Development of a Rapid Multiplex PCR Assay To Genotype Pasteurella multocida Strains by Use of the Lipopolysaccharide Outer Core Biosynthesis Locus

Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the "gold standard." The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains.

Anthranilate Hydroxylase from Aspergillus niger: Evidence for the Participation of Iron in the Double Hydroxylation Reaction

Evidence was obtained for the participation of iron in the double hydroxylation reaction catalyzed by anthranilate hydroxylase from Aspergillus niger (UBC 814). Omission of iron from the growth medium gave inactive preparations of anthranilate hydroxylase which could be reactivated by incubating the enzyme preparations with ferric citrate. The enzyme was susceptible to inhibition by metal chelating agents. The Ki for o-phenanthroline, which inhibited the enzyme activity non-competitively with respect to anthranilate, was calculated to be 0.9 mM. The inhibition by o-phenanthroline was counteracted by ferric complexes such as ferric-ethylenediaminetetraacetic acid and ferric citrate. Anthranilate afforded protection against inhibition by o-phenanthroline.

Antimicrobial susceptibility of Histophilus somni isolated from clinically affected cattle in Australia

This study investigated antimicrobial resistance traits, clonal relationships and epidemiology of Histophilus somni isolated from clinically affected cattle in Queensland and New South Wales, Australia. Isolates (n = 53) were subjected to antimicrobial susceptibility testing against six antimicrobial agents (ceftiofur, enrofloxacin, florfenicol, tetracycline, tilmicosin and tulathromycin) using disc diffusion and minimum inhibitory concentration (MIC) assays. Clonal relationships were assessed using repetitive sequence PCR and descriptive epidemiological analysis was performed. The H. somni isolates appeared to be geographically clonal, with 27/53 (47%) isolates grouping in one cluster from one Australian state. On the basis of disc diffusion, 34/53 (64%) isolates were susceptible to all antimicrobial agents tested; there was intermediate susceptibility to tulathromycin in 12 isolates, tilmicosin in seven isolates and resistance to tilmicosin in one isolate. Using MIC, all but one isolate was susceptible to all antimicrobial agents tested; the non-susceptible isolate was resistant to tetracycline, but this MIC result could not be compared to disc diffusion, since there are no interpretative guidelines for disc diffusion for H. somni against tetracycline. In this study, there was little evidence of antimicrobial resistance in H. somni isolates from Australian cattle. Disc diffusion susceptibility testing results were comparable to MIC results for most antimicrobial agents tested; however, results for isolates with intermediate susceptibility or resistance to tilmicosin and tulathromycin on disc diffusion should be interpreted with caution in the absence of MIC results.

The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop

Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large β-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker.

Beth Israel Hospital (Boston, MA) Records, undated, 1923, 1930-1964, 1988, 1991-1992

In 1916, the Jewish community of Boston established Beth Israel Hospital on Townsend Street in Roxbury to provide health care to immigrants in the area. Although accessible to everyone, the hospital provided Yiddish-speaking services for Eastern European Jewish immigrants and served kosher food, as well as conducted Jewish religious services. In 1928 the hospital entered into a teaching agreement with Harvard Medical School, Tufts University, and Simmons College. Shortly thereafter, the hospital moved to its current location in the Longwood area of Boston and expanded to a 220-bed operation. During 1935-1936, at the height of the Depression, Beth Israel spent 1.5 million dollars in free patient care and was only one of two local hospitals to offer health care to people on welfare. In 1996, Beth Israel Hospital merged with Deaconess Medical Center and became Beth Israel Deaconess Medical Center. This collection contains reports, pamphlets and hospital publications.

Programming rumen bacterial communities in newborn Merino lambs

Establishment of the rumen microbiome can be affected by both early-life dietary measures and rumen microbial inoculation. This study used a 2 × 3 factorial design to evaluate the effects of inclusion of dietary fat type and the effects of rumen inoculum from different sources on ruminal bacterial communities present in early stages of the lambs’ life. Two different diets were fed ad libitum to 36 pregnant ewes (and their lambs) from 1 month pre-lambing until weaning. Diets consisted of chaffed lucerne and cereal hay and 4% molasses, with either 4% distilled coconut oil (CO) provided as a source of rumen-active fat or 4% Megalac® provided as a source of rumen-protected fat (PF). One of three inoculums was introduced orally to all lambs, being either (1) rumen fluid from donor ewes fed the PF diet; (2) rumen fluid from donor ewes fed CO; or (3) a control treatment of MilliQ-water. After weaning at 3 months of age, each of the six lamb treatment groups were grazed in spatially separated paddocks. Rumen bacterial populations of ewes and lambs were characterised using 454 amplicon pyrosequencing of the V3/V4 regions of the 16S rRNA gene. Species richness and biodiversity of the bacterial communities were found to be affected by the diet in ewes and lambs and by inoculation treatment of the lambs. Principal coordinate analysis and analysis of similarity (ANOSIM) showed between diet differences in bacterial community groups existed in ewes and differential bacterial clusters occurred in lambs due to both diet and neonatal inoculation. Diet and rumen inoculation acted together to clearly differentiate the bacterial communities through to weaning, however the microbiome effects of these initial early life interventions diminished with time so that rumen bacterial communities showed greater similarity 2 months after weaning. These results demonstrate that ruminal bacterial communities of newborn lambs can be altered by modifying the diet of their mothers. Moreover, the rumen microbiome of lambs can be changed by diet while they are suckling or by inoculating their rumen, and resulting changes in the rumen bacterial microbiome can persist beyond weaning.

Phage therapy in livestock methane amelioration.

Viruses of prokaryotes (phages) are obligate microbial pathogens that can, in the lytic phase of development, infect and lyse their respective bacterial or archaeal hosts. As such, these viruses can reduce the population density of their hosts rapidly, and have been viewed as possible agents of biological control (phage therapy). Phage therapy is becoming increasingly important as a means of eradicating or controlling microbial populations as the use of antibiotics and chemical treatments becomes both less effective and less publicly acceptable. Phage therapy has therefore been raised as a potential strategy to reduce methane (CH4) emissions from ruminants, providing an innovative biological approach, harnessing the potent, yet targeted, biocidal attributes of these naturally occurring microbial predators.

Protection Conferred by Infectious Coryza Vaccines Against Emergent Avibacterium paragallinarum Serovar C-1

Infectious coryza is an upper respiratory disease of chickens caused by Avibacterium paragallinarum. Outbreaks of infectious coryza caused by Av. paragallinarum serovar C-1 isolates in coryza-vaccinated flocks in Ecuador and Mexico have been reported. In the current study, the protection conferred by four commercially available, trivalent infectious coryza vaccines in chickens challenged with a serovar C-1 isolate from an apparent coryza vaccine failure in a layer flock in Mexico was evaluated. Only one infectious coryza vaccine provided a good protection level (83%) in vaccinated chickens. These results might explain the infectious coryza outbreaks in vaccinated flocks that have been observed in the field.

A retrospective study of Babesia macropus associated with morbidity and mortality in eastern grey kangaroos (Macropus giganteus) and agile wallabies (Macropus agilis)

This is a retrospective study of 38 cases of infection by Babesia macropus, associated with a syndrome of anaemia and debility in hand-reared or free-ranging juvenile eastern grey kangaroos (Macropus giganteus) from coastal New South Wales and south-eastern Queensland between 1995 and 2013. Infection with B. macropus is recorded for the first time in agile wallabies (Macropus agilis) from far north Queensland. Animals in which B. macropus infection was considered to be the primary cause of morbidity had marked anaemia, lethargy and neurological signs, and often died. In these cases, parasitised erythrocytes were few or undetectable in peripheral blood samples but were sequestered in large numbers within small vessels of visceral organs, particularly in the kidney and brain, associated with distinctive clusters of extraerythrocytic organisms. Initial identification of this piroplasm in peripheral blood smears and in tissue impression smears and histological sections was confirmed using transmission electron microscopy and molecular analysis. Samples of kidney, brain or blood were tested using PCR and DNA sequencing of the 18S ribosomal RNA and heat shock protein 70 gene using primers specific for piroplasms. The piroplasm detected in these samples had 100 sequence identity in the 18S rRNA region with the recently described Babesia macropus in two eastern grey kangaroos from New South Wales and Queensland, and a high degree of similarity to an unnamed Babesia sp. recently detected in three woylies (Bettongia penicillata ogilbyi) in Western Australia.

Investigation of an emerging bacterial disease in wild Queensland groper, marine fish and stingrays with production of diagnostic tools to reduce the spread of disease to other states of Australia : final report

This project describes how Streptococcus agalactiae can be transmitted experimentally in Queensland grouper. The implications of this research furthers the relatedness between Australian S. agalactiae strains from animals and humans. Additionally, this research has developed diagnostic tools for Australian State Veterinary Laboratories and Universities, which will assist in State and National aquatic animal disease detection, surveillance, disease monitoring and reporting

First detection of extended-spectrum cephalosporin- and fluoroquinolone-resistant Escherichia coli in Australian food-producing animals

This study aimed to define the frequency of resistance to critically important antimicrobials (CIAs) [i.e. extended-spectrum cephalosporins (ESCs), fluoroquinolones (FQs) and carbapenems] among Escherichia coli isolates causing clinical disease in Australian food-producing animals. Clinical E. coli isolates (n = 324) from Australian food-producing animals [cattle (n = 169), porcine (n = 114), poultry (n = 32) and sheep (n = 9)] were compiled from all veterinary diagnostic laboratories across Australia over a 1-year period. Isolates underwent antimicrobial susceptibility testing to 18 antimicrobials using the Clinical and Laboratory Standards Institute disc diffusion method. Isolates resistant to CIAs underwent minimum inhibitory concentration determination, multilocus sequence typing (MLST), phylogenetic analysis, plasmid replicon typing, plasmid identification, and virulence and antimicrobial resistance gene typing. The 324 E. coli isolates from different sources exhibited a variable frequency of resistance to tetracycline (29.0–88.6%), ampicillin (9.4–71.1%), trimethoprim/sulfamethoxazole (11.1–67.5%) and streptomycin (21.9–69.3%), whereas none were resistant to imipenem or amikacin. Resistance was detected, albeit at low frequency, to ESCs (bovine isolates, 1%; porcine isolates, 3%) and FQs (porcine isolates, 1%). Most ESC- and FQ-resistant isolates represented globally disseminated E. coli lineages (ST117, ST744, ST10 and ST1). Only a single porcine E. coli isolate (ST100) was identified as a classic porcine enterotoxigenic E. coli strain (non-zoonotic animal pathogen) that exhibited ESC resistance via acquisition of blaCMY-2. This study uniquely establishes the presence of resistance to CIAs among clinical E. coli isolates from Australian food-producing animals, largely attributed to globally disseminated FQ- and ESC-resistant E. coli lineages.

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

Metabolism of DL-(+/-)-phenylalanine by Aspergillus niger.

A fungus capable of degrading DL-phenylalanine was isolated from the soil and identified as Aspergillus niger. It was found to metabolize DL-phenylalanine by a new pathway involving 4-hydroxymandelic acid. D-Amino acid oxidase and L-phenylalanine: 2-oxoglutaric acid aminotransferase initiated the degradation of D- and L-phenylalanine, respectively. Both phenylpyruvate oxidase and phenylpyruvate decarboxylase activities could be demonstrated in the cell-free system. Phenylacetate hydroxylase, which required reduced nicotinamide adenine dinucleotide phosphate, converted phenylacetic acid to 2- and 4-hydroxyphenylacetic acid. Although 4-hydroxyphenylacetate was converted to 4-hydroxymandelate, 2-hydroxyphenylacetate was not utilized until the onset of sporulation. During sporulation, it was converted rapidly into homogentisate and oxidized to ring-cleaved products. 4-Hydroxymandelate was degraded to protocatechuate via