Eric Leis

Optimizing, validating, and field testing a multiplex qPCR for the detection of amphibian pathogens

Amphibian populations worldwide are facing numerous threats, including the emergence and spread of infectious diseases. In the past 2 decades, Batrachochytrium dendrobatidis (Bd), a parasitic fungus, and a group of viruses comprising the genus Ranavirus have become widespread and resulted in mass mortality events and extirpations worldwide. In 2013, another novel fungus, B. salamandrivorans (Bsal), was attributed to dramatic declines in populations of fire salamander Salamandra salamandra in the Netherlands. Experimental infections demonstrated that Bsal is highly pathogenic to numerous salamander genera. In an effort to prevent the introduction of Bsal to North America, the US Fish and Wildlife Service (USFWS) listed 201 salamander species as injurious wildlife under the Lacey Act. To determine infection status and accurately assess amphibian health, the development of a sensitive and specific diagnostic assay was needed. We describe the optimization and validation of a multiplex quantitative polymerase chain reaction (qPCR) protocol for the simultaneous detection of Bd, Bsal, and frog virus 3-like ranaviruses. A synthetic genome template (gBlock®) containing the target genes from all 3 pathogens served as the positive control and allowed accurate quantification of pathogen genes. The assay was validated in the field using an established non-lethal swabbing technique to survey local amphibian populations throughout a range of habitats. This multiplex qPCR demonstrates high reproducibility, sensitivity, and was capable of detecting both Bd and ranavirus in numerous locations, species, and life stages. Bsal was not detected at any point during these sampling efforts.

Molecular characterization of a novel orthomyxovirus from rainbow and steelhead trout (Oncorhynchus mykiss)

Abstract A novel virus, rainbow trout orthomyxovirus (RbtOV), was isolated in 1997 and again in 2000 from commercially-reared rainbow trout (Oncorhynchus mykiss) in Idaho, USA. The virus grew optimally in the CHSE-214 cell line at 15°C producing a diffuse cytopathic effect; however, juvenile rainbow trout exposed to cell culture-grown virus showed no mortality or gross pathology. Electron microscopy of preparations from infected cell cultures revealed the presence of typical orthomyxovirus particles. The complete genome of RbtOV is comprised of eight linear segments of single-stranded, negative-sense RNA having highly conserved 5′ and 3′-terminal nucleotide sequences. Another virus isolated in 2014 from steelhead trout (also O. mykiss) in Wisconsin, USA, and designated SttOV was found to have eight genome segments with high amino acid sequence identities (89–99%) to the corresponding genes of RbtOV, suggesting these new viruses are isolates of the same virus species and may be more widespread than currently realized. The new isolates had the same genome segment order and the closest pairwise amino acid sequence identities of 16–42% with Infectious salmon anemia virus (ISAV), the type species and currently only member of the genus Isavirus in the family Orthomyxoviridae. However, pairwise comparisons of the predicted amino acid sequences of the 10 RbtOV and SttOV proteins with orthologs from representatives of the established orthomyxoviral genera and a phylogenetic analysis using the PB1 protein showed that while RbtOV and SttOV clustered most closely with ISAV, they diverged sufficiently to merit consideration as representatives of a novel genus. A set of PCR primers was designed using conserved regions of the PB1 gene to produce amplicons that may be sequenced for identification of similar fish orthomyxoviruses in the future.

Ligictaluridus michaelalicea n. sp. (Monogenea: Dactylogyridae) from flathead catfish ( Pylodictis olivaris ) in the upper Mississippi River, including remarks on taxonomy influencing monogenean treatment regulation in the United States

Ligictaluridus michaelalicea n. sp. (Monogenea: Dactylogyridae, Ancyrocephalinae) is described from the gills of Pylodictis olivaris (Siluriformes: Ictaluridae) from Wisconsin and Iowa portions of the upper Mississippi River. Diagnostic features include a relatively large, strongly curved tubular cirrus with minor terminal flare; an accessory piece with a prominent basal lobe and a simple, thick terminal limb featuring a thin lateral flange and blunt distal tip devoid of recurved hooks. The sinistral vagina, two prostatic reservoirs, and a terminal seminal vesicle of the vas deferens are prominent. The anchors, ventral and dorsal bars, and hooks are similar in overall form to those of other members of the genus. The description includes sequence data for the 18S rRNA gene, which aligned most closely with species of ancyrocephaline monogeneans, with the highest similarity being with Ligictaluridus pricei (Mueller, 1936). Other monogenean species identified from the flathead catfish examined included L. pricei and Ligictaluridus mirabilis (Mueller, 1937). L. michaelalicea n. sp. is the fourth species to be described from P. olivaris within its natural range in central and eastern North America. Implications resulting from taxonomic name changes, including species of Ligictaluridus, and United States Food and Drug Administration treatment regulations are discussed. An updated key to species of the genus Ligictaluridus is presented.

Comparison of Lethal and Nonlethal Sampling Methods for the Detection of Largemouth Bass Virus (LMBV) from Largemouth Bass in the Upper Mississippi River

Traditional methodologies to identify fish pathogens require euthanasia before the collection of tissue samples. While these methods are standardized and proven, there are instances where nonlethal alternatives would be preferred. Despite the need to develop nonlethal sampling techniques, few publications have focused on them and even fewer have used these approaches to identify viruses from infections occurring in wild fish populations. In this study, we compared the ability of nonlethal sampling techniques with traditional methods for the detection of Largemouth Bass virus (LMBV) from a wild population of Largemouth Bass Micropterus salmoides from the upper Mississippi River. Largemouth bass virus was isolated from 30% of the Largemouth Bass sampled using traditional methods where tissue samples were inoculated on Bluegill fry (BF-2) cells. Furthermore, when using tissue cell culture to isolate LMBV, there was no significant difference observed in the overall proportion that was positive between the mucus samples and the kidney and spleen samples. Mucus swabs analyzed with molecular methods (conventional PCR and quantitative PCR) were more sensitive than traditional tissue cell culture-based methods as they detected LMBV from >70% of the samples; limitations to these methods (i.e., carryover contamination) were also identified. The results of this study suggest that nonlethal sampling may be a useful option for detecting LMBV from fish populations.

Report of the Potential Fish Pathogen Pseudocapillaria (Pseudocapillaria) tomentosa (Dujardin, 1843) (Nematoda) from Red Shiner (Cyprinella lutrensis) Shipped from Missouri to Wisconsin

ABSTRACT:  A new host record for Pseudocapillaria (Pseudocapillaria) tomentosa (Dujardin, 1843) Moravec, 1987 from the intestine of red shiner, Cyprinella lutrensis, imported from a commercial facility in Missouri to a research facility in Wisconsin is described. Prevalence of infection was 85%. Intensity of infection was 1–26 worms per host. Supplementary information involving sequence data (18S ribosomal RNA gene) and new details of the male spicule are provided. The issue of parasite dispersal through the baitfish trade is briefly discussed.

Infections of Gyrodactylus crysoleucas and Gyrodactylus sp. (Monogenea) at a Golden Shiner (Notemigonus crysoleucas) Farm in Minnesota

ABSTRACT: Infections of Gyrodactylus are reported at a Minnesota baitfish farm producing golden shiner (Notemigonus crysoleucas) in earthen ponds. Intensities reached approximately 100 parasites per fish, with no apparent sign of disease. Two species were present: Gyrodactylus crysoleucas Mizelle and Kritsky, 1967, was common (90% of the Gyrodactylus population), while an unidentified Gyrodactylus sp., morphologically resembling Gyrodactylus wellborni Nowlin, 1968, but not confirmed as such, was less so (10% of the Gyrodactylus population). We provide supplemental morphological and molecular diagnostic information on these understudied parasites. The 2 species of parasites are easily confused diagnostically with Gyrodactylus salmonis Yin and Sproston, 1948, and Gyrodactylus colemanensis Mizelle and Kritsky, 1967, from salmonid fishes in North America, but can be differentiated on the basis of differences in shape and size of the haptoral hard parts and provided 18S and ITS sequences. The case history is ...