Gram positive cocco-bacteria (Micrococcaceae, Streptococcaceae)
causing systemic disease to intensively farmed marine fish in the Mediterranean.
A brief review.

Authored by Dr. PANOS VARVARIGOS
Freelance Veterinarian – Fish Pathologist
Athens, Greece.

 

 

    CONTENTS
Aspects of bacteriology
Epizootiology
Pathogenesis and diagnosis
Prevention and treatment
Economic implications
Regulations
Reference list


Staphylococcus sp.


Copyright © Dr. Panos Varvarigos.


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Aspects of bacteriology


Family: Micrococcaceae

Staphylococci are gram-positive cocci (about 0.5 to 1.5μm in diameter) occurring singly, in pairs, in tetrads, short chains of three or four cells, or most often in irregular "grape-like" clusters. Their name is derived from their latter frequent formation stemming from the Greek "staphylos" meaning grape.

They are nonmotile, nonsporeforming and usually catalase positive and unencapsulated. Although most species are facultative anaerobes, growth is more rapid and abundant under aerobic conditions. Most strains grow well on common noninhibitory media.

The cell wall contains peptidoglycan and teichoic acid. The diamino acid present in the peptidoglycan is L-lysine. Staphylococci are susceptible to lysis by the endopeptidase lysostaphin. They are generally resistant to lysis by the muramidase lysozyme.

Staphylococci are currently included together with micrococci and planococci in the family Micrococcaceae as a matter of convenience, since these genera are not closely related phylogenetically and should not be combined in one family. All three genera represented in the family Micrococcaceae are catalase-positive, gram-positive cocci with cell wall peptidoglycan containing L-lysine as the diamino acid.

Micrococci are generally resistant to lysostaphin. Among others, staphylococci and micrococci may be differentiated on the basis of anaerobic acid production from glucose, which is positive for most Staphylococcus species and negative for most Micrococcus species.

Colonies of most staphylococci, which have been isolated from fish on non-selective media, such as tryptic soy agar (TSA), are circular, smooth and raised with a butyrous consistency and a bright white, sometimes glistening, or pale white colour. Their diameter may be 0,5-2mm subsequent to 36-48 hours of incubation at room temperature of around 25°C. Such colonies may become sticky with age. Micrococci produce on TSA yellow colonies of similar shape.

Staphylococcus species are not possible to be distinguished on the basis of colony morphology. Colonies conforming to the description above should be Gram stained, sub-cultured and tested for genus and species biochemical properties (e.g. by means of the API STAPH commercial identification system). In human and veterinary medicine a few additional key characteristics must be examined for the identification of the most clinically significant species. Coagulase production is significant among these, that is, the ability to clot blood plasma. A rapid slide coagulase test may be performed. Most Staphylococcus species isolated from fish are coagulase negative.

Family: Streptococcaceae

The genera Streptococcus, Aerococcus, Leuconostoc, Pediococcus and Gemella comprise the members of this family. Nevertheless, the taxonomic status of the latter two genera is uncertain. They are gram-positive, cytochrome-negative coccoidal bacteria that usually grow in chains of various lengths and sometimes form tetrads. Under certain growth conditions these bacteria are elongated and appear rod-like when Gram stained. Their name is derived from their frequent chain formation stemming from the Greek "streptos" meaning twisted chain.

The most definitive difference between the families Streptococcaceae and Micrococcaceae is that catalase is most often absent from the cultures of the former and present in the cultures of the latter.

Streptococci and aerococci are mostly important in human and veterinary medicine. Streptococci are oxidase negative and catalase negative. Some streptococcal species contain specific polysaccharide antigens and are classified into distinct groups (Lancefield groups) according to the presence of these group-specific antigens (groups: A, B, C, D, E, F, G, H, K, L, M, N, O).

The group D streptococci are normal inhabitants of the human gastrointestinal tract, but may spread to cause wound infections and bacteremia. The medically important group D streptococci are divided into the enterococcal species (S. faecalis, S. faecium, S. durans, S. avium) and the non-enterococcal species. (S. bovis, S. equinus). The group D streptococci produce colonies that resemble those of staphylococci.

Complementary to the above grouping and a most useful characteristic for the identification of streptococci, is their haemolytic action on red blood cells (RBC's). Four distinct types of haemolytic action are described and characterise streptococcal colonies grown on the surface of blood agar:

Alpha-haemolysis: An indistint zone of partial destruction of rbc's appears around the colonies, often with a greenish to brownish discoloration of the medium.
Beta-haemolysis: A clear, colourless zone appears around the colonies, where the rbc's have been completely discoloured.
No-haemolysis: No apparent haemolytic activity or discolouration of rbc's is notable around the colonies.
Wide-zone alpha-haemolysis: A small halo of intact or partially lysed rbc's lies adjacent to the colonies, but a wider zone of complete haemolysis extends farther out into the medium. (Macroscopically may be confused with beta-haemolysis).

For the presumptive identification of the major categories of streptococci and aerococci, once isolated in pure culture, the catalase-negative, gram positive coccus is tested for haemolysis. In addition, the results of the tests that follow may be used:

Salt tolerance (6.5% NaCl) test: Visible growth within 24 hours after inoculation onto 6.5% NaCl agar indicates that the strain is salt tolerant, i.e., positive and it can be identified as an enterococcus.

Bile-esculin (BE) hydrolysis test: When an agar plate is incubated for 24-48 hours at 35-37°C subsequent to inoculation by the BE medium, which has been inoculated in turn with 3 colonies, a positive test produces blackening of the medium. Otherwise the test is negative. All group D streptococci (enterococci as well as non-enterococci) are BE positive.

Susceptibility to SXT test: Most group A and B streptococci are resistant to SXT when cultures are incubated in a normal atmosphere. (SXT disks containing 1.25 mg of trimethoprim and 23.75 mg of sulfamethoxazole.)

Presumptive identification of streptococci and aerococci

Category

Cellular arrangement

Haemolysis

Group antigen

SXT resistance

BE test

Salt tolerance

Group A strept.

chains

beta

A

yes

--

--

Group B strept.

chains

beta

B

yes

--

+

"Groupable" beta-haemolytic streptococci, not group A, B, or D

chains

beta

Not A, B, or D

no

--

--

Group D, enterococci

short chains, diplo

alpha, beta, none

D

yes

+

+

Group D, not enterococci

short chains, diplo

alpha, none

D

no

+

--

Viridans streptococci

chains

alpha, none

none

no

--

--

Pneumonococci

diplo, short chains

alpha

none

?

--

--

Aerococci

single cells, tetrads

alpha, none

none

?

variable

+

 

Since the first publication of fish streptococcicosis/streptococcosis in 1958, there has been confusion about the number and nature of the bacterial species involved (Austin and Austin, 1999). Serology has indicated that the most common fish pathogenic streptococci are representatives of enterococci. Generally, serological techniques have not improved the taxonomic status of the strains isolated form fish world-wide. Many characteristics are shared by the majority of fish pathogens and there is variance in the overall descriptions reported by different groups of workers. For example, traits of streptococci that have supposedly taxonomic significance, such as the ability to attack blood, have been reported to vary. Fish pathogenic strains of streptococci have been described, at one time or another, as either alpha or beta haemolytic or as non haemolytic (Austin and Austin, 1999).

Overall, the isolates recovered from fish may be clustered in the following taxa (Austin and Austin, 1999):

Enterococcus (Streptococcus) faecalis subsp. liquefaciens
Produces yellow colonies of 1-2mm in diameter on TSA (despite the fact that streptococci normally produce white colonies on TSA). Gram positive cocci, which do not produce catalase, H2S or indole and are VP negative (Voges Proskauer reaction).

Lactococcus garvieae (=Enterococcus seriolicida)
Alpha haemolytic, Gram positive cocci in short chains, which do not produce catalase, H2S, indole or oxidase. The VP reaction is positive. The isolates are salt tolerant and do not correspond to any of the serologically defined Lancefield groups of streptococci.

Lactococcus piscium
Represents a group of fish pathogenic lactococci/Group N streptococci. They comprise Gram positive, non-motile, short (ovoid) rods, which do not produce catalase or H2S. They grow at 5-30°C.

Streptococcus difficilis (=Str. agalactiae)
Causes meningo-encephalitis in cultured fish. Fairly unreactive and non-haemolytic, Gram positive cocci of varying diameters in small chains, which do not produce catalase and are not salt tolerant. The VP reaction is positive. Str. difficilis is similar to other group B, type Ib streptococci in terms of biochemistry.

Streptococcus iniae (=Str. shiloi)
This streptococcus was first isolated from an Amazon fresh water dolphin, Inia geoffrensis and was later described as a cause of mortality in fish. After incubation for 24h at 30°C, colonies on BHIA are 1mm in diameter and non-pigmented. They comprise Gram positive cocci in pairs and chains (pleomorphism has been observed), which do not produce catalase and are not salt tolerant. The VP reaction is negative and α-heamolysis is produced on bovine blood.

Streptococcus milleri
This streptococcus has been obtained from kidney samples of ulcerated koi carp. They comprise Gram positive cocci in chains, which are catalase and oxidase negative and are not salt tolerant. Weak β-heamolysis is produced on horse blood.

Streptococcus parauberis (=Str. uberis genotype II)
This streptococcus has been isolated from diseased farmed turbot in Spain. After incubation for 24h, whitish colonies of 1.5-2mm in diameter are produced, which are slightly α-haemolytic. They comprise non-motile Gram positive short rods/cocco-bacilli in pairs or short chains, which are catalase negative and are not salt tolerant.

Vagococcus salmoninarum
It has been isolated from diseased adult rainbow trout in Oregon, USA. Cultures comprise Gram positive short oval rods, which are catalase negative, H2S positive and are α-haemolytic. The VP reaction is negative.

 

        

Epizootiology (Fish susceptibility and transmission)


Streptococcosis

Reported epizootics of streptococcus septicemia of fish worldwide involve either group B streptococci or group D streptococci/enterococci. The disease caused by bacteria of the genus Streptococcus and the genus Enterococcus is termed streptococcosis or streptococcicosis and was first described among rainbow trout in Japan in 1958. Since then the disease has gained importance affecting a multitude of economically important farmed fish species.

Streptococcus sp. are causing infections in rainbow trout (Onchorynchus mykiss) (Munday et al., 1993). Lactococcus garvieae (=Enterococcus seriolicida) is infecting yellowtail (Seriola quinqueradiata) in Japan with serious economic implications (Kusuda et al., 1991) and Streptococcus iniae causes losses particularly in cultured ayu (Plecoglossus altivelis) also in Japan (Pier and Madin, 1976). Japanese eels, tilapia and coho salmon are also prone to infection. Streptococcosis has spread among rainbow trout farms in Australia, Israel, Italy and South Africa, but also in catfish, silver trout, sea trout, stingray, striped bass and striped mullet in the USA as well as Atlantic salmon farmed in Chile.

Infections caused by Lactococcus garvieae (Lactococcosis) to salmonid fish in Europe are becoming increasingly severe, particularly during the summer months and in those European countries that experience warm weather. Lactococcosis occurs in both fresh and seawater. The disease occurs also in Northern European countries, such as the UK and Norway.

There is evidence that potentially pathogenic streptococci are found throughout the year in the aquatic environment, that is, in the water, sediment and in the vicinity of fish net pens. Their abundance in seawater increases during the warmer seasons of the year. However, since the taxonomic status of fish pathogenic streptococci is unclear, it cannot be proved that the strains isolated from the farming environment are those causing fish disease; nonetheless, their presence is an indicator of a non-sanitary environment.

Besides the potential role of environmental contamination to fish health, it has been found that fish pathogenic streptococci may be present in fresh and frozen fish used for yellowtail diets. Hence, contaminated diets or raw materials may introduce the pathogens to fish populations and the environment. Food-borne infections may be the most important source for the spread of streptococcosis among cultured fish.

Further, it has been proved experimentally, that fish to fish transmission of the disease can be facilitated through contact with infected fish. Streptococcosis has been transmitted by means of cohabitation experiments (cohabiting an infected fish with healthy specimens of the same species). Fish which survive the disease may act as carriers. Wild fish may also carry the disease asymptomatically.

Some host specificity to streptococci exists, since different fish species show different sensitivity to infection (intensity of symptoms, extent of internal organ damage, mortality) by various bacterial strains.

Staphylococcosis

Staphylococcus epidermidis has been isolated from marine fish in Japan causing disease in red sea bream (Chrysophrys major) and yellowtail (Seriola quinqueradiata) showing exophthalmos and skin ulcers (Kusuda and Sugiyama, 1981). The bacteria were found to have pronounced antigenic differences to human related strains; hence the infections by Sta. epidermidis are considered to originate from the aquatic environment. In fresh water fish Staphylococcus aureus has been reported from silver carp (Hypophthalmichthys nobilis) in India causing corneal damage progressing to phthisis bulbi (Shah and Tyagi, 1986).

In Greece, sporadic disease with distinct pathogenicity is observed in fish affecting most often young sea bream (Sparus auratus) and sea bass (Dicentrarchus labrax). The disease is pronounced at the nursery stage, or soon after transportation to the on-growing sites. It has been investigated in a number of fish farms located in distinct regions and the causative agents have been identified to be Gram positive cocci of the genera Staphylococcus and Micrococcus. Although the relatively younger fish seem mostly susceptible to the disease, larger fish, even close to harvest size (>250g) have been found infected by staphylococci.

In particular, during the spring of 1997 severe outbreaks in both sea bream and sea bass occurred in several fish farms scattered virtually throughout all fish farming regions of Greece with daily mortality up to 2%.

Since 1997 several species of staphylococci have been isolated either from sea bass or sea bream grown at different farming locations in Greece. The most frequent were Staphylococcus epidermidis, S. xylosus, S. lentus, S. capitis, S. lugdunensis, S. hominis, S. warneri, S. cohnii, S. chromogenes and S. aureus. Subsequent to Gram staining, identification of them was based on the API STAPH (Biomerieux) system.

Mainly young fish of 0.5g to 30g suffer. Due to the appearance of the external skin and fin lesions, staphylococcosis is often confused with myxobacteriosis. Myxobacteria (Flexibacter sp.) commonly infect the surface wounds of fish subsequent to handling (Noga, 1996, Pepin and Emery, 1993). However, the condition does not recede after medicated bath treatments, which comprise a common measure against myxobacteriosis, thus, alarming the farmers.

Fish populations weakened by staphylococcosis soon become infected by other common warm water, Gram negative bacterial fish pathogens, such as Vibrio spp. causing vibriosis and/or Photobacterium damsela subsp. piscicida causing pasteurellosis in both bass and bream (Post, 1987). Fish resistance against parasitic infestations is compromised as well. In such cases, symptoms and lesions are confused and antibiotic treatments may improve health but do not offer a long-term cure. Reduction in mortality is short lived and disease returns in a matter of few days. In sea bream fingerlings infections by staphylococci often coincide with lymphocystis, hence the diagnosis of staphylococcosis is obscured.

Although staphylococci may be present in the environment throughout the year, the disease is exacerbated by the rise in seawater temperatures. It appears in the spring and is spread causing severe problems during the summer. Once established in a fish population, staphylococcosis is contagious and spreads easily among the fish of similar species or across the species that are grown on the same farm, that is, from sea bass to sea bream and vice versa. Other species on Greek farms that have been found infected include sharp snout sea bream (Puntazzo puntazzo) and dentex (Dentex dentex). Wild fish, mainly grey mullets (Mugil cephalus), that are usually scavenging around the cages, are apparently infected and have been frequently observed moribund showing similar symptoms.

As with the streptococcus infections world-wide, there is evidence also in Greece which points strongly towards the potential role that contaminated fish feeds may have regarding the spread of staphylococcus infections among the cultured fish populations. Preliminary microbiological studies of commercial fish feeds have shown high levels of contamination of these products by potentially pathogenic staphylococci.

 

        

Pathogenesis and diagnosis


The staphylococcal infections, which have been diagnosed so far on cultured marine fish in Greece, comprise infectious systemic diseases characterised by septicaemia. Similar symptoms and lesions prevail on all fish species infected.

Despite the increasing daily mortality and the number of dark coloured, lethargic, moribund fish swimming erratically close to the surface and the corners of the net cage, the infected population as a whole does not seem to loose its appetite and behaves rather normally. Apart from the dark colouration and ataxia, the sick fish show rather large external skin lesions covered by whitish mucus. By removing the mucus surface necrosis in the form of shallow skin ulcers are uncovered. The lips, mouth and lower jaws as well as the pectoral and the thoracic fins are inflamed and necrotic (mouth rot, fin rot). Exophthalmia, progressing inflammation and necrosis of the eye are not uncommon. In large sea bass, a rather common symptom is the inflamed and protruding anus and the inflamed and necrotic caudal fin.

Sea bass fry with deep ulcer uncovered when the mucous layer is removed

Sea bass fry showing skin inflammation (reddening)

Sea bream fry with inflamed and necrotic lips and mouth tissues

Sea bream with ulcerated skin

Deep ulcers and fin rot in infected sharp snout sea bream fry

 

Necropsy reveals that the stomach is invariably empty and the gut is distended to varying degrees containing a watery fluid. Often the spleen is enlarged and most often the gall bladder is distended and full of light yellow or dark green bile, especially in sparids (breams). The gills are usually anaemic with areas of necrosis. The liver is usually pale and may show inflamed, darkened areas. Congestion and haemorrhage of the brain is evident in small fry, mainly of sea bass.

Apparently, the staphylococci proliferate in the intestinal tract and cause enteritis with diarrhoea, which may spread the cocci in the water flushing and infecting other fish. Distension of the gall bladder may be a result of cholocystitis and accumulation of haemolysis products.

The staphylococci have been isolated almost in pure culture by plating material taken from the anterior kidney or the brain of the fish or from external necrotic lesions usually onto TSA agar, Blood agar and Chapman agar. Small about 1mm, white, round and regular colonies appear subsequent to incubation for 18 to 20 hours at 30-37oC.

Most of the species of staphylococci that have been isolated from fish are coagulase negative, namely, Staphylococcus epidermidis, S. xylosus, S. lentus, S. capitis, S. lugdunensis, S. hominis, S. warneri, S. cohnii, S. chromogenes. Only Staphylococcus aureus has been found to be coagulase positive.

The following table shows the biochemical attributes of five Staphylococcus species, which are often isolated.

Frequently isolated staphylococci from fish with some of their biochemical attributes:

Staphylococcus spp.

 Substrates

S. aureus

S. lentus

S. xylosus

S. epidermidis

S. capitis

Negative control

-

-

-

-

-

D-Glucose

+

+

+

+

+

D-Fructose

+

+

+

+

+

D-Mannose

+

+

+

±

+

Maltose

+

+

+

+

-

Lactose

±

+

+

+

-

D-Trehalose

+

+

+

-

-

D-Mannitol

±

+

+

-

-

Xylitol

-

+

-

-

-

D-Melibiose

-

+

-

-

-

Potassium nitrate

+

+

+

±

-

β-naphthyl-acid phosphate

+

-

+

+

+

Sodium pyruvate

±

+

+

±

+

Raffinose

-

+

+

-

-

Xylose

-

+

+

-

-

Sucrose

+

+

+

+

-

α-methyl-D-glucose

-

+

+

-

-

N-acetyl-glucosamine

+

+

+

±

-

Arginine

+

-

-

+

+

Urea

±

-

+

+

-

± variable reaction

It has been observed that the progress of the staphylococcal infections in caged fish populations is rather slow. Only few fish show ill-symptoms and die daily. Mortality persists. Stress-related acute outbursts may occur, killing increased numbers of fish in the course of a few days.

The disease spreads rapidly among the fish population as well as to neighbouring cages holding the same or other fish species. Daily mortality ranges between 0.1% and 2% and is related to the stocking density in the net cage. Usually, there is good response subsequent to the administration of the appropriate therapy. However, the condition often persists and mortality gradually reappears. Field evidence suggests that it is not easy to eradicate a staphylococcal infection when established in a fish population. Apparently, it is not possible to eliminate the pathogen from the fish or the environment. In addition, secondary infections by Gram negative bacteria (Vibrio spp., Aeromonas spp., Photobacterium spp., Tenacibaculum spp.) may require treatments with alternative antibiotics.

Diagnosis is based on the clinical signs and necropsy and is confirmed by the isolation of the bacteria on culture. The spread of a septicaemic ulcerative disease across different fish species on a farm is close to pathognomonic. Plating material from the internal organs (anterior kidney, spleen, liver) or from the brain on TSA medium results in the growth of usually small, convex, regular, white colonies after about 36 hours at room temperature (20-25°C). Stained squash preparations from these organs may reveal the presence of Gram positive cocci.

 

        

Prevention and treatment


There have been recent vaccine developments against streptococcosis/lactococcosis caused in salmonid fish by Lactococcus garvieae. Reports on vaccine developments refer also to formulations containing antigens against both Streptococcus iniae and Lactococcus garvieae, suitable for administration by immersion, injection and even oral application. However, these pathogens have not been confirmed to produce disease in the fish species that are cultured in Greece. Hence, vaccination against disease caused by Gram positive coccobacteria (staphyloccocci or micrococci) is not an option yet in Greece.

Good husbandry ensuring clean nets and overall farm hygiene as well as supply of good quality certified fish feeds, which are fresh and have been properly stored are the means for prevention.

When treatment is considered necessary, potentiated sulfonamides, and oxytetracycline are usually effective. Occasionally, erythromycin, ampicillin and amoxycillin may be good alternatives. Sensitivity testing is always required prior to deciding the preferred therapy.

 

        

Economic implications


With the exception of the Spring and Summer of year 1997, when staphylococci produced infections throughout Greece, experience suggests that Gram positive coccobacteria (staphyloccocci or micrococci) are only occasionally present as secondary pathogens, especially in weakened fish. Even in cases of young fry suffering from staphylococcus or micrococcus septicaemia, mortality is rather low and the condition improves soon after the administration of the proper therapy. Overall, there is no hard evidence that such pathologies should be considered crucial for the Greek aquaculture industry.

        

Regulations


Although there is no proof that contamination of fish destined for human consumption by Gram positive bacteria may pose any risk to consumer's health, these bacteria are indicators of sub-standard product quality. This may be due to poor environmental conditions or contaminated fish feeds used.

It would be advisable that such incidences are investigated further.

There are currently no regulations in place.

 

    

Reference list


(1)
Pier, G.B. and Madin, S.H. (1976) Streptococcus iniae sp. nov., a beta-haemolytic streptococcus isolated from an Amazon freshwater dolphin, Inia geoffrensis. International Journal of Systematic Bacteriology 26, 545-553.

(2)
Kusuda, R. and Sugiyama A. (1981) Studies on the characters of Staphylococcus epidermidis isolated from diseased fishes. Part 1. On the morphological, biological and biochemical properties. Fish Pathol. 16, 15-24.

(3)
Manual of Clinical Microbiology (1985) Fourth Edition. American Society for Microbiology, Washington, D.C., pp. 143-175.

(4)
Shah, K.L. and Tyagi, B.C. (1986) An eye disease in silver carp, Hypophthalmichthys molitrix, held in tropical ponds, associated with the bacterium Staphylococcus aureus. Aquaculture 55, 1-4.

(5)
Post, G. (1987) Textbook of Fish Health. Revised and Expanded Edition. T.F.H. Publications Inc.

(6)
Kusuda, R., Kawai, K., Salati, F., Banner, C.R. and Fryer, J.I. (1991) Enterococcus seriolicida sp. nov., a fish pathogen. International Journal of Systematic Bacteriology 41, 406-409.

(7)
Munday, B.L., Jack, D.L., Schmidtke, L. (1993) Pathogenicity of the species Streptococcus causing disease in rainbow trout (Onchorynchus mykiss). Bulletin of the European Association of Fish Pathologists 13(1), 25-27.

(8)
Pepin, J.P. and Emery, E. (1993) Marine Cytophaga-like bacteria (CLB) isolated from diseased reared sea bass (Dicentrarchus labrax) from French Mediterranean coast. Bulletin of the European Association of Fish Pathologists 13(5), 165-167.

(9)
Noga, E.J. (1996) Fish Disease: Diagnosis and Treatment. Mosby-Year Book Inc.

(10)
Gould, C. (2000) Lactococcosis - a disease waiting i the wings. Fish Farmer 23(3), 43.

(11)
News report. (2000) First appearance of Lactococcus in Britain. Fish Farmer 23(6), 5.

(12)
Padilla, D., Real, F., Rodriguez, V., Gomez, S., and Acosta, F. (2001) Histological changes in Staphylococcus xylosus experimentally infected red porgy (Pagrus pagrus). Poster presented at the 10th International Conference of the European Association of Fish Pathologists "Diseases of Fish and Shellfish", held at Trinity College, Dublin, Ireland. 9-14 September 2001.

 

        


Author: Dr. Panos Varvarigos
Freelance Veterinarian – Fish Pathologist, Athens, Greece.


AquaHealthTM
Laboratory.

Reproduction of this website (or parts of it) is illegal and strictly forbidden.
No rights can be derived from this website.

Disclaimer:

Every effort has been made to ensure that the information is accurate until the date of last editing. It is based upon the accumulated personal experience of applied veterinary work. The author cannot take responsibility for incorrect interpretation or any resulting consequences. The contents may be used as an educational guide and are definitely not meant to become a stand-alone diagnostic tool or operations manual.

 

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