Leonie J. Barnett

A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp

BackgroundLeptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often flu like symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT.MethodsThe polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples.Results and ConclusionsThe PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

Leptospiral Antibodies in Flying Foxes in Australia

The sera of 271 pteropid bats (or flying foxes) collected from Queensland, New South Wales, Western Australia, and the Northern Territory were screened against a reference panel of 21 Leptospiraspp. using the microscopic agglutination test (MAT). Sera were collected from December 1997 through August 1999. The MAT panel represented those serovars previously isolated in Australia, as well as exotic serovars found in neighboring countries. Leptospiral antibodies were detected in 75 (28%) of the sera and represented seven serovars, one of which, L. interrogansserovar cynopteri has been regarded as exotic to Australia. Sixty sera were reactive to one serovar, 12 sera were reactive to two serovars, and three sera were reactive to three serovars. The L. kirschneriserovar australis was most frequently identified (60.2%). The findings suggest a previously unrecognized role of pteropid bats in the natural history of leptospirosis. The potential exists for establishment of infection in new host species, the transmission of new serovars to known host species, and for changes in virulence of leptospires as a result of passage through these species.

Leptospirosis serology in the Common Brushtail Possum (Trichosurus vulpecula) from urban Sydney, Australia

The common brushtail possum (Trichosurus vulpecula) is indeed a common marsupial in major cities of Australia. This species is known to be susceptible to leptospirosis and often lives in close contact with humans, raising concerns about the potential for transmission of this disease in urban areas. A total of 192 brushtail possum blood samples were collected from 136 individuals in suburban areas of metropolitan Sydney from November 2002 to November 2004. Sera were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test. Leptospiral antibodies were detected in 9.6% (13/136) of tested brushtail possums and represented two serovars; antibodies to Leptospira interrogans serovar Hardjo were most frequently identified (11/136). A representative of the exotic serogroup Ballum, most likely serovar Arborea, was found in two of 136 brushtail possums. Exposure to leptospirosis seemed to be associated with age, as older animals had a higher incidence, but there was no distinction in relation to gender. Antibody prevalence varied between the different sampling sites and seropositive animals were clustered and restricted to a few sites. These data support the possible role of brushtail possums as a maintenance host for Leptospira spp. in urban environments and also identified them as a previously unknown and potential source of serovar Arborea.

A review of the currently recognised opecoelid cercariae, including the identification and emergence ecology of Cercaria capricornia XII (Digenea: Opecoelidae) from Nassarius olivaceus (Gastropoda: Nassariidae) in Central Queensland, Australia

A new opecoelid cercaria, Cercaria capricornia XII, is reported from Nassarius olivaceus in Capricornia, Central Queensland, Australia. Combined molecular and morphological data indicate that this cercaria is a member of the subfamily Opecoelinae. Cercaria capricornia XII is the first known opecoeline cercaria reported from a nassariid gastropod. Cercaria capricornia XII can be distinguished from other opecoelid cercariae by the combination of the presence of a 2-pointed stylet, body length and width, and the size of the tail. The emergence pattern for C. capricornia XII in captivity was erratic; rapid emergences of thousands of cercariae were interspersed by periods that sometimes exceeded a month in which no emergence occurred. There was no detectable pattern to or stimulus of the emergence. The molluscan host range of opecoelids is analysed in detail. Gastropods from the Buccinoidea, Cerithioidea and Rissooidea are hosts to both opecoeline and plagioporine cercariae, but the dominant subfamily infecting the Cerithioidea and Rissooidea is the Plagioporinae. The dominant marine host gastropod superfamily for opecoeline cercariae is the Buccinoidea; the family Nassariidae is contained in the Buccinoidea. The range of gastropod superfamilies known as hosts of plagioporines is much broader than that for opecoelines, which may be explained by the relative size of the two opecoelid families and perhaps by indications that the Plagioporinae is polyphyletic.

Vigilance to Limit the Bidirectional Introduction and Co-circulation of Virus Serotypes Aims to Reduce Risk of Severe Dengue Disease in North East India

Dengue is a serious re-emerging arboviral disease that presents a global human health challenge. Infection is caused by one of several recognised serotypes of dengue virus, a member of the mosquito-transmitted Flaviviridae family of human pathogens, which also includes yellow fever, Zika and West Nile viruses. The greatest burden of dengue is borne by countries in the Asia Pacific region, in particular South East Asia and the Indian subcontinent where infection breaks out with increasing frequency and intensity. The World Health Organization South East Asia Region has a population of 1.3 billion people and includes 11 countries, of which Thailand, Myanmar and India have the highest reported incidence of dengue. Infection outcomes vary from the commonly asymptomatic or a self-limiting fever to more severe manifestations such as dengue haemorrhagic fever and dengue shock syndrome. Although primary infection with one serotype elicits lifelong immunity against homologous reinfection, severe dengue is linked to an antibody-related immunopathological response that is triggered by secondary infection with a heterologous serotype. The principal vectors of transmission are the day-biting mosquitoes Aedes aegypti and Ae. albopictus, the respective preference of which for urban and rural habitats has facilitated the rapid and continued spread of dengue across tropical and sub-tropical climatic zones. The narrow elevated corridor of North East India was considered historically to be non-endemic for dengue and as such separated areas of high endemicity in the Bengal Delta and the other zones of India from those in Thailand and Myanmar. In these regions the predominant serotype is usually distinct, so a breach of this geographical bottleneck would potentially enable the co-circulation of virus serotypes, thus posing an increased risk of severe disease upon secondary infection. This alarming scenario may now be realized following the notification of clinical cases in the states of Arunachal Pradesh, Assam and Manipur in recent years. While incidence is still sporadic in North East India limitation of local outbreaks is required through vigilant adherence to the integrated implementation of clinical management, diagnosis, surveillance and vector control in order to prevent the transcontinental spread of dengue.

A review of the currently recognised zoogonid cercariae, including the identification and emergence ecology of Cercaria capricornia XI (Digenea: Zoogonidae) from Nassarius olivaceus (Gastropoda: Nassariidae) in Central Queensland, Australia

In the present study, we review the known zoogonid cercariae, summarise their life-cycles and first intermediate host distributions, and present a new cercaria, Cercaria capricornia XI (Digenea: Zoogonidae), which was found in one of three nassariid gastropods, Nassarius olivaceus (Bruguière), surveyed in the intertidal zone in the Capricornia region of Central Queensland, Australia. Morphological data and molecular analysis of the ITS2 rDNA region support placement of this cercaria in the family Zoogonidae but do not allow any further resolution of its identity. There are now fifteen cercariae described as belonging to the Zoogonidae; thirteen of these, including the present species, infect neogastropods as first intermediate hosts and two use vetigastropods. This study reinforces the pattern that the Nassariidae is by far the most commonly reported family for the Zoogonidae. Given its richness we predict that the Nassariidae will prove to harbour many more zoogonid species.

Phenotypic plasticity of six unusual cercariae in nassariid gastropods and their relationships to the Acanthocolpidae and Brachycladiidae (Digenea)

Species-level relationships of a complex of six putative acanthocolpid cercariae with ventral keels from nassariid gastropods from Capricornia, Queensland (Cercaria capricornia I-VI) were explored using mitochondrial and nuclear DNA sequence data. Analysis of the large subunit of nuclear ribosomal DNA (rDNA), the internal transcribed spacer 2 rDNA and subunit 3 of the nicotinamide-adenine-dinucleotide dehydrogenase mitochondrial gene indicates that the six distinct morphotypes previously identified relate to only three discrete genotypes. Consequently, the six cercarial morphotypes that were inferred to represent six species and distinguished previously based on morphology and behaviour, are considered here to be comprised of three species (i.e. Cercaria capricornia I and II are considered the same species, C. capricornia types III to V are considered to belong to a second species and C. capricornia VI is considered to comprise the third species in this complex). This recognition of reduced diversity following molecular analysis runs contrary to the more frequently reported uncovering of cryptic diversity, especially when larval trematodes are examined. Phylogenetic analysis of these morphologically unusual cercariae supports their inclusion in the Acanthocolpidae. Bayesian inference analysis identifies three distinct clades: (a) Stephanostomum+Monostephanostomum; (b) the Brachycladiidae and Pleorchis; and (c) Tormopsolus and the present cercariae. Genus-level identification of these cercariae remains uncertain given the current lack of available comparative genetic data on nucleic acid databases.