Axel Colling

Development and validation of a TaqMan ® PCR assay for the Australian abalone herpes-like virus

The recent emergence of a herpes-like virus in both farmed and wild populations of abalone in Victoria, Australia, has been associated with high mortality rates in animals of all ages. Based on viral genome sequence information, a virus-specific real-time TaqMan assay was developed for detection and identification of the abalone herpes-like virus (AbHV). The assay was shown to be specific as it did not detect other viruses from either the Herpesvirales or the Iridovirales orders which have genome sequence similarities. However, the TaqMan assay was able to detect DNA from the Taiwanese abalone herpes-like virus, suggesting a relationship between the Taiwanese and Australian viruses. In addition, the assay detected < 300 copies of recombinant plasmid DNA per reaction. Performance characteristics for the AbHV TaqMan assay were established using 1673 samples from different abalone populations in Victoria and Tasmania. The highest diagnostic sensitivity and specificity were 96.7 (95% CI: 82.7 to 99.4) and 99.7 (95% CI: 99.3 to 99.9), respectively, at a threshold cycle (C(T)) value of 35.8. The results from 2 separate laboratories indicated good repeatability and reproducibility. This molecular assay has already proven useful in confirming presumptive diagnosis (based on the presence of ganglioneuritis) of diseased abalone in Victorian waters as well as being a tool for surveillance of wild abalone stocks in other parts of Australia.

Real-time RT-PCR for detection of equine influenza and evaluation using samples from horses infected with A/equine/Sydney/2007 (H3N8).

Equine influenza (EI) virus (H3N8) was identified in the Australian horse population for the first time in August 2007. The principal molecular diagnostic tool used for detection was a TaqMan real-time reverse transcription-polymerase chain reactions (RT-PCR) assay specific for the matrix (MA) gene of influenza virus type A (IVA). As this assay is not specific for EI, we developed a new EI H3-specific TaqMan assay targeting the haemagglutinin (HA) gene of all recent EI H3 strains. The IVA and the EI H3 TaqMan assays were assessed using in vitro transcribed RNA template, virus culture, diagnostic samples from the outbreak and samples from experimentally infected horses. The EI H3 TaqMan assay had a higher diagnostic sensitivity (DSe) when compared to the IVA TaqMan assay and also when using a conventional PCR for EI H3 as a standard of comparison. The performance of both TaqMan assays was compared with an antigen detection ELISA and virus isolation using nasal swabs collected daily from horses experimentally infected with the outbreak strain A/equine/Sydney/2888-8/2007. The EI H3 TaqMan assay was the most sensitive of the assays, able to detect EI from day 1 or 2 post-challenge, as early as virus isolation, and before clinical signs of disease were observed.

Molecular epidemiology of foot-and-mouth disease viruses from South East Asia 1998–2006: The Lao perspective

Foot-and-mouth disease (FMD) causes sporadic disease outbreaks in the Lao Peoples Democratic Republic (Lao PDR) and appears to be endemic within a livestock population largely susceptible to infection. As Lao PDR is a major thoroughfare for transboundary animal movement, regular FMD outbreaks occur causing economic hardship for farmers and their families. The dominant serotype causing outbreaks between 1998 and 2006 was type O. Using phylogenetic analysis, type O isolated viruses were divided into two topotypes: South East Asia (SEA) and the Middle East-South Asia (ME-SA). Type A virus was reported only in 2003 and 2006 and type Asia 1 only in 1996 and 1998.

Microsphere Suspension Array Assays for Detection and Differentiation of Hendra and Nipah Viruses

Microsphere suspension array systems enable the simultaneous fluorescent identification of multiple separate nucleotide targets in a single reaction. We have utilized commercially available oligo-tagged microspheres (Luminex MagPlex-TAG) to construct and evaluate multiplexed assays for the detection and differentiation of Hendra virus (HeV) and Nipah virus (NiV). Both these agents are bat-borne zoonotic paramyxoviruses of increasing concern for veterinary and human health. Assays were developed targeting multiple sites within the nucleoprotein (N) and phosphoprotein (P) encoding genes. The relative specificities and sensitivities of the assays were determined using reference isolates of each virus type, samples from experimentally infected horses, and archival veterinary diagnostic submissions. Results were assessed in direct comparison with an established qPCR. The microsphere array assays achieved unequivocal differentiation of HeV and NiV and the sensitivity of HeV detection was comparable to qPCR, indicating high analytical and diagnostic specificity and sensitivity.

Henipavirus microsphere immuno-assays for detection of antibodies against Hendra virus

Hendra and Nipah viruses (HeV and NiV) are closely related zoonotic pathogens of the Paramyxoviridae family. Both viruses belong to the Henipavirus genus and cause fatal disease in animals and humans, though only HeV is endemic in Australia. In general and due to the acute nature of the disease, agent detection by PCR and virus isolation are the primary tools for diagnostic investigations. Assays for the detection of antibodies against HeV are fit more readily for the purpose of surveillance testing in disease epidemiology and to meet certification requirements in the international movement of horses. The first generation indirect ELISA has been affected by non-specific reactions which must be resolved using virus neutralisation serology conducted at laboratory bio-safety level 4 containment (PC4). Recent developments have enabled improvements in the available serology assays. The production of an expressed recombinant truncated HeV G protein has been utilised in ELISA and in Luminex-based multiplexed microsphere assays. In the latter format, two Luminex assays have been developed for use in henipavirus serology: a binding assay (designed for antibody detection and differentiation) and a blocking assay (designed as a surrogate for virus neutralisation). Equine and canine field sera were used to evaluate the two Luminex assays relative to ELISA and virus neutralisation serology. Results showed that Luminex assays can be effective as rapid, sensitive and specific tests for the detection of HeV antibody in horse and dog sera. The tests do not require PC4 containment and are appropriate for high throughput applications as might be required for disease investigations and other epidemiological surveillance. Also, the results show that the Luminex assays detect effectively HeV vaccine-induced antibodies.

Flavivirus detection and differentiation by a microsphere array assay

Flaviviruses of the Japanese encephalitis virus (JEV) serocomplex include major human and animal pathogens that have a propensity to spread and emerge in new geographic areas. Different genotypes or genetic lineages have been defined for many of these viruses, and they are distributed worldwide. Tools enabling rapid detection of new or emerging flaviviruses and differentiation of important subgroups have widespread application for arbovirus diagnosis and surveillance, and are crucial for detecting virus incursions, tracking virus emergence and for disease control. A microsphere suspension array assay was developed to identify JEV serocomplex flaviviruses of medical and veterinary importance. Assay performance was evaluated using representative virus strains as well as clinical and surveillance samples. The assay detected all JEV serocomplex viruses tested in this study with an apparent analytical sensitivity equal or better than the reference real-time or conventional RT-PCR assays and was able to identify mixed virus populations. The ability to identify mixed virus populations at a high analytical sensitivity would be pertinent in the Australian context when attempting to detect exotic JEV or West Nile virus (WNV), and differentiate from endemic Murray Valley encephalitis virus and WNV-Kunjin virus. The relatively low cost, the ability to identify mixed virus populations and the multiplex nature makes this assay valuable for a wide range of applications including diagnostic investigations, virus exclusions, and surveillance programs.

Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia.

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller’s stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories.

A comparative evaluation of feathers, oropharyngeal swabs, and cloacal swabs for the detection of H5N1 highly pathogenic avian influenza virus infection in experimentally infected chickens and ducks.

Oropharyngeal and cloacal swabs have been widely used for the detection of H5N1 highly pathogenic avian Influenza A virus (HPAI virus) in birds. Previous studies have shown that the feather calamus is a site of H5N1 virus replication and therefore has potential for diagnosis of avian influenza. However, studies characterizing the value of feathers for this purpose are not available, to our knowledge; herein we present a study investigating feathers for detection of H5N1 virus. Ducks and chickens were experimentally infected with H5N1 HPAI virus belonging to 1 of 3 clades (Indonesian clades 2.1.1 and 2.1.3, Vietnamese clade 1). Different types of feathers and oropharyngeal and cloacal swab samples were compared by virus isolation. In chickens, virus was detected from all sample types: oral and cloacal swabs, and immature pectorosternal, flight, and tail feathers. During clinical disease, the viral titers were higher in feathers than swabs. In ducks, the proportion of virus-positive samples was variable depending on viral strain and time from challenge; cloacal swabs and mature pectorosternal feathers were clearly inferior to oral swabs and immature pectorosternal, tail, and flight feathers. In ducks infected with Indonesian strains, in which most birds did not develop clinical signs, all sampling methods gave intermittent positive results; 3–23% of immature pectorosternal feathers were positive during the acute infection period; oropharyngeal swabs had slightly higher positivity during early infection, while feathers performed better during late infection. Our results indicate that immature feathers are an alternative sample for the diagnosis of HPAI in chickens and ducks.

Molecular detection of Mikrocytos mackini in Pacific oysters using quantitative PCR

Mikrocytos mackini is an internationally regulated pathogen and causative agent of Denman Island disease in Pacific oysters Crassostrea gigas. Recent phylogenetic breakthroughs have placed this parasite within a highly divergent and globally distributed eukaryotic lineage that has been designated a new taxonomic order, Mikrocytida. The discovery of this new radiation of parasites is accompanied by a heightened awareness of the many knowledge gaps that exist with respect to the general biology, epizootiology, and potential impact of mikrocytid parasites on hosts, ecosystems, and commercial fisheries. It has also highlighted current shortcomings regarding our ability to detect these organisms. In this study, we developed a species-specific, sensitive, and quantitative method for detecting M. mackini DNA from host tissues using probe-based real-time qPCR technology. A limit of sensitivity between 2 and 5 genome copy equivalents was achieved in a reaction matrix containing ≥ 40 ng/μL host gDNA without inhibition. This detection proved superior to existing methods based on conventional PCR, histology or gross pathology and is the first species-specific diagnostic test for M. mackini. Quantitative assessment of parasite DNA using this assay remained accurate to between 10 and 50 copies identifying that during infection, M. mackini DNA was significantly more prevalent in hemolymph, labial palp, and mid-body cross-sections compared to mantle or adductor muscle. DNA extracted from a mid-body cross-section also provided the highest likelihood for detection during diagnostic screening of infected oysters. Taken together, these findings provide strong analytical evidence for the adoption of qPCR as the new reference standard for detecting M. mackini and give preliminary insight into the distribution of the parasite within host tissues. Standardised operating methodologies for sample collection and qPCR testing are provided to aid in the international regulatory diagnosis of M. mackini and serve as a useful platform for the future development of multiplexed or alternate mikrocytid species detection.

Assessment of a Rabies Virus Rapid Diagnostic Test for the Detection of Australian Bat Lyssavirus

Australian bat lyssavirus (ABLV) is closely related to the classical rabies virus and has been associated with three human fatalities and two equine fatalities in Australia. ABLV infection in humans causes encephalomyelitis, resulting in fatal disease, but has no effective therapy. The virus is maintained in enzootic circulation within fruit bats (Pteropid spp.) and at least one insectivorous bat variety (Saccolaimus flaviventris). Most frequently, laboratory testing is conducted on pteropodid bat brains, either following a potential human exposure through bites, scratches and other direct contacts with bats, or as opportunistic assessment of sick or dead bats. The level of medical intervention and post-exposure prophylaxis is largely determined on laboratory testing for antigen/virus as the demonstrable infection status of the in-contact bat. This study evaluates the comparative diagnostic performance of a lateral flow test, Anigen Rabies Ag detection rapid test (RDT), in pteropodid variant of ABLV-infected bat brain tissues. The RDT demonstrated 100% agreement with the reference standard fluorescent antibody test on 43 clinical samples suggesting a potential application in rapid diagnosis of pteropodid variant of ABLV infection. A weighted Kappa value of 0.95 confirmed a high level of agreement between both tests.