A variant of the Y strain of Trypanosoma cruzi which exhibits a high frequency (≅90%) of epimas... more A variant of the Y strain of Trypanosoma cruzi which exhibits a high frequency (≅90%) of epimastigote-to-trypomastigote transformation when cultured under nor.
Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general,... more Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general, and Tasmanian Atlantic salmon reovirus (TSRV) specifically, were developed, and their diagnostic sensitivity and specificity were determined and compared with virus isolation in cell culture. Intralaboratory and interlaboratory comparison of PCR (conventional hemi-nested RT-PCR & RT-qPCR) and virus isolation in cell culture using finfish cell lines, CHSE-214 and EPC, was carried out for the detection and identification of TSRV using field samples of farmed Atlantic salmon Salmo salar, L. from various aquaculture sites around Tasmania. The interlaboratory comparison of diagnostic methods was carried out between two laboratories, AAHL-CSIRO and DPIPWE-Tasmania. A total of 144 fish from nine sites (12–33 fish per site) were sampled from two regions of Tasmania (Tamar River estuary in the north and Huon River estuary in the south-east) during late spring to early summer of 2009, and the dat...
We are investigating whether koi herpesvirus is a potentially good biological control agent for c... more We are investigating whether koi herpesvirus is a potentially good biological control agent for carp (Cyprinus carpio) in Australia. Our initial results show that: * Mortality is higher in juvenile compared with older carp. * Carp-goldfish hybrids are less susceptible to KHV, and we are currently determining the prevalence of hybrids in selected wild populations of carp in Australia. * We are examining wild-caught immature carp for less virulent cross-reactive viruses that may confer protection from KHV. * KHV has been found to have no effect on three native fish species that have been tested – Murray cod, golden perch and silver perch. Two other species are currently being tested. * Infected carp excrete KHV for 1 to 2 days before clinical signs of disease in the fish. We have also reviewed past successful viral biological control programs of vertebrate pests, and have concluded that management and development of a successful program is underpinned by (i) a thorough understanding o...
The growing global demand for seafood together with the limited capacity of the wild-capture sect... more The growing global demand for seafood together with the limited capacity of the wild-capture sector to meet this demand has seen the aquaculture industry continue to grow around the world. A vast array of aquatic animal species is farmed in high density in freshwater, brackish and marine systems where they are exposed to new environments and potentially new diseases. On-farm stresses may compromise their ability to combat infection, and farming practices facilitate rapid transmission of disease. Viral pathogens, whether they have been established for decades or whether they are newly emerging as disease threats, are particularly challenging since there are few, if any, efficacious treatments, and the development of effective viral vaccines for delivery in aquatic systems remains elusive. Here, we review a few of the more significant viral pathogens of finfish, including aquabirnaviruses and infectious hematopoietic necrosis virus which have been known since the first half of the 20th century, and more recent viral pathogens, for example betanodaviruses, that have emerged as aquaculture has undergone a dramatic expansion in the past few decades.
The use of swabs relative to organs as a sample collection method for the detection of Tasmanian ... more The use of swabs relative to organs as a sample collection method for the detection of Tasmanian salmon reovirus (TSRV) in farmed Tasmanian Atlantic salmon, Salmo salar L., was evaluated by RT-qPCR. Evaluation of individual and pooled sample collection (organs vs swabs) was carried out to determine the sensitivity of the collection methods and the effect of pooling of samples for the detection of TSRV. Detection of TSRV in individual samples was as sensitive when organs were sampled compared to swabs, and in pooled samples, organs demonstrated a sensitivity of one 10-fold dilution higher than sampling of pooled swabs. Storage of swabs at 4 °C for t = 24 h demonstrated results similar to those at t = 0. Advantages of using swabs as a preferred sample collection method for the detection of TSRV compared to organ samples are evident from these experimental trials.
In 1995, and again in 1998, high mortalities of pilchards Sardinops sagax neopilchardus were seen... more In 1995, and again in 1998, high mortalities of pilchards Sardinops sagax neopilchardus were seen over their entire geographical range on the Australian coastline. A virus with typical herpesvirus morphology was identified as the causative agent, although the source of the virus remains unknown. At the time of the mortality events, the only available diagnostic test available for the detection of the virus was electron microscopy and hence, development of a rapid diagnostic test for detection and identification of the virus was required. Initial sequence data for Pilchard herpesvirus (PHV) was acquired by comparing the highly conserved region of the ORF 62 gene of other piscine herpesviruses (Ictalurid herpesvirus 1 [Channel catfish virus, CCV] and Salmonid herpesvirus 2 [Oncorhynchus masou virus, SaHV2, OMV]), and designing primers that successfully amplified a fragment of PHV. Here we describe the use of polymerase chain reaction (PCR) technology to detect PHV in infected tissues. Sequence analysis of amplified fragment resulting from this PCR is different from all known herpesviruses and can therefore distinguish PHV from all known viruses.
An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by se... more An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by sequencing the 18S-ITS-1 region of the small subunit rRNA operon obtained from flat oysters Ostrea angasi shown to be infected by histological examination. Sequence data alignment with homologous genes from 2 other isolates of Bonamia (New Zealand and France) revealed high levels of nucleotide identity with both isolates. However, the Australian Bonamia is shown to be more closely related to the New Zealand isolate, suggesting the existence of an oceanic subgroup of Bonamia.
ABSTRACT Experimental infection models using immersion and injection challenges were developed to... more ABSTRACT Experimental infection models using immersion and injection challenges were developed to investigate the effects of various physicochemical treatments on the abalone herpes virus (AbHV), an emerging virus causing viral ganglioneuritis in abalone in Australia. To determine stability at different temperatures, the virus was held at 4, 15, or 25oC for 1, 5, and 12 days prior to immersion challenge of naïve abalone. Mortality curves indicated that when held for one day in sea water at 4oC and 15oC the virus remained infectious and highly pathogenic. In addition, the virus retained partial infectivity after 5 days held at 4oC. Histological examination of abalone tissues following viral exposure confirmed the presence of lesions typical of abalone viral ganglioneuritis in animals showing morbidity signs. An additional experiment was performed to determine the virucidal efficacy of three disinfectants (calcium hypochlorite, Buffodine and the non-ionic surfactant Impress). The disinfectants were used at various doses and durations to treat AbHV prior to injection and immersion challenges. Results showed that Buffodine and the non-ionic surfactant Impress were effective at inactivating the virus with no detectable adverse effects on the abalone’s health. In addition, calcium hypochlorite showed a virucidal effect when used on lower titers of virus prior to immersion challenge.
Page 1. * Corresponding author E-mail: mark.crane@csiro.au This paper was presented in the First... more Page 1. * Corresponding author E-mail: mark.crane@csiro.au This paper was presented in the First International Symposium on Viral Nervous Necrosis of Fish held in Hiroshima, 2006. 魚病研究 Fish Pathology, 42 (4), 173179, 2007. 12 ...
ABSTRACT The development of molecular diagnostic assays with increased sensitivity compared with ... more ABSTRACT The development of molecular diagnostic assays with increased sensitivity compared with conventional histological techniques is highly desirable for effective management of bonamiosis in cultured oyster stocks and wild populations. A real-time TaqMan PCR assay was developed for the specific detection of Bonamia species in infected oyster tissues. The TaqMan assay was shown to be significantly more sensitive than histopathology. Although a real-time TaqMan PCR assay is comparable with conventional PCR in terms of sensitivity, it offers the advantages that it is a rapid test and has a very low risk of sample cross-contamination. Furthermore, it can be optimised to quantify the parasite load in samples. The assay detected Bonamia isolates from Australia, New Zealand, Europe, Canada, Chile and the USA and therefore demonstrated genus specificity as tested in this study.
An aquatic birnavirus, first isolated in Australia from farmed Atlantic salmon in Tasmania in 199... more An aquatic birnavirus, first isolated in Australia from farmed Atlantic salmon in Tasmania in 1998, has continued to be re-isolated on an infrequent but regular basis. Due to its low pathogenicity, there has been little urgency to undertake a comprehensive characterisation of this aquatic birnavirus. However, faced with possible incursions of any new aquatic birnaviruses, specific identification and differentiation of this virus from other, pathogenic, aquatic birnaviruses such as infectious pancreatic necrosis virus (IPNV) are becoming increasingly important. The present study determined the nucleic acid sequence of the aquatic birnavirus originally isolated in 1998, as well as a subsequent isolate from 2002. The sequences of the VP2 and VP5 genes were compared to that of other aquatic birnaviruses, including non-pathogenic aquatic birnavirus isolates from New Zealand and pathogenic infectious pancreatic necrosis virus isolates from North America and Europe. The deduced amino acid (aa) sequences indicate that the Australian and New Zealand isolates fall within Genogroup 5 together with IPNV strains Sp, DPL, Fr10 and N1. Thus, Genogroup 5 appears to contain aquatic birnavirus isolates from quite diverse host and geographical ranges. Using the sequence information derived from this study, a simple diagnostic test has been developed that differentiates the current Australian isolates from all other aquatic birnaviruses, including the closely related isolates from New Zealand.
ABSTRACT A continuous cell line, designated SBT-E1, was established from tissues of a southern bl... more ABSTRACT A continuous cell line, designated SBT-E1, was established from tissues of a southern bluefin tuna (Thunnus maccoyii) fingerling. Maximum cell proliferation rates occurred at 25 °C in Liebovitz's L-15 medium containing 10–20% FBS. Survival of SBT-E1 after short-term cryostorage was 70.8 ± 12.8%. The species of origin was confirmed by sequencing a region of the mitochondrial gene for cytochrome c oxidase, subunit I (cox1). The SBT-E1 karyotype, with a modal chromosome number of 2n = 46, differs from the constitutional karyotype of the parent species (2n = 48), with the apparent loss of either whole or partial chromosomes and the presence of small marker chromosomes. The SBT-E1 cell line represents the first reported cell line from any species of tuna.
A variant of the Y strain of Trypanosoma cruzi which exhibits a high frequency (≅90%) of epimas... more A variant of the Y strain of Trypanosoma cruzi which exhibits a high frequency (≅90%) of epimastigote-to-trypomastigote transformation when cultured under nor.
Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general,... more Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general, and Tasmanian Atlantic salmon reovirus (TSRV) specifically, were developed, and their diagnostic sensitivity and specificity were determined and compared with virus isolation in cell culture. Intralaboratory and interlaboratory comparison of PCR (conventional hemi-nested RT-PCR & RT-qPCR) and virus isolation in cell culture using finfish cell lines, CHSE-214 and EPC, was carried out for the detection and identification of TSRV using field samples of farmed Atlantic salmon Salmo salar, L. from various aquaculture sites around Tasmania. The interlaboratory comparison of diagnostic methods was carried out between two laboratories, AAHL-CSIRO and DPIPWE-Tasmania. A total of 144 fish from nine sites (12–33 fish per site) were sampled from two regions of Tasmania (Tamar River estuary in the north and Huon River estuary in the south-east) during late spring to early summer of 2009, and the dat...
We are investigating whether koi herpesvirus is a potentially good biological control agent for c... more We are investigating whether koi herpesvirus is a potentially good biological control agent for carp (Cyprinus carpio) in Australia. Our initial results show that: * Mortality is higher in juvenile compared with older carp. * Carp-goldfish hybrids are less susceptible to KHV, and we are currently determining the prevalence of hybrids in selected wild populations of carp in Australia. * We are examining wild-caught immature carp for less virulent cross-reactive viruses that may confer protection from KHV. * KHV has been found to have no effect on three native fish species that have been tested – Murray cod, golden perch and silver perch. Two other species are currently being tested. * Infected carp excrete KHV for 1 to 2 days before clinical signs of disease in the fish. We have also reviewed past successful viral biological control programs of vertebrate pests, and have concluded that management and development of a successful program is underpinned by (i) a thorough understanding o...
The growing global demand for seafood together with the limited capacity of the wild-capture sect... more The growing global demand for seafood together with the limited capacity of the wild-capture sector to meet this demand has seen the aquaculture industry continue to grow around the world. A vast array of aquatic animal species is farmed in high density in freshwater, brackish and marine systems where they are exposed to new environments and potentially new diseases. On-farm stresses may compromise their ability to combat infection, and farming practices facilitate rapid transmission of disease. Viral pathogens, whether they have been established for decades or whether they are newly emerging as disease threats, are particularly challenging since there are few, if any, efficacious treatments, and the development of effective viral vaccines for delivery in aquatic systems remains elusive. Here, we review a few of the more significant viral pathogens of finfish, including aquabirnaviruses and infectious hematopoietic necrosis virus which have been known since the first half of the 20th century, and more recent viral pathogens, for example betanodaviruses, that have emerged as aquaculture has undergone a dramatic expansion in the past few decades.
The use of swabs relative to organs as a sample collection method for the detection of Tasmanian ... more The use of swabs relative to organs as a sample collection method for the detection of Tasmanian salmon reovirus (TSRV) in farmed Tasmanian Atlantic salmon, Salmo salar L., was evaluated by RT-qPCR. Evaluation of individual and pooled sample collection (organs vs swabs) was carried out to determine the sensitivity of the collection methods and the effect of pooling of samples for the detection of TSRV. Detection of TSRV in individual samples was as sensitive when organs were sampled compared to swabs, and in pooled samples, organs demonstrated a sensitivity of one 10-fold dilution higher than sampling of pooled swabs. Storage of swabs at 4 °C for t = 24 h demonstrated results similar to those at t = 0. Advantages of using swabs as a preferred sample collection method for the detection of TSRV compared to organ samples are evident from these experimental trials.
In 1995, and again in 1998, high mortalities of pilchards Sardinops sagax neopilchardus were seen... more In 1995, and again in 1998, high mortalities of pilchards Sardinops sagax neopilchardus were seen over their entire geographical range on the Australian coastline. A virus with typical herpesvirus morphology was identified as the causative agent, although the source of the virus remains unknown. At the time of the mortality events, the only available diagnostic test available for the detection of the virus was electron microscopy and hence, development of a rapid diagnostic test for detection and identification of the virus was required. Initial sequence data for Pilchard herpesvirus (PHV) was acquired by comparing the highly conserved region of the ORF 62 gene of other piscine herpesviruses (Ictalurid herpesvirus 1 [Channel catfish virus, CCV] and Salmonid herpesvirus 2 [Oncorhynchus masou virus, SaHV2, OMV]), and designing primers that successfully amplified a fragment of PHV. Here we describe the use of polymerase chain reaction (PCR) technology to detect PHV in infected tissues. Sequence analysis of amplified fragment resulting from this PCR is different from all known herpesviruses and can therefore distinguish PHV from all known viruses.
An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by se... more An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by sequencing the 18S-ITS-1 region of the small subunit rRNA operon obtained from flat oysters Ostrea angasi shown to be infected by histological examination. Sequence data alignment with homologous genes from 2 other isolates of Bonamia (New Zealand and France) revealed high levels of nucleotide identity with both isolates. However, the Australian Bonamia is shown to be more closely related to the New Zealand isolate, suggesting the existence of an oceanic subgroup of Bonamia.
ABSTRACT Experimental infection models using immersion and injection challenges were developed to... more ABSTRACT Experimental infection models using immersion and injection challenges were developed to investigate the effects of various physicochemical treatments on the abalone herpes virus (AbHV), an emerging virus causing viral ganglioneuritis in abalone in Australia. To determine stability at different temperatures, the virus was held at 4, 15, or 25oC for 1, 5, and 12 days prior to immersion challenge of naïve abalone. Mortality curves indicated that when held for one day in sea water at 4oC and 15oC the virus remained infectious and highly pathogenic. In addition, the virus retained partial infectivity after 5 days held at 4oC. Histological examination of abalone tissues following viral exposure confirmed the presence of lesions typical of abalone viral ganglioneuritis in animals showing morbidity signs. An additional experiment was performed to determine the virucidal efficacy of three disinfectants (calcium hypochlorite, Buffodine and the non-ionic surfactant Impress). The disinfectants were used at various doses and durations to treat AbHV prior to injection and immersion challenges. Results showed that Buffodine and the non-ionic surfactant Impress were effective at inactivating the virus with no detectable adverse effects on the abalone’s health. In addition, calcium hypochlorite showed a virucidal effect when used on lower titers of virus prior to immersion challenge.
Page 1. * Corresponding author E-mail: mark.crane@csiro.au This paper was presented in the First... more Page 1. * Corresponding author E-mail: mark.crane@csiro.au This paper was presented in the First International Symposium on Viral Nervous Necrosis of Fish held in Hiroshima, 2006. 魚病研究 Fish Pathology, 42 (4), 173179, 2007. 12 ...
ABSTRACT The development of molecular diagnostic assays with increased sensitivity compared with ... more ABSTRACT The development of molecular diagnostic assays with increased sensitivity compared with conventional histological techniques is highly desirable for effective management of bonamiosis in cultured oyster stocks and wild populations. A real-time TaqMan PCR assay was developed for the specific detection of Bonamia species in infected oyster tissues. The TaqMan assay was shown to be significantly more sensitive than histopathology. Although a real-time TaqMan PCR assay is comparable with conventional PCR in terms of sensitivity, it offers the advantages that it is a rapid test and has a very low risk of sample cross-contamination. Furthermore, it can be optimised to quantify the parasite load in samples. The assay detected Bonamia isolates from Australia, New Zealand, Europe, Canada, Chile and the USA and therefore demonstrated genus specificity as tested in this study.
An aquatic birnavirus, first isolated in Australia from farmed Atlantic salmon in Tasmania in 199... more An aquatic birnavirus, first isolated in Australia from farmed Atlantic salmon in Tasmania in 1998, has continued to be re-isolated on an infrequent but regular basis. Due to its low pathogenicity, there has been little urgency to undertake a comprehensive characterisation of this aquatic birnavirus. However, faced with possible incursions of any new aquatic birnaviruses, specific identification and differentiation of this virus from other, pathogenic, aquatic birnaviruses such as infectious pancreatic necrosis virus (IPNV) are becoming increasingly important. The present study determined the nucleic acid sequence of the aquatic birnavirus originally isolated in 1998, as well as a subsequent isolate from 2002. The sequences of the VP2 and VP5 genes were compared to that of other aquatic birnaviruses, including non-pathogenic aquatic birnavirus isolates from New Zealand and pathogenic infectious pancreatic necrosis virus isolates from North America and Europe. The deduced amino acid (aa) sequences indicate that the Australian and New Zealand isolates fall within Genogroup 5 together with IPNV strains Sp, DPL, Fr10 and N1. Thus, Genogroup 5 appears to contain aquatic birnavirus isolates from quite diverse host and geographical ranges. Using the sequence information derived from this study, a simple diagnostic test has been developed that differentiates the current Australian isolates from all other aquatic birnaviruses, including the closely related isolates from New Zealand.
ABSTRACT A continuous cell line, designated SBT-E1, was established from tissues of a southern bl... more ABSTRACT A continuous cell line, designated SBT-E1, was established from tissues of a southern bluefin tuna (Thunnus maccoyii) fingerling. Maximum cell proliferation rates occurred at 25 °C in Liebovitz's L-15 medium containing 10–20% FBS. Survival of SBT-E1 after short-term cryostorage was 70.8 ± 12.8%. The species of origin was confirmed by sequencing a region of the mitochondrial gene for cytochrome c oxidase, subunit I (cox1). The SBT-E1 karyotype, with a modal chromosome number of 2n = 46, differs from the constitutional karyotype of the parent species (2n = 48), with the apparent loss of either whole or partial chromosomes and the presence of small marker chromosomes. The SBT-E1 cell line represents the first reported cell line from any species of tuna.
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Papers by Mark Crane