Evaluation of quantitative real-time PCR for rapid assessments of the exposure of sentinel fish to Myxobolus cerebralis

Abstract

Pathogen-free rainbow trout (Oncorhynchus mykiss) aged 735 degree days were experimentally exposed to a low dose of infectious Myxobolus cerebralis (20 triactinomyxons fish(-1)). Three time periods were chosen for sampling that included 10 days (d), 67 d, and 5 months (mo) post exposure. Five diagnostic assays were used: (1) conventional single-round polymerase chain reaction (PCR), (2) nested PCR, (3) real-time TaqMan PCR, (4) pepsin-trypsin digest, and (5) histopathology. M. cerebralis was detected among individual rainbow trout by all of the PCR diagnostic tests employed at each of the three sampling time points. This result demonstrates that any of these three diagnostic approaches are capable of detecting the parasite from infected fish tissues under the conditions tested. Real-time PCR provided good biological evidence that parasite replication increases temporally as shown by quantification values that were significantly different (P<0.0001) at 10 d as compared to 67 d and 5 mo postexposure. Although sampling at 10 d by real-time PCR may be too early to accurately predict quantities of the parasite that will be present at 5 mo, it does forecast the proportions of fish that are likely to be infected at 67 d and 5 mo postparasite exposure. Real-time PCR could potentially be used as a quantitative diagnostic PCR tool to predict parasite load and outcome of M. cerebralis infection.

Molecular Identification and Real-time Quantitative PCR (qPCR) for Rapid Detection of Thelohanellus kitauei, a Myxozoan Parasite Causing Intestinal Giant Cystic Disease in the Israel Carp

“…Pathogen monitoring is an integral part of developing management strategies to reduce the impact of disease on free-ranging and wild fish populations as well as farmed fish populations [15], and real-time PCR has been used as a useful tool for the detection and quantification of a variety of myxozoan parasites [12,13]. Moreover, the number of water-borne parasites in water samples may be considered an indirect indicator of infection risk or intensity, and real-time PCR analysis of water samples may prove useful as a novel method to replace the traditional use of susceptible host fish for both the detection and quantification of specific parasites [15,16,19-21].…”

Quantitative Polymerase Chain Reaction (PCR) for Detection of Aquatic Animal Pathogens in a Diagnostic Laboratory Setting

“…A good target gene choice requires that one knows (1) the extent of sequence variation within the target primer and probe sites, (2) whether the target gene is single or multi-copy, and (3) the extent of similarities with nontarget sequences from other pathogens or sources. Ribosomal DNA genes (e.g., small subunit rDNA) are a common choice for qPCR assay development (Corbeil et al 2003;Kelley et al 2006;Funk et al 2007;Phelps and Goodwin 2007;True et al 2009) because of their widespread use in phylogenetic analysis of bacterial and parasitic pathogens. Less commonly, internal or nontranscribed spacer (ITS and NTS) regions are chosen as a target (Ulrich et al 2007;Foltz et al 2009).…”

In vivo exposure of susceptible and non-susceptible fish species to Myxobolus cerebralis actinospores reveals non-specific invasion behaviour

“…Quantitative real-time PCR (qPCR) methods have recently become a valuable tool to estimate parasite load and infection course of Myxobolus cerebralis (Cavender et al 2004, Kelley et al 2006. By using this technique, with an in vivo exposure approach and detection of invading stages on fish tissues and parasite abundance after sporogenesis, we intended to shed light on host-specificity during the invasion of M. cerebralis actinospores.…”