ORIGINAL ARTICLE Hematological and histopathological assessment of pacu (Piaractus mesopotamicus) after treatment of pathogens with veterinary medicinal products S P Carraschi1,2 & T Florêncio1,2 & N F Ignácio1,2 & C V Ikefuti1 & C Cruz3,2 & M J T Ranzani-Paiva4 Received: 8 June 2016 /Accepted: 30 September 2016 /Published online: 7 October 2016 # Springer-Verlag London 2016 Abstract Fish species are impacted by multiple pathogens, and they are exposed to the chemicals used to treat these diseases at several stages during the aquaculture production cycle. This study performed hematological and histopatholog- ical evaluations of pacu (Piaractus mesopotamicus) that had been naturally infected with Aeromonas sp., Streptococcus sp., Ichthyophthirius multifiliis, Trichodina heterodentata, and Anacanthorus penilabiatus and treated with enrofloxacin and toltrazuril or with florfenicol and thiamethoxam. After 7 days of treatment from nine fishes were collected blood, via caudal puncture, and samples of the gills, liver, and kid- neys. Following toltrazuril and enrofloxacin treatment, fish exhibited leukocytosis with lymphocytosis. With thiamethoxam and florfenicol treatment, the fish showed an increase in hematocrit, hemoglobin level, mean corpuscular volume, and mean corpuscular hemoglobin concentration and a decrease in red blood cells. The infected control fish presented aneurysms and a disruption of the secondary lamel- lae, which can cause death. The drugs used in this study stim- ulated the immune systems in the fish or caused electrolyte imbalances, which were temporary. Keywords Disease . Parasites . Bacteria . Histology . Hematology Background Despite advances in aquaculture techniques, the produc- tion of pacu (Piaractus mesopotamicus), a native fish species from Brazil, is impacted by multiple pathogens. The pacu is one of the most important species used in Brazilian fish farming due to its rapid growth rate, easy adaptation to artificial feeding, and high consumer appre- ciation, as well as its ecological and commercial value (Biller-Takahashi et al. 2015). Commercially grown fish, such as the pacu, are often parasitized by members of the Monogenoidea, which re- sults in economic losses, a problem that is especially prevalent in the Neotropics where ecological characteris- tics facilitate the rapid and constant spread of various parasites (Schalch et al. 2006). Under high-temperature conditions combined with high concentrations of organic mat ter in the water, the monogenean paras i te , Anacanthorus penilabiatus, can cause high levels of fish mortality. Another common parasite in aquaculture is Trichodina heterodentata. Fish infected by this parasite present abnormal epithelial proliferation in the skin and gills and severe aberrations in their lamellae (Yemmen et al. 2010; Pádua et al. 2012). According to Xu et al. (2007), heavy Trichodina infections cause epidermal in- juries that lead to an increase in Streptococcus infections. Under lower temperatures, Ichthyophthirius multifiliis, a ciliated protozoan, causes even greater mortality in larvae and fingerlings, which can become even worse in con- junction with Aeromonas hydrophila (Liu and Lu 2004). * S P Carraschi patycarraschi@gmail.com 1 Centro de Aquicultura da UNESP, Via de Acesso Prof. Dr. Paulo Donato Castellane, s/ no, Zona Rural, Jaboticabal, São Paulo 14884-900, Brazil 2 Núcleo de Estudos e Pesquisas Ambientais em Matologia (NEPEAM) da UNESP, Jaboticabal, São Paulo, Brazil 3 Fundação Educacional de Barretos (UNIFEB), Barretos, SP, Brazil 4 Instituto de Pesca, São Paulo, SP, Brazil Comp Clin Pathol (2017) 26:105–114 DOI 10.1007/s00580-016-2351-9 http://crossmark.crossref.org/dialog/?doi=10.1007/s00580-016-2351-9&domain=pdf Species of the bacterial genera Aeromonas, which are gram-negative, and Streptococcus, which are gram-posi- tive, are among those that cause the largest losses in fish farming, independent of the species or developmental stage of the fish. Aeromonas spp. damage the gills, cause hemorrhagic septicemia, and predispose fish to infection by other pathogens (Carraschi et al. 2012); and Streptococcus spp. cause anorexia, lethargy, and erratic swimming behaviors due to its effects on the central nervous system (Azad et al. 2012). These pathogens are also important to human health; Aeromonas spp. are often found in the feces of patients with foodborne diarrhea (Vila et al. 2003), and water is the princi- pal mode for direct human infection, as well as for the con- tamination of food (Janda and Abbott 2010). Streptococcus sp. is a common colonizer of humans that causes septicemia and meningitis and is the leading cause of invasive bacterial disease in neonates and children (Eickhoff et al. 1964; Ragunathan et al. 2009). In aquaculture, fish are exposed to the chemicals used for disease control at several stages during the production cycle. The use of antimicrobials, such as florfenicol (FFC) (Yáñez et al. 2014) and enrofloxacin (EF) (Bowser et al. 1990), and antiparasitics, such as toltrazuril (TOL) (Jaafar and Buchmann 2011) and thiamethoxam (TH) (Carraschi et al. 2014), has become a common practice. However, before using a chemi- cal in an aquatic system, it is necessary to study its efficacy, toxicity, and the residual of the substance and to understand its possible hematological and histopathological effects in fish following treatment. Histological techniques are important tools in the eval- uation of the sublethal effects of contaminants or patho- gens, and pathological changes can serve as biomarkers. Furthermore, histological changes provide a rapid, inter- mediate methodology to identify stressors and detect their effects on tissues (Bernet et al. 1999; Moon et al. 2006; Figueiredo-Fernandes et al. 2007). In addition, he- matological analyses aid in determining the physiological status of an organism (Seriani et al. 2013) and can reflect environmental indicators and the toxic effects of chemicals (Gabriel et al. 2007; Ghaffar et al. 2014). Therefore, it is necessary to identify the effects of both the pathogens and the drugs used to treat them to enable the identification and monitoring of disease and the pos- sible toxic effects in animals following treatment. The aim of this study was to perform a hematological and histopathological evaluation of pacu (P. mesopotamicus) using the following chemical treatments for infected fish: (a) fish naturally infected with Aeromonas sp., Streptococcus sp., I. multifiliis, T. heterodentata, and A. penilabiatus and treated with EF and TOL; and (b) fish naturally infected with Aeromonas sp., Streptococcus sp., and A. penilabiatus and treated with FFC and TH. Material and methods There were 20 fish per trial, for a total of 60 fish per treatment, which were maintained under mesocosm con- ditions in 600-L tanks. Each experiment, there were 60 fish each treatment, totalizing 240 fish each assay. Cohabitation conditions were used for the infection of all fish, and parasites on the surface of the fish and the gills were counted. In addition, the occurrence of bacte- ria on the epidermal surface before the start of the ex- periment and after the treatment were also evaluated, with ten fish used as samples for each time period (Carraschi et al. 2014). Pacus (31.40 ± 2.77 g) that were naturally infected with parasites and bacteria were treated with an antibiotic and a parasiticide in 600-L tanks. In the first experiment, the fish that showed infections with I. multifiliis (at least 100 ictio fish−1), T. heterodentata (at least 40 T. heterodentata fish−1), A. penilabiatus (at least 50 monogeneans fish−1), Aeromonas sp., and Streptococcus sp. (bacterial infections were identified based on biochemical tests) were treated with 3.0 mg TOL L−1, as a parasiticide, for 5 days (Bayer HealthCare©, Sao Paulo, Brazil) and 90 mg EF kg−1 (Baytril©, Sao Paulo, Brazil), as an antibiotic in their feed ration, for 7 days. In this experiment, the treatments were healthy control fish (HC), infected control fish (InC), infected and treated fish with TOL and EF (Tr), and healthy fish exposed to the drugs treat- ment TOL and EF (ExC). TOL was directly applied to the water in the tanks (500 L) in which fish were kept, and fish were exposed to the drug for an hour each day, 5 days. In the second experiment, other fish showing natural infec- tions with A. penilabiatus (over 200 parasites fish−1), Aeromonas sp., and Streptococcus sp. were treated with 75 mg TH L−1 (Novartis©, Sao Paulo, Brazil), as a parasiti- cide, for 4 days, and 10.0 mg FFC kg−1 (MSD©, Sao Paulo, Brazil), as an antibiotic in their feed ration, for 7 days. In this experiment, the treatments were healthy control fish (HC), infected control fish (InC), infected and treated fish with TH and FFC (Tr), and healthy fish exposed to the drugs treat- ment TH and FFC (ExC). TH was applied to the water, with 2 h of exposure per day, 4 days. The antibiotics were dissolved in 2% vegetable oil and added to commercial fish food (containing 40 % protein), which was offered for 7 days (1.5 % body weight). The concentrations have been chosen accordingly to previ- ous assays in the laboratory conditions where it was evaluated several concentrations in the efficacy for the same pathogens (Carraschi et al. 2014). In both experiments, the temperature was kept between 25 and 30 °C, the dissolved oxygen was >5.0 mg L−1, the elec- trical conductivity stayed between 180 and 220 μS cm−1, and the pH ranged from 7.0 to 8.0. 106 Comp Clin Pathol (2017) 26:105–114 In the end of the treatment, 20 fish from each treatment, from both experiments, were sampled and were evaluated for the parasite and bacterial counts. The samples from skin and gills were evaluated under microscope and counted the para- sites. The bacteria were confirmed by the observation of a high bacterial count, visualized under ×1000 magnification. For the identification of Aeromonas sp., samples from skin, liver, and kidney were cultivated in phenol red starch ampicil- lin media and transferred to nutrient agar media. Subsequently, the samples were Gram stained and subjected to a catalase and oxidase reaction. The colonies were observed to be gram-positive and catalase and oxidase positive, thus providing a positive identification as genre Aeromonas sp. For the identification of Streptococcus sp., samples from liver, kidney, and brain were cultivated in blood agar plates, and suggestive colonies were later transferred to dextrin agar. The suggestive colonies had clear color and transparent halos indicating hemolysis. Before, the sam- ples were submitted to Gram staining and subjected to a catalase and oxidase reaction. The colonies were thus identified as gram-positive and catalase and oxidase neg- ative, indicating genre Streptococcus sp. In the first experiment, TOL was 100 % effective at con- trolling T. heterodentata infections and 39.80 % effective at controlling A. penilabiatus infections, but it was not possible to evaluate the efficacy of TOL against I. multifiliis because there was no infestation found after 7 days in either the treated or the nontreated fish. EF was effective (80 %) in reducing the bacterial density of Aeromonas sp. and Streptococcus sp. In the second experiment, THwas 81.86% effective in control of A. penilabiatus infections and FFC was effective (60 %) in reducing the bacterial density of Aeromonas sp. and Streptococcus sp. Once the treatments, i.e., feed combined with antibiotics, were complete (7 days), tissue samples were collected from the nine fish per treatment for hematological and histopatho- logical analyses. Histopathological analysis From each experiment, nine animals from each treatment (three from each replicate) were euthanized via immersion in a benzocaine bath and used for histopathological analyses. Gill, liver, and kidney samples were collected from fish in both experiments. Samples were immersed in a buffered (0.1 M PBS, pH 7.2) aqueous solution of 10 % formaldehyde for 24 h, and after fixation, they were subjected to dehydration and diaphanization and embedded in Histosec (Merck) wax. The samples were then cut on an automatic microtome (Leica, RM-2155) into 5-mm slices. Staining was performed using hematoxylin–eosin (HE) and periodic acid–Schiff (PAS) methods (Behmer et al. 1998). Hematological analysis For hematological analyses, blood samples from the same nine fish (1 ml), each experiment, were collected via caudal puncture with a heparinized syringe and needle. The blood samples were used to determine the following parameters: hematocrit (Ht) (using a microhematocrit tech- n i q u e ) , h emog l o b i n (Hb ) c o n t e n t ( u s i n g t h e cyanmethemoglobin method), red blood cell (RBC) count (using a Neubauer chamber and an index), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concen- tration (MCHC) (Ranzani-Paiva et al. 2013). Immediately after the blood was collected, the blood smears were performed, and after dry, the slides were stained with May–Grünwald–Giemsa–Wright (MGGW) stain, and total and differential leukocyte (WBC) and thrombocyte (Tromb) counts were conducted using an indirect method (Hrubec and Smith 1998). The total protein content from the plasma was determined using a refractometer. The mean values were subjected to an analysis of variance (ANOVA) and compared via Tukey’s tests at a 95 % signifi- cance level using the program Statistica. Results Effects of enrofloxacin and toltrazuril treatment Histopathology Gills contain four groups of arches, and each is composed of primary lamellae with secondary lamellae (Fig. 1a), which contain chloride, pillar, mucous, and lining cells. Animals from the InC treatment group exhibited disorders in the secondary lamellae that can compromise survival (Fig. 1c, d), while treated fish presented an increase in the height of the interlamellar epithelium. Fish in the ExC treat- ment group also exhibited disorders of the secondary lamellae (Fig. 1b). The liver is composed of sinusoid capillaries and hepato- cytes in a coronal arrangement. Hepatocytes have a hexagonal arrangement, a clear cytoplasm (or acidophilic, depending on the state of the cell), glycogen stores, and a nucleus that is slightly displaced to the periphery. Fish from the InC and ExC treatment groups showed an increase in sinusoid capil- lary diameter and hepatocyte hypertrophy (Fig. 2a). Treated fish exhibited increased glycogen storage (Fig. 2b), and gly- cogen stores were reduced in the ExC group, which was the opposite of the trend observed for the capillaries, indicating an increase in glycogen metabolism. The kidney is composed of proximal and distal tubules and glomeruli, which constitute Bowman’s capsules, and of fen- estrated capillaries. It is surrounded by melano-macrophage Comp Clin Pathol (2017) 26:105–114 107 centers and hematopoietic and lymphoid tissues. Fish from all treatment groups showed histological features that were sim- ilar to those of the control (Fig. 2d). Hematology The Ht, MCV, and MCHC values were not significantly dif- ferent among the treatment groups. Fish from the ExC treat- ment group had significantly higher RBC counts than those of the treated and InC fish. Additionally, the InC group showed a 19.0 % reduction in Hb compared with that of the HC group (Table 1). WBC and lymphocyte levels were significantly higher in treated fish. Eosinophil levels were lower in the ExC treatment group and higher in the HC group, while the other variables remained unchanged (Table 2). After treatment with thiamethoxam and florfenicol Histopathology Fish in the InC treatment group exhibited aneurysms in their gills and increased thickness of the interlamellar epithelium (Fig. 1e–g). The ExC treatment group showed less intensive aneurysms and a disruption of the secondary lamellae Fig. 1 Gills ofP. mesopotamicus. a HC. CVS central venous sinus, PL primary lamellae, SL secondary lamellae. b Tr with TOL + ENRO. Lines: increase in interlamellar epithelium. c, d InC group treated with TOL + ENRO. Monog monogenetic, Ictio I. multifiliis. e, f InC group treated with THI + FFC. DSL disruption of secondary lamellae. g ExC group treated with TH + FFC. Aneu aneurism. h Tr with TH + FFC. All staining is with HE except in e (PAS staining) 108 Comp Clin Pathol (2017) 26:105–114 (Fig. 1g). In contrast, there were no changes in the treated fish (Fig. 1h). The ExC, InC, and treated fish showed low glycogen levels, and glycogen stores were polarized in the hepatocytes: they were located in the region of the cells away from the capillaries. The treated fish also showed congestion in their sinusoid capillaries and hepatocyte hypertrophy, which indi- cates an increase in the organelles involved in metabolism (Fig. 2d). There was no change in the histomorphology of the kid- neys from all treatments. Hematology The treated fish showed a 19.0 % increase in Ht, a 35.0 % increase in Hb, and an 18 % decrease in RBC, all of which differed significantly from the values in the HC group, and the MCV and MCHC values were significantly higher in the treated fish. The increase in Ht, Hb, and MCV in the treated fish and ExC groups (Table 3) suggests an electrolytic imbalance. The ExC fish showed a 30.0 % decrease in thrombocytes, and in the InC group, leukopenia, lymphocytopenia, eosinopenia, and neutrophilia were observed (Table 4). Discussion Histopathology Gills are sensitive to changes in the water caused by xenobi- otics because they have a large surface area and a short diffu- sion distance (Nero et al. 2006; Brunelli et al. 2011). The pathogens I . mult i f i l i is , T. heterodentata , A. penilabiatus, and bacteria can cause changes in the gills that can impact oxygen uptake, metabolism, and the intake Fig. 2 a Liver samples from the InC group treated with TOL + ENRO. Arrows indicate increase in sinusoid capillary diameter. b Liver samples from Tr with TOL + ENRO. Hepatocytes with glycogen. c Liver samples from the group treated with TH+FFC. Asterisk: hypertrophy d Kidney samples from the group treated with TH+FFC. TH: tissue hematopoietic. TD: tubule distal. TP: tubule proximal: Arrowhead: melano-macrophages. Staining is with PAS in B and HE in A, C, and D Table 1 Hematological variables of Piaractus mesopotamicus after treatment with toltrazuril and enrofloxacin (average ± standard error) Treatments Ht (%) Hb (g dL−1) RBC (106 mm−3) MCV (fL) MCHC (g dL−1) InC 26.44 ± 0.66 7.60 ± 0.15B 1.64 ± 0.16B 172.11 ± 8.29 28.87 ± 0.78 Treated 27.11 ± 1.04 8.31 ± 0.25AB 1.56 ± 0.21B 166.05 ± 6.78 30.89 ± 1.05 HC 27.94 ± 1.05 9.27 ± 0.26A 1.83 ± 0.25AB 155.33 ± 10.11 33.56 ± 1.59 ExC 27.66 ± 0.74 9.01 ± 0.35A 1.92 ± 0.22A 145.13 ± 5.53 32.72 ± 1.45 Different letters indicate significant differences in the column according to Tukey’s test (P < 0.05) Ht hematocrit,Hb hemoglobin content, RBC red blood cells,MCVmedium corpuscular volume,;MCHCmedium corpuscular hemoglobin concentration, InC infected control, ExC exposed control, HC healthy control Comp Clin Pathol (2017) 26:105–114 109 and excretion of various molecules. I. multifiliis has been shown to cause hyperplasia and hypertrophy of the secondary lamellae in Oreochromis mossambicus (Subasinsghe 1990), Ictalurus punctatus (Maki et al. 2001), and Rhamdia quelen (Carneiro et al. 2006), and A. hydrophila can cause these same conditions in pacu (Carraschi et al. 2012). Hypertrophy and hyperplasia of the lamellae were ob- served in the treated fish (TH + FFC and EF + TOL) and have also been observed in Cyprinus carpio exposed to carbamaz- epine (40 at 80 mg L−1) (Malarvizhi et al. 2012) and Gambusia holbrooki exposed to sublethal doses of tetracy- cline (5, 50, and 500 ng L−1) (Nunes et al. 2015). These changes are reversible and act as an adaptive defense response because the decrease in gas exchange area also acts to prevent the intake of chemicals (Fernandes and Mazon 2003; Lupi et al. 2007). Powell et al. (1995) suggested that the thickening of the interlamellar epithelium in trout (Oncorhynchus mykiss) ex- posed to chloramine-T occurs via ionic regulation due to de- creases in Na+, Cl−, and Ca2+ in the bloodstream. These types of changes manifest as an innate defense mechanism that or- ganisms use to survive in adverse conditions. According to Mallatt (1985), the hyperplasia of mucous cells provokes the hypersecretion of mucous, which protects tissue structure under adverse environmental conditions and during exposure to toxic agents. Mucosubstances have polyanions that can act as a protective barrier against the ab- sorption of xenobiotics or invasion by pathogens. These changes were observed in the InC and treated fish, and they protect the gill epithelium from exposure to pathogens and drugs. The disruption of the secondary lamellae observed in the ExC and InC groups in both experiments has been previously shown to be caused by drug exposure or by pathogens, such as A. hydrophila, in P. mesopotamicus (Carraschi et al. 2012). The changes observed in the InC treatment (T. heterodentata, I. multifiliis , A. penilabiatus , Aeromonas sp., and Streptococcus sp.) can cause hypoxia, respiratory failure, and ionic and acid base imbalances. Furthermore, after expo- sure, organisms can remain more susceptible to secondary infections and can die (Hawkins et al. 1984; Yasser and Naser 2011). The aneurysms observed in the InC (A. penilabiatus, Aeromonas sp., and Streptococcus sp.) and ExC (TH + FFC) fish occurred due to an increase of blood in the lamellae, which can cause the damage to the pillar cells and the loss of vascular integrity (Nunes et al. 2015). Hyperplasia, epithe- lial hemorrhaging, and increased mucous production can dis- turb the respiratory function of the gills, and these changes can be caused by monogeneans (Hayes and Ferguson 1989). Aneurysms have also been observed by Nunes et al. (2015) in G. holbrooki exposed to tetracycline and in Salmo trutta Table 2 Absolute number of leukocytes in Piaractus mesopotamicus treated with toltrazuril and enrofloxacin (average ± standard error) Cells (μL−1) Infected control Treated Healthy control Exposed control Tromb 24,690.64 ± 2795.03 23,275.89 ± 3792.84 22,923.29 ± 1835.32 31,552.28 ± 3474.33 WBC 15,498.46 ± 1969.61B 25,031.90 ± 3921.9A 12,859.73 ± 1284.0B 10,247.5 ± 1558.5B Mono 814.37 ± 134.90A 519.16 ± 124.95AB 464.59 ± 135.91AB 278.22 ± 82.51B Neut 283.59 ± 123.13 218.80 ± 81.95 346.22 ± 108.10 172.21 ± 70.24 Eos 155.16 ± 65.13AB 134.36 ± 41.55AB 330.49 ± 118.78A 10.55 ± 5.31B Lymph 13,924.39 ± 1818.46B 23,720.49 ± 3572.9A 11,541.87 ± 1282.1B 9723.78 ± 1472.2B EGC 57.98 ± 26.40 124.16 ± 58.09 77.77 ± 51.94 0.00 ± 0.00 ImLeu 262.97 ± 92.53 338.70 ± 193.85 98.78 ± 45.94 62.78 ± 34.89 Different letters indicate significant differences in the line according to Tukey’s test (P < 0.05) WBC white blood cells (total leukocytes), Mono monocytes, Neut neutrophils, Eos eosinophils, Lymph lympho- cytes, SGC special granulocytic cells, ImLeu immature leukocytes, Tromb thrombocytes Table 3 Hematological variables of Piaractus mesopotamicus after treatment with thiamethoxam and florfenicol (average ± standard error) Treatments Ht (%) Hb (g dL−1) RBC (106 mm−3) MCV (fL) MCHC (g dL−1) InC 27.94 ± 1.06B 9.27 ± 0.27D 1.83 ± 0.08AB 155.32 ± 10.12BC 33.56 ± 1.59C Treated 33.22 ± 0.62A 15.91 ± 0.53A 1.56 ± 0.07B 216.80 ± 11.40A 47.82 ± 0.86A HC 27.83 ± 0.71B 11.70 ± 0.60C 1.92 ± 0.08A 146.99 ± 7.85C 41.94 ± 1.54B ExC 30.88 ± 0.89AB 13.77 ± 0.55B 1.64 ± 0.05B 189.97 ± 8.56AB 44.69 ± 1.63AB Different letters indicate significant differences in the column according to Tukey’s test (P < 0.05) Ht hematocrit,Hb hemoglobin content, RBC red blood cells,MCVmedium corpuscular volume,MCHCmedium corpuscular hemoglobin concentration, InC infected control, ExC exposed control, HC healthy control 110 Comp Clin Pathol (2017) 26:105–114 exposed to salicylic acid. However, this disruption of the sec- ondary lamellae is reversible, and after water purification, the morphology and function of the tissues return to normal (Lupi et al. 2007). The liver is the primary detoxifying organ in fish, and he- patic changes suggest a type of defense mechanism and that the substances used for chemical storage can be used for de- toxification (Olsson et al. 1996; Nunes et al. 2015). Hypertrophy in the hepatocytes indicates an increase in the organelles responsible for metabolism, as verified in S. trutta exposed to salicylic acid (Nunes et al. 2015) and P. mesopotamicus infectedwithA. hydrophila and treated with 10 mg FFC kg−1 (Carraschi et al. 2012). No critical or irreversible damage to the liver was found. This is consistent with the observations of Maki et al. (2001) in I. punctatus infested with I. multifiliis, which suggested that this parasite does not damage the kidney, liver, or spleen. Low levels of glycogen were observed in the fish in the ExC treatment in both experiments that can be used as a his- topathological indicator of environmental quality (Teh et al. 1997); the results observed in the treated (TH + FFC) and InC fish (A. penilabiatus, Aeromonas sp., and Streptococcus sp.) may reflect the results of environmental stress caused by path- ogens or xenobiotics. The changes observed in the liver are reversible and non- specific and suggest an initial effect of the drug treatment on metabolism. The histopathological changes in the treated fish did not compromise their development, and the use of drugs during the production cycle may successfully treat disease. Hematology Normal variations due to intrinsic or extrinsic factors or dis- eases affecting blood cell function and number may be evalu- ated via clinical hematology. Obtaining even a small blood sample may reveal information helpful in guiding treatment options (Grant 2015). The anemia obse rved in the InC group (T. heterodentata, I. multifiliis, A. penilabiatus, Aeromonas sp., and Streptococcus sp.) is a characteristic of the pres- ence of the pathogens, primarily that of the monogenean A. penilabiatus, which enters the mouth and affixes to the gill epithelium, causing disruption of the vessels (Jerônimo et al. 2014). This change and the low RBC levels observed in the InC group compromise the trans- portation of oxygen via Hb to the tissues. Without treat- ment and under chronic exposure, fish will experience respiratory deficiencies that can cause death. The reduction in RBC and Hb contents in the InC group (T. heterodentata, I. multifiliis, A. penilabiatus, Aeromonas sp., and Streptococcus sp.) indicates that bacteria are using the iron from the hemoglobin of the erythrocytes. Bacteria cause eryth- rocyte hemolysis, which releases more hemoglobin and heme groups to the bloodstream (Wooldridge and Williams 1993). This ability is associated with a bacterial virulence factor and involves siderophores, which are iron-transport cofactors se- creted by bacteria (Massad et al. 1991). A low iron concen- tration is the principal change that occurs when bacteria in- vade a host and produce virulence factors (Litwin and Calderwood 1993). Anemia occurs due to the inhibition of erythropoiesis or an increase in the erythrocyte destruction rate in hematopoietic organs or in circulation. A decrease in RBC and Hb content in C. carpio following exposure to diazinon has also been report- ed by Svoboda et al. (2001). TOL, a derivative of triazinetrione, and EF, a quinolone, cause lymphocytosis in P. mesopotamicus, similar to the ef- fects of trichlorfon in C. carpio parasitized by Argulus (Ranzani-Paiva et al. 1987) and of endosulfan in Clarias gariepinus (Yekeen and Fawole 2011). Sublethal doses of cypermethrin (0.30 mg L−1) and carbofuran (0.8 μg L−1) also cause leukocytosis in Labeo rohita (Adhikari et al. 2004), but 1.0 mg L−1 diflubenzuron, a derivative of benzoylurea, has been shown to not change hematological parameters in Table 4 Absolute number of leukocytes in Piaractus mesopotamicus treated with thiamethoxam and florfenicol (average ± standard error) Cells (μL−1) Infected control Treated Healthy control Exposed control Trombo 28,273.22 ± 2704.29A 25,067.26 ± 1536.21AB 28,156.93 ± 1304.94A 19,506.04 ± 1892.06B WBC 6214.98 ± 983.82B 8049.44 ± 1106.8AB 13,138.433 ± 2642A 10,749.83 ± 1009.4AB Mono 500.66 ± 114.42 493.73 ± 104.69 331.93 ± 65.72 636.23 ± 82.91 Neut 678.29 ± 193.11A 384.76 ± 8060AB 146.16 ± 34.67B 496.35 ± 96.75AB Eos 132.26 ± 44.43B 202.48 ± 46.46AB 594.26 ± 189.81A 398.954 ± 79.42AB Lymp 4859.37 ± 728.37B 6783.04 ± 943.5AB 11,760.77 ± 2418.9A 7961.35 ± 1358.82AB EGC 0.00 ± 0.00B 80.26 ± 23.03AB 86.773 ± 31.33AB 108.67 ± 34.02A ImLeu 83.00 ± 19.83 164.34 ± 71.21 139.28 ± 28.75 142.47 ± 30.71 Different letters indicate significant differences in the line according to Tukey’s test (P < 0.05) WBC white blood cells (total leukocytes), Mono monocytes, Neut neutrophils, Eos eosinophils, Lymph lymphocytes, EGC especial granulocytic cells, ImLeu immature leukocytes, Tromb thrombocytes Comp Clin Pathol (2017) 26:105–114 111 R. quelen infected with Lernaea cyprinacea (Mabilia and Souza 2006). The effects of the drugs (ExC: TOL + EF) or the pathogens alone (InC: T. heterodentata, I. multifiliis, A. penilabiatus, Aeromonas sp., and Streptococcus sp.) did not stimulate leu- kocyte formation, but the infected fish that were treated with TOL and EF exhibited leukocytosis and lymphocytosis. The main function of leukocytes is in the defense against bacteria or foreign bodies entering the tissues (Ranzani-Paiva and Silva-Souza 2004). Jerônimo et al. (2014) observed leukocy- tosis and lymphocytosis in P. mesopotamicus with >2200 A. penilabiatus. Thus, the organism’s defenses are stimulated when the exposure of the fish to drugs and pathogens decreases. The larger number of eosinophils found in the HC group from both experiments is an inherent feature of P. mesopotamicus be- cause they are resident cells (Ranzani-Paiva et al. 1999). Eosinopenia occurred in the ExC group (TOL + EF), which showed a 96.0 % decrease in cells, suggesting a disturbance caused by the drugs and in the InC treatment group (A. penilabiatus, Aeromonas sp., and Streptococcus sp.), which showed a 77.8 % decrease caused by the pathogens. Eosinopenia is a decrease in the production and release of eosin- ophils from the bone marrow (Savari et al. 2011), such as that caused by cortisol in C. carpio (Wojtaszek et al. 2002). Hematocrit percentages reflect the proportion of RBCs in the blood relative to the white blood cells (WBCs) and plasma. Because RBCs are responsible for the transport of oxygen and carbon dioxide (Ranzani-Paiva et al. 2013), the greater Ht and lower RBC levels in the treated fish (TH + FFC) suggest that some stressor and/or respiratory dysfunction was present that suppressed RBCs. This has also been observed by Hashimoto et al. (2016) in Nile tilapia infected with a monogenean and treated with an essential oil from Lippia sidoides. The increases in MCVand MCHC observed in the treated fish (TH + FFC) have also been found in L. rohita exposed to cypermethrin (Adhikari et al. 2004) and in C. carpio exposed to dichlorvos (Svobodova 1975) and diazinon (Svobodova et al. 2001). The increase in MCHC and the decrease in RBCs in the treated fish (TH + FFC) indicate hemolysis, according to Thrall et al. (2006). Cypermethrin causes hemolysis in L. rohita (Adhikari et al. 2004), which is also caused by diazinon in C. carpio (Banaee et al. 2008) and by potassium perman- ganate in Oreochromis niloticus (Salawu et al. 2013), and C. carpio fed with praziquantel have been shown to have decreased RBC, Hb, and MCV values (Sudová et al. 2008). Increases in Hb and MCV are related to a higher capacity of the blood to transport oxygen to meet energy demands (Labarrére et al. 2012). Reduced thrombocyte counts indicate damage to the coag- ulation capacity and defense mechanisms of an organism (Martins et al. 2008). It can be inferred that the low thrombocyte counts found in the fish exposed at TH and FFC (ExC) may have been associated with the migration of these cells, as verified by Hashimoto et al. (2016) in Nile tilapia infected with a monogenean and treated with an essen- tial oil from L. sidoides. According to Tavares-Dias et al. (2001), lymphocytopenia is an indicator of stress. Lymphocytes are white blood cells tha t remain in c i rcula t ion for a long t ime, and lymphocytopenia most often occurs when there is stress or in response to steroids (Gad 2007). Neutrophils are granulocytes that are responsible for organ- ismal defense. They phagocytose small objects (e.g., bacteria) and participate in inflammatory processes (Savari et al. 2011). The presence of neutrophilia observed in the InC group (A. penilabiatus, Aeromonas sp., and Streptococcus sp.) has also been shown to be caused in situations involving infection (Martins et al. 2008) or stress management (Jerônimo et al. 2011). Hashimoto et al. (2016) also reported the presence of neutrophilia in Nile tilapia infected with a monogenean and treated with an essential oil from L. sidoides. The immunosuppression observed in the InC (A. penilabiatus, Aeromonas sp., and Streptococcus sp.) group caused by pathogens has also been verified by Achuthan Nair and BalakrishnanNair (1983) inChanna striatus infested with Alitropus typus, by Höglund et al. (1992) in Anguilla anguilla infested with Anguillicola crassus, and by Tavares Dias et al. (2002) in O. niloticus with ichthyophthiriasis. TOL, EF, FFC, and TH caused reversible histopathological changes; thus, these drugs have the potential for use in Brazilian aquaculture. However, the changes observed in the InC fish can lead to death, so more detailed studies of dosage adjustments and residue quantification must be performed to inform the regulatory process. The use of drugs at standard- ized concentrations, exposure times, and number of treatment days will improve the success of the production cycle and decrease the losses caused by pathogens. TOL, TH, FFC, and EF caused reversible and minor hema- tological and histopathological changes, while the pathogens caused more serious hematological and histopathological changes that can compromise P. mesopotamicus survival. The drug-related data presented here can be used in viability studies as part of the registration process for their use in com- mercial aquaculture. Acknowledgments To FAPESP, scholarship no. 09888-6/2010 and supporting grant no. 08453-9/2011. State of Sao Paulo Research Foundation process nos. 2008/51900-3 and 2011/08453-9. 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