PISCINE MYCOBACTERIOSIS IN GOLD FISH (CARASSIUS AURATUS) AND KOI

BACKGROUND Rhodes, 2009). Diagnosis is based on history, gross pathology, microscopic evaluation of organ smears for Piscine Mycobacteriosis is a chronic progressive acid fast rods (AFR) followed by cultural, disease affecting many species of wild and cultured fish histopathological and molecular investigations. inhabiting both fresh water and marine environments (Jacobs, 2009; Gauthier and Rhodes, 2009). It is This disease is especially a problem for ornamental fish considered as the most prevalent chronic disease in industry due to several reasons. The presence of clinical tropical and subtropical fresh water aquarium/ornamental disease directly affects their physical appearance, fish (Antonio et al., 2000; Gauthier and Rhodes, 2009) and thriftiness and the reproductive performance resulting in has been reported in native and imported ornamental fish economic losses to the ornamental fish industry. Further, stocks in many countries including Thailand, Italy, Czech the disease will slowly and silently spread among the fish Republic, Slovenia, Poland, Iran, India and Sweden. An in a farm due to the longevity of the ornamental fish and increasingly diverse array of Mycobacterium spp., has the chronic nature of the infection that transmits both been implicated in piscine mycobacteriosis (Gauthier and horizontally and vertically as well as through water, Rhodes, 2009) all belonging to Nontuberculous making it difficult to get rid of the infection once Mycobacteria (NTM) also designated as Mycobacteria established. Further, the presence of mycoabcteriosis can other than Tuberculosis (MOTT), or Atypical adversely affect the international trade of aquarium fish Mycobacteria (ATM) (Primm et al., 2004; Al-Ghafli and (Passantino et al., 2008). Moreover, many of the NTM Al-Hajoj, 2017). Among them, Mycobacterium fortuitum, associated with fish infections are reported to cause M. marinum and M. chelonae are the most frequently cutaneous nodular and ulcerative lesions in humans isolated NTM from fish (Decostere et al., 2004). (Caputo et al., 2010) posing a public health risk to those who handle aquarium fish or inadvertently get exposed to Mycobacteriosis in ornamental fish is characterized by aquarium water contaminated with mycobacteria. non specific clinical signs that include progressive emaciation, chronic non healing ulcers, pigmentary In the recent past, there have been published reports on changes, scale loss and death of fish over a period of the occurrence of mycobacteriosis in Sri Lankan months to years. Often, the infection is clinically ornamental fish. Both slow growing and fast growing inapparent and may not produce clinical signs (Gauthier Mycobacteria have been isolated from the internal organs and Rhodes, 2009; Floyd, 2013). Granulomatous of apparently healthy and clinically diseased fish collected inflammation is the classic histopathological from farms located in different districts of Sri Lanka manifestation of piscine mycobacteriosis which is grossly (Dissanayake et al., 2009; Dissanayake et al., 2017). visible as grey or white nodules in multiple internal organs Perera et al (2010) identified M. fortuitum as the (Chinabut, 2009), especially in the kidney and the spleen. commonest species isolated from guppies showing Significant variation in the size and structural clinical disease based on PCR-RFLP of the rpoB gene. organization of granulomas are seen, from highly Edirisinghe et al (2014) reported the occurrence of NTM organized lesions with concentric layers of epithelioid in aquarium water collected from different locations and cells to poorly organized inflammation (Gauthier and M. fortuitum was the most frequently isolated species. SUMMARY: Piscine mycobacteriosis is a systemic, chronic, progressive disease in fish caused by the nontuberculous mycobacteria (NTM). It has significant economic and public health impacts on commercial culture of ornamental fish. Clinical manifestation and the pathology of the infection in ornamental fish may vary depending on the species of fish affected and the species of the pathogenic NTM involved. This clinical communication describes mycobacteriosis in two different ornamental species; gold fish (Carassius auratus) and koi carp ( Cyprinus carpio). http://doi.org/10.4038/slvj.v64i1A.20

In this report, we describe the clinical manifestations, For the confirmation of the species of Mycobacteria, gross and histopathological lesions associated with total DNA was extracted from the isolate by QIAamp mycobacteriosis in a group of gold fish that had been DNA mini kit (Qiagen, Germany) and a fragment of maintained in a fiberglass tank in a fish disease diagnosis 360bp of the rpoB gene was amplified by PCR according a laboratory, and a koi carp reared in a home garden pond.
protocol described by Lee et al. (2000). After verification of PCR amplicons by gel electrophoresis (1% agarose gel CASE PRESENTATION stained with ethidium bromide), PCR product was subjected to Restriction Fragment Length Polymorphism Case 1: A group of thirty gold fish (Carassius auratus) (RFLP) analysis with the restriction enzymes Msp I and purchased from a fish breeding center had been Hae III according to protocol described by Lee et al maintained in a fiberglass tank in the Center for Aquatic (2000). Further, PCR amplicons were sequenced and the Animal Disease Diagnosis and Research (CAADDR), to resulting consensus sequences were aligned with public be used in an experiment once they attain maturity. They nucleotide sequences by nucleotide BLAST analysis. were fed with commercial pellet feed and the water changes were done weekly. After about one month, low Examination of the wet mounts of skin scrapings and grade mortality was observed in the group, and a gill clips revealed only few gill flukes (Dactylogyrus spp). maximum of 1-2 fish died per day that continued over a Mesophilic, motile Aeromonas spp. was isolated from the period of three weeks. The moribund fish displayed erratic kidney on TSA. AFR were observed in the kidney, liver, swimming, lethargy, slight to moderate abdominal spleen of two fish examined. In others, none of the tissue distention, scale loss, haemorrhages on the skin, faded smears other than smear of intestinal contents had AFR. In pigmentation and reduced appetite ( Figure 1A). Clinical histopathological sections of kidney, liver and the spleen, signs were not consistent in all moribund fish and some multiple granulomas of varying sizes with eosinophilic died suddenly without showing any signs of the disease.
necrotic centers were observed ( Figure 3A), but AFR were either absent or rarely visible within those ( Figure  In March 2016, five moribund fish showing typical 3B). Off white colonies suggestive of mycobacteria signs of the condition were collected from the affected appeared 7 days post inoculation in the kidney and liver tank and subjected to a detailed necropsy to find out the samples. These colonies were ZN positive, hence sub aetiological agent involved. The fish were euthanized cultured on Ogawa egg medium and identified by the using buffered MS-222 at a dose rate of 150 mg/L. At morphology (growth rate and pigment production) and necropsy, there were numerous, white, small, miliary biochemical characteristics (Nitrate reduction, Urease, granulomas in the kidney, liver, spleen, intestines and growth on MacConkey agar without Crystol Violet, gonads ( Figure 1B). These visceral granulomas were Tween 80 hydrolysis, catalase and NaCl tolerance). rpoB sharply demarcated from surrounding parenchymatous gene PCR confirmed the genus level identification as tissues. Mild ascites (accumulation of blood tinged fluid) Mycobacterium yielding an amplicon of 360 bp (Figure was also observed in all five fish examined. For routine 4A). Both phenotypic characteristics and PCR-RFLP bacterial isolations, samples from kidney were cultured patterns ( Figure 4B) supported the identification of the on trypticase soy agar and incubated overnight at room isolate as M. fortuitum. The sequence comparisons temperature. Since the gross pathology was suggestive of revealed 99% homology to piscine mycobacteriosis, squashed tissue smears from the kidney, liver and spleen, and a smear of intestinal contents were prepared and stained with Ziehl Neelsen stain to look Case 2: A koi carp was presented to CAADDR with the for acid fast rods (AFR).
complaints of lethargy, reduced appetite and erratic swimming. This koi carp was the only survivor out of Pieces of tissues from internal organs with granulomas fifteen koi carps (Cyprinus carpio koi) that had been were fixed in 10 % buffered formalin and processed maintained in a garden pond of a hobbyist. This garden routinely for histological evaluation. Sections were pond was an established one with no recent introductions stained using both hematoxylin and eosin (H&E staining) of new fish and chlorinated municipal water was used for and Ziehl Neelsen (ZN) stain. For the culture of water changes. According to the owners, low grade mycobacteria, tissue samples of liver, kidney, intestine, mortality (2-3 fish per day on some days) was observed gonads and heart were collected aseptically from each that continued over a period of one week and the fish, transferred into sterile 1.5 ml microcentrifuge tubes, moribund fish showed erratic swimming, reduced homogenized and decontaminated by adding an equal appetite, lethargy and labored breathing with gasping air volume of 1M NaOH (w/v) and incubated for 30 minutes at the water surface. Owners tried to treat the fish with 0 antibiotics and other chemicals, but all fish except the at 37 C according to a procedure described previously submitted koi carp succumbed to death. Upon submission (Dissanayake et al., 2009). Decontaminated samples were to the laboratory, wet mounts of skin scrapings and gill cultured on Ogawa egg medium and incubated aerobically clips examined revealed a heavy infestation of Trichodina at ambient temperature for two months. Inoculated media spp., (12-15 per field) and Dactylogyrus spp., (gill flukes). were observed daily for 7 days and then on every other day Zeihl Neelson stained smear of fresh faeces showed during rest of the incubation period.
numerous acid fast rods. The fish was transferred to a well infected tissue either in the very early or in the later stages aerated treatment tank and maintained in a bath of 2% of infection. Another diagnostic dilemma associated with NaCl and 0.5mg/L Potassium permanganate for a period mycobacterisos is the lack of concordance between the of three days. On the third day of admission, fish was presence of AFR in organ smears and positive culture found dead. Necropsy and the subsequent investigations isolation results. In both these cases, irrespective of the performed were markedly similar to case No. 1. At presence of AFR, it was possible to isolate M. fortuitum necropsy, a considerable amount of clear ascitic fluid in from the kidney and liver tissues. Negative growth results the abdominal cavity and very few atypical nodular in some organ samples may be either due to a low number lesions that were not well demarcated from the of live mycobacteria which may have been killed during surrounding parenchymatous tissues were observed in the decontamination (Shukla et al., 2013). However, on the kidney, liver and spleen. Visceral organs were soft and other hand, in the absence of clinical signs and granuloma, irregular. Squashed smears made from the kidney, liver isolation of mycobacteria from fish tissues may also be a and the spleen showed clumps of AFB (Figure 2). result of mere colonization of internal organs with Histopathological lesions included mild to moderate mycobacteria present in aquatic environment. granulomatus inflammation, but the number of organized granulomas were markedly low compared to case No: 1.
Unfortunately, there is no effective treatment for Mesophilic, motile Aeromonas spp., was isolated from the mycobacteriosis in fish. Depopulation of the affected kidney of this fish as well. Off white colonies suggestive stock followed by disinfection of tanks is the only reliable of mycobacteria appeared 6 days post inoculation in means of control. Treatment with antimycobacterial drugs kidney and liver samples. This isolate was subsequently is not advocated since the full disease resolution takes identified phenotypically, biochemically and later by time and promotes the resistance development. Antibiotic PCR-RFLP as M. fortuitum. The amplified sequence treatment would not be cost effective for the farmers matched with unless for individual, valuable fish (Strike et al., 2017). 99% sequence identity.
The remaining fish of case no: 1 are being maintained in isolation and antibiotic treatment was not attempted. In

DISCUSSION AND CLINICAL SIGNIFICANCE
case no: 2, the owners were advised to disinfect and dry the pond. M. fortuitum was first isolated from the tropical fish neon tetra in 1953 (Ross and Brancato, 1959). It has since Maintaining a population of Mycobacterium positive been reported in different species of ornamental fish ornamental fish stock makes a little sense to ornamental including Cyprinids and has received a remarkable fish farmers since they are debilitated and prone to have attention as a fish pathogen. Gold fish and koi carps are chronic health problems and poor performance in terms of two commonly cultured ornamental fish varieties in Sri growth and reproduction. Infected fish will be a constant Lanka and therefore Mycobacterium infections in such source of Mycobacteria to other fish inhabiting the same species could result in devastating impacts on the local tank and also to their offsprings (Floyd, 2013). These two sales as well as exports of ornamental fish. Sri Lanka cases in general support two important phenomena. First, imports ornamental fish brood stocks to improve the ornamental fish infected with mycobacteria die off mostly quality of existing stocks, from countries where piscine not due to the pimary pathogen but due to secondary mycobacteriosis is prevalent, increasing the risk of infections by other bacteria and parasites and poor water transboundary transmission of this disease. However, it is quality conditions that make them stressful. And secondly, not yet clear whether this disease has been introduced to any fish from a population experiencing low grade Sri Lanka unintentionally through fish imports or it was mortality accompanied by emaciation, faded there unnoticed and undiagnosed. Therefore, considering pigmentation and weight loss should be suspected for the economical importance of the ornamental fish industry mycobacteriosis. In intensive culture of ornamental fish, and associated international trade, early and accurate high organic loads, poor water quality and high stocking diagnosis of mycobacterial infections and subsequent density exacerbate the condition. Adoption of good management of affected stocks should be considered as husbandry practices will minimize the risk of infection. crucial aspects in ornamental fish health management.
Piscine mycobacterisis is a minor zoonosis. Given the The two cases described in the present study show two fact that ornamental fish and aquarium water are natural different clinical manifestations which were non reservoirs of pathogenic NTM, the close contact between pathognomonic of the infection posing a diagnostic aquarium fish and people offers favourable conditions for challenge. Routine laboratory diagnosis of piscine mycobacterial transmission (Kušar et al., 2017). mycobacteriosis at the field largely depends on the Therefore, it is of prime importance to educate those examination of visceral organ smears with bench top ZN involved in ornamental fish farming; farmers, staining for AFR. However, as observed in both these aquaculturists, hobbyists and veterinarians on the cases, AFR may not be visible in all affected fish even in potential zoonotic risk associated with exposure to the presence of granulomatous lesions in visceral organs. ornamental fish infected with mycobacteria. It has been reported that mycobacteria can be detected in Mycobacterium fortuitum subsp. fortuitum Piscine mycobacteriosis in gold fish and koi carp