Bloquear blindajes

The Gram-negative motile bacteria Aeromonas (Fig. 8.3) are probably the most common bacterial disease that infects wild and cultured tilapia.

They cause ‘fin rot’ or ‘skin rot’ diseases and may lead to heavy mortality in cultured tilapia (Roberts and Sommerville, 1982). Infected fish usually have a dark colour, lose their appetite and develop ulcers or hyperaemia at the bases of pectoral and pelvic fins (Scott, 1977), ascites and exophthalmia (M.K. Soliman, Alexandria, 2005, personal com- munication). Internal symptoms include a pale liver and the presence of many focal haemorrhagic necroses in the liver, heart and skeletal muscles and over the visceral and peritoneal surfaces. Mas- sive losses of tilapia due to Aeromonas infection have been reported worldwide. Considerable attention has therefore been paid to this disease, for the sake of reducing its potential in tilapia farms.

Outbreaks of caudal fin rot caused by

Aeromonas hydrophila have caused 80–100% morta- lity in farmed Nile tilapia in South China (Liu et al., 1993). Infected fish showed slow movements, had rotted caudal fins, swam near the surface and had a poor appetite. Similar infection of Nile tilapia by

A. hydrophila (biotype 1) was reported in wintering

ponds in some districts of China (Wang and Xu, 1985). The major symptoms included the presence of circular or elliptic erosion of the skin (‘rotten skin’) and the dorsal fin. In early infection stages, the body surface was haemorrhagic. Aeromonas

hydrophila were also isolated from Nile tilapia dur-

ing fish disease outbreaks in various aquaculture farms and projects in the Philippines between 1994 and 1996 (Yambot, 1998). The occurrence of the disease was observed in low-volume and high-density aquaculture of Nile tilapia during the rainy season and cold months when water temper- ature was low. Infected fish suffered from skin lesion, ulceration, fin rot, body discoloration, mouth sore, eye opacity, exophthalmia, dislodged eyeball and sluggishness. High mortality was also observed in cage culture of Nile tilapia during the rainy season and cold months. The bacteria were isolated from the liver, kidney, spleen, gall bladder and opaque eyes of infected fish.

Garcia et al. (1999) isolated A. hydrophila,

A. schubertii and A. sobria from cultured

O. mossambicus and hybrid tilapia (O. mossambicus× O. niloticus) in Venezuela. The bacteria were isolated

from the kidney, liver and dermal lesions of infected fish. Aeromonas salmonicida also caused mass mortality of wild common carp and O. mossambicus in Kalyani reservoir in India (Reddy et al., 1994). The infection of blue tilapia with A. hydrophila was also recorded in Mexico (Constantino Casas et al., 1997). Stress and

Fig. 8.3. Aeromonas hydrophila isolated from

lack of proper culture conditions were the major contributors to the development of the disease.

Experimental infection of Mozambique tilapia held in seawater with A. hydrophila via intra- muscular injection has been tested (Azad et al., 2001). Experimentally infected fish developed ulceration leading to open wounds, dermal necro- sis and liquefying muscular degeneration, with infiltration of cellular and serum factors to the site of injection. The major histopathological manifes- tations were focal necrosis, loss of submucosa and sloughing of the intestinal microvilli. Similar experimental infection of tilapia by Aeromonas was reported by Liu et al. (1990) in Taiwan.

A number of studies have been conducted on the resistance of carp to Aeromonas, based on non- specific immunity functions, including intra-species and inter-species variations (Cai and Sun, 1994, 1995). Studies on potential heterosis for disease resistance of tilapia are almost lacking, despite the fact that resistance of tilapia to bacterial infection may vary from one tilapia species to another. Only very recently, Cai et al. (2004) studied the resistance of Nile tilapia, blue tilapia and their hybrids (male blue tilapia× female Nile tilapia) to

Aeromonas sobria. Based on the median lethal dose

(LD50), a function of non-specific immunity, the

authors found that the hybrids had the highest resistance to the disease, while blue tilapia were the least resistant.

Several methods of treatment of and pro- tection from Aeromonas disease have been tested, with varying effectiveness. Jirawong (2000) studied the toxicity of copper sulphate (CuSO4) to A. hydrophila. He found that a concentration of

0.5–1.0 ppm CuSO4killed 90–99% of the bacteria

within 2–8 h, while 100% were killed within 2 h at 2 ppm. The immune response of tilapia (Oreochromis sp.) immunized with formalin-killed

A. hydrophila by intramuscular injection (IM) and

direct immersion (DI) was significantly increased when the fish were fed diets containing ascogen (5 g/kg feed) (Ramadan et al., 1994). The survival of challenged fish after IM and DI vaccination was 89.80% and 40.82% in ascogen-fed groups com- pared to 75.51% and 10.20% in the vaccinated, non-ascogen-treated groups, respectively.

Protective vaccination against Aeromonas in other tilapias has also been successful. Ruangpan

et al. (1986) found that Nile tilapia vaccinated

with formalin-killed bacterial vaccines showed a 53–61% protection within 1 week after vaccination,

while 100% of the fish were protected within 2 weeks. Non-specific immune systems of tilapia have been shown to be stimulated by different polysaccharides. Wang and Wang (1997) exam- ined the efficacy of 11 polysaccharides in the protection of blue tilapia and grass carp against bacterial infection by A. hydrophila and

Edwardsiella tarda. They found that four glycans,

namely Bar (glycan extracted from barley), Krestin, scleroglucan and Zymosan, significantly increased the survival rates of tilapia (80, 60, 70 and 60%, respectively) and grass carp (60, 70, 90 and 60%, respectively). The non-specific immune response of Nile tilapia clones was also investi- gated (Sarder et al., 2001). Serum lysozyme activity, phagocytosis and natural resistance to

A. hydrophila infection by bacterial challenge were

compared between fully inbred clones (IC) of Nile tilapia (produced by gynogenesis and sex reversal) and crosses between these lines (outbred clones). The results showed a positive correlation between the level of infection and the non-specific immune parameters measured. Cumulative mortality also showed that the cross between resistant IC and IC susceptible to A. hydrophila resulted in progeny with intermediate levels of resistance to those of their parents.

8.5.2. Pseudomonas

Pseudomonas are Gram-negative bacteria that have

been reported to infect cichlid fishes in different geographical regions. Pseudomonas fluorescens were found to cause chronic mortality in farmed Nile tilapia in Japan (Miyashita, 1984). Infected fish were characterized by fine white nodules in the spleen and abscesses in the swim bladder. The infection occurred mainly in winter and spring, with peak mortality at low water temperatures (15–20°C). Miyazaki et al. (1984) also found that pond-farmed Nile tilapia infected with Pseudomonas in Japan suffered from exophthalmia, dark body coloration, nodular lesions, focal necrosis in the liver, spleen, kidney and gills, inflamed swim blad- der, abscesses in the eyes, spleen and swim bladder and granuloma formation. Pseudomonas spp. have also been recorded in red tilapia, blue tilapia and their hybrids in brackish-water aquaculture in the Philippines, causing dermal lesions and increased mortality in infected fish (Aban et al., 1999).

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8.5.3. Vibriosis

Vibriosis, a bacterial disease caused by the Gram-negative bacteria Vibrio, can affect fresh- water and saltwater fish. This bacterium could constitute a serious health hazard for cultured tilapia. Several incidences of Vibrio infections have been reported in tilapia, with varying levels of damage. The susceptibility of tilapia to vibriosis depends on tilapia species, bacteria spe- cies and strains, environmental conditions and culture systems. Sakata and Hattori (1988) iso- lated and characterized three strains of Vibrio

vulnificus from infected, pond-cultured Nile tilapia

in Kagoshima, Japan. Different Vibrio species, including V. vulnificus, V. harveyi and V. mimicus, have also been isolated from diseased red tilapia, blue tilapia and their hybrids in brackish-water floating cages in the Philippines (Aban et al., 1999). Infected fishes showed body lesions and high mortality. This secondary infection was attributed to the great fluctuation of water sali- nity (18–35‰) at cage sites. In Saudi Arabia, it was reported that the outbreak of vibriosis among tilapia in fish farms in the eastern region was mainly due to stress and external parasite infec- tion (FRRC, 2001). Disease symptoms included exophthalmia, scale loss, haemorrhage and increased mucus secretion. In a similar case, Saeed (1993) reported an outbreak of Vibrio sp. in

Oreochromis spilurus reared in seawater tanks in

Kuwait. Infected fish suffered from lethargy, dark coloration, dermal necrosis and mortality. Terra- mycin, added to the water at a concentration of 45 mg/l for 7 days, successfully controlled the disease and improved fish survival.

The susceptibility of Nile tilapia to a

V. vulnificus strain with a high degree of virulence

for eels has been investigated (Fouz et al., 2002). Infected fish developed a septicaemia similar to eel vibriosis. This result suggested that when tilapia are co-cultured with eels they should be vacci- nated against V. vulnificus (biotype 2) to prevent the occurrence of the disease and improve fish survival under adverse environmental conditions. In another study, the non-specific immunity of red and black strains of Nile tilapia was compared following challenge with the bacterium Vibrio

parahaemolyticus (Balfry et al., 1997). The results

revealed a significant effect of tilapia strain on immune response (e.g. serum lysozyme and phagocytic activities). Phagocytic activity increased

while lymphocyte numbers decreased following the bacterial challenge.

8.5.4. Streptococcosis

Streptococcosis is a disease caused by the Gram-positive, non-motile bacteria Streptococcus spp. This disease is a major cause of damage to the global aquaculture industry, with an economic loss exceeding US$150 million annually (Perera et al., 1994, 1997; Shoemaker and Klesius, 1997). These bacteria are opportunistic pathogens that are widely spread in aquaculture environments because of their dependence on stress to assert patho- genicity (Bunch and Bejerano, 1997). They have been isolated from pond water, bottom mud, organic manure used for pond fertilization and contaminated fish (Bunch and Bejerano, 1997).

Streptococcus gains access to the fish body by inges- tion, injured skin and experimental injection (Evans et al., 2000; McNulty et al., 2003).

One other major concern about Streptococcus is that it can be transmitted from fish to humans.

Streptococcus iniae has been reported to infect people

who have handled fresh, whole fish (mostly tilapia) from fish farms in Toronto, Canada (Getchell, 1998). The patients, who were of Asian origin, cleaned and ate the fish, and some of them had injured their hands during fish cleaning, when the bacteria may have entered their bodies through the wounds. This particular accident indicates that tilapia infected with Streptococcus should be care- fully handled and treated.

The infection of tilapia by Streptococcus sp. has been widely reported, but S. iniae is among the most serious pathogens affecting the tilapia culture indus- try. Stressful culture conditions, including low or high water temperature, high salinity and alkalinity (pH> 8), low dissolved oxygen, high nitrite concen- tration and high stocking density, increase the sus- ceptibility of tilapia to streptococcal infection (Chang and Plumb, 1996a, b; Bunch and Bejerano, 1997; Perera et al., 1997; Shoemaker et al., 2000).

Streptococcal outbreaks have been recorded in farmed tilapia worldwide. Tung et al. (1987) reported epizootics of streptococcal infections in cage-cultured Mozambique tilapia in Taiwan, which resulted in heavy mortalities, reaching 50–60% within 1 month. The major pathological signs of the disease included haemorrhage, exophthalmia, with corneal opacity, and dark

body coloration, with nodular or abscess forma- tion on the trunk and/or peduncle muscles (Figs 8.4 and 8.5). Similar disease symptoms and mass mortality were found in the same species infected with S. iniae in brackish-water ponds in India (Mukhi et al., 2001). When healthy fish were injected with the pathogen (105–8cells/ml), 10% mortality was observed within 7 days of infection. Incidences of infection caused by Streptococcus sp. and Enterococcus sp. have also been associated with endemic mortality of tilapia hybrids in commercial freshwater farms in Colombia (Pulido et al., 1999). In addition, Berridge et al. (1998) reported periodi- cal outbreaks of streptococcosis caused by S. iniae and Streptococcus difficile in hybrid tilapia (O. aureus×

O. niloticus) in Texas, USA. The authors observed

that the rapid and marked mortality of market fish were associated with transferring fish from production units to market tanks.

McNulty et al. (2003) studied the haematologi- cal changes in Nile tilapia infected with S. iniae by

naris inoculation. Infected fish had increased pig- mentation, eye opacity, erratic swimming and leth- argy. The authors suggested that two stressors may have resulted in the infection: the loss of the oxygen carrying capacity and an increase in iron levels.

Several drugs have been tested for the treat- ment of Streptococcus. Antibiotic treatment is gener- ally ineffective, and the need for a proper vaccine has become a must (Klesius et al., 2000). However, Darwish and Griffin (2002) found that oxytetracy- cline was effective in controlling S. iniae in blue tilapia (O. aureus). Oxytetracycline was incorpo- rated into the feed at 0, 25, 50, 75 and 100 mg/kg body weight. The 75 and 100 mg dose signifi- cantly increased the survival of the infected fish from 7% in the infected non-medicated to 85 and 98%, respectively.

In addition, recent studies have shown the suc- cess of passive immunization in Nile tilapia by vac- cination with anti-S. iniae whole sera (Klesius et al., 2000, 2001; Shelby et al., 2002a). Immunized fish developed significant antibacterial responses and a sharp reduction in abnormal behaviour and mor- phology. Furthermore, Shelby et al. (2002b) found that Nile tilapia intraperitoneally injected with anti-Streptococcus whole sera developed a secondary antibody response and immunity to S. iniae, with 100% survival after challenge with S. iniae.

8.5.5. Staphylococcosis

Staphylococcus epidermidis is another bacterium that

has been reported to infect tilapia. Huang et al. (1999) described the epizootiology and pathoge- nicity of S. epidermidis in cultured tilapia (Oreochromis spp.) in Taiwan. Diseased fish showed spleno- megaly with diffusion of several white nodules and lesions in the spleen and anterior kidney. When blue tilapia (O. aureus) were challenged with viable

152 Chapter 8

Fig. 8.4. Severe haemorrhage and fin erosion in Nile tilapia caused by streptococcal infection (photo provided by M.K. Soliman).

Fig. 8.5. Exophthalmia (pop-eye) in Nile tilapia caused by streptococcal infection (photo provided by M.K. Soliman).

Stress and Diseases 153

S. epidermidis and its supernatant, apoptosis was

predominantly detected in the lymphocytes and macrophages in the spleen and kidney, and occa- sionally in the brain, liver, gonads, mesentery, stomach, intestine and skeletal muscles (Huang

et al., 2000). This particular study indicated that

the pathogenicity of S. epidermidis for tilapia is due to the toxicity of the bacterial product, which induces the apoptosis.

8.5.6. Mycobacteriosis

Mycobacteriosis or ‘fish tuberculosis’ is a chronic bacterial disease caused by Mycobacterium spp. The disease can infect a wide range of freshwater and marine fish. The infection of wild and cultured tilapia by Mycobacterium is well documented. Three pathogenic agents, M. marinum, M. fortuitum and

M. chelonae, have been reported to cause the dis- ease (Chen et al., 1998). Mycobacterium fortuitum has been reported in the tilapias Sarotherodon andersonii and Tilapia sparrmanii from the Okavangu swamp in Botswana (Roberts and Matthiessen, 1979). The infection of farmed Nile tilapia in intensive culture in Kenya with M. fortuitum (Roberts and Sommerville, 1982) and the infection of tilapia hybrids (O. niloticus × O. mossambicus × O. aureus) with M. marinum (Wolf and Smith, 1999) was also reported. Affected fish showed focal granulomata in the liver, spleen, kidney and viscera, a high proportion of epithelial macrophages and more peripheral lymphocytes.

The effects of the extracellular products (ECP) of Mycobacterium spp. on the non-specific

immune response of Nile tilapia have been exam- ined by Chen et al. (1998). The fish were immu- nized by injecting their swim bladders with ECP, Freund’s complete adjuvant (FCA) and Freund’s incomplete adjuvant (FIA). The results revealed that ECP from Mycobacterium spp. and the adju- vants used in the study provided a good stimula- tion of the non-specific immune response in Nile tilapia.

8.5.7. Edwardsiellosis

Edwardsiellosis is a bacterial disease caused by

Edwardsiella tarda, which are short-rod, Gram-

negative bacteria belonging to the family Entero- bacteriaceae. Edwardsiella tarda have been isolated from reptiles, fishes, amphibians and freshwater and integrated aquaculture (Muratori et al., 2000). The pathogenicity of E. tarda to fish increases under stressful conditions, especially at a high water temperature (> 30°C) and high organic con- tents (even at a low water temperature).

The infection of tilapia by E. tarda has been reported by a number of authors. Miyashita (1984) recorded chronic mortalities in Nile tilapia in some farms in Japan due to infection by E. tarda and Pseudomonas. Muratori et al. (2000) also isolated

E. tarda from the skin, gills, fins, intestines and

muscles of Nile tilapia reared in an integrated fish farm fertilized with pig manure. External surfaces (skin, gills and fins) were more affected than other organs. Pathological signs of the disease include haemorrhage, corneal opacity (Fig. 8.6) and chronic mortality.

The resistance of tilapia to E. tarda infection has also been investigated, with contradictory results. When Nile tilapia were intraperitoneally injected with a protein-bound polysaccharide preparation (PS-K), at a dose of 0.1 mg/g body weight, maximal resistance was developed in the fish 1 week after the injection (Park and Jeong, 1996). PS-K-injected fish showed increased phagocytic activity of the pronephros cells. These results suggested that PS-K activated the non- specific immune system of injected tilapia. Simi- larly, Wang and Wang (1997) evaluated the effectiveness of 11 polysaccharides in the protec- tion of blue tilapia against E. tarda. They found that four glycans (Bar, Krestin, Scleroglucan and Zymosan) improved fish resistance and survival rates. In conflict with the above results, Lio-Po and Wakabayashi (1986) found that Nile tilapia immunized with formalin-killed E. tarda through intraperitoneal injection were not effectively pro- tected from infection by E. tarda.