The Use of Polymerase Chain Reaction Assay to Diagnose Proliferative Gill Disease in Channel Catfish (Ictalurus Punctatus)

Abstract

Abstract. To assess the potential of a polymerase chain reaction (PCR) assay as a diagnostic tool in the detection of proliferative gill disease (PGD) in channel catfish (Ictalurus punctatus), PCR assays were compared with the traditional diagnostic methods of gill wet mounts and histology. A PCR assay using primers for Aurantiactinomyxon ictaluri, the actinospore associated with PGD, was performed with tissues from fish from commercial ponds. Using histology as the ''gold standard,'' the sensitivity, specificity, and accuracy of the PCR assay were all Ͼ90%. In comparison, the wet mount examinations had a lower sensitivity and specificity. Using the chi-square test and a test for strength of association, there was a significant, strong association between results obtained by PCR and those obtained by the other 2 methods. These results demonstrate that the PCR assay is a good diagnostic tool for the detection of PGD.

A Real-Time Polymerase Chain Reaction Assay for the Detection of the Myxozoan Parasite Henneguya Ictaluri in Channel Catfish

“…Target regions of the 18S SSU rRNA genes were amplified from genomic DNA of H. ictaluri actinospores and specific pathogen-free (SPF) channel catfish by use of H. ictaluri-specific PCR primers 19,24 and universal 18S SSU rRNA primers (Elibol-Fleming B: 2006, Effects of Edwardsiella ictaluri infection on transcriptional expression of selected immune relevant genes in channel catfish, Ictalurus punctatus. Doctoral dissertation.…”

“…Actinospores of each species were pooled separately by species, were enumerated, and a subsample was taken for molecular confirmation by sequencing. 11,20,24 DNA extraction from actinospores Actinospores were suspended in 20 ml of nuclease-free H 2 O and gently stirred with a magnetic stir bar. The concentration of actinospores/ml was determined by counting the number of actinospores in 10 separate 10-ml samples.…”

“…11 A unique H. ictaluri-specific TaqMan probe was designed based on the 103-bp region from nt 1516 to nt 1619 amplified by the H. ictaluri-specific primers A1-1 and A1-2 19,24,25 (Table 1). The 23-bp probe sequence (59-TCAGCCTTGATGTTGC-CACCTCA-39) beginning at nt 1573 was commercially prepared.…”

“…3, 4) in hematoxylin and eosin (HE)-stained tissue sections or by H. ictalurispecific polymerase chain reaction (PCR). 19,24,26 Research has shown that although PCR is more sensitive than histologic examination and more reliable in detecting early stages of infection, 24 it is unable to objectively quantify level of infection and is relatively time consuming in that it requires postreaction processing. Quantitative real-time PCR uses PCR chemistry in conjunction with fluorogenic probes to detect and monitor the amplification of reaction products after each amplification cycle.…”

“…19,20 The complex life cycle involves a myxospore stage in channel catfish and an actinospore stage in the benthic oligochete Dero digitata (Bellerud BL: 1993, Doctoral dissertation). 2,5,11,17,19,20,[23][24][25][26] Damage to gill tissue is thought to result from the development of an intense inflammatory reaction by the fish in response to penetration and proliferation of the actinospore stage of the parasite life cycle 19,20,23,26 (Fig. 1).…”