Toxoplasmosis epidemic in a population of urbanised allied rock-wallabies (Petrogale assimilis) on Magnetic Island (Yunbenun), North Queensland.

A mortality event involving 23 allied rock-wallabies (Petrogale assimilis) displaying neurological signs and sudden death occurred in late April to May 2021 in a suburban residential area directly adjacent to Magnetic Island National Park, on Magnetic Island (Yunbenun), North Queensland, Australia. Three allied rock-wallabies were submitted for necropsy, and in all three cases, the cause of death was disseminated toxoplasmosis. This mortality event was unusual because only a small, localised population of native wallabies inhabiting a periurban area on a tropical island in the Great Barrier Reef World Heritage Area were affected. A disease investigation determined the outbreak was likely linked to the presence of free-ranging feral and domesticated cats inhabiting the area. There were no significant deaths of other wallabies or wildlife in the same or other parts of Magnetic Island (Yunbenun) at the time of the outbreak. This is the first reported case of toxoplasmosis in allied rock-wallabies (Petrogale assimilis), and this investigation highlights the importance of protecting native wildlife species from an infectious and potentially fatal parasitic disease.

A novel alphaherpesvirus and concurrent respiratory cryptococcosis in a captive koala (Phascolarctos cinereus).

A novel alphaherpesvirus was detected in a captive adult, lactating, female koala (Phascolarctos cinereus) admitted to James Cook University Veterinary Emergency Teaching & Clinical Hospital in March 2019, showing signs of anorexia and severe respiratory disease. Postmortem examination revealed gross pathology indicative of pneumonia. Histopathology demonstrated a chronic interstitial pneumonia, multifocal necrotising adrenalitis and hepatitis. Intranuclear inclusion bodies were detected by light microscopy in the respiratory epithelium of the bronchi, bronchioles, alveoli, and hepatocytes, biliary epithelium and adrenal gland associated with foci of necrosis. Cryptococcus gattii was isolated from fresh lung on necropsy, positively identified by PCR, and detected histologically by light microscopy, only in the lung tissue. A universal viral family-level PCR indicated that the virus was a member of the Herpesviruses. Sequence analysis in comparison to other known and published herpesviruses, indicated the virus was a novel alphaherpesvirus, with 97% nucleotide identity to macropodid alphaherpesvirus 1. We provisionally name the novel virus phascolarctid alphaherpesvirus 3 (PhaHV-3). Further research is needed to determine the distribution of this novel alphaherpesvirus in koala populations and establish associations with disease in this host species.

Epizootics of Streptococcus agalactiae infection in captive rays from Queensland, Australia.

The aim of this study was to describe two epizootics of high mortalities from infection with Streptococcus agalactiae, occurring in captive rays held in a marine display aquarium in south-east Queensland, Australia, in 2009 and 2010. Five different species of rays were affected, including mangrove whiprays (Himantura granulata), estuary rays (Dasyatis fluviorum), eastern shovelnose rays (Aptychotrema rostrata), white-spotted eagle rays (Aetobatus narinari) and blue-spotted mask rays (Neotrygon kuhlii). This report describes the history of both epizootics including collection, quarantine and husbandry of rays, the disease epizootics, clinico-pathological features of the disease, antimicrobial therapy, autogenous vaccine production, and laboratory studies including clinical and histopathology, bacteriology, PCR, molecular serotyping and sequencing of the bacterium S. agalactiae.

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.

Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia.

Ninety-three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Rüppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S. agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S. agalactiae; genotyping of selected S. agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S. agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.

Toxoplasmosis in Indo-Pacific humpbacked dolphins (Sousa chinensis), from Queensland.

OBJECTIVE: To describe the clinical signs, gross pathology, serology, bacteriology, histopathology, electron microscopy and immunohistochemistry findings associated with toxoplasmosis in four Indo-Pacific humpbacked dolphins (Sousa chinensis) that stranded in Queensland in 2000 and 2001. DESIGN: Clinical assessment, gross necropsy, and laboratory examinations. PROCEDURE: Necropsies were performed on four S. chinensis to determine cause of death. Laboratory tests including serology, bacteriology, histopathology and transmission electron microscopy were done on the four dolphins. Immunohistochemistry was done on the brain, heart, liver, lung, spleen and adrenal gland from various dolphins to detect Toxoplasma gondii antigens. RESULTS: Necropsies showed all of four S. chinensis that stranded in Queensland in 2000 and 2001 had evidence of predatory shark attack and three were extremely emaciated. Histopathological examinations showed all four dolphins had toxoplasmosis with tissue cysts resembling T. gondii in the brain. Tachyzoite stages of T. gondii were detected in the lungs, heart, liver, spleen and adrenal gland, variously of all four dolphins. Electron microscopy studies and immunohistochemistry confirmed the tissues cysts were those of T. gondii. All four dolphins also had intercurrent disease including pneumonia, three had peritonitis and one had pancreatitis. CONCLUSION: Four S. chinensis necropsied in Queensland in 2000 and 2001 were found to be infected with toxoplasmosis. It is uncertain how these dolphins became infected and further studies are needed to determine how S. chinensis acquire toxoplasmosis. All four dolphins stranded after periods of heavy rainfall, and coastal freshwater runoff may be a risk factor for T. gondii infection in S. chinensis. This disease should be of concern to wildlife managers since S. chinensis is a rare species and its numbers appear to be declining.

Interlaboratory comparison exercise for the analysis of PCDD/Fs in samples of digested sewage sludge.

Five UK laboratories participated in a study designed to explore the principal sources of interlaboratory variation in the analysis of PCDD/Fs in sewage sludge. Samples of wet sludge, dry sludge, toluene extract of sludge and cleaned extract of sludge were prepared by an organising laboratory. The samples were analysed in duplicate by each laboratory along with a solution of PCDD/F standards and reference sediment. Mean coefficients of variation between laboratories were 45% for the wet sludge, 33% for the dry sludge, 32% for the extract of sludge, 36% for the cleaned extract of sludge, 32% for the reference sediment and 28% for the standard solution. The results were subjected to statistical analysis, which showed that there was no specific part of the analysis that introduced a dominant part of the variation. The spread of data generated from the analysis of wet sludge samples was not appreciably greater than the spread for the analysis of cleaned extracts. Thus the drying, extraction and clean up processes in the PCDD/F analysis of wet sludge did not have a dramatic effect on the interlaboratory variation.

Bacterial DNA ligases.

DNA ligases join breaks in the phosphodiester backbone of DNA molecules and are used in many essential reactions within the cell. All DNA ligases follow the same reaction mechanism, but they may use either ATP or NAD+ as a cofactor. All Bacteria (eubacteria) contain NAD+-dependent DNA ligases, and the uniqueness of these enzymes to Bacteria makes them an attractive target for novel antibiotics. In addition to their NAD+-dependent enzymes, some Bacteria contain genes for putative ATP-dependent DNA ligases. The requirement for these different isozymes in Bacteria is unknown, but may be related to their utilization in different aspects of DNA metabolism. The putative ATP-dependent DNA ligases found in Bacteria are most closely related to proteins from Archaea and viruses. Phylogenetic analysis suggests that all NAD+-dependent DNA ligases are closely related, but the ATP-dependent enzymes have been acquired by Bacterial genomes on a number of separate occasions.

The intrinsically unstable life of DNA triplet repeats associated with human hereditary disorders.

Expansions of specific DNA triplet repeats are the cause of an increasing number of hereditary neurological disorders in humans. In some diseases, such as Huntington's and several spinocerebellar ataxias, the repetitive DNA sequences are translated into long tracts of the same amino acid (usually glutamine), which alters interactions with cellular constituents and leads to the development of disease. For other disorders, including common genetic disorders such as myotonic dystrophy and fragile X syndrome, the DNA repeat is located in noncoding regions of transcribed sequences and disease is probably caused by altered gene expression. In studies in lower organisms, mammalian cells, and transgenic mice, high frequencies of length changes (increases and decreases) occur in long DNA triplet repeats. These observations are similar to other types of repetitive DNA sequences, which also undergo frequent length changes at genomic loci. A variety of processes acting on DNA influence the genetic stability of DNA triplet repeats, including replication, recombination, repair, and transcription. It is not yet known how these different multienzyme systems interact to produce the genetic mutation of expanded repeats. In vitro studies have identified that DNA triplet repeats can adopt several unusual DNA structures, including hairpins, triplexes, quadruplexes, slipped structures, and highly flexible and writhed helices. The formation of stable unusual structures within the cell is likely to disturb DNA metabolism and be a critical intermediate in the molecular mechanism(s) leading to genetic instabilities of DNA repeats and, hence, to disease pathogenesis.

Length of CTG.CAG repeats determines the influence of mismatch repair on genetic instability.

We showed previously that mutations in methyl-directed mismatch repair of Escherichia coli reduced the occurrence of large deletions in (CTG.CAG)(175) repeats contained on plasmids. By contrast, other workers reported that mutations in mismatch repair increase the frequency of small-length changes in the shorter (CTG.CAG)(64). Using plasmids with a variety of lengths and purity of (CTG.CAG) repeats, we have resolved these apparently conflicting observations. We show that all lengths of (CTG.CAG) repeats are subject to small-length changes (eight repeats) in (CTG.CAG)(n) occur more readily in cells with active mismatch repair. The frequency of large deletions is proportional to the tract length; in our assays they become prominent in tracts greater than 100 repeats. Interruptions in repeat purity enhance the occurrence of large deletions. In addition, we observed a high level of incidence of deletions in (CTG.CAG) repeats for cultures passing repeatedly through stationary phase during long-term growth experiments of all strains (i.e. with active or inactive mismatch repair). These results agree with current theories on mismatch repair acting on DNA slippage events that occur in DNA triplet-repeats.

Two superhelix density-dependent DNA transitions detected by changes in DNA adsorption/desorption behavior.

The adsorption behavior of covalently closed circular plasmid DNA at the mercury/water interface was studied by means of AC impedance measurements. The dependence of the differential capacitance (C) of the electrode double layer on the potential (E) was measured in the presence of adsorbed DNA. It was found that the C-E curves of supercoiled DNA at native and highly negative superhelix densities (sigma), relaxed covalently closed circular DNA, and nicked DNA differed from each other. A detailed study of topoisomer distributions ranging from -sigma of 0 to 0.11 revealed two supercoiling-dependent transitions, at about -sigma = 0.04 (transition TI) and 0.07 (transition TII). Transition TI was detected by measuring the height of the adsorption/desorption peak 1 (at about -1.2 V against the saturated calomel electrode) and the decrease of capacitance (DeltaC) at -0.35 V. This transition may be due to a sudden change in the ability of the DNA to respond to the alternating voltage, probably caused by changes in the DNA tertiary and/or secondary structure. Transition TII was detected by measuring peak 3* (at about -1.3 V), which was absent in topoisomers with -sigma less than 0.05. This transition is due to changes in the DNA adsorption/desorption behavior related to increased accessibility of bases at elevated negative superhelix density. Opening of the duplex at highly negative superhelix density was also detected by the single-strand selective probe of DNA structure, osmium tetroxide, 2, 2'-bipyridine. Our results suggest that electrochemical techniques provide sensitive experimental analysis of changes in DNA structure.

Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV.

BACKGROUND: Mammalian cells deficient in the XRCC4 DNA repair protein are impaired in DNA double-strand break repair and are consequently hypersensitive to ionising radiation. These cells are also defective in site-specific V(D)J recombination, a process that generates the diversity of antigen receptor genes in the developing immune system. These features are shared by cells lacking components of the DNA-dependent protein kinase (DNA-PK). Although the XRCC4 gene has been cloned, the function(s) of XRCC4 in DNA end-joining has remained elusive. RESULTS: We found that XRCC4 is a nuclear phosphoprotein and was an effective substrate in vitro for DNA-PK. Human XRCC4 associated extremely tightly with another protein(s) even in the presence of 1 M NaCl. Co-immunoprecipitation and adenylylation assays demonstrated that this associated factor was the recently identified human DNA ligase IV. Consistent with this, XRCC4 and DNA ligase IV copurified exclusively and virtually quantitatively over a variety of chromatographic steps. Protein mapping studies revealed that XRCC4 interacted with ligase IV via the unique carboxy-terminal ligase IV extension that comprises two tandem BRCT (BRCA1 carboxyl terminus) homology motifs, which are also found in other DNA repair-associated factors and in the breast cancer susceptibility protein BRCA1. CONCLUSIONS: Our findings provide a function for the carboxy-terminal region of ligase IV and suggest that BRCT domains of other proteins may mediate contacts between DNA repair components. In addition, our data implicate mammalian ligase IV in V(D)J recombination and the repair of radiation-induced DNA damage, and provide a model for the potentiation of these processes by XRCC4.

Transcription increases the deletion frequency of long CTG.CAG triplet repeats from plasmids in Escherichia coli.

Induction of transcription into long CTG.CAG repeats contained on plasmids in Escherichia coli is shown to increase the frequency of deletions within the repeat sequences. This elevated genetic instability was detected because active transcription into the triplet repeat influenced the growth transitions of the host cell, allowing advantageous growth for cells harboring plasmids with deleted repeat sequences. The variety of deletion products observed in separate cultures suggests that transcription altered the metabolism of the DNA in a manner that produced random length changes in the repeat sequence. For cultures containing plasmids without active transcription into the triplet repeat, or those maintained in exponential growth, deletions occurred within the repeat at a lower frequency (5-20-fold lower). In these incubations the extent of deletions was proportional to the number of cell divisions and many repeat lengths were observed within each culture, suggesting that the decrease in average repeat length at long incubation times was due to multiple small deletions. These observations show that deletions within long CTG.CAG repeats contained on plasmids in E.coli occur via more than one pathway and their level of genetic instability is altered by the enzymatic processes occurring upon the DNA.

Relationship between Escherichia coli growth and deletions of CTG.CAG triplet repeats in plasmids.

Instabilities that are intrinsic to natural repetitive DNA sequences produce high frequencies of length changes in vivo. Triplet repeats cloned in plasmids in Escherichia coli undergo expansions and deletions, and this instability is affected by multiple factors. We show that CTG-CAG repeats in plasmids can influence the growth of E. coli, which affects the observed stabilities. At extended growth periods, the observed frequencies of deletions were dramatically increased if the cells passed through stationary phase before subculturing. Deletions were particularly pronounced for a plasmid containing the longest repeat, 525 bp in total, with the CTG sequence as the lagging strand template for replication. Measurements of cell growth showed that the lag phase associated with E. coli growth was increased for cultures containing plasmids with long CTG-CAG repeats, particularly when the CTG-containing strand was the lagging template. High frequencies of deletions were observed because of a growth advantage of cells containing plasmids with deleted triplet repeats. Incubation conditions that reduced the bacterial growth-rate produced a decreased extent of deletions, presumably because they alleviated the growth advantage of cells harboring plasmids with deleted triplet repeats. The experimental observations were simulated by a model in which shorter triplet repeats provided a growth advantage due to a shorter lag phase. We demonstrate that the accumulation of deletions within repeating sequences during growth of E. coli can be prevented, and discuss these findings in relation to the studies of repetitive DNA sequences. These are the first observations to show a direct influence between a plasmid-based DNA sequence or structure and factors controlling bacterial growth.

Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases.

Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH). When plasmids containing (CTG)180 were grown for about 100 generations in mutS, mutL, or mutH strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat. The deletions occur only in the (CTG)180 insert, not in DNA flanking the repeat. While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed. We propose that the E. coli mismatch repair proteins recognize three-base loops formed during replication and then generate long single-stranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA. Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants. Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases.