Quantitative polymerase chain reaction offers improved diagnostic and analytical sensitivity in identifying differential microsporidium (Loma morhua) infections among family lines of cultured Atlantic cod (Gadus morhua)

Abstract

The failed attempt to establish Atlantic cod as an alternate aquaculture species in eastern Canada is due in part to hatchery outbreaks of the microsporidium Loma morhua during selective breeding programs. The sporadic nature of microsporidian epizootics during fish culture emphasizes the need for streamlined quantitative diagnostic approaches that help promote development of disease mitigation strategies. The current gold standard for assessing infection status relies on proficiency with parasitological confirmation of hypertrophied host cells (xenomas) that house infectious microsporidian spores. Parasite quantification is complicated with certain host organs showing better diagnostic utility, xenomas being heterogeneously distributed throughout these tissues, variable numbers of spores occurring within xenomas, and with pre-xenoma life-stages being difficult to identify using gold standard procedures. Herein, individuals from ten cod families with differential susceptibility to L. morhua were analyzed using a quantitative PCR (qPCR) assay, developed in accordance to the Minimum Information Required for Quantitative Experiments (MIQE) guidelines, to assess assay utility in quantifying infections relative to the gold standard procedure. Heterogeneous parasite distribution within spleens necessitated development of a tissue disruption protocol (FastPrep homogenizer; MP Biomedical) yielding a homogenous preparation for standardized DNA extractions. qPCR primers and a hydrolysis probe targeting a region of large subunit ribosomal DNA (rDNA) enable L. morhua to be distinguished from congeneric species infecting non-gadid hosts, but not those infecting gadid hosts. Parasite rDNA copies were normalized to Atlantic cod glyceraldehyde-3-phosphate dehydrogenase genomic DNA and to spleen weight. The qPCR assay demonstrates linearity (R2 = 0.971 to 0.999; range) throughout a large range of five log10 plasmid concentrations (102 to 106 titrated in host DNA), low variability (CV = 11.2–23.2%; n = 11 separate reactions), and the lower limit of detection is 10 plasmid copies. Quantification of parasite rDNA among ten cod families with differential infections resulted in a positive correlation with gold standard xenoma enumeration/confirmation (Pearson Correlation, r = 0.71), thereby validating effectiveness of qPCR in the absence of parasitological expertise. Furthermore, three fish lacking morphologically discernible spleen xenomas were identified as false negatives based on qPCR detection of 141–242 rDNA copies per 200 ng of spleen DNA, thereby confirming infection. This finding extends qPCR assay utility to detection of even subclinical infections. This qPCR assay improves diagnostic and analytical sensitivity in detecting L. morhua infections, facilitates empirical assessment of preventative/therapeutic treatment of microsporidian infections, and supports studies to identify biomarkers that promote selection of broodstock fishes that resist microsporidian disease.