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

Standish, Isaac; Leis, Eric; Schmitz, Noel; Credico, Jeena; Erickson, Sara M.; Bailey, Jennifer; Kerby, Jacob L.; Phillips, Kenneth; Lewis, Teresa D.

doi:10.3354/dao03230

Cited by 30 publications

(29 citation statements)

References 76 publications

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“…A synthetic gene block (gBlock ® ; Figure ) was utilized for optimization and quantification purposes. The gBlock was rehydrated, quantified and diluted as described previously (Standish et al, ), resulting in a standard curve of 10‐fold dilutions ranging from 10 7 to 1 copy reaction −1 . Initial steps were taken to optimize and validate the assay prior to use.…”

Section: Methodsmentioning

confidence: 99%

Vagococcus salmoninarum II—qPCR, tropism and egg‐associated transmission

Standish

Leis

Erickson

et al. 2020

Journal of Fish Diseases

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Vagococcus salmoninarum was identified as the causative agent of a chronic epizootic in broodstock “coaster” brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery. The epizootic spanned more than a year, was unresponsive to multiple florfenicol treatments, and resulted in >50% mortality of the affected fish. The decision was made to cull the remaining fish during spawning, which presented an opportunity to more thoroughly examine V. salmoninarum sampling methods, organ tropism and vertical transmission. A newly developed qPCR targeting the pheS gene was used in concert with bacterial culture to show that V. salmoninarum indeed disproportionately affects females and has a tropism for female reproductive tissues. The study demonstrates that some female reproductive tissues (e.g. ovarian fluid, unfertilized eggs) are also an effective option for non‐lethal detection. Despite the widespread presence of V. salmoninarum in ovarian fluid and on egg surfaces, we found no evidence of intra‐ova transmission.

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Section: Methodsmentioning

confidence: 99%

Vagococcus salmoninarum II—qPCR, tropism and egg‐associated transmission

Standish

Leis

Erickson

et al. 2020

Journal of Fish Diseases

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show abstract

“…Field studies suggested that co-occurring Bd and Ranavirus can, in some cases, have positive effects on each other [ 8 , 61 , 165 , 166 ]. Still, this interaction was not confirmed in other cases [ 61 , 159 , 164 , 167 ] or even turned negative [ 165 , 168 ] (Table 2 ). A lower Bd or Ranavirus infection intensity in individuals co-infected by both parasites compared to single infections may arise from an enhanced immune response of the host or the interspecific competition between the agents [ 166 ].…”

Section: Co-infection By Heterologous Parasitesmentioning

confidence: 94%

Host–multiparasite interactions in amphibians: a review

et al. 2021

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Parasites, including viruses, bacteria, fungi, protists, helminths, and arthropods, are ubiquitous in the animal kingdom. Consequently, hosts are frequently infected with more than one parasite species simultaneously. The assessment of such co-infections is of fundamental importance for disease ecology, but relevant studies involving non-domesticated animals have remained scarce. Many amphibians are in decline, and they generally have a highly diverse parasitic fauna. Here we review the literature reporting on field surveys, veterinary case studies, and laboratory experiments on co-infections in amphibians, and we summarize what is known about within-host interactions among parasites, which environmental and intrinsic factors influence the outcomes of these interactions, and what effects co-infections have on hosts. The available literature is piecemeal, and patterns are highly diverse, so that identifying general trends that would fit most host–multiparasite systems in amphibians is difficult. Several examples of additive, antagonistic, neutral, and synergistic effects among different parasites are known, but whether members of some higher taxa usually outcompete and override the effects of others remains unclear. The arrival order of different parasites and the time lag between exposures appear in many cases to fundamentally shape competition and disease progression. The first parasite to arrive can gain a marked reproductive advantage or induce cross-reaction immunity, but by disrupting the skin and associated defences (i.e., skin secretions, skin microbiome) and by immunosuppression, it can also pave the way for subsequent infections. Although there are exceptions, detrimental effects to the host are generally aggravated with increasing numbers of co-infecting parasite species. Finally, because amphibians are ectothermic animals, temperature appears to be the most critical environmental factor that affects co-infections, partly via its influence on amphibian immune function, partly due to its direct effect on the survival and growth of parasites. Besides their importance for our understanding of ecological patterns and processes, detailed knowledge about co-infections is also crucial for the design and implementation of effective wildlife disease management, so that studies concentrating on the identified gaps in our understanding represent rewarding research avenues.

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“…Extracted DNA from skin swabs was used to screen for Batrachochytrium dendrobatidis ( Bd ), B. salamandrivorans ( Bsal ), and the ranavirus frog virus 3 (FV3). Multiplex quantitative PCR was conducted as described by Standish et al (2018).…”

Section: Methodsmentioning

confidence: 99%

Nephroblastoma in a Common Mudpuppy Necturus maculosus simultaneously Present with a Mollicute Bacterium of the Genus Acholeplasma

et al. 2021

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In March 2017, a wild‐caught female common mudpuppy Necturus maculosus from Iowa, USA, with an enlarged posterior abdomen was submitted for diagnostic assessment. The cause of the abdominal distension was a large fluid‐filled abdominal mass, diagnosed as a nephroblastoma. Parasites and numerous bacteria were isolated and identified from the mudpuppy but were determined to be incidental. Samples of the neoplasm inoculated onto an American toad Anaxyrus americanus cell line (BufoTad) yielded cytopathic effect during several passages. However, standard molecular testing of the cell culture supernatant failed to identify any viruses. Next‐generation sequencing identified the replicating agent as a bacterium of the genus Acholeplasma. Immunohistochemistry confirmed the presence of Acholeplasma within the nephroblastoma, including within tumor cells. This is the first report of nephroblastoma and the second report of neoplasia in this species. The results also suggest that certain bacteria of the genus Acholeplasma might be oncogenic.

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

Cited by 30 publications

References 76 publications

Vagococcus salmoninarum II—qPCR, tropism and egg‐associated transmission

Vagococcus salmoninarum II—qPCR, tropism and egg‐associated transmission

Host–multiparasite interactions in amphibians: a review

Nephroblastoma in a Common Mudpuppy Necturus maculosus simultaneously Present with a Mollicute Bacterium of the Genus Acholeplasma

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