Joanne Connolly

Infectivity and persistence of an outbreak strain of Salmonella enterica serotype Typhimurium DT160 for house sparrows (Passer domesticus) in New Zealand

Abstract AIM: To examine the infective dose, incubation period and disease progression of an isolate of Salmonella enterica serotype Typhimurium definitive type 160 (DT160) originating from a naturally-infected house sparrow (Passer domesticus) during an outbreak of the disease in New Zealand. METHODS: Thirty-six house sparrows captured from the wild and free of Salmonella spp were divided into six groups of six birds, housed individually, and inoculated orally with phosphate buffered saline (PBS) or 101, 102, 103, 105, 2 × 108 colony forming units (cfu) of the outbreak strain of S. Typhimurium DT160. The birds were observed for 10 days for clinical signs and/or mortality, and faecal samples were collected to determine excretion of S. Typhimurium. The birds were eutha- nised 11 days post-inoculation (p.i.) and a wide range of tissue samples were collected for histopathological examination, and culture and typing of Salmonella spp. Macro-restriction profiling by pulsed-field gel electrophoresis (PFGE) using XbaI was performed for the epidemiological typing of S. Typhimurium DT160 isolates. RESULTS: Mortality in house sparrows inoculated with S. Typhimurium DT160 was dose-dependent, and 2/6 birds inoculated with 105 cfu and all six birds inoculated with 2 × 108 cfu died during the study. Infected sparrows displayed few clinical signs, apart from diarrhoea and/or polyuria, fluffed plumage, and sitting on the floor of the cage. Faecal excretion of DT160 occurred briefly in two birds inoculated with 102 cfu and four birds inoculated with 103 cfu, on most days in five birds inoculated with 105 cfu, and continuously in six birds inoculated with 2 × 108 cfu. DT160 was isolated from the livers of three birds which received 103 cfu, five birds dosed with 105 cfu, and all six birds given 2 × 108 cfu. Following necropsy, histopathological lesions similar to those seen in the natural disease were observed in the liver or spleen of three birds which received 103 cfu, and all birds dosed with ≥105 cfu. CONCLUSION: The results indicate that an isolate of S. Typhimurium DT60 originating from house sparrows in New Zealand is pathogenic to these birds and that the response is dose- dependent. The persistence and excretion of the pathogen may last for at least 10 days. This confirms that sparrows infected with DT160 could be a source of infection to humans and other in-contact animals.

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

Haemolytic anaemia associated with Theileria sp. in an orphaned platypus.

CASE REPORT The clinical and laboratory findings in an orphaned juvenile female platypus (Ornithorhynchus anatinus) that presented with a severe anaemia and tick infestation are reported. The animal developed a terminal septicaemia and died. Antemortem clinical pathology, postmortem histopathology and 18S rDNA sequencing supported a diagnosis of extravascular haemolytic anaemia secondary to Theileria ornithorhynchi infection. CONCLUSION Although T. ornithorhynchi infection is common in the platypus, this is the first case in which it has been shown to cause a haemolytic anaemia in this species and molecular characterisation of the organism has been described. A review of the previous literature concerning T. ornithorhynchi and possible treatment options for future cases are discussed.

A review of mucormycosis in the platypus (Ornithorhynchus anatinus)

Many infectious agents and parasites have been reported from the platypus (Ornithorhynchus anatinus), but most do not cause serious disease. The fungus, Mucor amphibiorum, is the only disease agent known to cause significant morbidity and mortality in the free-living platypus in Tasmania. Infection has also been reported in free-ranging cane toads and green tree frogs from mainland Australia, but not confirmed in platypuses from the mainland. This paper reviews mucormycosis in the platypus and includes the epidemiology, clinical features, mycology, pathology as well as possible surveillance, treatment and/or control modalities. The emergence and geographical spread of mucormycosis as a disease entity in Tasmanian platypuses from 1982 till 2005 are discussed. The host, agent and environment factors of the traditional epidemiological paradigm are discussed as they contribute to the conditions that lead to the selection or emergence of Mucor amphibiorum as a pathogen in a population of platypuses.

Investigation into the characteristics, triggers and mechanism of apnoea and bradycardia in the anaesthetized platypus (Ornithorhynchus anatinus)

By relating respiratory and heart rate changes observed in some platypuses under isoflurane anaesthesia to those produced by various stimuli to the trigeminal nerve we propose that in platypuses the changes that can result from isoflurane gas in the nasal cavity are most appropriately described by the term ‘nasopharyngeal response’.

GENOTYPIC ANALYSIS OF MUCOR FROM THE PLATYPUS IN AUSTRALIA

Mucor amphibiorum is the only pathogen known to cause significant morbidity and mortality in the free-living platypus (Ornithorhynchus anatinus) in Tasmania. Infection has also been reported in free-ranging cane toads (Bufo marinus) and green tree frogs (Litoria caerulea) from mainland Australia but has not been confirmed in platypuses from the mainland. To date, there has been little genotyping specifically conducted on M. amphibiorum. A collection of 21 Mucor isolates representing isolates from the platypus, frogs and toads, and environmental samples were obtained for genotypic analysis. Internal transcribed spacer (ITS) region sequencing and GenBank comparison confirmed the identity of most of the isolates. Representative isolates from infected platypuses formed a clade containing the reference isolates of M. amphibiorum from the Centraal Bureau voor Schimmelcultures repository. The M. amphibiorum isolates showed a close sequence identity with Mucor indicus and consisted of two haplotypes, differentiated by single nucleotide polymorphisms within the ITS1 and ITS2 regions. With the exception of isolate 96-4049, all isolates from platypuses were in one haplotype. Multilocus fingerprinting via the use of intersimple sequence repeats polymerase chain reaction identified 19 genotypes. Two major clusters were evident: 1) M. amphibiorum and Mucor racemosus; and 2) Mucor circinelloides, Mucor ramosissimus, and Mucor fragilis. Seven M. amphibiorum isolates from platypuses were present in two subclusters, with isolate 96-4053 appearing genetically distinct from all other isolates. Isolates classified as M. circinelloides by sequence analysis formed a separate subcluster, distinct from other Mucor spp. The combination of sequencing and multilocus fingerprinting has the potential to provide the tools for rapid identification of M. amphibiorum. Data presented on the diversity of the pathogen and further work in linking genetic diversity to functional diversity will provide critical information for its management in Tasmanian river systems.

Investigation into individual health and exposure to infectious agents of platypuses (Ornithorhynchus anatinus) in two river catchments in northwest Tasmania

Abstract Changes in the health of individuals within wildlife populations can be a cause or effect of population declines in wildlife species. Aspects of individual platypus (Ornithorhynchus anatinus) health have been reported. However, holistic studies investigating potential synergistic effects of both pathogens and environmental factors are needed to expand understanding of platypus individual health. We collected baseline data on the health of platypuses in two Tasmanian river catchments (including evidence of the potentially fatal fungal disease mucormycosis) and on individual, demographic, and geographic patterns associated with health data results. We examined 130 wild platypuses from the Inglis River Catchment and 24 platypuses from the Seabrook Creek Catchment in northwest Tasmania between 29 August 2011 and 31 August 2013. More than 90% of captured platypuses were infected with ticks, Theileria spp., and trypanosomes. Evidence of exposure to other infections, including Salmonella spp., Leptospira spp., and intestinal parasites, was low (<10%). Three platypuses had single fungal granulomas in the webbing of a forefoot, but no evidence of mucormycosis was found in any of the study animals. Possible subclinical hepatopathies or cholangiohepatopathies were found in six platypuses. Exposure to infectious agents did not cluster geographically, demographically, or in individuals, and there was minimal evidence of morbidity resulting from infection. This study has provided important baseline data for monitoring the effects of threatening processes, including mucormycosis, on the health of infected populations.

Novel use of in-stream microchip readers to monitor wild platypuses

A variety of techniques have been used to monitor platypus populations to assess the impacts of the threats they face, but each technique has limitations. In this study we investigated the novel use of in-stream microchip readers, to remotely monitor the movements of microchipped wild platypuses. Over 13 months, we recorded movements of 18 microchipped individuals past nine fixed locations in the Inglis Catchment in northwest Tasmania, using three units of which all were capable of detecting Trovan® unique microchips and two were additionally capable of detecting ISO microchips. Each site was monitored one or two times, for durations of 8–39 days. We undertook direction of movement investigations during two monitoring periods, by placing the antennas from two systems in the same creek within 3 m of each other. In a total of 264 days of monitoring, 528 platypus observations were made from 18 individual platypuses, consisting of 13 of 18 (72%) platypuses captured at the monitoring sites within 16 months prior to monitoring, two platypuses captured at other sites in the same time period, and three of seven (43%) individuals microchipped 3–5 years previously. This number of platypus observations, in combination with the stable number of platypuses observed per day, the range of movement behaviours recorded and the results of the direction of movement investigations, indicates that at appropriate sites, in-stream microchip readers are an effective method of monitoring the movements and survivorship of microchipped wild platypuses.

Evaluation of Two Multiplex PCR–High-Resolution Melt Curve Analysis Methods for Differentiation of Campylobacter jejuni and Campylobacter coli Intraspecies

SUMMARY Campylobacter infection is a common cause of bacterial gastroenteritis in humans and remains a significant global public health issue. The capability of two multiplex PCR (mPCR)–high-resolution melt (HRM) curve analysis methods (i.e., mPCR1-HRM and mPCR2-HRM) to detect and differentiate 24 poultry isolates and three reference strains of Campylobacter jejuni and Campylobacter coli was investigated. Campylobacter jejuni and C. coli were successfully differentiated in both assays, but the differentiation power of mPCR2-HRM targeting the cadF gene was found superior to that of mPCR1-HRM targeting the gpsA gene or a hypothetical protein gene. However, higher intraspecies variation within C. coli and C. jejuni isolates was detected in mPCR1-HRM when compared with mPCR2-HRM. Both assays were rapid and required minimum interpretation skills for discrimination between and within Campylobacter species when using HRM curve analysis software.

A NEED FOR DYNAMIC HEMATOLOGY AND SERUM BIOCHEMISTRY REFERENCE TOOLS: NOVEL USE OF SINE WAVE FUNCTIONS TO PRODUCE SEASONALLY VARYING REFERENCE CURVES IN PLATYPUSES (ORNITHORHYNCHUS ANATINUS)

Abstract Seasonal changes in hematology and serum biochemistry results, described by separate reference intervals for different seasons, have been reported in many animals. We developed a novel method to investigate seasonal variation in values and a reference tool (the reference curve) based on sine wave functions that, for suitable variables, represents data more appropriately than a fixed reference interval. We applied these techniques to values observed in blood samples from 126 adult wild platypuses (Ornithorhynchus anatinus; 58 females and 68 males). Samples were collected under isoflurane anesthesia from animals captured in the Inglis Catchment in northwest Tasmania. In general, packed cell volume (PCV), red cell count (RCC), and hemoglobin (Hb) values appeared to be lower than those in two studies that previously reported platypus hematology reference intervals. This likely resulted from reduced stress-related splenic contraction or isoflurane-associated splenic sequestration of red blood cells in our study. Reference curves were described for five variables (PCV, RCC, Hb, albumin, and magnesium). We found evidence that this seasonal variation may result from metabolic changes associated with seasonal variations in environmental temperature. These observations suggest that it is important for researchers reporting platypus hematology and serum biochemistry to look for seasonal changes in their data to ensure it is appropriately interpreted.