Florfenicol is a palatable broad-spectrum antibiotic and has been conditionally approved by the USFDA for use in aquaculture (USFWS 2015). Florfenicol therapeutics differs from country to country but includes control of mortality due to diseases associated with the warm water bacterial pathogens like Edwardsiella ictaluri, Streptococcus iniae, S. agalactiae, Flavobacterium columnare, Francisella asiatica and Aeromonas hydrophila (Gaunt et al. 2004; 2010; Matthews et al. 2013; Soto et al. 2016; de Oliveira et al. 2018; Assane et al. 2019). The studies of Barreto et al. (2018) demonstrated the use of vegetable oil as a successful coating agent in the top-coating of FFC medicated feed. Since drug metabolism is considered temperature dependent, this study was conducted at the ideal growth temperature of 28.6 ± 1.68ºC. Our experimental results demonstrated the margin of safety of oral FFC administration to O. niloticus juveniles at 0–10 times the lowest therapeutic dose (10 mg/kg biomass/day) for 30 days. Our results are consistent with those found in similar studies that evaluated the safety of FFC administered in feed to other freshwater finfish (Straus et al. 2012; Gaikowski et al. 2013; Bowker et al. 2013). The feed consumption and growth of FFC-dosed O. niloticus were significantly reduced in a dose-related fashion, particularly in the 3X-10X groups. The fish consumed approximately most of the feed offered during the dosing period (0X: 100%, 1X: 98.71% and 3X: 95.75%), often breaking the surface of the water while feeding. The fish tend to feed by gulping the feed on the surface followed by releasing the feed inside the water column and subsequently re-gulping it. Previous works (Bowker et al. 2013; Gaikowski et al. 2013) also elucidated similar feeding behaviour among the experimental fish. Further, the extended feeding of FFC-diets beyond 10 days significantly decreased the feed consumption in the higher dosed groups. On the contrary, FFC therapy in S. salar (Inglis et al. 1991), Perca flavescens (Bowker et al. 2013) and hybrid striped bass, female Morone chrysops x male M. saxatilis (Straus et al. 2012) did not alter feed consumption. No gross or microscopic lesions were observed during the experimental tenure. The observed significant differences in feed intake among the treatment groups during the dosing and post-dosing regimen possibly related to the palatability and dose-dependent toxicity of FFC. The clinical implication of the declined feed consumption is likely of minimal importance as decreased feed consumption was only comprehended at higher levels and only after administration for longer than the proposed 10-day period. Nevertheless, the fish were able to mount biological responses during the post-dosing period and the feed intake recovered in a dose-dependent manner. The FFC-dosing at the 1X dose did not cause loss of equilibrium, gasping, flashing and hyperactivity. Contrarily, the 5X and 10X dosed O. niloticus had opercular pigmentation and a black peritoneal layer. Likewise, Gaikowski et al. (2013) reported body discolouration in FFC fed O. niloticus.
The mortalities during the first 10 days of dosing, i.e., therapeutic dosing period, were observed only in the 10X group possibly due to FFC-intoxication. The cumulative mortalities of 3.33% in 1X to 18.33% in 10X groups on day 30 of dosing corroborate the works of Gaikowski et al. (2013), who asserted the chances of FFC-intoxication due to prolonged FFC feeding. Hentschel et al. (2005) opined that any drug at a higher concentration than its permissible limit is toxic to the host organism thereby rendering several intoxication symptoms in fish. Our results on the elevated mortality with the increase in FFC-dose and dosing period supported the earlier observations (Hentschel et al. 2005; Gaikowski et al. 2013). Contrarily, Gaikowski et al. (2003) did not observe any mortality in Ictalurus punctatus during the experimental period. The necropsy observation on the alterations in the internal organs such as swelled kidney and spleen in the 5X and 10X groups probably indicated the concern of FFC toxicity upon oral dosing. The therapeutic dose group (1X) revealed a slight decrease in biomass, i.e., 1.24 folds hike, on day 30 FFC-dosing compared to the control (1.45 folds). An insignificant dose-dependent decrease in fold change in biomass of the 1X, 3X and 5X groups compared to control was observed, which coincided with the decreased feed intake of the respective treatment groups. The biomass increased for all the treatment groups with the termination of FFC-dosing, thereby indicating the recovery of fish.
The mean serum glucose level of the O. niloticus (74.00 ± 5.00 mg/dL) was concomitant with the values recorded by Bittencourt et al. (2003). The 30 days of FFC-dosing increased the glucose levels significantly in all the treatment groups, indicating the FFC induced stress and altered carbohydrate metabolism (Sopinka et al. 2016; Julinta et al. 2019). Even at the lowest therapeutic dose, the 30 days of FFC-dosing raised the glucose levels significantly. On day 10 FFC-dosing, the glucose levels of the 1X groups were significantly high, which signified that the therapeutic FFC dose and dosing period (10 days) may be stressful to the normal O. niloticus. The degree of glucose increment was comparatively higher in 5X and 10X groups. Further, the elevated glucose levels in FFC doses higher than the therapeutic dose and the extended dosing period gave conclusive indications on FFC as a stress inducer. Though the glucose levels abridged significantly on day 13 post-FFC-dosing, the levels were still significantly higher than the initial levels recorded on day 0. These results suggested that the FFC-induced stress and the physiological changes persevered. Also, the fish could not recover fully even after 13 days of termination of FFC-dosing, which would influence the growth and farm production of O. niloticus. Possibly, more time would be required for the fish to revert to their initial conditions, which is a cause for concern for the aquaculturists.
At the therapeutic dose, the serum calcium levels reduced significantly on day 30 FFC-dosing, indicating an imbalance in osmolarity and ionic concentration. Nevertheless, within 2 weeks of cessation of FFC-dosing, the calcium levels recovered to normal. Even, the fish offered the higher doses followed a similar trend in a dose-dependent manner. Likewise, the chloride levels reduced but insignificantly on day 30 FFC-dosing in the 1X group and recovered completely on day 13 post-FFC-dosing. The higher dosed groups also followed a similar trend except for the 10X group, which failed to recover within the 2 weeks of termination of dosing. These results suggested that the effects of FFC were more on calcium than on chloride ions. Our results corroborate the observations of decreased serum calcium levels in pigs (Liu et al. 2003) and chicks (Klaudia and Alina 2015) when injected with FFC. Although aquaculture reports are not available on the relationship between serum calcium and FFC, antibiotics like tetracyclines tend to bind to calcium reducing its availability in serum (Guidi et al. 2018). The works of Zhang et al. (2016) on FFC established a relationship between FFC and free available chlorine (FAC), which according to them readily combines with FFC and transforms it for easy removal. The transformation kinetics of FAC, thus, hinted at a similar mechanism in fish blood. Noticeably, the increased output of faecal matter may have a close linkage with decreased serum calcium and chloride levels in O. niloticus. It has been documented that the increase in faecal output elevated the output of faecal carbonates and bicarbonates, leading to a disruption in osmotic balance and acid-base homeostasis (Wilson and Grosell 2003).
The recorded serum creatinine levels (0.08 ± 0.02–0.09 ± 0.01 mg/dL) in the control group were concomitant with the results of earlier studies (Julinta et al. 2019). The creatinine levels increased significantly in all the treatment groups with the maximum in the 5X and 10X groups. Subtle fluctuations in creatinine levels (0.20 ± 0.01–0.22 ± 0.03 mg/dL) were observed in the 1X group between day 10 and day 30 FFC-dosing. The increase in serum creatinine levels implied kidney damage and a reduction or loss of renal function (Julinta et al. 2019). Contrarily, in an earlier study by Reda et al. (2013), the FFC at the growth-promoting dose (5 mg/kg fish) significantly reduced the serum creatinine levels in O. niloticus. The creatinine levels in the 10X group increased by almost 4.41 folds on day 10 and 5.75 folds on day 30 FFC-dosing signifying the nephrotoxicity of FFC. Although the creatinine levels reduced on day 13 post-FFC-dosing in all the treatment groups, the levels were still significantly higher than on day 0. These observations suggested only a slight improvement in the renal functions of O. niloticus in 2 weeks of suspension of FFC-dosing. Perhaps, the fish would require more time to recoup.
The alterations in serum ALT and AST levels are indicative of liver tissue impairment or damage caused by drugs or stress (Julinta et al. 2019; Bojarski et al. 2020). The current study documented serum ALT levels in the range of 37.00 ± 1.73 to 38.67 ± 3.51 IU/L in the control group, which are analogous to previous studies (Julinta et al. 2019; Dawood et al. 2020). The significant increase in ALT levels in the 1X group on day 20 and day 30 FFC-dosing indicated the FFC induced liver tissue impairment or damage upon extended FFC-dosing. Nevertheless, the hike observed on day 10 FFC-dosing at the therapeutic dose (1X) was insignificant compared to control, suggesting minimal liver damage, similar to the observations of Reda et al. (2013). Contrarily, the dose-dependent elevated ALT levels as observed in the 3X-10X groups on day 10, 20 and 30 FFC-dosing hinted at the hepatotoxicity of FFC with increased dose and dosing period. The 10X group demonstrated about 5 folds increase in ALT levels on day 30 FFC-dosing. Yet, the ALT levels, more or less, recouped on day 13 post-FFC-dosing, except for the 10X group, which had significantly higher levels than on day 0. Notably, the pronounced impact of FFC on liver enlargement was noted during necropsy. The serum AST levels of control (77 ± 4.58–80.33 ± 0.58 IU/L) were concomitant with the studies of Hastuti and Subandiyono (2020). A significant increase in AST levels was observed in all the treatment groups with the highest in the 10X group on day 30 FFC-dosing, thus confirming the hepatotoxicity of FFC. The observed significant hike in AST levels in the 1X group during the FFC-dosing period also signified that the FFC even at the therapeutic dose may impair the liver tissues or cause metabolic damage. In contrast, Reda et al. (2013) observed a significant decline in serum AST levels when fed FFC at a lower dose (5 mg/kg fish) in O. niloticus. Although the higher doses (3X-10X) showed a significant rise in serum AST levels, there existed insignificant differences among them, thus suggesting persevering hepatotoxicity of FFC at elevated doses and portentous hepatic dysfunction in O. niloticus. It has been reported that amphenicols (Memik 1975) and oxytetracycline (Julinta et al. 2019) are hepatotoxic so also our study with FFC. Also, the FFC can cause an increase in the weight of the liver (Elia et al. 2016). Our results confirm the findings of Er and Dik (2014), who observed a hike in AST levels upon FFC application in Oncorhynchus mykiss. Except for the 10X group, all the treatment groups recuperated within 2 weeks of suspension of FFC-dosing. The serum ALP levels of control (12.00 ± 1.73–13.00 ± 2.65 IU/L) were similar to the observations of Hrubec and Smith (2000). The significant increase in ALP levels at the therapeutic dose (1X) on day 10 FFC-dosing hinted at the possibility of FFC induced liver inflammation and hepatotoxicity (Labarrère et al. 2013; Soltanian et al. 2018). The ALP levels increased in a dose-dependent fashion with the highest in the 10X group on day 30 FFC-dosing. These results are in agreement with the observations recorded in fish during the misuse of FFC (Shiry et al. 2020) and goat (Shah et al. 2016) upon FFC injection. Although the ALP levels reduced with the cessation of FFC-dosing in all the treatment groups, their levels were still significantly higher than on day 0. These observations suggested persisting liver inflammation in FFC-dosed fish.
The oral FFC-dosing not only affected the serum biomarkers of healthy O. niloticus but also induced relative cytotoxicity. Our study used giemsa and safranin stains for assessing the blood cellular morphological changes upon FFC-dosing. The results showed that the safranin staining method is a good alternative to the giemsa method and has the advantage that abnormalities in other blood elements, in particular WBCs and thrombocytes, are better identified. Giemsa staining did not provide for comprehensible granulations in leucocytes. The safranin staining granted efficient visualization of smudge cells and granulations in leucocytes. However, considering the diagnosis of erythrocytes in a blood smear, the giemsa stain proved to be much superior to safranin. In a comparative study of Leishman and giemsa staining, Sathpathi et al. (2014) suggested the superiority of giemsa staining for a thick blood smear. In the present study, the giemsa staining produced distinct and evident erythrocytic morphology with subsequent alterations and abnormalities. Deformities like rupturing of nuclear membrane and vacuolation were prominent with giemsa stain. However, this did not translate into reduced sensitivity or reduced accuracy of safranin in light microscopy. This comparison, alongside cellular morphological alterations, also hinted at the effectiveness of time. Staining with safranin is shorter, which in many scenarios can be helpful.
The blood cell morphological changes were mostly restricted to the erythrocytes in our study. The predominant increase in mature and immature lymphocytes in all the treatment groups suggested a toxic or stress-related effect of FFC on the lymphoid cells and cell proliferation (Gaokowski et al. 2013). Although there was no direct evidence of hematopoietic or lymphopoietic tissue degradation, the sudden increase in lymphocytes hinted at the stress the fish endured. Likewise, Umamaheswari et al. (2019) in their studies with amoxicillin on Labeo rohita demonstrated a significant increase in lymphocytes. The haematopoietic tissues are normally sensitive to antibiotics. This could be the reason for the increased incidence of WBCs (phagocytic response). The works of Passantino et al. (2004) indicated the morphology of mature fish erythrocytes (more elongated) and immature erythrocytes (less elongated). Our study noted an increased incidence of immature erythrocytes in the therapeutic group throughout the dosing period. Chico et al. (2018) in their works on O. mykiss RBCs coined the term “shape-shifted RBCs (shRBCs)” for normal RBCs, which when exposed to certain stimuli produce apparent morphological and molecular alterations. These shape-shifting RBCs are also often observed in fish under the stressed conditions (Lewis et al. 2010; Chico et al. 2018). Such alterations in RBCs, viz., teardrop-shaped and spindle-shaped, were frequently observed in our study. The morphological changes in fish erythrocytes further confirmed the cytotoxic effect of FFC, which may result in chromosomal disparities (Ghaffar et al. 2015). The increased incidence of erythrocytes with eccentric nucleus was observed possibly due to the higher production of caspase-activated DNAase or oxidative stress to the mitochondrion causing disruption and breakage in the cytoskeleton (Ghaffar et al. 2018). Changes like extruding nucleus, reduced cytosol density (lighter staining) and vacuolations were also observed under light microscopy. Such changes are comparable to those of Chico et al. (2018). Similar to the findings of this study with FFC, antibiotics like amikacin have been known to reduce cell size, increase deformability and osmotic fragility in erythrocytes (Lijana and Williams 1986). The increased incidence of ruptured cells at the higher doses in our study supported the results of Blaskó et al. (1986), possibly due to the attachment of antibiotics on the erythrocytic membrane and hindrance in cation transport. Smudge cells are associated with high lymphocyte counts and hence, the observations of increased incidence of smudge cells and lymphocytes are related. Cytotoxicity was prominent in the 5X and 10X groups with distinguishably irregular RBCs, ruptured cells and eccentric nuclei. Cytotoxicity of FFC at the higher concentrations has been well demonstrated in goats and reptiles (Saganuwan 2019). Nevertheless, the therapeutic dose did not show any signs of cell rupture hinting at the safety of FFC at the test dose. Upon cessation of FFC-dosing, the blood smear produced healthier cellular elements. Increased prominence of mature erythrocytes was also seen. The erythrocytic abnormalities like eccentric nuclei were not seen on day 13 post-FFC-dosing in O. niloticus of the higher dosed groups. The elimination of stress also terminated the formation of shRBCs. Although such erythrocytic aberrations decreased, the nucleus to cytoplasm ratio was still high among erythrocytes of all the treatment groups. Also, the increased prominence of lymphocytes persevered. The number of mature lymphocytes was still high on day 13 post-FFC-dosing in all the groups. With the observance of such blood cell morphological changes particularly in fish erythrocytes, the impact of FFC on blood cells need further studies to elucidate the mechanisms. It is believed that due to such significant deformities in blood cell morphology, the fish erythrocytes could be targeted as an efficient blood biomarker for future studies on the safety of approved aquacultural antibiotics.