Characterization of various pathogens

General characteristics:

• The bacterium Vibrio anguillarum is a polarly flagellated, Gram-negative, curved rod.
  
• The edged are round and it appears individually or in chains of two. During divisions, the curvature is mostly oppositely arranged, so that a wavy structure develops in long segmentation chains.
  
• The length of young cells varies between 1.2 and 2.1µm and the width between 0.4 and 0.6µm.
  
• Motile, usually one end has a single flagellum, but there are also lophotrichous types. The length of the flagellum is two or three times the length of the bacterial cell.
  
• Form no capsules or spores.
  
• Colonies on nutrient agar are pale white or yellowish-brown. They are semi-transparent or opaque. They are round, with protrusions smooth borders and a luster.
  
• Vibrio anguillarum with type A, B and C as the etiological agent for all disease and lisses.
  
• It grows in a wide temperature range 5-35⁰c and optimum lies near 26⁰c.
  
• pH range of possible growth is 6-9.6 while growth does not take place at pH 5.
  
• Growth is possible at a salt concentration of 0-5% in nutrient broth, type C strain can also be detected in fresh water fish.
  
• Sensitivity is exhibited to streptomycin, chloramphenicol aureomycin, terramycin, tetracycline, erythromycin, neomycin novohiocin, polymyxin B, vibriost static agent 0/129.
  
• The causative agent, of this Vibriosis disease: V. anguillarum, was first described in 1909 as the aetiological agent of the ‘red pest of eels’ in the Baltic Sea.
  
• Before this report, described epizootics in migrating eels (Anguilla vulgaris) dating back to 1817 that implicated a bacterium named Bacillus anguillarum.
  
• The pathology of the disease and the characteristics of the bacterium in these two reports suggested that the etiological agents were the same.
  
• Vibriosis was not reported in North America until 1953, when V. anguillarum was isolated from chum salmon (Oncorhynchus keta).
  
• Vibrio anguillarum belongs to one of the halophilic groups of vibrios and survives at different salinities. Studies have shown that it is able to survive in sea water for more than 50 months.
  

Vibrio parahaemolyticus:

General characteristics:

When ingested, V. parahaemolyticus causes watery diarrhea often with abdominal cramping, nausea, vomiting, fever and chills. Usually these symptoms occur within 24 hours of ingestion. Illness is usually self-limited and lasts 3 days. Severe disease is rare and occurs more commonly in persons with weakened immune systems. V. parahaemolyticus can also cause an infection of the skin when an open wound is exposed to warm seawater. Most people become infected by eating raw or undercooked shellfish, particularly oysters. Less commonly, this organism can cause an infection in the skin when an open wound is exposed to warm seawater.

Edwardsiella tarda:

General Characteristics:

• Edwardsiella
  tarda is a Gram-negative bacterium associated with
  freshwater ecosystems.
  
• Rod shaped, facultatively anaerobic, motile with peritrichous flagella, size is 0.6-2µm.
  
• Growth on a nutrient agar plate is slow, with a semitransparent colony swelling up in the moist, lustrous, gray circle about 1mm in diameter in 24 hours at 25⁰c.
  
• In nutrient broth form uniform turbidity with slight white deposit, no film formation.
  
• Temperature range of possible growth is 15-42⁰c, the optimum temperature is 30.8⁰c (Hoshina, 1962).
  
• pH range is 5.5-9.0, no survival at pH 4.5 or 9.5.
  
• salt concentration range is 0-4%, but some strains do not grow at 4.5%.
  
• Known to colonize a wide variety of
  amphibians, reptiles, and fish,
  
• E tarda
  can also cause disease
  in these animals, as exemplified by emphysematous putrefactive
  disease of catfish.
  

Edwardsiellosis:  
This disease is also known as Emphysematous Putrefactive Disease (EPD) and is caused by Edwardsiella tarda. This disease is much less common than ESC and is more of a problem in older channel catfish, but fingerlings are still susceptible. Clinically, lesions are initially observed as 3 to 5 mm red cutaneous foci on the flanks and caudal peduncle. They are caused from fistulas originating deep in the skeletal muscle. There is petechiation and malodorous liquefactive necrosis of the viscera with fibrinous peritonitis. Catfish affected with this disease will continue to eat even if they are severely affected. There may be posterior paresis in the later stages of the disease. Definitive diagnosis is based on identification of the bacterium within the lesions and the attendent clinical findings. A fluorescent antibody test is available for identification of the bacterial agent, using kidney as the target tissue.

labeled

Renibacterium salmoninarum:

General Characteristics:

• Gram negative bacilli which lack flagella (non-motile).
  

• Very small (0.03-0.05×0.5^-1.0µm in size).
  

• Spores do not form. The bacteria are non-acid fast. Moreover, they are catalase positive with protein dissolution.
  

• 1-cystine is required as a nutrient.
  

• Asporogenic, without capsule formation.
  

• Tends to form diploforms; chains can appear in old laboratory cultures and metachromatic granules are often observed (Smith, 1964).
  

• Grows very slowly at 5⁰c, but well at 15⁰c.
  

• It does not grow on the usual laboratory culture media, since 1-cystine HCl is necessary for its growth.
  

Disease:

Renibacterium salmoninarum causes kidney disease in salmonids. The disease develops gradually and can take a long time to build up in the herd with slowly growing mortalities. The disease is quite latent and is seldom diagnosed in fish younger than 6-12 month old fish. The disease has been detected in fish in freshwater as well as saline waters and in all age groups. The different species of salmonids have different level of susceptibility. The Arctic charr in Iceland is quite tolerant compared to salmon but so is the Icelandic salmon compared with Norwegian salmon. Internal changes due to this bacterium have been found in Arctic charr in Iceland without any obvious external symptoms.

Mycobacterium marinum:

General Characteristics:

• Gram-positive bacilli, size varies with the culture conditions even in the same species, but generally it is 0.3-0.7µm wide and 1-4µm long. Many strains are stated to attain lengths up to 12µm and more.
  
• Growth often does not take place at 37⁰c. Optimum temperature for growth is 18-25⁰c, but some species can tolerate up to 40⁰c. This bacterium does not grow at normal body temperature. That is why it remains localized to the cooler skin surface.
  
• Aerobic and non-motile.
  
• Acid fast, difficult to stain by conventional pigments.
  
• Mycobacterium marinum
  (M. marinum) is a slowly growing bacterium that may cause disease in fish and people.
  
• The bacterium is normally found in bodies of fresh or salt water in many parts of the world.
  
• Skin infection with Mycobacterium marinum is relatively rare and is usually acquired from swimming pools, aquariums, or fish-handling.
  

History:

Flexibacter columnarjs:

General Characteristics:

• Long, gram-negative bacilli.
  
• Usually 4-8×0.5^-0.7µm in size.
  
• Uniform in size and shape, but deformed bacteria. For example, extremely long curved or looped bacteria appear with the elapse of time.
  
• Moreover, involuted forms and spherical or amorphous particles of irregular size also appear.
  
• These differ from microcystis, and do not germinate even if transmitted to new media.
  
• The bacteria have specific motility. One tip of the bacteria attaches to the substratum, and the other end moves as through waving. Gliding motion is also evident.
  
• They are flexible and creep or glide in nutrient substrates.
  
• The movement can be easily seen when samples of diseased tissue in which bacteria have grown are placed on glass slides and observed microscopically.
  
• Temperature range is 5-30⁰c, but warm water strains may develop even at 35-37⁰c.
  
• Growth occurs well in salt concentration of 0.5%, but does not occur at 2%.
  
• pH range, in which growth is possible, is 6.5-8.5 and the optimum pH is 7.5.
  
• does not form micocysts.
  

Disease:

‘Columnaris’ disease, caused by the bacterial pathogen ‘Flexibacter columnarjs’ (Leadbetter 1974), is adisease of many freshwater fishes and has a worldwidedistribution. This bacterium has not been listed in the’ Approved Lists of Bacterial Names’ (Skerman et al.1980, Moore et al. 1985), nor has it been validated in

the International Journal of Systematic Bacteriology. Thus, its name must be cited in quotation marks. ‘Columnaris’ disease has been described in many freshwater fishes (Bullock et al. 1971, MC Carthy 1975), and can be of economical importance in intensive fish farming (Amend 1982). Low virulence bacterial strains become pathogenic for salmonids at water temperatures exceeding 20°C, while high virulence strains may be pathogenic at temperatures above 15°C. Mortality rates range from ca 10 to 100 % depending on water temperature (Holt et al. 1975). The clinical signs

and lesions are usually restricted to the body surface and take the form of s l n erosions and gill necrosis. Characteristic ‘saddleback’ lesions can be found in advanced cases in catfish. A systemic infection may occur in severe cases, depending on the virulence of the strains (Snieszko & Bullock 1976, Amend 1982).

Columnaris, is a common bacterial disease that affects the skin or gills of freshwater fish and is caused most commonly by Flexibacter columnaris. This disease is also known by a wide variety of synonyms including the following: mxyobacterial disease, peduncle disease, saddleback, fin rot, cotton wool disease, and black patch necrosis. This bacteria is usually pathogenic at temperatures greater than 59 ^oF. Both mortality and acuteness of the disease will increase at higher water temperatures. Virulence mechanisms associated with this disease are not well understood, however, the mineral content of the water is thought to be important, since this bacteria has been shown to be less pathogenic in soft water as compared to hard water. Other risk factors include physical injury, low dissolved oxygen, organic pollution and high nitrite levels. This is primarily an epithelial disease, i.e., it causes erosions and necrosis of the skin and gills which may become systemic. It often presents as whitish plaques that may have a red peripheral zone on the head or back (hence the name saddleback) and/or the fins (hence, fin rot) and especially the caudal fin (hence, peduncle disease). Fragments of the fin rays may remain after the epithelium has sloughed, leaving a ragged appearance. Lesions rapidly progress to ulcers, which may be yellow or orange due to masses of pigmented bacteria. Ulcerations spread by radial expansion and may penetrate into deeper tissues, producing a septicemia. Gill infections are less common but more serious. Columnaris begins at the tips of the lamellae and causes a progressive necrosis that may extend to the base of the gill arch. Definitive diagnosis is dependent upon the isolation of the bacterial agent in the presence of attending clinical lesions. It should be noted that a presumptive identification of Flexibacter columnaris can be made by examination of wet mounts and observation of long thin bacterial rods which a characteristic flexing or gliding motion. In addition to Flexibacter columnaris, other bacterial agents which have been implicated in this disease include: Flexibacter psychrophilia as well as Cytophaga and Flavobacterium branchiophila.

Cytophaga psychrophila:

General Characteristics:

• C. psychrophila as a gram-negative, flexible rod which exhibited creeping motility.
  
• Size is 0.75×1.5^-7.5µm.
  
• The organism produced neither fruiting bodies nor microcysts, was strictly aerobic, and grew only at low temperatures. Temperature range is 4-23⁰c with no growth occurring at 30⁰c. The optimum temperature for development is stated to be 20⁰c.
  
• Salt tolerance is 0-0.8%. Growth is possible even at 1%, depending on bacterial strain, but not at 2%.
  
• Chlortetracycline, bacitracin, chloramphenicol, erythromycin, neomycin, penicillin, tetracycline and dihydrostreptomycin impair its growth ( Pacha, 1968;Pacha and Ordal, 19670)
  
• It exhibits proteolytic properties on Cytophaga agar or in liquid culture medium and hydrolyzes gelatin, casein, albumin and tributyrin as well as autoclaved cells of Escherichia coli.
  
• The cultures grow well on casein hydrolysate culture medium but on citrate medium.
  

Disease:

Cytophaga psychrophila (Order Cytophagales), the causative agent of cold-water disease, is a wide-spread pathogen of cultured salmonid fishes in North America

(Pacha & Ordal 1970, Schachte 1983). Epizootics typically occur at between 4 and 15 “C in underyearling fish (Conrad & DeCew 1967), but yearling coho Oncorhynchus kisutch and chinook salmon 0. tshawytscha may also be affected (Holt 1972, Wood 1974). The bacteria often infected the external surfaces, resulting in ulceration of the dermis and necrosis of the underlying muscle (Wolke 1975). The disease may also progress to septicemia with necrosis observed in internal organs (Wolke 19751, and bacteria can be isolated from these infected tissues (Wood 1974). Though Wood & Yasutake (1956) reported Little inflammatory response in the visceral organs, Borg (1960) reported a mild mononuclear infiltration associated with the disease. A chronic form of the infection resulting in lordosis and scoliosis was observed in juvenile coho salmon (Conrad & DeCew 1967, Holt 1972), and Wood (1974) surmised that these lesions were caused by bacterial destruction of muscle bundles adjacent to vertebrae which resulted in unequal tension on the spinal column. Spiral swimming behavior has been observed in fish from hatcheries where cold-water disease is enzootic (Yasutake 1965), and we have observed this syndrome at several hatcheries in Oregon and Washington, USA. Presented here are histological, bacteriological, and epizootiological data that indicate that this ataxic swimming behavior was due to chronic inflammation in the cranial cavity and anterior vertebrae associated with C. psycl~rophila infections.

Yersinia ruckeri:

General Characteristics: