A Synergy of Oxytetracycline and Erythromycin Antibiotics Improves Growth, Health and Resistance to Streptococcus agalactiae in African Catfish, Clarias gariepinus

The aquaculture industry, particularly Catfish farming, has experienced significant growth in achieving food security [1].  According to Boyd et al. [2], aquaculture has exhibited rapid expansion in recent decades, establishing itself as an industry of significant economic importance. Parallel to the increase and intensification of Catfish culture, disease outbreaks in local production have significantly increased [3]. Bacterial infections, particularly streptococcus spp., have emerged as major pathogens in intensive fish farms [4]. In addressing the problem of bacterial menace in fish farms antibiotic use have being employed [5]. Among antibiotics, oxytetracycline (OTC), and erythromycin (ERY) have being extensively used in aquaculture for therapeutic, methaphylatic and prophylactic purposes [6].  In other to assess and measure aquatic animal health as well as the physiological effects of antibiotics and other toxicants, biochemical, hematological, enzymological, and histopathological biomarkers are routinely analyzed to assess the mode of action of toxicants [7]. Blood parameters can show the physiological status of organisms exposed to environmental toxins because they respond fast to environmental or physiological changes [8]. Hematological indicators, such as hematocrit (HCT), red blood cell (RBC) count, and white blood cell (WBC) count, are extensively employed to determine toxic stress caused by environmental pollutants [9]. Similarly, changes in enzyme activities of fish exposed to chemical contaminants can be employed as logical candidates in environmental biomonitoring, disease diagnosis, severity of cell damage, and organ malfunction [10]. Transaminases (alanine aminotransferase (ALT) and aspartate aminotransferase (AST), among others found in all tissues, are recognised as potential biomarkers for determining the toxicity of a chemical after chronic exposure [11]. Despite the good qualities of oxytetracycline and erythromycin, to the best of our knowledge, research on many antibiotic supplements are mostly based on the use of the individual [6]. However, more benefits could be obtained by combining suitable and compactible ones and investigating the possibility of synergistic effects. It is envisaged that the findings of this study would provide avenues for stakeholders such as fish farmers and aquaculture drug dealers to use this strategy to prevent drug resistance while also contributing to food security.

Test substance and stock preparation

Oxytetracycline (OXY) and Erythromycin (ERY), were purchased from a certified pharmacy in Tamale Metropolis and stock solution prepared in a ratio 1:1 by dissolving 75 mg/Kg each of OXY and ERY in an appropriate amount of distilled water.

Preparation of experimental diets

A 2 mm commercial fish feed (Ranaan fish feed: 45% protein, 11% fat, 20% fibre, 9.5% ash, 1.3% phosphorus) was purchased from the open market. To prepare the experimental feed, 1 kg each of the commercial feed was weighed into four separate bowls. To one of the measured feeds, 100 ml of distilled water was added and mixed thoroughly, representing the control diet. The experimental diets were prepared by adding to the commercial feed different concentrations (1%, 2%, 3%) of the antibiotic mixture mixed with 1 Kg of the commercial feed and remolded to represent 1% OXY/ERY gKg¹, 2% OXY/ERY gKg¹ and 3% OXY/ERY gKg¹ respectively.

Experimental fish and design

A total of 300 Catfish without abdominal distension, ragged fins, or hemorrhage of a mean weight of 100 ± 1.0 g were obtained from Water Research Institute (Tamale, Ghana) for use in the experimental site. The fish were put into eight groups and permitted to acclimate in concrete circular tanks with about 80 liters of water for a. Throughout the acclimatization period fishes were fed twice with the control diet on daily basis at 2% of their body mass. The feed was administered in two equal rations at 8:30 am and 4:00 pm daily. Fish were then assigned to the respective groups (i.e. CT, 1% OXY/ERY gKg¹, 2% OXY/ERY gKg¹ and 3% OXY/ERY gKg¹) in triplicate with each tank containing 25 fishes. By assessing the individual body weight of the fish in each group biweekly, the amount of food given out was adjusted. About 30% of the water was renewed on daily basis to maintain the quality, thus of average temperature of 26 ± 2.0°C, pH of 6.5 ± 0.28, and dissolved oxygen concentration of 6.10 ± 0.35 mg/L.

Data on growth

Parameters that were assessed as growth indices included the: initial and final weight, weight gain, feed conversion ratio (FCR), hepatosomatic index (HSI), viscerosomatic index (VSI) and condition factor (K) as have been described by (Saiyad Musthafa et al., 2018).

a) Weight gain (WG) = (final body weight (g) + dead fish weight (g) - initial body weight (g))/initial body weight (g) * 100

b) Feed conversion ratio (FCR) = diet intake of total feed (g)/ (final body weight (g) + dead body weight (g) - initial body weight (g)) * 100

c) Condition factor (CF) = (100 * [total body weight (g)]/ [total body length (cm)]³)

d) Viscerosomatic index (VSI) = (100 *[viscera weight (g)]/ [total bodyweight (g)])

e) Hepatosomatic index (HSI) = (100 *[liver weight (g)]/ [total body weight (g)]).

Blood and tissue sample collection

Blood samples were taken from the treated fish and the control (i.e., 3 gathered from each tank replicates) each from the treatment group and the control group were taken at 4 and 8 weeks to assess the impact of feeding trial on the health (blood profile) and liver health (liver toxicity).  Whole blood (0.5-1 mL) was taken using 2 mL disposable syringe from the caudal section of fish as following steps for blood collection as outlined in other fish [12]. Blood samples taken were decanted into a polypropylene specimen tube with dipotassium EDTA for analysis.

Bacteria acquisition and challenge test

From the University for Development Studies, Nyankpala Spanish Laboratory Complex, Streptococcus agalactiae suspension was obtained, cultured, rinsed with phosphate-buffered saline, and reconstituted to correspond to an optical density of 1.0. One milliliter of the cultured Streptococcus agalactiae suspension was added to 9 mL of distilled water and serially diluted to constitute different concentrations. A pre-challenge experiment was conducted an LD₅₀ was determined to be 1 × 10⁷ CFU/mL. At the end of the feeding trial, fish from the different treatments in triplicates (i.e., 14 fish per replicate) were infected with Streptococcus agalactiae suspension using the LD₅₀ determined by intraperitoneal injection with 0.2 mL of the bacterial suspension, and mortalities were monitored for 14 days.

Cumulative mortality and survival were computed using the formula according to Saiyad Musthafa et al. [13] as:

1. Cumulative mortality (%) = Total mortality in each treatment after challenge / Total number of fish challenged for same treatment × 100
2. Relative per cent survival (RPS) = 1- (% of mortality in the treated group) / (% of mortality in the control group) × 100.

Data analysis

SPSS version 16.0 was used to analyze differences (p < 0.05) in growth, hematological, and toxicity parameters between control and OXY/ERY groups. One-way ANOVA with Duncan range test was used to compare treatment means. Data is presented as means with standard error in tables and chats.

Growth parameters

Fish fed OXT/ERY gKg¹ supplemented diets had higher final weight, WG, FCR, CF, higher hepatosomatic and lower viscerosomatic indexes, improved (lower) feed conversion ratio, and condition factor than those fed the control diet. The fish group fed 3% OXY/ERY had statistic similarities of FCR and CF to those fed the control diet (P < 0.05, Table 1). 

Table 1: Growth performance and feed utilization fed test diet.

Where, WG= weight gain; FBW= final body weight; VSI = viscerosomatic index; HSI = hepatosomatic index; FCR = feed conversion ratio; K = condition factor. Means ± SE (Duncan rang test, n = 3) with different superscript letters in the same column denote significant difference; OXY/ERY gKg¹.

Haematological profile of African catfish

White blood cell (WBCs): The effects of OXY/ERY gKg¹ supplemented diets on the WBCs of Catfish after 4 and 8 weeks of feeding is shown in (Figure1). After 4 weeks of experimental feeding, fish fed OXY/ERY supplemented diets exhibited significantly higher levels of WBCs compared to the control group (P < 0.05). Among the OXY/ERY supplemented groups, fish fed 1% OXY/ERY supplementation showed significantly higher WBC compared to the others (P < 0.05).  At 8 weeks, a similar trend was observed with a decreasing trend in the treated diet. It is worth noting that WBCs tend to be higher with continues feeding for eight weeks compared to 4 weeks. 

 Figure 1: WBC levels of catfish at four and eight weeks (OXY/ERY gKg¹).

Note: Bars with the same alphabet per treatment at either 4 or 8 weeks are not statistically different. 

Red blood cell (RBC): RBC ranged from 20.7 x 10² L to 60.6 x 10² L and 23.07 x 10² L to 46.66 (x 10² L^-1) at 4 and 8 weeks respectively with the least recorded in the control and the best recorded in 1% OXY/ERY gKg¹ fish group (P < 0.05) (Figure 2). It was observed in the RBC levels a reverse trend in the OXY/ERY treated groups as RBC was decreasing with increasing level of OXY/ERY at 8 weeks.

Figure 2: RBC levels of Catfish at four and eight weeks (OXY/ERY gKg¹).

Note: Bars with the same alphabet per treatment at either 4 or 8 weeks are not statistically different.

Haemoglobin (HGB) level: Figure 3 shows the levels of HGB in Catfish fed control and OXY/ERY gKg¹ treated meals.  Significantly higher levels of HGB were observed at 4 and 8 weeks in comparison to the control (P < 0.05). Regarding treatment groups, a decreasing trend was noticed with increasing concentrations. HGB were similar (P < 0.05) at 3% OXY/ERY compared to the control at week 8 It was obvious that fish fed 1% OXY/ERY exhibited the best incremental effects (P < 0.05) on HGB compared to all other groups in the study both at 4 and 8 weeks of measure. 

 Figure 3: HGB levels of Catfish at four and eight weeks (OXY/ERY gKg¹).

Note: Bars with the same alphabet per treatment at either 4 or 8 weeks are not statistically different. 

Levels of hematocrit (HCT): Mean levels of HCT of the experimental fish at week 4 of feeding trial and at week 8 is presented in Figure 4.   A declining trend in the OXY/ERY gKg¹ dietary inclusion at the 4^th and 8^th week was observed. With the exception of 3% OXY/ERY group showing similarity with CT groups at week 8, all other OXY/ERY supplemented fish group at week 4 and 8 recorded significantly higher HCT levels in comparison to the control group (P < 0.05) 8 with control group. 1% OXY/ERY presented the best effect. 

 Figure 4: HCT levels of Catfish at four and eight weeks (OXY/ERY gKg¹).

Note: Bars with the same alphabet per treatment at either 4 or 8 weeks are not statistically different. 

Liver health test of African Catfish 

AST levels of tested fish: Figure 5 shows levels of AST in Catfish fed control and OXY/ERY gKg¹ inclusions. The highest AST level was found in the OXY/ERY-treated diet at 3% OXY/ERY, while the lowest was found in the control group at 4 and 8 weeks (P < 0.05). All in all, AST levels in OXY/ERY supplemented diets were observed increasing at both week 4 and 8 compared to the control group. 

 Figure 5: AST levels of Catfish at four and eight weeks (OXY/ERY gKg¹). 

Note: Bars with the same alphabet per treatment at either 4 or 8 weeks are not statistically different. 

ALP levels of tested fish: Average levels of ALP in fish given a control and OXY/ERY gKg¹ supplemented diets are represented in Figure 6. At weeks 4 and 8, ALP levels within the OXY/ERY supplemented diet groups were found to be significantly higher (P < 0.05) than the control with 3% OXY/ERY recording the highest.

 Figure 6: ALP levels of Catfish at four and eight weeks (OXY/ERY gKg¹).

Note: Bars with the same alphabet per treatment at either 4 or 8 weeks are not statistically different.

ALT levels of tested fish: In Figure 7 is ALT levels of fish fed both control and OXY/ERY gKg¹ inclusion meal. A clear observation was noticed with a rising trend of increasing percentage of OXY/ERY supplemented diet (i.e., 1% OXY/ERY, 2% OXY/ERY, and 3% OXY/ERY) in each group at week 4 and 8 with 3% OXY/ERY recording the highest (P < 0.05). 

 Figure 7: ALT levels of Catfish at four and eight weeks (OXY/ERY gKg¹).

Note: Bars with the same alphabet per treatment at either 4 or 8 weeks are not statistically different.

Challenge test

The cumulative mortality (%) of OXY/ERY gKg¹ and control fed Catfish following the infestation with the bacteria Streptococcus agalactiae after 14 days of observation is shown in Figure 8. Among the treated fish, those fed 1% OXY/ERY had the lowest cumulative mortality rate, whereas those fed control meal had the highest. However, there were significant differences in cumulative mortalities between the OXY/ERY and control fed groups (P < 0.05). 

Figure 8: Cumulative mortality (%) of Catfish, fed different doses of with control (0% OXY/ERY gkg¹) and OXY/ERY supplemented diets (1% OXY/ERY gkg¹, 2% OXY/ERY gkg¹, and 3% OXY/ERY gkg¹) after 14 days post-challenge with Streptococcus agalactiae. Each line graph represents the mean ± SE of three biological replicates (n = 3).

Studies on the administration of a mixture of antibiotics in sustainable aquaculture have demonstrated that it significantly improves growth performance and feed utilization in cultured fish due to synergistic effects [14]. Pharmaceuticals have been used to promote growth, health, and resistance to infections and diseases under pathological conditions, but they can also have harmful effects on non-target organisms because to their unique mechanism of action such as, high water solubility, and resistance to biodegradation [15]. The results of the current study demonstrate that OXY/ERY supplementation in the diets of Catfish could contribute to higher weight gain, condition factor, and decreased feed conversion ratio in Catfish. The data also shows that lower supplementation levels of 1% OXY/ERY are superior for overall growth parameters measured, with 1% OXY/ERY diet producing the best outcomes. We hypothesize that increased performance is related to better feed utilization, which results from enhanced metabolism. This finding supports other studies that have demonstrated improvements in growth and feed utilization by including lower doses of veterinary drugs [16]. Although larger doses of OXY/ERY had a favorable effect on Catfish growth and feed consumption, declining performance with increasing OXY/ERY concentration could be attributed to lower feed palatability due to the bitterness of OXY/ERY [6,17].

In many aquaculture operations, the health status of fish is frequently assessed using hematological parameters [18]. These metrics may also enable for the early detection of changes in fish under stressful conditions. WBCs are engaged in the regulation of immunological processes or defense mechanisms, and their increase in levels denote as a protective response of fish to environmental and pathological stress [19]. In general, leucocyte counts give a sensitive indication of toxicant stress, and their count is directly proportional to the potency of the toxicant. RBC determines the status of oxygen transport to tissues in the organism, and haematocrit (HCT) determines the volume of RBC [8,20]. HGB and HCT can also serve as indicators of anemia and fluid volume imbalance [21]. In this study, we found that fish fed the OXY/ERY supplemented meal considerably enhanced the haematological variables compared to those fed the control. However, as compared to the control, the diet treated with 1% OXY/ERY produced the best outcomes. This shows that the experimental fish fed group has the potential to boost resistance to infections and diseases under pathological conditions by driving a large increase in WBC levels. Oxytetracycline and erythromycin are thought to contain metabolites including 4-dimethylamino-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-hexahydroxy-6-methyl-1,11-dioxo-2-naphthacencarboxamide (32.3.2) and Saccharopospora erythraea [6] which could explain for the triggered immunostimulation in the investigated fish [22]. Farmed fish react to production stressors by deploying additional RBCs, HGB, and HCT, allowing them to better adapt to harsh environment [23]. Catfish fed OXY/ERY diets had significantly higher levels of RBCs, HGB, and HCT than control fish after the feeding trial, indicating that OXY/ERY supplementation could help fish better adjust to environmental and production stressors and improve their chances of survival [24]. Erythrocytes in teleosts are typically elliptical and biconcave, and they play a vital role in oxygen delivery to body tissues [25,26]. Prolonged exposure to antibiotics can damage the organisms, causing in poor osmoregulation, anaemia, and possibly a drop in RBC count [27].

Transaminases play a crucial part in protein and carbohydrate metabolism, and any change in the above-mentioned metabolism might cause modifications in glutamate oxalacetate transaminase and glutamic pyruvate transaminase activity [28]. Toxicants accumulate in fish's gills, liver, and kidneys over time can cause changes in their enzymatic activity [29]. As a result, any changes in fish AST, ALT, and ALP activities may serve as appropriate biomarkers in the identification of tissue and organ injury [30]. The observed increase in AST, ALT, and ALP levels in the blood in the current study (i.e. with 1% OXY/ERY < 2% OXY/ERY < 3% OXY/ERY feed supplementation) indicate damaged tissues or that the organism is trying to reduce OXY/ERY-induced stress by raising the rate of metabolism. Other study found a similar enzymatic reaction in fish C. carpio treated to diclofenac and clofibric acid [31]. Higher levels of AST and ALT activity in fish may possibly indicate cumulative toxicity or a potential method for meeting higher energy demand during sustained and extended toxic stress. Inhibition of AST and ALT activity could be mediated by enzyme loss from the soluble areas of hepatocytes [32].

 A simulated infection test with a target pathogen could be used to assess the effectiveness of antibiotic-supplemented diets offered to fish in increasing their resistance to microbial infections [33]. The results of this current research suggest that OXY/ERY supplementation can lower mortalities (i.e. with 1% OXY/ERY > 2% OXY/ERY > 3% OXY/ERY feed supplementation) to infection of Streptococcus agalactiae in Catfish compared to the control. This could be the result of the enhanced synthesis of haematological variables in fish exposed to OXY/ERY diet, as previously indicated [19]. Furthermore, synergy of bioactive compounds in a compactible antibiotic mixture can stimulate the secretion of immunological substances, which can prevent the growth of harmful bacteria and offer health benefits to the host [34]. In previous studies, antibiotics administered separately or in combination have been shown to reduce fish mortality from a variety of diseases and infections [35].

These research findings suggest that the use of OXY/ERY antibiotics in Catfish culture can greatly enhance weight gain, with 1% OXY/ERY having the best effect. The application of 1%, 2%, and 3% OXY/ERY can dramatically boost haematological variables and resistance to diseases such as infections of Streptococcus agalactiae in Catfish, although the best effects are obtained with the application of 1% OXY/ERY. It has been discovered that the use of OXY/ERY can be harmful to the liver in Catfish, particularly after 8 weeks of treatment, by increasing the leakage of enzymes. As a result, the levels of these liver enzymes could be employed as biomarkers of OXY/ERY application to identify its toxicity to Catfish in the field of environmental biomonitoring.

The authors wish to thank Faculty of Biosciences, UDS and the Department of Aquaculture and fisheries Sciences, UDS.

All authors wish to declare no issue of competing of interest during the manuscript preparation,

Data will be made available upon request from the corresponding author.

The study was approved by the University for Development Studies ethical committee and for the study’s findings can be shed.

No external funding was received during the preparation of the manuscript. All authors contributed to finance the study.

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