White County Highway Garage Project Development and outcomes

In this presentation we will discuss the conducted project needs assessment; site search and selection process; ancillary benefits to the county and the surrounding communities; site challenges; design development strategies and lessons learned; the anticipated time line to duplicate this project; and the contractual methods used to deliver the project. We will then wrap up with a photographic campus tour of the final outcomes and improvements

Experimental Canine Leptospirosis Caused by Leptospira Interrogans Servars Pomona and Bratislava

Objective—To evaluate gross, histopathologic, and serum biochemical findings caused by Leptospira interrogans serovars pomona and bratislava inoculated in dogs. Animals—Twenty-seven 8-week-old female Beagles. Procedure—Dogs were randomly assigned to challenge or control groups. Challenge groups were conjunctivally inoculated on 3 successive days with 5 X 107 L interrogans serovar pomona (n = 12) or serovar bratislava (11). Clinical signs were recorded throughout the experiment, and clinical pathology assays, bacteriologic culture, and necropsies (6 or 7 dogs necropsied at each time point) were done on postinoculation day (PID) 7, 10, 14, and 20. Results—Infection could not be confirmed in any serovar bratislava–inoculated dog, and control dogs remained healthy throughout the experiment. Positive culture and fluorescent antibody test results were confirmed in 11 of 12 serovar pomona–inoculated dogs. Fever and lethargy starting at PID 7 were the most common clinical signs in serovar pomona–infected dogs. On day 10, gross lesions included multifocal renal and pulmonary hemorrhage and perirenal edema. Serovar pomona–inoculated dogs had histopathologic lesions including hepatitis, interstitial nephritis, and pneumonia at PID 7, 10, 14, and 20. Increases in BUN, anion gap, and bilirubin concentration occurred on PID 10, 14, and 20. Platelet counts in dogs with positive results of bacteriologic culture were decreased from baseline values on PID 10, 12, and 14. Conclusions and Clinical Relevance— Conjunctival inoculation with L interrogans serovar pomona resulted in a high rate of infection with concomitant hemorrhagic and inflammatory lesions of the kidneys, liver, and lungs

First isolation of 'Brachyspira hampsonii' from pigs in Europe

Swine dysentery in Europe is classically attributed to Brachyspira hyodysenteriae. However, other Brachyspira species have been increasingly associated with intestinal disorders in pigs. This case report describes the first diagnosis of a “Brachyspira hampsonii” infection in European pigs. In a routine quarantine monitoring protocol, two gilts were presented for necropsy, in which soft watery non-haemorrhagic colonic content was found. Microbial culture from the colonic content and from faecal samples revealed the presence of strongly haemolytic, ring-phenomenon positive spirochetes indicative for Brachyspira hyodysenteriae. A diagnostic commercial PCR could not confirm the presence of B. hyodysenteriae. Phenotypic characterisation and PCRs targeting the 16S rRNA, 23S rRNA, nox, hlyA and tlyA genes of different swine-related Brachyspira spp. were performed. Phylogenetic analysis of sequences of the partial nox and 16S rRNA genes and multi locus sequence typing demonstrated that the isolates in this case were “B. hampsonii” isolates. This case report shows that the diagnosis of infections caused by new, emerging Brachyspira species is not self-evident and that the combination of microbial culture and PCR is recommended, completed with more extensive genotyping if necessary

Conservation of the S10-spc-α Locus within Otherwise Highly Plastic Genomes Provides Phylogenetic Insight into the Genus Leptospira

S10-spc-α is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-α locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-α locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-α locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously

Comparison of Two Multilocus Sequence Based Genotyping Schemes for Leptospira Species

Two independent multilocus sequence based genotyping schemes (denoted here as 7L and 6L for schemes with 7 and 6 loci, respectively) are in use for Leptospira spp., which has led to uncertainty as to which should be adopted by the scientific community. The purpose of this study was to apply the two schemes to a single collection of pathogenic Leptospira, evaluate their performance, and describe the practical advantages and disadvantages of each scheme. We used a variety of phylogenetic approaches to compare the output data and found that the two schemes gave very similar results. 7L has the advantage that it is a conventional multi-locus sequencing typing (MLST) scheme based on housekeeping genes and is supported by a publically accessible database by which genotypes can be readily assigned as known or new sequence types by any investigator, but is currently only applicable to L. interrogans and L. kirschneri. Conversely, 6L can be applied to all pathogenic Leptospira spp., but is not a conventional MLST scheme by design and is not available online. 6L sequences from 271 strains have been released into the public domain, and phylogenetic analysis of new sequences using this scheme requires their download and offline analysis

Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials

Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance