Introduction

Murrels, commonly called snakeheads belonging to the family Channidae (Ophiocephalidae), constitute the most common and dominant group of air breathing freshwater fishes and are highly regarded as food fish in the South and Southeast Asian countries48. It has long been commercially cultured in Thailand, Taiwan, and the Philippines. There are several species of murrels belonging to the genus Channa (syn. Ophiocephalus), but only one species, namely Channa striatus also called striped murrel, enjoys a good deal of popularity as food fish in many parts of India20. Besides the high quality of their flesh in terms of taste and texture, they also have good market value due to the low fat, fewer intramuscular spines, and medicinal qualities17.

Bacteria of the genus Aeromonas are wide spread in fresh, brackish, estuarine and marine water13. Motile aeromonads are associated with tail and fin rot hemorrhagic septicemia and epizootic ulcerative syndrome (EUS) in a variety of freshwater and marine fish of the world40. They are frequently isolated from both healthy and diseased fish as well as from other aquatic animals. Under predisposing factors such as poor water quality, high ammonia as a result of high stocking density and feeding, ectoparasites, inadequate handling and stressful conditions, this organism found a portal of entry into the fish host31. Motile aeromonads are considered to be one of the most important bacteria among the etiological agents of fish diseases33. The outbreaks of motile Aeromonas associated diseases can reach epidemic proportions among the aquatic animals, leading to massive mortality rates22.

There are several studies on fish bacteria identification, experimental infection or disease resistance4112 but little relates the haematological parameters to bacterial experimental infection. The haematological parameters are an important tool of diagnosis that reveals the state of health of fish83729. Blood tissue of fish gives clue about physiology and environmental conditions of fish35. Knowledge of hematology is very important since it deals with the morphology, physiology and the biochemistry of blood. By analyzing blood cell characteristics, disease status can be identified3. Bruno and Munro11 have stated that hematological indices aid in the diagnosis and assessment of disease in fish. In fisheries, it is important to find out illness and parasites as the source of these causes may not be generally detectable in early period of the infection. However it is also possible early diagnosis of illnesses in case of evaluating hematological data, particularly blood parameters38.

This study evaluated the haematological changes in Channa striatus intramuscularly administered with 2.4 x 108 CFU/ mL of A.hydrophila in the caudal region originally isolated from naturally infected C. striatus.

Materials and Methods

A total of 90 striped murrels (C. striatus) of average mean length (15 ± 2 cm) and average weight (180 ± 3.5 g) were collected from fish market, Melapalayam, Tirunelveli (8.44°N, 77.44°E), Tamilnadu, India in the month of February 2010 (Figure:1). They were transported to the Centre for Aquaculture Research and Extension (CARE) Aquafarm in live condition with oxygenated water in plastic bags (10 l) and they were acclimatized in cement tanks (3 m × 12 m × 1 m) for 7 days before assay and fed with commercial diet. During this period, the water temperature was maintained at 28 ± 1.5 °C, dissolved oxygen 5.8 mg/L and pH 7.1-7.4.

Fig. 1: Infected Channa straitus.

Pathogenic A. hydrophila strain was isolated from infected C.striatus, further purified by streaked on selective medium, Aeromonas Isolation agar (Hi-media). The isolate was identified by their reaction to standard test following the Bergey’s manual7. The pathogenicity of A. hydrophila was performed following the method of Lafrentz25, it was confirmed by injection of A. hydrophila on healthy C.striatus, it caused 100% mortality within 72 hours (mean death time was 52.7 hours) with development of clinical symptoms.

This experiment consisted of two treatments in triplicates: non-injected control fish (C) (n=30); fish injected with 2.4 x 108 CFU/ mL of A.hydrophila (T) (n=60) intramuscularly in the caudal region according to the Matushima and Mariano30 and Martins28 method.

Forty-eight hours after injection, the fish were sacrificed and blood sample was collected by vein puncture using 1ml syringe. Before collecting the blood sample, the needle was treated with 0.5% EDTA to avoid coagulation36. To determine the count of erythrocytes, blood sample was taken with an erythrocytes pipette and diluted (1/200) with the Hayem solution, loaded in haemocytometer and examined in light microscope (Nikon-Eclipse E400 microscope, Germany) with a magnification of 400x9. Leukocytes counting was performed by transporting blood sample (diluted in WBC diluting fluid) with a leukocytes pipette into counting lamella and examined as for erythrocytes91016.

The amount of hemoglobin was determined according to cyanomethemoglobin procedure9. Non-clotted blood (20microlitre) was diluted with Drabkin solution (5mL) and left stand for 10 min. The absorbency of the mixture was read at 540 nm and the amount for hemoglobin was calculated using hemoglobin standard solution5. Non-clotted blood was transferred into PCV tube and centrifuged at 12,500 rpm for 5 min and the ratio of blood components in plasma was determined212. For differential leukocyte count, six blood smears per fish were prepared from fresh blood, air-dried, stained with Leishman-Giemsa’s stain and fixed in methanol. In each sample, three visual fields at 1,000 X were identified for the leukocyte count19. The percentage of neutrophil (NEU), eosinophil (EOS), lymphocyte (LYM) and monocyte (MON) tissues was determined. The derived blood indices of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were calculated using standard formulae. Hematological data were analyzed with SPSS 7.5 for Windows by using one way analysis of variance.

Results

The hematological parameters of the experimental fish (T) were compared with control fish (C) are presented in Table: 1, during this assay, no mortality was observed after experimental infection. Fish injected with 2.4 x 108 CFU/mL of A. hydrophila (T) showed a gradual decrease in Haemoglobin (HB,g/dl), Haematocrit (PCV,%), Mean Corpuscular Haemoglobin (MCH, pg), Mean Corpuscular Haemoglobin Concentration (MCHC,g/dl), Red blood cell (RBC,103/mL) which were significantly lower (P < 0.05) from values of the control fish (C) which indicated poor physiological blood production. Mean Corpusular Volume (MCV, fl), White Blood Cells (WBCs, 103/mL), Lymphocytes (LYM, 103/mL), Monocytes (MON, 103/mL) and Eosinophils (EOS, 103/mL) of A.hydrophila injected fish (T) which were increased significantly (P < 0.05) than values of the control fish (C). The number of lymphocytes in fish injected with 2.4 x 108 CFU/mL of A.hydrophila (T) was significantly (P < 0.05) higher than that of the control fish (C).

Table: 1. Changes in the hematological parameters of Channa striatus injected with 2.4 x 108 CFU/mL of A. hydrophila. (Values expressed in Mean ± S.D of 3 replicates) (P<0.05).

Discussion

The predominance of A. hydrophila in epizootic ulcerative syndrome (EUS) affected fish has also been reported previously by Kumar24 in India, Tonguthai45 in Thailand, Wong and Leong49 in Malaysia, Dana15 in Indonesia, Roberts39 in Myanmar and Balasurya6 in Srilanka. Lio-Po26 reported that several species of bacteria and fungi were found to be associated with EUS affected snakehead C. straiatus and that 89% of the total isolates were A. hydrophila. A. hydrophila can often be isolated from ulcers or internal organs of EUS-affected fish2732. Some of these A. hydrophila strains have been characterised as virulent464323 or cytotoxic50. Sabina41 have reported that A. hydrophila is one of the important pathogens of fish in freshwater and brackish water.

The results presented in this study have revealed an interesting pattern showing that the level of HB, values of PCV, MCH and MCHC and the number of RBCs were significantly decreased in fish injected with A.hydrophila when compared to the control fish. The decreased HB trend may be a result of the swelling of the RBC as well as poor mobilization of HB from the spleen to other hemopoeitic organs42. These data support the present finding that the significant decrease in RBC and HB content is possibly due to hypochromic microcytic anemia caused by A. hydrophila. Similarly, decreased red blood corpuscles and PCV were found in coho salmon (Oncorhynchus kisutch) infected with Vibrio anguillarum18; in Asian cichlid fish (Etroplus suratensis) with epizootic ulcerative syndrome34 in rainbow trout (Oncorhynchus mykiss) with ulcerous dermatitis36; in rainbow trout experimentally infected with Aeromonas sobria and A. caviae37; in carp (Cyprinus carpio) experimentally infected with A. hydrophila (Harikrishnan et al., 2003) and in Nile tilapia experimentally infected with Streptococcus iniae14.

In this experiment, the PCV level significantly decreased in fish injected with A.hydrophila. For instance, the pearl spot fish Etroplus suratensis when infected with EUS becomes anaemic and then suffers a significant reduction in RBC, HB, and PCV levels34.

In this present study, fish injected with 2.4 x 108 CFU/mL of A.hydrophila showed increased MCV, WBCs, LYM, MON and EOS. Pathiratne and Rajapakshe34 have reported that increased WBCs were found in Asian cichlid fish (Etroplus suratensis) with epizootic ulcerative syndrome. Total leucocytes count suggested severe leucocytosis of 24.0 +0.25x 103 WBC/ mL in control to 37.5 +0.35 x 103 WBC/ mL in fish injected with 2.3 x 108 CFU/mL of A.hydrophila. This fact shows more production of leucocytes in A.hydrophila injected fish enhancing the fish defense mechanisms.

In this present study, an increase in MCV were observed in fish injected with 2.4 x 108 CFU/mL of A.hydrophila, it may be attributed to the swelling of the erythrocytes, resulting in a macrocytic anaemia. An increase in MCV is also linked to the swelling of the RBC as a result of a hypoxic condition or impaired water balance (osmotic stress) or macrocytic anaemia in fishes exposed to stress47 this would increase the affinity for oxygen in the blood44. The decreased level of MCH and MCHC were observed in fish injected with 2.4 x 108 CFU/mL of A.hydrophila in the present study clearly indicates that the concentration of HB in the RBC was much lower in the infected fishes than in the control fishes, thereby indicating an anaemic condition. The MCHC, as a good indicator of RBC swelling is neither influenced by the blood volume nor by the number of cells in the blood, so can be interpreted incorrectly when new cells with different HB concentration are released into the blood circulation44. A significant decrease in the MCHC after A. hydrophila infection is probably an indication of RBC swelling and/or a decrease in HB synthesis. The higher lymphocytes count observed in fish injected with A.hydrophila in this study has also been recorded in infected brown trout and rainbow trout.

As the aquaculture industry expands, tools to monitor the health status of fish using standardized and inexpensive methods will be needed. Evaluation of hematological analyses will enhance the culture of fish by facilitating early detection of infectious disease and identification of sub-lethal conditions affecting production performance. This will contribute to more specific, timely and effective disease treatments in the future.