of animals submitted with lesions/number of attested animals killed) and confirmation risk (number of animals with laboratory-confirmed lesions/number of animals submitted with lesions), adjusting for the risk profile of the animals slaughtered, including potential confounding factors such as their age and sex, whether they were purchased or homebred, the test history of their herd, the prevalence of bovine tb in the area and the season of slaughter. Approximately 3.7 million cattle were slaughtered in 42 Irish export-licensed factories during the two years. Complete data were available for 2,374,987 animals from 84,510 attested herds in 2845 District Electoral Divisions. Samples from 7398 animals with suspected tb lesions were submitted for laboratory examination; 4767 (64.4 per cent) were positive, 2011 were negative and 620 were inconclusive. The average unadjusted submission risk for all the factories was 22 per 10,000, ranging from 0 to 58 per 10,000. The unadjusted factory confirmation risk (excluding factories that had sent in fewer than 10 lesions) varied between 34.3 per cent and 86.3 per cent. The unadjusted and adjusted submission and confirmation risks were highly correlated, and animal-related factors (including their characteristics and origin) therefore did not contribute to the variations in factory-level submission and confirmation risks.

Quantifying badger exposure and the risk of bovine tuberculosis for cattle herds in county Kilkenny, Ireland.

The objectives of the study were to quantify the levels of badger exposure for cattle and to test the hypothesis that increased badger exposure does not increase the risk of bovine tuberculosis (BTB) in a herd. Information that became available from the targeted removal of badgers over the study period, and from a badger-removal project in county Kilkenny, during 1996-1999 was used. The specific location of cattle within each farm, and the length of time that cattle spent in each farm field during the grazing season, and in the barnyard during winter, was used to build an exposure coefficient to quantify the amount of badger exposure that cattle encountered either on pasture or in the barn. The study design was a matched case-control study in which the control herds were selected using incidence density sampling. During the 4-year study period, 543 badgers were removed and of these 96 badgers were classified as tuberculosis positive; 96 BTB herd breakdowns occurred. There was a significant association between case herds and having a higher badger sett exposure coefficient during 1996-1998. No significant association between case herds and having a higher exposure coefficient based on the number of badgers, or the number of tuberculous badgers, during September 1997-December 1999 was found.

Breakdown severity during a bovine tuberculosis episode as a predictor of future herd breakdowns in Ireland.

A retrospective cohort study of Irish cattle herds investigated whether the severity of a herd's bovine tuberculosis (BTB) breakdown was a predictor of the hazard of a future BTB breakdown in that herd. Data on 10,926 herds not having had BTB in 1995 (the "non-exposed" group) were obtained using a 10% random sample from all herds without BTB in 1995. Data on 6757 herds that had a new BTB breakdown in 1995 (the "exposed" group) were obtained and categorized into five increasing exposure-severity classes based on the total number of standard reactors (to the single intra-dermal comparative cervical tuberculin test) detected during the breakdown. Exposed herds were deemed to be free of BTB after they passed a 6-month check test; non-exposed herds were deemed free as of the date of the first negative herd-test in 1995. In the 5 years after 1995, 18% of the non-exposed herds had a BTB breakdown, whereas 31% of the exposed herds had a subsequent breakdown. Relative to the hazard for non-exposed herds, the hazard for the first future singleton standard reactor breakdown, was 1.6-times higher for exposed herds with only 1 standard reactor in 1995, and 1.8-times higher for those exposed herds with 4-8 standard reactors during the 1995 episode. When the outcome for future breakdowns was 2 or more standard reactors, the hazard ratios ranged from 1.6 for exposed herds with only 1 standard reactor in 1995 up to 2.9 in exposed herds with 8 or more standard reactors during the 1995 episode. The latter hazard ratio varied over time, decreasing to 1.7 after 3 years of risk. The hazard of a future BTB breakdown increased directly with number of cattle in the herd, a positive history of previous BTB in the herd, and the local herd prevalence of BTB. The presence of confirmed BTB lesions in reactor cattle was not predictive of the future breakdown hazard when the effects of other factors were controlled.

Characterization of the minimal DNA binding domain of the human papillomavirus e1 helicase: fluorescence anisotropy studies and characterization of a dimerization-defective mutant protein.

The E1 helicase of papillomaviruses is required for replication of the viral double-stranded DNA genome, in conjunction with cellular factors. DNA replication is initiated at the viral origin by the assembly of E1 monomers into oligomeric complexes that have unwinding activity. In vivo, this process is catalyzed by the viral E2 protein, which recruits E1 specifically at the origin. For bovine papillomavirus (BPV) E1 a minimal DNA-binding domain (DBD) has been identified N-terminal to the enzymatic domain. In this study, we characterized the DBD of human papillomavirus 11 (HPV11), HPV18, and BPV E1 using a quantitative DNA binding assay based on fluorescence anisotropy. We found that the HPV11 DBD binds DNA with an affinity and sequence requirement comparable to those of the analogous domain of BPV but that the HPV18 DBD has a higher affinity for nonspecific DNA. By comparing the DNA-binding properties of a dimerization-defective protein to those of the wild type, we provide evidence that dimerization of the HPV11 DBD occurs only on two appropriately positioned E1 binding-sites and contributes approximately a 10-fold increase in binding affinity. In contrast, the HPV11 E1 helicase purified as preformed hexamers binds DNA with little sequence specificity, similarly to a dimerization-defective DBD. Finally, we show that the amino acid substitution that prevents dimerization reduces the ability of a longer E1 protein to bind to the origin in vitro and to support transient HPV DNA replication in vivo, but has little effect on its ATPase activity or ability to oligomerize into hexamers. These results are discussed in light of a model of the assembly of replication-competent double hexameric E1 complexes at the origin.

Characterization of the DNA-binding properties of the origin-binding domain of simian virus 40 large T antigen by fluorescence anisotropy.

The affinity of the origin-binding domain (OBD) of simian virus 40 large T antigen for its cognate origin was measured at equilibrium using a DNA binding assay based on fluorescence anisotropy. At a near-physiological concentration of salt, the affinities of the OBD for site II and the core origin were 31 and 50 nM, respectively. Binding to any of the four 5'-GAGGC-3' binding sites in site II was only slightly weaker, between 57 and 150 nM. Although the OBD was shown previously to assemble as a dimer on two binding sites spaced by 7 bp, we found that increasing the distance between both binding sites by 1 to 3 bp had little effect on affinity. Similar results were obtained for full-length T antigen in absence of nucleotide. Addition of ADP-Mg, which promotes hexamerization of T antigen, greatly increased the affinity of full-length T antigen for the core origin and for nonspecific DNA. The implications of these findings for the assembly of T antigen at the origin and its transition to a non-specific DNA helicase are discussed.

Characterization of recombinant HPV6 and 11 E1 helicases: effect of ATP on the interaction of E1 with E2 and mapping of a minimal helicase domain.

To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K(m)(ATP) of 12 microm for HPV6 E1 and 6 microm for HPV11 E1. Interestingly, the value of K(m)(ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain.

Identification of domains of the human papillomavirus type 11 E1 helicase involved in oligomerization and binding to the viral origin.

The E1 helicase of papillomavirus is required, in addition to host cell DNA replication factors, during the initiation and elongation phases of viral episome replication. During initiation, the viral E2 protein promotes the assembly of enzymatically active multimeric E1 complexes at the viral origin of DNA replication. In this study we used the two-hybrid system and chemical cross-linking to demonstrate that human papillomavirus type 11 (HPV11) E1 can self-associate in yeast and form hexamers in vitro in a reaction stimulated by single-stranded DNA. Self-association in yeast was most readily detected using constructs spanning the E1 C-terminal domain (amino acids 353 to 649) and was dependent on a minimal E1-E1 interaction region located between amino acids 353 and 431. The E1 C-terminal domain was also able to oligomerize in vitro but, in contrast to wild-type E1, did so efficiently in the absence of single-stranded DNA. Sequences located between amino acids 191 and 353 were necessary for single-stranded DNA to modulate oligomerization of E1 and were also required, together with the rest of the C terminus, for binding of E1 to the origin. Two regions within the C-terminal domain were identified as important for oligomerization: the ATP-binding domain and region A, which is located within the minimal E1-E1 interaction domain and is one of four regions of E1 that is highly conserved with the large T antigens of simian virus 40 and polyomavirus. Amino acid substitutions of highly conserved residues within the ATP-binding domain and region A were identified that reduced the ability of E1 to oligomerize and bind to the origin in vitro and to support transient DNA replication in vivo. These results support the notion that oligomerization of E1 occurs primarily through the C-terminal domain of the protein and is allosterically regulated by DNA and ATP. The bipartite organization of the E1 C-terminal domain is reminiscent of that found in other hexameric proteins and suggests that these proteins may oligomerize by a similar mechanism.

Role of the ATP-binding domain of the human papillomavirus type 11 E1 helicase in E2-dependent binding to the origin.

Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37 degrees C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.

Understanding the molecular mechanism of viral resistance to peptidomimetic inhibitors of ribonucleotide reductase.

Herpes simplex virus (HSV) encodes a ribonucleotide reductase which provides high levels of deoxynucleotides necessary for replication of viral DNA in infected cells. The enzyme is composed of two distinct subunits, R1 and R2, whose association is required for enzymatic activity. Compounds that mimic the C-terminal amino acids of the HSV ribnucleotide reductase R2 subunit inhibit the enzyme by preventing the association of R1 and R2. Moderate resistance to one of these inhibitors, BILD 733, has been generated in cell culture. This resistance is the result of two point mutations in R1, P1090L and A1091S. Here we report on the binding of additional peptidomimetic inhibitors with altered functional groups to these mutants. This study has made it possible, in the absence of a crystal structure for this enzyme, to define the molecular mechanism by which these two mutations cause the observed resistance. Mutation of proline 1090 to leucine causes a conformational shift in the R1 inhibitor binding site. Mutation of alanine 1091 to serine weakens a specific binding interaction with the hydrophobic carboxy terminus of both R2 and inhibitors. Potential limitations on the degree of viral resistance possible by each resistance mechanism are discussed.

Meropenem versus tobramycin plus clindamycin for treatment of intraabdominal infections: results of a prospective, randomized, double-blind clinical trial.

The efficacy of meropenem was compared to that of the combination of tobramycin plus clindamycin (T/C) in a multiinstitutional clinical trial of treatment for patients suffering intraabdominal infection. Among the 177 patients enrolled and randomized, 127 were clinically evaluable and 86 were microbiologically evaluable. Analysis of data on an intent-to-treat basis for all randomized patients and on the basis of a successful outcome (absence of any infection) for clinically evaluable patients failed to detect any difference in efficacy between the two treatments. Infection was cleared in 92% of meropenem- and 89% of T/C-treated clinically evaluable patients. Eradication of pathogens also was similar in the two treatment groups. Overall, adverse drug experiences were comparable between the two treatment groups, with the exception of an increase in serum creatinine level (which occurred more frequently in patients receiving T/C). Meropenem appears to be efficacious for the treatment of intraabdominal infections.

Treatment of nonocclusive mesenteric infarction after cardiopulmonary resuscitation.

Nonocclusive mesenteric infarction is a potentially lethal complication that may follow successful resuscitation of cardiopulmonary arrest. This case demonstrates that the manifestations may be delayed and that urgent surgical intervention may be lifesaving despite the precarious status of these patients. Vigilance for several days is mandatory.

Thrombotic complications of heparin therapy: including six cases of heparin--induced skin necrosis.

Thrombotic complications of heparin administration were observed in eight patients during a two year period. At sites of subcutaneous heparin injection, six patients developed areas of the skin and subcutaneous necrosis. Systemic thrombotic events and thrombocytopenia were observed in two of these patients when they received intravenous heparin and in two other patients who did not have primary skin necrosis. The complications included peripheral ischemia in three patients (two requiring amputation), myocardial infarction in two, and a cerebral infarction in one. All patients were receiving heparin for at least six days before complications occurred. Seven patients received heparin of bovine origin. Heparin-induced in vitro platelet aggregation was present in all six of the eight patients tested. (It was marked in four of these patients). It is theorized that skin necrosis and the other thrombotic complications observed are the result of heparin-induced in vivo platelet aggregation followed by intravascular thrombosis. Heparin-induced skin necrosis is a rare but serious hazard encountered with prophylactic heparin regimens. If heparin-induced thrombosis is present, the further use of heparin is contraindicated in most instances.