Natural Concurrent Infections with Black Spot Disease and Multiple Bacteriosis in Farmed Nile Tilapia in Central Kenya

Nile tilapia (Oreochromis niloticus) is the most cultured and available fish for Kenyan consumers, and therefore, any tilapine disease deprives them the valuable source of protein. Nile tilapia farm was diagnosed with severe concurrent black spot disease and multiple bacteriosis using gross lesions and parasitological, histopathology, and standard bacteriological procedures. A total of 25 fish were sampled and inspected, and all of them had raised, macroscopic 1 mm-sized black spot lesions. The mean intensity of black spots per fish was 728 with an abundance of 2–1740 metacercariae cysts per fish. A high intensity of black spot infestation was observed in the fins (43.9%), skin and underlying muscles (18.3%), and gills (18%). In addition, histopathological data confirmed presence of a metacercaria of Neascus spp. as the aetiological agent of black spot disease. Furthermore, a thick fibrous capsule around the metacercaria, black pigment melanomacrophages, and moderate muscle atrophy were observed. The most prevalent bacteria isolated were Aeromonas, Enterobacter cloacae, Klebsiella pneumoniae, and Micrococcus luteus. Physicochemical parameters of pond water were temperature (28.2°C), dissolved oxygen (4.2 mgl−1), pH (8.5), ammonia free nitrogen (15.8 mgl−1), alkalinity (112 mgl−1), hardness (68 mgl−1), nitrites (0.058 mgl−1), nitrates (58 mgl−1), and phosphates (0.046 mgl−1). However, the levels of nitrates, nitrites, alkalinity, and ammonia free nitrogen exceeded the recommended limits. In conclusion, these findings suggest that coinfections by these organisms coupled by water quality-related stress can be associated with low-grade mortality observed in postfingerling tilapia as well as reduced growth. The authors recommended immediate destocking, thorough disinfection, and control of piscivorous birds. Moreover, attention ought to be geared towards prevention of parasitic infestations in fish so as to minimize fish deaths related to secondary bacteriosis. Further experimental studies should be carried out to elucidate the relationship of these pathogens.


Introduction
Nile tilapia (Oreochromis niloticus) is a resilient species, which grows in a diverse range of aquatic environment, enduring extreme limits of dissolved oxygen, temperature, and contaminants [1]. Nile tilapia possesses advantageous characteristics for pisciculture, including ability to grow rapidly [2], excellent feed conversion efficiency [3], high resistance to disease [4], and ability to reproduce in captivity [5]. is justifies the dominance of Nile tilapia in global-scale production. e roughness and/or resilient nature and disease resistance make it an ideal aquatic "zebu". However, recent epidemics such as tilapia lake virus have threatened the species and consequently have outlined the importance of protecting the species through increased disease surveillance [6].
Farmed tilapine fish are exposed to single or multiple pathogens such as parasitic, bacterial, or mixed infections leading to diseases and mortalities [7]. Even though multiple or concurrent infections are common naturally, most studies on tilapia diseases focus on isolation of single pathogen [8]. Poor understanding of coinfections may explain this observation [9].
Black spot disease or black grub disease is caused by the metacercarial stage of several genera of digenean flukes among the families Heterophyidae and Diplostomatidae [20]. Species of the genera Apophallus, Crassiphiala, Uvulifer [21], Bolbophorus [22], and more generally as Neascus-type trematodes [23,24] have been documented in many freshwater and saltwater fish. e pathogenesis of black spot disease follows penetration of larvae forms (cercariae) of the parasites into the skin of a fish, where they encyst and develop into metacercariae [25]. e metacercariae provokes the host to form a fibrous capsule around the parasite. e host immune and/or inflammatory response results into infiltration of melanomacrophages through the fibrous wall of the cysts, hence causing the typical appearance of black spots [26]. e black spot lesions are usually visible grossly. e objective of this study was to describe the occurrence of natural coinfection of black spot disease with multiple bacteriosis in Nile tilapia (Oreochromis niloticus) in a smallscale farm in Central Kenya that was experiencing mortality and aesthetic rejection of fish.

Study Area and Case History.
e study was conducted between the months of February and November 2018, in a farm located in Kibingo, Kirinyaga Central subcounty, Central Kenya, on longitude 37°15.256E and latitude 00°29.085S and at an altitude of 1652 m above sea level. e farm owns a single 336 m 2 sized ultraviolet-treated plasticlined pond, stocked with mixed sex Nile tilapia. e disease history was obtained from the owner. e initial fish stocking density was 1000 fingerlings; however, current stocking density was unknown due to inbreeding, partial harvests, and mortalities. e disease was surmised when the owner noted raised black skin lesions on fish with low mortality (especially the postfingerlings) and high morbidity and harvested fish rejected by consumers.

Water Quality Assessment.
Water parameters such as temperature, dissolved oxygen, and pH were measured in situ using waterproof handheld HANNA Multiprobe meters 9142 and 98127, respectively (Hanna Instruments Inc., USA), at three different sites on the pond. Water samples were collected and analysed for ammonia free nitrogen, alkalinity, hardness, nitrites, nitrates, and phosphates following standard methods for examination of water and wastewater developed by Boyd and Tucker [27].

Fish Sampling, Necropsy, and Parasitological
Examination. Following owner's consent, twenty-five fish were harvested using a seine net. ese were then transported alive in buckets with source water to County Veterinary Laboratory, Kerugoya, for necropsy and analysis.
Postmortem examination was performed using standard procedures as described by Noga [28] and Roberts [29]. e fish were stunned with a single blow to the back of the head and pithed to separate the central nervous system from the spinal cord. An external examination of individual fish was performed to check for occurrence of ectoparasites and gross lesions. Prior to dissection, individual fish weight (g) and total length were recorded for calculation of the condition factor. e condition factor was calculated by a formula given by Froese [30]: K � (Wx100/L 3 ), where "K" is Fulton's condition factor, "W" is the wet weight in grams, and "L" is the total body length in centimetres. e sex of the fish was also recorded.

Histopathological Examination.
Tissue specimens were taken from the skin and underneath muscle from the flanks of few O. niloticus that were heavily infested with black spot lesions. e samples were fixed in 10% buffered formalin overnight. e tissue sections were then gradually dehydrated in 70-100% ethanol, cleared in xylene, and finally embedded in paraffin wax through standard procedures [31,32]. Tissue specimens were sectioned at 3-5 µm and stained with haematoxylin and eosin (H&E). Slides were then observed under a light microscope.

Bacteriological Examination.
Out of the 25 fish collected, 10 fish were randomly selected for bacteriological isolation, which was done on aseptically collected kidney swab from individual fish. ese swabs were streaked separately and aseptically onto plates containing nutrient agar, 10% sheep blood agar, and MacConkey agar. e inoculated plates were incubated aerobically at room temperature (24°C-26°C), in an inverted position. After 24-48 hours of incubation, the plates were examined, and colony morphology on the plates was recorded. e isolates were identified using colony morphology, Gram staining characteristics, conventional biochemical tests (catalase reaction, cytochrome oxidase, methyl red, citrate utilization, urea degradation, sulphur-indole-motility, and sugars fermentation) following Austin and Austin [11], Bergey's Manual of determinative bacteriology [33], and Markey et al. [34]. Further characterization of Gramnegative lactose fermenters was conducted using the Analytic Profile Index 20E (API 20E) microbial identification strips according to manufacturer's protocol (Bio-Mérieux Marcy-l'Étoile, France).

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Data Analysis.
e disease prevalence, mean abundance (average number of black spot lesions seen in all the fish), and mean intensity (mean number of black spot lesions per fish infested) were calculated as described by Bush et al. [35]. e collected data were validated, entered, and stored in Microsoft Excel ® spreadsheet, which was also used to calculate means and proportions. Inferential statistics were performed using Statistical Package for the Social Sciences (SPSS ® ), version 22.0. e chi square test was used to compare proportions. Pearson correlation matrix was used to check for association. All tests were tested at a level of 0.05 for significance.

Physicochemical Parameters of Pond Water and on Farm
Observations. e pond water colour was dark green, an indication of overfertilization. e average values of other parameters measured are summarized in Table 1. From the table, the pond water temperature, pH, dissolved oxygen, water hardness, and phosphates levels measured were within the recommended/desired range for rearing tilapia. However, levels of nitrates, nitrites, alkalinity, and ammonia free nitrogen were above the recommended limits.

Fish Samples and eir Biodata.
Of the 25 fish sampled, 16 were females and the rest were males. e mean weight, standard, and total lengths of the fish samples were 119 ± 4.7 g, 15.2 ± 0.2 mm, and 19 ± 0.3 mm, respectively. e mean condition factor (K) of fish was 1.73 ± 0.03, with no significant difference between males and females.

Macroscopic and Necropsy Findings.
On macroscopic examination, all the 25 fish examined were infested with macroscopic 1 mm raised black spot lesions, with no other apparent clinical signs (behavioural) of parasitism or bacteriosis. e lesions were extensively distributed throughout the body surface including the fins, operculum, mouth, skin, gills, and eyes (Figure 1(a)). On dissecting the fish, more conspicuous black spots were also observed on the epidermis, dermis, and musculature and on the gill rakers and filaments of fish (Figures 1(b) and 1(c)). ere were lesions in the vertebral bone; however, no skeletal deformity was observed. ere were no significant findings seen in other visceral organs. Table 2 shows distribution of lesions in various organs and their corresponding percentage. e fins were the most infested.
e mean intensity of black spots per fish was 728.48 3 with an abundance range of 2-1740 metacercariae cysts per fish. ere was a significant negative correlation between intensity and body condition (R 2 � −0.49).

Histopathological Findings.
Histopathological findings revealed that the aetiological agent causing this disease was a metacercaria of Neascus spp., a digenean trematode. e parasite localized in the stratified epithelial tissue and musculature (Figure 2(a)). Histopathological analysis further revealed a thick fibrous capsule around the encysted metacercariae with the periphery of the cyst containing black pigment due to melanomacrophages responsible for the formation of dark spots (Figure 2(b)). Moderate muscle atrophy was also observed.

Discussion
Although black spot disease (BSD) does not cause much pathological effect, it affects the aesthetic appeal of fish, therefore causing rejection at market level [25]. However, the black spots can be quite pathogenic if they are located in sensitive areas such as gills. Gill infestation leads to respiratory distress [39], while fish with eye infestation may be blind.
Coinfections occur when hosts are infected by two or more different pathogens either concurrently or as secondary invaders, so that two or more pathogenic agents are active together in the same host [40].
is study confirmed such coinfections of metacercarial stages of Neascus spp. causing "black spot" disease with several bacteria genera including Aeromonas, Enterobacter, Klebsiella, and Micrococcus in farmed Nile tilapia collected from Kirinyaga County. e results are contrary to a previous study in Winam Gulf of Lake Victoria, Kenya, by on et al. [41] who reported a prevalence of 0.7% in wild Nile tilapia. e differences in prevalence rate may be due to the fact that parasitic infestations might be  [36][37][38]. Key: a � pond value was above the recommended range; b � pond value was below the recommended range; and c � pond value was within the recommended range.
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is is so because farmed fish is associated with stressful culture conditions including overcrowding and poor water quality, thus making them more susceptible to diseases [42,43]. Moreover, on et al. [41] focused on parasitological isolation of endohelminths of wild Nile tilapia, and therefore, the bacteriological status of the sampled fish was not investigated. e findings are, however, partially in line with a previous study in Pacific Northwest by Arkoosh et al. [23] who reported coinfection of black spot disease and Renibacterium salmoninarum in juvenile salmon (Oncorhynchus species). In this study, several bacteria taxa including Aeromonas spp., Klebsiella pneumoniae, Enterobacter cloacae, and Micrococcus luteus were isolated from the kidney of examined fish. e discrepancy in bacteria taxa in the current study and that of Arkoosh et al. [23] could be due to culture environment of the fish species; tilapia is a warm water species, while salmon is a cold water fish. e bacteria isolated in this study are ubiquitous in aquatic environments and have been reported previously in Kenya [13,14]. Some of these including Aeromonas spp. and Klebsiella pneumoniae are pathogenic to fish [11], while Micrococcus luteus have been developed as fish probiotics [44]. Recovery of these bacteria from the kidney is an indication of infection after overcoming the fish defence mechanisms.
In this study, all the sampled fish had a condition factor of more than one. A condition factor value of more than one implies a good fish health condition and proportional growth, which is recommendable in a fish farm [45]. However, histopathological sections revealed moderate muscle atrophy; which explains the negative correlation between parasite intensity and condition factor. is perhaps suggests that this was a case of recent infection, considering that none of the fish had skeletal abnormality.
Physicochemical parameters of the water and nutrients and presence of pollutants have a positive influence on the occurrence of parasitic and bacterial populations and communities in fish cultured environments [46][47][48][49]. e fish pond recorded slightly high levels of calcium carbonates (alkalinity) and high levels of nitrites, nitrates, and ammonia free nitrogen than the recommended range for rearing tilapia. A study by Ismail et al. [50] demonstrated that presence of Aeromonas hydrophila, Enterobacter cloacae, and Micrococcus spp., among other bacteria was greatly influenced by water temperature, levels of nitrites, phosphates, sulphide, and ammonia. On the other hand, Isyagi et al. [36] reported that consistently high levels of ammonia nitrogen above the recommended limit are associated with   [48], including bacteria and parasites. is could be the first report on natural concurrent infection of Neascus spp. and multiple bacteriosis. However, the relationship among these pathogens is not fully understood. In a probably similar pathogenesis cascade as the current study, Sandell et al. [51] reported stunted growth and low survival chances among juvenile salmons due to concurrent infections of Neascus spp. and Renibacterium salmoninarum. Some researchers have postulated that coinfections of some ectoparasites simultaneously with ubiquitous bacteria have a mutual benefit relationship [52,53], unlike in this case where there were mortalities. A number of studies have shown synergetic interaction of parasitic and bacterial coinfections [54,55]. ese studies have shown higher mortality rates in concurrent bacterialparasitic infections.
is synergistic effect has been explained as a result of stress caused by parasites that lowers   [57] work shows that parasitic infestation in fish vary greatly from one system to another, and this may be influenced by physicochemical properties of culture water and occurrence of intermediate hosts. Moreover, climatic/topographical conditions of the region, seasonality, and host parasite relationship may also play a role in the epidemiology of parasites of fish.

Conclusion
In conclusion, our findings show that synergistic coinfections by metacercariae of Neascus spp. and multiple bacteria taxa coupled with poor water quality were responsible for the reported mortality in postfingerling Nile tilapia. e findings of this study may be of significance to aquaculture, especially at a time of scanty information on diseases of farmed fish in developing countries, including Kenya.

Recommendations
It ought to be noted that presence of snails and piscivorous birds is one of the risk factors that propagate the life cycle of the Neascus spp. While there are no practical means to treat BSD, the authors recommend immediate destocking, thorough disinfection (including use of molluscicides), and controlling piscivorous birds. Moreover, attention ought to be given towards prevention of parasitic infestations in fish so as to minimize fish deaths related to secondary bacteriosis and economic loss due to aesthetic reasons. Further experimental infection challenge studies are recommended in order to evaluate the associations of these organisms so as to comprehend their significance on fish.

Data Availability
Data associated with this paper are retrievable from the online repository at http://doi.org/10.17632/w2h8mhy3f5.2.

Conflicts of Interest
e authors have no conflicts of interest regarding publication of this paper.