Cyprinid herpesvirus 2 (CyHV2) has been associated with epidemic mortality among cultured populations of goldfish Carassius auratus. As the principal target tissues are hematopoietic cells in the ...kidney and spleen, the disease is designated herpesviral hematopoietic necrosis (HVHN). Originally described from Japan, the virus is present in at least five other countries and probably has a global distribution in goldfish. Preventing the further spread of the virus via control programs that exploit sensitive viral detection methods is critical. We developed a conventional polymerase chain reaction (PCR) test based on unique sequences found in the putative helicase gene of CyHV2 and completed initial steps toward the validation of this test. The helicase CyHV2 PCR has an analytic sensitivity of at least 78 copies of the target sequence per reaction in serially diluted plasmid and 84 copies/μg of DNA from the kidney and spleen of goldfish experimentally infected with CyHV2. The analytic specificity of the helicase CyHV2 PCR was demonstrated by the lack of amplification of genomic DNA from cyprinid herpesvirus 1, cyprinid herpesvirus 3, and ictalurid herpesvirus 1 (IcHV1). The helicase CyHV2 PCR effectively detected DNA from CyHV2 from goldfish over a broad geographic range, including Japan, California, Ohio, and Pennsylvania. The performance of the helicase CyHV2 PCR was compared with that of the previously described real‐time TaqMan PCR for CyHV2 on a set of 37 samples of DNA from goldfish after experimental or natural exposure to CyHV2. The two tests had very strong agreement (kappa coefficient = 0.907) in classifying fish as positive or negative for CyHV2. The helicase CyHV2 PCR therefore complements the real‐time PCR test as a conventional diagnostic method for preventing the further spread of CyHV2.
Molecular epidemiology of koi herpesvirus Kurita, J.(National Research Inst. of Aquaculture, Tamaki, Mie (Japan). Tamaki Station); Yuasa, K; Ito, T ...
Fish Pathology,
2009, Letnik:
44, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Three regions of koi herpesvirus (KHV) genomic DNA were compared for 34 samples from Japan, six from Indonesia, two from Taiwan, one from the Philippines, 13 from the Netherlands, one from the UK, ...one from the USA and one from Israel. The analyzed genomic regions included known PCR-detection targets (Sph1-5, 9/5 and the thymidine kinase gene). The KHVs from Asian countries were very homogeneous, although two variants were noted based on a single nucleotide polymorphism. In contrast, seven variants were found in KHVs from outside of Asia, and although closely related to one another, they were clearly distinct from those from Asian. The results suggest that a clear genetic distinction exists between Asian and European (including each single isolate from the USA and Israel) types of KHV, and that unique types of KHV were independently introduced or emerged in the respective geographic locations.
Since 1998, episodes of mass mortality have occurred in populations of common carp Cyprinus carpio carpio in Israel and in populations of koi Cyprinus carpio koi in Israel and the USA. A herpesvirus ...isolated from infected fish has been shown in experimental studies to induce disease and mortality similar to those observed in outbreaks at infected farms. Initial characteristics of the virus show that it is clearly different from Herpesvirus cyprini (CHV), the most commonly known herpesvirus from cyprinid fish. The koi herpesvirus (KHV) has 31 virion polypeptides. Twelve of the virion polypeptides of KHV have similar molecular weights to those of CHV and 10 are similar to those of channel catfish virus (CCV). Both virion polypeptide and restriction fragment length polymorphism analyses of genomic DNA showed that the first KHV isolates from Israel and the USA were identical. In contrast, the genomic DNA restriction fragments clearly distinguish KHV from CHV and CCV. A polymerase chain reaction (PCR) assay to detect the virus in koi tissues was developed with sequences obtained from 1 restriction fragment of KHV DNA. The PCR assay effectively detected a 484 base pair sequence from KHV but did not amplify genomic DNA from either CHV or CCV. The PCR assay detected as little as 1 pg of KHV DNA mixed with 100 ng of host DNA. Viral sequences were amplified from koi obtained from field collections and from koi that were experimentally exposed to 10(2) TCID50 ml(-1) of KHV via the waterborne route. All KHV exposed fish dying of infection between 8 and 10 d post exposure or surviving to 14 d post exposure were found to be positive by PCR, while unexposed control koi were all negative. The assay also showed the presence of KHV DNA in tissues of koi obtained from farms in Israel. The PCR assay should assist virus isolation procedures and histologic and electron microscopic analyses now commonly used to detect KHV infection. Current studies are examining the possibility of using the PCR to detect KHV DNA in live fish and the relative sensitivity and specificity of the KHV PCR assay compared with other diagnostic tests.
Megalocytiviruses have been associated with epizootics resulting in significant economic losses in public aquaria and food-fish and ornamental fish industries, as well as threatening wild fish ...stocks. The present report describes characteristics of the first megalocytivirus from a wild temperate North American fish, the threespine stickleback Gasterosteus aculeatus. Moribund and dead fish sampled after transfer to quarantine for an aquarium exhibit had amphophilic to basophilic intracytoplasmic inclusions (histopathology) and icosahedral virions (transmission electron microscopy) consistent with an iridovirus infection. Phylogenetic analyses of the major capsid, ATPase, and DNA polymerase genes confirmed the virus as the first known member of the genus Megalocytivirus (family Iridoviridae) from a gasterosteid fish. The unique biologic and genetic properties of this virus are sufficient to establish a new Megalocytivirus species to be formally known as the threespine stickleback iridovirus (TSIV). The threespine stickleback is widely distributed throughout the northern hemisphere in both freshwater and estuarine environments. The presence of megalocytiviruses with broad host specificity and detrimental economic and ecologic impacts among such a widely dispersed fish species indicates the need for sampling of other stickleback populations as well as other North American sympatric marine and freshwater ichthyofauna.
Bivalve molluscs concentrate
Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction ...(PCR) system was developed to allow for large scale quantitative detection of
Cryptosporidium spp. in mussels (
Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log
10 improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected
C. parvum in <
5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of
C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of
C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of
C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.
Six species of the genus Myxobolus (Myxozoa) from the marine environment were collected from two species of mullet (Mugil cephalus and Liza ramada) in Ichkeul Lake, Tunisia. Four of these species ...were described previously (Myxobolus bizerti, Myxobolus ichkeulensis, Myxobolus spinacurvatura, and Myxobolus episquamalis) and two (Myxobolus exiguus and Myxobolus muelleri) are redescribed. The small subunit ribosomal (18S rDNA) sequences of these six myxozoans were obtained and compared to traditional criteria used in the identification and taxonomy of myxozoan species (such as spore morphology, host specificity, and tissue tropism). A distance analysis of 1,600–1,700 base pairs of the 18S rDNA of the six species indicates that they formed a monophyletic group separate from Myxobolus spp. found as parasites of freshwater fish. The sequence analyses also confirm that these morphologically different Myxobolus spp. that infect mullet represent different species. Lastly, M. exiguus and M. muelleri, which were found in the same host, exhibit clear differences in spore morphology but sequencing of two different regions of the 18S rDNA show they are closely related. These results demonstrate the utility of DNA sequence data in providing more detailed relationships among the Myxobolus species based upon existing morphological taxonomic approaches. We suggest that future descriptions of Myxobolus spp. provide both careful spore descriptions as part of the traditional criteria but also 18S rDNA sequence data that will aid in situations where morphological details may be absent or misleading.
Nucleospora salmonis is an intranuclear microsporidian parasite of salmonids that causes a chronic lymphoblastosis and a leukemic-like condition in both cultured and wild salmonids. Loop-mediated ...isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. Using the LAMP method, a protocol for detection of
N. salmonis was developed. A set of four primers, two inner and two outer were designed based on the sequence of ribosomal RNA intermediate spacer of this parasite. Optimum time and temperature conditions for detection of
N. salmonis by LAMP were 60 min at 63 °C, respectively. The detection-limit (DL) using LAMP was found to be similar to polymerase chain reaction (PCR). In this study, we have developed a highly sensitive and rapid diagnostic procedure for detection of
N. salmonis infections in cutthroat trout(
Oncorhynchus clarki) and rainbow trout (
Oncorhynchus mykiss).
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► UV irradiation at doses of 10–80mJ/cm2 inactivated waterborne infective stages of Myxobolus cerebralis. ► Attachment and penetration of infective stages of M. cerebralis to rainbow ...trout were unaffected by UV irradiation. ► UV-irradiated M. cerebralis initially replicated but then aborted development in rainbow trout. ► UV-irradiated M. cerebralis provided protection to subsequent exposures to fully infective parasites.
Myxobolus cerebralis is a microscopic metazoan parasite (Phylum Myxozoa: Myxosporea) associated with salmonid whirling disease. There are currently no vaccines to minimise the serious negative economical and ecological impacts of whirling disease among populations of salmonid fish worldwide. UV irradiation has been shown to effectively inactivate the waterborne infective stages or triactinomyxons of M. cerbralis in experimental and hatchery settings but the mechanisms by which the parasite is compromised are unknown. Treatments of triactinomyxons with UV irradiation at doses from 10 to 80mJ/cm2 either prevented (20–80mJ/cm2) or significantly inhibited (10mJ/cm2) completion of the parasite life cycle in experimentally exposed juvenile rainbow trout (Oncorhynchus mykiss). However, even the highest doses of UV irradiation examined (80mJ/cm2) did not prevent key steps in the initiation of parasite infection, including attachment and penetration of the epidermis of juvenile rainbow trout as demonstrated by scanning electron and light microscopy. Furthermore, replication of UV-treated parasites within the first 24h following invasion of the caudal fin was suggested by the detection of concentrations of parasite DNA by quantitative PCR comparable to that among fish exposed to an equal concentration of untreated triactinomyxons. Subsequent development of parasites treated with an 80mJ/cm2 dose of UV irradiation however, was impaired as demonstrated by the decline and then lack of detection of parasite DNA; a trend beginning at 10days and continuing thereafter until the end of the study at 46days post parasite exposure. Treatments of triactinomyxons with a lower dose of UV irradiation (20mJ/cm2) resulted in a more prolonged survival with parasite DNA detected, although at very low concentrations, in fish up to 49days post parasite exposure. The successful invasion but only short-term survival of parasites treated with UV in rainbow trout resulted in a protective response to challenges with fully infective triactinomyxons. Prior treatments of juvenile rainbow trout with UV-treated triactinomyxons (10 and 20mJ/cm2) resulted in a reduced prevalence of infection and significantly lower concentrations of cranial myxospores (two direct measures of the severity of whirling disease) compared with trout receiving no prior treatments when assessed 5months post parasite exposure to fully infective triactinomyxons.
This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank ...exposure experiments to evaluate clams that had filtered
Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect
Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting
Cryptosporidium parvum oocysts in clams. In the wild clam study,
Cryptosporidium and
Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of
C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.
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