Investigation of the prevalence and characterisation of infection by Kudoa thyrsites and K. paniformis in South African marine fish species

Thesis (PhD)--Stellenbosch University, 2019.ENGLISH ABSTRACT: Post-mortem myoliquefaction and formation of black pseudocysts in marine fish muscle are associated with infection by Kudoa thyrsites and/or K. paniformis. These quality defects become apparent only upon filleting, resulting in economic losses. Methods to detect infection include microscopy and polymerase chain reaction (PCR) techniques. These methods are time consuming, expensive, and destructive to the sample. Near-infrared (NIR) spectroscopy is a rapid, non-destructive method, used for identifying nematodes-infected fish. The aims of this study were to investigate the prevalence of K. thyrsites and K. paniformis in South African (SA) sardine (Sardinops sagax ocellatus), kingklip (Genypterus capensis) Cape hake (Merluccius capensis and M. paradoxus), and to use NIR spectroscopy to discriminate between infected and uninfected samples. A real-time quantitative PCR (qPCR) method was designed for use as qualitative determination of K. thyrsites. A total of 536 fish samples (296 SA sardine, 70 kingklip and 170 Cape hake) were analysed with qPCR. The SA sardine samples had K. thyrsites prevalence of 92%, Cape hake 79%, and SA kingklip 40%. Results showed that there was no significant difference in K. thyrsites prevalence between male and female (SA sardines, kingklip and Cape hake), season (SA sardines) and area of capture (SA sardines). Cape hake samples showed conflicting results for the relationship between K. thyrsites prevalence and size. A higher prevalence for smaller (39.63 ± 10.88 cm) than larger sized (49.46 ± 13.94 cm) samples was found in one study, while the other independent study showed no significant relationship to size. A limited number of Cape hake samples were used, resulting in larger sized fish to be poorly represented in this study. Infection by K. thyrsites had no significant effects (P > 0.05) on moisture and ash contents of SA sardine. Similarly, the moisture, ash and protein contents of infected Cape hake did not differ from uninfected samples. Moisture, ash and protein contents were therefore not useful in prediction of K. thyrsites infection. No NIR spectral differences between K. thyrsites infected and uninfected fish sample were observed when NIR spectroscopy and principal component analysis (PCA) were investigated. In addition, NIR spectroscopy, in combination with soft independent modelling of class analogy (SIMCA) and partial least square discriminant analysis (PLS-DA), was unable to distinguish infected from uninfected samples. Fish samples used in this study were frozen, resulting in water loss, protein degradation and aggregation, thus possibly masking the chemical and textural changes associated with K. thyrsites infection. NIR spectroscopy, in combination with SIMCA and PLS-DA, respectively, was therefore not a useful method to predict K. thyrsites infection in frozen fish samples. Further studies are suggested where the relationships between level of infection (number of K. thyrsites spores), myoliquefaction, and texture analyses, in combination with NIR spectroscopy, are investigated. However, since SA sardine has a high prevalence of infection, such a study may require more than 500 samples in order to ensure an equal amount of infected and uninfected samples for the development of reliable NIR classification models. For Cape hake, it is suggested to investigate the use of imaging spectroscopy in an attempt to differentiate between infected and uninfected samples based on optical properties of fish muscle and black pseudocysts.AFRIKAANSE OPSOMMING: Die nadoodse afbreking van spierweefsel en vorming van swart pseudoblaas in seevis word geassosieer met besmetting van Kudoa thyrsites en/of K. paniformis. Hierdie agteruitgang van viskwaliteit word eers waargeneem tydens filering wat bydrae tot ekonomiese verliese. Metodes om besmetting te bepaal behels mikroskopiese en polymerase ketting reaksie (PKR) tegnieke. Hierdie metodes is tydrowend, duur en destruktief tot die produk. Naby-infrarooi (NIR) spektroskopie is ‘n vinnige metode wat gebruik word om vis wat met nematodes besmet is te identifiseer. Die doelstellings van hierdie studie was om die teenwoordigheid van of K. thyrsites en/of K. paniformis in Suid-Afrikaanse sardien (Sardinops sagax ocellatus), stokvis (Merluccius capensis en M. paradoxus) en koningklip (Genypterus capensis) te bepaal, asook om NIR spektroskopie te gebruik om tussen besmette en onbesmette monsters te onderskei. ‘n Reëele kwantitatiewe PKR metode was ontwikkel om kwalitatief die teenwoordigheid van K. thyrsites te bepaal. Totaal van 536 visse (296 sardien, 70 koningklip and 170 stokvis) was geanaliseer met diè PKR metode. Suid-Afrikaanse sardien, stokvis en koningklip was besmet met K. thyrsites. Meeste (92%) van die sardien monsters was met K. thyrsites besmet, terwyl 83% van die stokvis monsters besmet was. Slegs 40% van die koningklip monsters was besmet. Resultate het geen noemenswaardige verskille in K. thyrsites besmetting tussen manlike en vroulike monsters (sardien, stokvis en koningklip), seisoen (sardien) en area van vangs (sardien) gewys nie. Monsters van stokvis het ‘n hoër voorkoms van K. thyrsites infeksie in kleiner (39.63 ± 10.88 cm) as groter monsters (49.46 ± 13.94 cm) getoon in die een studie; terwyl resultatke in ‘n onafhanklike studie geen verskil getoon het nie. ‘n Beperkte aantal monsters was gebruik. Gevolglik was groter stokvis swak verteenwoordig in hierdie studie. Resultate van besmetting met K. thyrsites het geen noemenswaardige effek (P > 0.05) op vog en as inhoud van sardien getoon nie. Soortgelyk, daar was geen noemenswaardige verskil in proteïen inhoud tussen besmette en onbesmette stokvis nie. Vog, as en proteïen inhoud was dus nie ‘n goeie voorspelling van K. thyrsites infeksie nie. Geen NIR spektrale verskille tussen K. thyrsites besmette en onbesmette vis monsters was waargeneem wanneer NIR spektroskopie en hoof komponent analises (PCA) ondersoek was nie. NIR spektroskopie, in kombinasie met sagte onafhanklike modelleerwerk van klas ooreenstemming (SIMCA) en gedeeltelik minste kwadraat ondreskeid analise (PLS-DA), was ook nie in staat om tussen besmette en onbestemme monsters te ondoerskei nie. Vis monsters gebruik in hierdie studie was bevrore, wat gelei het tot vogverlies en denaturasie van proteïne. Hierdie chemiese en struktureële veranderinge het moontlik die chemiese en tekstuur veranderinge wat geassosieer word met K. thyrsites besmetting verbloem. NIR spektroskopie, in kombinasie met SIMCA en PLS-DA, was dus nie suksesvol in die voorspelling van K. thyrsites infeksie in bevrore vis-monsters nie. Verdere navorisng word aanbeveel waar die verhouding tussen die vlak van besmetting (aantal K. thyrsites spore), nadoodse afbreking in speirweefsel, en die tekstuur van vis, in kombinasie met NIR spektroskopie, ondersoek word. Aangesien daar in sardien ‘n hoë voorkoms van besmetting bestaan, sal verdere navorsing meer as 500 monsters vereis om sodoende te verseker dat ‘n gelyke aantal besmette asook onbesmette monsters bekikbaar is vir die ontwikkeling van betroubare NIR klassifikasie modelle. Betreffende stokvis word navorisng deur die toepassing van beeldspektroskopie voorgestel in ‘n poging om tussen besmette en onbesmette monsters te onderskei, gebaseer op optiese verskille van visspierweefsel en swart pseudoblaas