Histological assessment of blood cockles (Anadara granosa) using different stains and fixatives.

Blood cockles (Anadara granosa) or locally known as ‘kerang’ are mollusc belonging to Bivalvia class. It is an important fisheries commodity and a delicacy in Malaysia. In RMK-9, one of the objectives of Malaysia’s aquaculture industries is to maximize the production of bivalves up to 130,000 MT per year, which includes cockles, green mussels, clams and oysters. Cockles become one of the important bivalves cultured in Malaysia as it had very good market value in Malaysia, Thailand and Singapore. According to Phillips and Muttarasin (1985), 80% of the cockles marketed in Thailand were imported from Malaysia. In spite of the diversity and its economic importance, the knowledge of bivalves specifically cockle medicine should be developed in order to meet the requirement of diagnostic work which is necessary in determining the cause of diseases infecting the animals. Up to date, there is no comprehensive information of the histology of cockles. Thus, this study was undertaken to provide basic histological descriptions of normal and anomalies microstructures in Anadara granosa by using different fixatures and stains. Forty live cockles were divided into two groups; one group was fixed with Davidson solution and the other fixed with 10% buffered formalin. The tissues were then processed for 18 hours, and later embedded with paraffin wax and sectioned at 5 µm thickness. There were stained with four stains namely, Haematoxylin and Eosin, Masson’s Trichome, Periodic Acid Schiff’s and Van Gieson’s stains. The sections were evaluated under a computer attached-compound light microscope at low and high magnification. The organs were assessed for microscopic structures staining affinity (Cox et al., 2006). Haematoxylin and Eosin stain was considered the better stain to be used for mantle, foot complex and haemocytes tissues. Combination of Haematoxylin and Eosin and Periodic Acid Schiff’s stains can be used to stain digestive system and palp. Gills could be stained with Masson’s Trichome and Van Gieson’s, while for gonads Masson’s Trichome would be the preferred choice. Van Gieson’s on the other hand, was the choice for foreign body detection. Gills of cockles could be divided into three parts: frontal, intermediate and abfrontal zone similar to Mytella falcate (David et al., 2008). The intermediate zone have a homogenous densely stained structure embedded in the filament which highly indicative that it provided supportive frame to the frontal zone

Wound healing potential of aloe vera in climbing perch (Anabas testudineus).

Fresh gel portion of Aloe vera leaves was evaluated for the wound healing potential in climbing perch (Anabas testudineus). Fifteen adult climbing perch were divided into three groups. Group 1 received Aloe vera gel, group 2 received Betadine® antiseptic ointment (positive control) and group 3 received no treatment (negative control). Wound was created at the flank area using a punch biopsy (8 mm in diameter). One topical application was applied on the wound. Assessment of healing progress was carried out on day 10. Gross observation and histological studies were carried out. There was no significant difference in wound reduction among the groups. However, group 2 showed highest rate of healing followed by group 1 and group 3, and groups 1 and 3 had similar rate of healing. Histological examination on cellular activity of healing process showed increased thickness of epithelium layer, infiltration of inflammatory cells, presence of fibroblast cell and rearrangement of the cell

Abiotic and biotic control of Argulus sp. among goldfish (Carassius auratus)

Argulus spp. are branchiuran crustaceans with a direct life cycle, therefore current aquaculture practice with a high density of potential hosts provides ideal conditions for effective transmission of this obligate ectoparasite. Chemicals are usually used to treat parasite infestation of fishes. However chemical treatments result in side-effects including environmental pollution, parasite resistance, alteration of zooplankton ecosystem, changes in certain hematological parameters and contamination of host organs. Thus there is a need for alternative measures to control parasitic diseases in the aquaculture industry. To date, there are no studies reported on biological control of fish lice, in particular the use of Thai silver barb (TSB) (Pontius gonionotus) to control Argulus population. In this study, the effectiveness of abiotic and biotic control methods for an ectoparasite infestation was examined. Therefore, an abiotic study was designed firstly to examine the effect of water current on parasite abundance and incidence of the fish lice, Argulus sp. among captive goldfish (Carassius auratus). Secondly, in a biotic study, the effectiveness of using TSB to reduce the parasite number was investigated and thirdly, to compare both the abiotic and biotic control methods in their effectiveness to reduce parasite number. The mean abundance and incidence of the ectoparasite, Argulus sp., on goldfish was measured. In the abiotic study goldfish infected with Argulus sp. was mixed with other susceptible goldfish hosts and kept in tanks with fast flowing or stagnant water. There was no difference in the parasite mean abundance and incidence between fast flowing and stagnant water. Thai Silver Barb which is a predator of the Argulus was introduced to the parasite infected goldfish for the biotic study. The mean parasite abundance was significantly lowered (P<0.05) after introducing the predator TSB into the tanks. Combination of both control methods did not further improve the effectiveness in reducing Argulus population. Hence this study showed that utilizing this method of biotic control among goldfish is definitely a future control solution. This study also showed that water current seemed to reduce Argulus transmission and population. The predator effect of TSB was lessened by the fast flowing current. The major finding in this study indicated that TSB was a very effective biological control where with only two TSBs, 96% of Argulus was removed in less than 24 hours. The number of TSB used in the biotic control did not affect the predator efficiency. It is suggested that the biological control tested in this study can be used to reduce Argulus population among goldfish in aquaculture settings

Histopathological changes of skin and gills of catfish (Clarias gariepinus) exposed to organophosphate toxicant

The usage of pesticide in the industry will help increase crop yield and meet consumer demand for Malaysia, a country that is undergoing economic revolution through the agricultural. However, the excessive and inappropriateness use of pesticides can affect the environment, water quality and public health. In this experiment, 40 adult African catfish (Clarias gariepinus) were divided and exposed into sublethal concentrations of Malathion (0.8, 1.6 and 3.0 ppm) and a control group for 48 hours. At the end of the study, all fish were sacrificed before skin and gill samples were collected to determine the effect of malathion on the gills and skin. The histopathological lesions in the gills and skin were scored and statistically analysed to compare between group. Histological examination of the gill showed epithelial lifting at secondary lamella, hyperplasia of primary epithelium, fusion of secondary lamella and infiltration of inflammatory cells with peeling and rupture of epithelial cells of secondary lamellae. In the skin, the changes observed were hyperplasia of mucus cell and shrinkage of the club cells after the exposure to malathion. There were significant differences between treatment and control groups. Thus, malathion causes concentration-independent and nonspecific lesions in the gills and skin of African catfish

Histopathology of goldfish (Carassius auratus) exposed to chlorine toxicant

Chlorine is widely used as disinfectant in Malaysian fish farms. As we know chlorination as a method for water disinfection is practiced by most water municipal treatment in Malaysia (Abdullah et al., 2003) due to its efficiency and cost effectiveness. Like many toxins in water, chlorine is more toxic to fish than humans (Mahjoor and Loh, 2008). Thus, the objectives of the current study were to assess pathological changes in organs, to ascertain behavioral response of goldfish when exposed to acute chlorine toxicity and to determine the Median Lethal Concentration (LC50) of free chlorine. Juveniles of Oranda goldfish, Carassius auratus, were exposed to chlorine in a 96 hour static renewal test to determine the LC50 of free chlorine. The goldfish behavioral responses were recorded. Moribund fish were promptly sacrificed and processed for histopathology. Using SPSS 16.0 probit analysis, the LC50 of free chlorine in juvenile goldfish was determined to be at 0.3 ppm. Goldfish showed signs of dyspnea, lethargy and increased mucus production. Histopathologically, the gills indicated secondary lamellar edema, exfoliation of interlamellar cells’ membrane and congestion in the capillary lumen. Kidney parenchyma manifested generalised degeneration, but with marked desquamation of tubules and dilated tubular lumen in the posterior kidney. Spleen showed congested blood vessels and hemosiderosis while liver showed some vacuolative changes in the parenchyma suggestive of liver necrosis. Even though the findings in gills, liver and kidney were not specific for chlorine toxicity, hemosiderosis in the spleen could be used to differentiate with other toxicities and diseases. Current findings were in agreement with earlier report by Mahjoor and Loh (2008) and also supported Zeitoun (1977) observation that the cause of death in chlorine toxicity was due to hemolytic anemia

Histopathological evaluation of visceral organs and brain of African catfish (Clarias gariepinus) exposed to sublethal concentrations of malathion

Organophosphate is a neurotoxin that inhibits acetylcholinesterase. Exposure to organophosphate toxicants can be through inhalation, absorption and ingestion of food treated with an organophosphate herbicide or insecticide. Malathion is among the most frequently used organophosphate insecticides. Malathion has been associated with reproductive malfunction, neurotoxicity, kidney and liver damage and birth defects. The objective of this study was to determine the effect of malathion on the catfish species C. gariepinus. One hundred and ten mature catfish, C. gariepinus with a body length of about 15cm were obtained from Taman Pertanian Universiti, Universiti Putra Malaysia. Prior to experimental trial, the fish were acclimatised for one week in a clean fibreglass tank provided with aeration, biophysical filtration and treated with Aqua-Med® to remove external bacteria and parasites. The fish were exposed to four different malathion concentrations of 1.6, 3.2, 6.4 and 8.5 ppm in a controlled laboratory condition. The LC50 of malathion at 48 hours was determined to be 3.2 ppm. In the acute exposure study, 40 catfish with an average length of 18.0 ± 2 cm were divided equally into four tanks. Tank 1 contained the control group, where the fishes were bathed in dechlorinated water provided with aeration and biophysical water filter. The fish in the other three tanks were exposed to malathion concentrations of 0.8, 1.6 and 3.0 ppm for 48 hours. The fishes were then euthanized with an overdose of MS222 and their visceral organs viz., kidney and liver tissues were fixed in 10% buffered formalin and the brain tissue in Bouin’s solution. All samples were histologically processed and stained with H & E stains. Histopathological evaluations were carried out using lesion scores of 0 to 3 and the significant differences were tested by one-way ANOVA. The kidneys of the malathion-treated group showed necrosis, infiltration of inflammatory cells, and capillary congestion while the controls did not. There was no difference (p>0.05) in kidney lesions between malathion-treated groups. Liver necrosis, infiltration of inflammatory cells, congestion and haemorrhage were evident in the malathion-treated group, which was greatest (p0.05) in other lesions among treated groups. The control catfish did not show any lesion. The malathion-treated catfish also showed brain necrosis, congestion and infiltration of inflammatory cell, which were not significantly (p>0.05) different among treatment groups. The control group did not show any brain lesion. The study showed that at concentrations below LD50 and exposure time of 48 hours, malathion is moderately toxic to C. gariepinus

Fatty acid compositions in mucus and roe of haruan, Channa striatus, for wound healing

1. Fatty acid profiles in the external mucus extract and roe of Channa striatus were determined using gas chromatography (GC). 2. The mucus samples were collected by inducing hypothermic stress (−20°C) for about 1 hr, and the roe were collected from gravid females at night soon after they liberated their eggs in a spawning program. 3. All mucus and roe samples were freeze-dried, except a part of roe which was not. 4. The mucus extract contained unsaturated fatty acid (oleic acid, C18:1 and linoleic acid, C18:2) as a major component, 21.25% and 22.47% of total lipid. 5. For the freeze- and nonfreeze-dried roe, the major components of fatty acid were somewhat similar to the mucus but with higher percentages: 58.56%, 26.08% and 45.76%, 20.94%. Interestingly, the nonfreeze-dried roe contained a large proportion of arachidic acid, C20:0 (22.16%), which was totally absent in the freeze-dried roe samples. 6. This profiling of the fatty acid mucus extract and roe is useful in strengthening the earlier claims that haruan possesses a potential remedy for wound healing (Mat Jais et al. 1994). Therefore, we are discussing the possibility of getting an optimum amount of the essential fatty acid for wound healing from various other parts of the fish without sacrificing the fish

Pathogenicity of Aeromonas hydrophila in high-value native Pangasius catfish, Pangasius nasutus (Bleeker)

Pangasius catfish, Pangasius nasutus, is a promising candidate for aquaculture due to its high market value. However, the presence of pathogenic bacteria in Aeromonas hydrophila is a major concern in P. nasutus farming in this country. This study determines the pathogenicity of A. hydrophila in P. nasutus. A total of 80 P. nasutus juveniles were intraperitoneally injected with 0, 103 , 105 , and 107 CFU mL-1 of A. hydrophila and monitored until 240 hr. The infected moribund fish’s kidneys, livers, and spleens were collected for histopathological analysis. The LD50-240hr value was found at 0.8 × 104 CFU/ml of A. hydrophila. The percentage of mortality in 0, 103 , 105 , and 107 CFU/ml infected groups were found to be at 0, 40, 60, and 90, respectively. The infected fish showed congestion at the base of the fin, ascites, enlarged gall bladder, and swollen spleen. It is the earliest report on A. hydrophila’s pathogenicity in high-value native fish, P. nasutus

Efficacy of whole cell inactivated Vibrio harveyi vaccine against vibriosis in a marine red hybrid tilapia (Oreochromic niloticus x Oreo mossambicus) model

Vibrio harveyi causes vibriosis in various commercial marine fish species. The infection leads to significant economic losses for aquaculture farms, and vaccination is an alternative approach for the prevention and control of fish diseases for aquaculture sustainability. This study describes the use of formalin-killed Vibrio harveyi (FKVh) strain Vh1 as a vaccine candidate to stimulate innate and adaptive immunities against vibriosis in a marine red hybrid tilapia model. Tilapia are fast growing; cheap; resistant to diseases; and tolerant to adverse environmental conditions of fresh water, brackish water, and marine water and because of these advantages, marine red hybrid tilapia is a suitable candidate as a model to study fish diseases and vaccinations against vibriosis. A total of 180 healthy red hybrid tilapias were gradually adapted to the marine environment before being divided into two groups, with 90 fish in each group and were kept in triplicate with 30 fish per tank. Group 1 was vaccinated intraperitoneally with 100 µL of FKVh on week 0, and a booster dose was similarly administered on week 2. Group 2 was similarly injected with PBS. Skin mucus, serum, and gut lavage were collected weekly for enzyme-linked immunosorbent assay (ELISA) and a lysozyme activity assay from a total of 30 fish of each group. On week 4, the remaining 60 fish of Groups 1 and 2 were challenged with 108 cfu/fish of live Vibrio harveyi. The clinical signs were monitored while the survival rate was recorded for 48 h post-challenge. Vaccination with FKVh resulted in a significantly (p < 0.05) higher rate of survival (87%) compared to the control (20%). The IgM antibody titer and lysozyme activities of Group 1 were significantly (p < 0.05) higher than the unvaccinated Groups 2 in most weeks throughout the experiment. Therefore, the intraperitoneal exposure of marine red hybrid tilapia to killed V. harveyi enhanced the resistance and antibody response of the fish against vibriosis