We report a molecular-based approach for diagnosing fish infections and the associated diseases in the most predominantly cultured fish species from some selected farms in Ghana. This was undertaken following the urgent need for a fast and reliable method of disease diagnosis in the country to help curb the fast rise in mass mortalities resulting from diseases in recent years. Thus, in this study, naturally occurring diseased tilapia and catfish with clinical signs (erratic swimming, lethargy, darkening of the skin, exophthalmia, skin lesions and ulcerations, eroded barbells, ascites, septicemia, abscess, gill necrosis, enlargement of liver) associated with bacterial diseases were investigated by PCR in order to identify the etiological agents. In aquaculture, diseases are known to be agents of mortality in all life stages of fish, once conditions are not favorable. However, juveniles or early growth stages are the most susceptible stages for diseases in fish (Houde, 1987; Sifa and Mathias, 1987). Morphometric analysis of diseased fish samples in this study corroborates these assertions, as diseased samples, particularly tilapia specimens were predominantly among the juveniles.
Fish are by their nature submerged in their environment, thus tend to have the percentage of bacterial genera present in their skin mucus almost the same as those in the environment. The results from this study showed that, almost all the bacterial pathogens detected on the fish were also present in the water in which they were cultured. The fish mucosal immunity made up of the skin and gut serve as the first barrier to infections (Maaser and Kagnoff, 2002) but also constitute a large area for the possible invasion of pathogens. Thus, the skin, the gut and by extension the diseased portion (dp) recorded significantly high bacterial invasion (P< 0.05) of 32.33%, 31.44% and 34.25% respectively. The diseased portions, as labelled in this study were mainly ulcerative skin lesions and lesion from effective parts such as the eyes, fins, and gills necrosis, also exposed to the fish environment as the skin. The other organs, mainly internal (liver, kidney,) harbored less bacterial pathogens (Fig 3) supporting the claim that detection of pathogens in the gills and mucosal areas of fish are more sensitive than in the muscle and other internal organs. Hence, an important aspect of the pathogenesis of some infections like columnaris constitutes the adherence of the bacteria to the gills (Decostere et al., 1999).
Direct isolation of bacteria from diseased fish without prior culturing was possible in a single-plex PCR reaction. Unlike in many research works where Taq buffer and polymerase were used in similar reactions (del Cerro et al., 2002; Mata et al., 2004), this work used a syber-mix reaction protocol which made it a better option as it reduced bench time and further increased the ease and speed in diagnosis. The protocol was highly sensitive with a minimum of 1pg of template DNA. The multiplex PCR (M-PCR) protocol developed was able to simultaneously determine the presence of target pathogens (S. agalactiae, S. iniae and S. aureus). Thus, in a single reaction, three important pathogenic species of bacteria predominantly involved in disease outbreak in aquaculture farms could be identified. The protocol represents a good and fast diagnostic tool as it also gave opportunity of knowing when only one, two, all three or none of the target species are present in a sample (Fig 6). The m-PCR protocol was found to be 100% specific to target species as there was no amplification of any bacterial DNA after series of reactions using the same primer sets against different bacteria species. It also demonstrated a 100% concordance with traditional phenotypic identification systems. Sequenced data confirmed the specificity of target species with 90-100% percentage identity from blast result from NCBI. Application of this protocol was practically easy, less tedious and enhanced the duration for diagnosis and the number of samples analyzed in minimum sample volume. Laboratory testing is the ultimate means of diagnosing fish infections, hence, this protocol could better help in rapid identification of disease pathogens. Particularly, considering the fact that all previous studies on bacterial infections in fish in Ghana using mainly culture-based methods, identified most pathogens to genus level and could not trace it down to a particular species or strain as causative agent of specific diseases (Takyi et al., 2012; Ampofo, 2000)
Multiple or co- infections were confirmed in the study by the presence of more than one pathogenic bacterium in an individual fish. Tilapia samples showed multiple infections with all six target species; S. iniae, S. agalactiae, S. aureus, Aeromonas hydrophila, Edwardsiella ictaluri, and Flavobacterium columnare. Also, in the catfish, all six species of bacteria were recorded with significantly higher (P<0.05) prevalence rates and intensity of infections compared to tilapia. These multiple infections in the catfish were highly consistent with the advanced state of the clinical signs (septicemia) shown in the diseased samples obtained. Multiple infections here thus substantiate the assertion that disease is caused by the synergistic interactions between two or more taxa, and not by a single-taxon (Kotob et al, 2017). Diagnosed individuals in this study were consistent with those reported in other studies (Mohamed et al., 2014; Iregui et al., 2014; Marcusso et al., 2015).
The genus Streptococcus is one of the most important Gram-positive bacteria (Conroy, 2009; Jimenéz, 2010) in aquaculture systems. Within this, the species S. agalactiae is the most prevalent worldwide (Sheehan et al., 2009). S. iniae is also a predominant pathogen in both tilapia and catfish. From the results, S. iniae, was the most predominant species affecting 93.33% of catfishes and 88% of tilapia fish samples followed by S. agalactiea. These species of Streptococcus isolated in the study are typical pathogens implicated in warm-water streptococcosis. Streptococcus infections particularly S. iniae, are known to be associated with outbreaks that occur at water temperatures above 150C. As observed, all the ponds and cages recorded a temperature higher than 150C and hence were affected by warm-water streptococcosis. Particularly, the catfishes were cultured in a pond of relatively high temperature and slightly above the recommended values, hence had severe multiple bacterial infections as compared to tilapia. When Budiati et al., (2015) assessed the microbial quality of catfish and tilapia from wet markets and ponds, they observed that higher bacterial load in fishes corresponded to high temperatures of pond. Similar assessment was made by Kouamé et al., (2014) when they studied the aquaculture potential of fish in various rearing systems in Ivory Coast and noted that, high temperature of the ponds had effects on fish growth rate.
With regards to prevalence, S. aureus was second to S. iniae in catfish. It affected 86.67% of catfishes and 14.78% of Tilapia. Staphylococci in fish and fish products has been traced from handlers and fish farmers (Singh and Kulsherestha, 1993). specifically, catfish used in this study as observed had experienced several human handlings during sorting, harvesting and packaging of ideal size for the market with those below marketable sizes returned to ponds. Also coupled with the fact that S. aureus was the only bacterial pathogen that was not recorded from the culture water samples obtained for this study, humans could therefore be confirmed as the direct source of S. aureus on the fish.
Aeromonas hydrophila is the causative agent of motile aeromonad septicemia (MAS). The symptoms of MAS include swelling of tissues, dropsy, red sores, necrosis, ulceration and haemorrhagic septicemia (Ibrahem et al., 2008; Swann. And White, 1914). Almost all these signs were shown by the diseased catfish. The presence of A. hydrophila, and similarly of E. ictalluri by itself, is not indicative of disease but stress often considered to be a contributing factor in the outbreak of disease caused by these bacteria (Nils kautsky et. al, 2000). Elevated water temperature, decreased dissolved oxygen concentration, or increased ammonia and carbondioxide concentrations have been shown to promote stress in fish and trigger MAS (Ibrahem et al., 2008; Swann and White, 1914) and from this study, co-infection with other pathogenic bacteria further promotes this condition.
In tilapia culture, four major bacterial diseases have so far been identified as most important to aquaculture production namely: Streptococcus agalactiae, Streptococcus iniae, Flavobacterium columnare and Francisella sp (Komar et al., 2018). Results from this study contributes to these findings as three of these pathogens were found to be prevalent in Ghanaian aquaculture facilities. Although at various prevalence rates (46%, 39%, 46%, 74%, 75%, 100%), all farms recorded the presence of these pathogens. Farms from which samples were taken were a good representation of large scale, mid to low scale operation systems in Ghana thus, corresponding to intensive to semi-intensive systems of fish farming. Farming intensity, good husbandry practices and maintaining optimum water quality was found to be a challenge in some of the farms, hence most of the bacterial infections triggered by suboptimal water quality and environmental conditions like Flavobacterium columnare, Aeromonas hydrophila, and Edwardsiella ictaluri were prevalent in this study.
Generally, pathogen load in samples obtained from ponds (all the catfishes and some tilapia samples) were higher than those from the cages. The source of these high prevalence of target pathogens was also traceable to the pond water as there was less flushing in the pond compared to the increased flushing in the cages. This reflected in the slightly anoxic conditions recorded in most of the ponds. Generally, frequent flushing of ponds reduces the accumulation of benthic communities in the pond, which in turn reduces bacterial load in the water, and subsequently in the fish. However, under poor husbandry practices, where change of fish pond water is rarely done, excessive accumulation of waste feed and high fecal material results in increased bacterial populations, and as an indicator the pH would be acidic (Mente et al., 2006). It was observed from the farms, particularly from the catfish pond, that it had huge accumulation of organic material and was visibly green colored, an evidence of huge quantity of algal bloom, hence water quality indicated values within the anoxic and acidic ranges as well as high conductivity and salinity (Table 3), This poor water quality therefore significantly correlated with the high prevalence of co-infections (Table 6).
Prevalence rate of bacterial pathogens among diseased and healthy fish samples were not significant. However, quantitative PCR analysis detailing the actual concentrations of pathogens in both diseased and healthy fish are reported elsewhere. The public health implication in consuming physically healthy but infected fish from various farms may be of high concern and the proper treatment of fish prior to consumption cannot be overemphasized owing to the zoonotic potential of these species. Hence, aquaculture production in Ghana, focused on increasing consumption of fish using methods that minimizes risks to public health is highly critical, particularly that which ensures maintaining good water quality and minimum contamination by handlers during, feeding, grading and netting.