Pathogenesis of leptospirosis: the influence of genomics

Leptospirosis is the most widespread zoonosis worldwide and is caused by serovars of pathogenic Leptospira species. The understanding of leptospiral pathogenesis lags far behind that of many other bacterial pathogens. Current research is thus directed at identification of leptospiral virulence factors. Saprophytic Leptospira species are environmental organisms that never cause disease. Comparative genomics of pathogens and saprophytes has allowed the identification of more than 900 genes unique to either Leptospira interrogans or Leptospira borgpetersenii; these genes potentially encode virulence-associated proteins. However, genes of unknown function are over-represented in this subset of pathogen-specific genes, accounting for 80% and 60% of open reading frames, respectively. This finding, together with the absence of virulence factor homologues among the proteins of known function, suggests that Leptospira possesses unique virulence mechanisms. Whole genome microarray studies have identified genes whose expression is differentially regulated under a range of simulated in vivo conditions, such as physiological temperature and osmolarity, low iron levels, and the presence of serum. The subset of genes identified by these studies is likely to include virulence factors. However, most such genes encode proteins of unknown function, consistent with the hypothesis that leptospiral virulence genes do not have homologues in other bacterial species. The recent development of mutagenesis systems for pathogenic Leptospira spp. has allowed the screening of defined mutants for attenuation of virulence in animal infection models and has identified definitively for the first time a range of virulence factors, including lipopolysaccharide, flagella, heme oxygenase, and the OmpA-family protein, Loa22. Interestingly, inactivation of a number of genes hypothesised to encode virulence factors based on in vitro virulence-associated properties did not result in attenuation of virulence, suggesting a degree of functional redundancy in leptospiral pathogenic mechanisms.     

Serological detection of multiple retroviral infections in cattle: bovine leukemia virus, bovine syncytial virus and bovine visna virus

Individual experimental animals used in our studies on bovine leukemia virus (BLV) are routinely screened for the presence of antibodies to the three bovine lymphotropic retroviruses. We utilized these screening methods to examine frozen sera from eight herds for antibodies to BLV, bovine visna virus (BVV) and bovine syncytial virus (BSV). Serum samples from 235 animals in four dairy and four beef herds were analyzed. Detection methods used included indirect fluorescent antibody tests of virus-infected cell cultures (BLV, BSV, BVV) and agar gel immunodiffusion (BLV). Sera from the BLV-infected animals in the dairy herds showed the highest single (50%, 49/97) and multiple (30%, 29/97) infections compared with 5% (7/138) and less than 1% (1/138), respectively in the beef herds. Single BVV infections were not detected in the dairy herds, but 11% (11/97) of the sera contained antibodies to BVV plus BLV or BSV. Five sera from beef cattle had antibodies only to BVV and four were obtained from one herd. Only one beef serum of the 138 tested demonstrated multiple antibodies (BLV, BVV).     

Long-lasting enhancement of CYP activity after discontinuation of repeated administration of phenobarbital in dogs

We investigated how long in vivo hepatic cytochrome P450 (CYP) activity is enhanced even after discontinuation of repeated oral administration of phenobarbital (PB) in dogs using antipyrine clearance, which reflects hepatic CYP activity. A single antipyrine (5 mg/kg) was administered intravenously before and 34 days after the repeated oral administration of PB (5 mg/kg, bid) and 2, 4, 6, and 8 weeks after the discontinuation of PB in 5 dogs. Antipyrine clearance was increased by the repeated administration of PB, and remained increased 2 and 4, but not 6 and 8 weeks after the discontinuation of PB. The result suggests that hepatic CYP activity was enhanced by the repeated administration of PB, and this enhancement may last for at least 4 weeks even after its discontinuation.     

Mechanical comparison of an interlocking nail locked with conventional bolts to extended bolts connected with a type-IA external skeletal fixator in a tibial fracture model

OBJECTIVE: To compare the structural properties of interlocking nails (ILNs) locked with bolts (ILNb) to ILN locked with extended bolts connected with a type-IA external skeletal fixator (ILN-ESF) in a fracture gap model. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Synthetic tibial bone substitutes. METHODS: Custom-made synthetic tibial bone substitutes were implanted with standard ILNs locked with either bolts or extended bolts connected to an external skeletal fixation (ESF). Constructs were tested in torsion, bending, and axial compression (n=4/testing mode). Data, consisting of construct compliance and associated deformation, were compared using t-tests. RESULTS: The ILN-ESF construct compliance and deformation were significantly less than those of the ILNb construct in torsion, bending, and compression (P<.001). Slack was present in the ILNb construct under torsion and bending, but not in the ILN-ESF construct, regardless of testing mode. CONCLUSIONS: Substitution of locking bolts with extended bolts connected to an ESF significantly reduced the construct compliance and overall deformation in torsion, bending, and compression. Furthermore, the inherent slack of the ILNb was eliminated by the use of an ESF in torsion and bending. CLINICAL RELEVANCE: The improvement in structural properties of the ILN-ESF constructs could diminish interfragmentary motion at the fracture site and potentially improve bone healing.     

Genetic analysis of virulence factors of Mannheimia (Pasteurella) haemolytica A1

Sixteen 3-week-old calves were intratracheally inoculated with Mycoplasma bovis. Follow-up consisted of regular bronchoalveolar lavages (BALs) and clinical examinations. Animals were slaughtered from 4 to 21 days after inoculation. Counts were made of the mycoplasmas and other bacteria systematically isolated from the BAL liquids and lung lobes after slaughter. On the 6th day, spectinomycin 20mg/kg was given intramuscularly in three repeated doses at 24h intervals to six randomly chosen calves. All animals had developed a persistent M. bovis infection with a maximum BAL count on the 6th day (start of treatment). Co-occuring Pasteurella multocida infection was found in most animals with a maximum rate on the 14th day. The extent of lung surface lesions varied widely (0-64%) but was greater in the later slaughtered calves. Average counts of M. bovis and P. multocida in the BAL liquids were lower in treated calves than in untreated ones but the difference was not statistically significant. However, M. bovis and P. multocida counts in the lungs of the treated group were significantly lower than in the untreated group (p=0.003 and 0.009, respectively).     

Effects of sugar cane extract on pseudorabies virus challenge of pigs

This experiment aimed to evaluate the efficacy of sugar cane extract (SCE) on the modulation of porcine immunity against pseudorabies virus (PrV) infection. Twelve-week-old experimental pigs were fed with SCE (500 mg/kg of body weight per day) for 3 days and challenged with PrV (2 x 10(5) TCID(50)) on the second day. Pigs that were only challenged with PrV and without SCE-treatment served as controls. The leukocyte functional assays were performed on the 7th and 14th day post-PrV challenge. Our results showed a significant enhancement (P<0.05) of natural killer cytotoxicity, lymphocyte proliferation, phagocytic function of monocytes, and interferon-gamma (IFN-gamma) production of CD4(+) and gammadelta T cells in the SCE-treated pigs compared with the controls. In addition, SCE administration reduced the severity of clinical signs and brain lesion in the course of disease in PrV-challenged pigs. SCE-treated pigs showed a 12% growth enhancement compared with untreated controls. SCE administration had an immunostimulating effect on porcine immunity that may subsequently enhance protective activities against PrV infection which may be extensively applied in field for the prevention of infections.     

Herd-level risk factors for subclinical Salmonella infection in European finishing-pig herds

Our objective was to find herd factors associated with pigs testing seropositive for Salmonella. Data were collected from 359 finishing-pig herds in Germany, Denmark, Greece, The Netherlands and Sweden, between 1996 and 1998. Pigs fed non-pelleted feed (dry or wet) had 2- and 2.5-times lower odds of seropositivity, compared to pigs fed pelleted feed. The protective effect of non-pelleted feed over pelleted feed may be ascribed to the structure and composition. Also, pigs that were given whey (to drink or as the liquid part of the diet) had 2.6-times lower odds to test seropositive than pigs not getting whey. Pigs produced in batches in herds with hygienic-lock facilities had >3-times lower odds for testing seropositive compared to pigs in herds where only one or neither factor was present. In herds where the caretaker(s) washed hands consistently before tending to the animals, pigs had 1.5-times lower odds of seropositivity than pigs in herds where the caretaker did not. Pigs which were able to have snout contact with pigs in neighbouring pens (because pen separations were either open or too low) had 1.7-times higher odds to test seropositive compared to pigs for which such contact was prevented. Pigs in herds recruiting from more than three supplier herds had three-times higher odds to test seropositive than pigs in herds which breed their own replacement stock or recruit from a maximum of three supplier herds.     

Antihyaluronidase action of ellagic acid effectively prevents polyspermy as a result of suppression of the acrosome reaction induced by sperm-zona interaction during in vitro fertilization of porcine oocytes

The present study was conducted to examine the effects of three tannin relatives (tannic acid, TA; gallic acid, GA; and ellagic acid, EA) on antihyaluronidase and reactive oxygen species (ROS) scavenging activity, in vitro fertilization (IVF) parameters, and the acrosome reaction (AR) induced by sperm-zona interaction. Among the three tannin relatives, TA and EA showed the strongest potency for blocking the hyaluronidase activity of boar sperm, with concentration-dependent inhibition over the range of 2-10 microg/ml. In contrast, ROSs were effectively scavenged by TA and GA, but not EA. When cumulus-free oocytes were inseminated in IVF medium containing 5 microg/ml of the tannin relatives, polyspermy was significantly reduced by TA and EA (32 and 29%, respectively) compared with oocytes treated with or without GA (51 and 69%, respectively) under conditions that maintained a high sperm penetration rate (P<0.05). Interestingly, induction of the AR by treatment of preincubated sperm with progesterone was blocked by TA and GA as a result of their higher levels of ROS scavenging activity, while EA, which possessed weak ROS scavenging activity, did not disturb induction of the AR with progesterone. However, the incidence of AR induced by sperm-zona interaction was significantly decreased by the strong antihyaluronidase actions of TA and EA compared with that in the absence of these compounds. Treatment with the compounds caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization nor a reduction in acrosomal proteolytic activity or the number of zona-bound sperm. These findings suggest that the antihyaluronidase action of EA effectively prevents polyspermy by suppression of AR functionality induced by sperm-zona interaction and that hyaluronidase intervention is therefore required during porcine IVF.