Introduction

Piscine mycobacteriosis is not an uncommon cause of chronic morbidity and mortality in ornamental fish populations, especially on ornamental fish farms, where disease causes production problems when broodstock are affected. Many uncertainties exist regarding mycobacteriosis. Among these are a good understanding of the disease's epidemiology relative to both horizontal and vertical transmission; a reliable method for ante-mortem diagnosis; and an understanding of how fish with incompetent immune systems, such as very young fry, react to mycobacterial infections.

The purposes of this ongoing study are numerous and include the following:

1.  To identify water quality and system factors which may correlate with higher incidence of mycobacteriosis;

2.  To identify shedding and/or spread of mycobacterial organisms via the gametes, either by culture of eggs and sperm obtained either by manual stripping or by necropsy;

3.  To observe and identify infected offspring from broodstock populations with a known history of mycobacteriosis, especially with regard to age and onset of disease;

4.  To use CB-18 processing methods in an attempt to develop an ante-mortem diagnostic technique for broodstock.

Mycobacteriosis and Mycobacterial Detection in Fish

First indications of mycobacterial infection in a fish population are often historical, with evidence of chronic mortality. Signs, though not specific, include inappetance, lethargy, abnormal swimming behavior, isolation, reduced growth and vigor, ulcerations or erosions of body or finnage, exophthalmia, and ascites. Scrapes of ulcerations may reveal acid fast bacilli with bench top AFB staining kits. Intradermal skin testing is an inconclusive ante-mortem method due to wide variations in immune reactions. Positive diagnosis usually requires necropsy, which is not a desirable option when working with valuable broodstock. Gross necropsy findings often demonstrate initial granulomatous lesions in kidney, liver, and spleen, but in more advanced cases, granulomas can be found throughout many other organs including testes and ovaries. Histopathologic examination of tissues reveals acid fast organisms usually within granulomas; however, in advanced cases, acid-fast organisms are located throughout the parenchyma of many organs and not only within granulomas. History and gross necropsy, coupled with histopathologic findings, including identification of acid fast bacilli within affected tissues are often enough evidence to support a diagnosis of mycobacteriosis. Culture of affected tissues on mycobacterial supportive media, such as Lowenstein-Jensen agar, is confirmatory, although negative growth does not preclude a diagnosis of mycobacterial disease.

CB-18 Processing vs. Standard Methods^4,5

Technological improvements in sample preparation and culture, as well as in isolation and identification, are permitting greater sensitivity in detection. Nevertheless, numerous impediments to laboratory detection still exist. These can be divided into three general groups. First, specimens and standard processing methods reduce detection. Specimens often contain numerous contaminating organisms which necessitate a decontamination step. However, many solutions used for decontamination compromise the viability of the mycobacteria, and/or inhibit nucleic acid amplification (as for PCR). Secondly, mycobacteriosis in early to mid stages of the disease is characterized by relatively few organisms, along with intermittent shedding. Finally, physiological characteristics of the organisms make their efficient concentration in samples difficult. Among these characteristics are: a) aggregation, cording, and clumping; b) surface tension caused by a waxy cell wall; c) buoyancy; d) slow growth; and e) a thick cell wall. Surface tension and buoyancy reduce numbers expected with centrifugation due to the fact that they are poured with the supernatant fraction following centrifugation. Few organisms exacerbate slow growth. Properties of the cell wall lead to difficulties lysing the organisms for PCR amplification.

Many of these processing problems often lead to differences in results between laboratories, so that one laboratory may obtain positive culture results while another laboratory may consistently obtain negative results from the same source.

Thornton has developed a new processing method, which uses a zwitterionic detergent, N,N-dimethyl-N-(n-octadecyl)-N-(3-carboxypropyl) ammonium inner salt (Chemical Abstract Service number 78195-27-4), also known as C₁₈-carboxypropylbetaine (CB-18) to circumvent many of the above problems. CB-18 is actively sequestered by Mycobacterium sp. and subsequently alters its physical properties by alleviating the innate buoyant density. This new processing method has been proven to be more efficient in detection of mycobacteria and was the method used for processing samples for this study.

A word of caution, however, is warranted. As detection methods for mycobacteria, and in fact all pathogens become more sophisticated, clinicians and researchers must remember that presence of pathogen does not equate with disease. Careful and thoughtful evaluation of affected and exposed animals and systems is essential.

Mycobacterial "Ecology"³

Nontuberculous mycobacteria are relatively common environmental bacteria, being found everywhere including in drinking water supplies, swimming pools, coastal waters, and aquaculture facilities. In one study, nontuberculous mycobacteria, specifically MAIS (Mycobacterium avium/intracellulare/scrofulaceum) complex, were found in relative abundance in acid, brown water swamps in the southeastern coast. High numbers of bacteria were correlated with warmer temperature, low pH, low dissolved oxygen, high soluble zinc, high humic acid, and high fulvic acid. Fish pathogenic forms have been found in similar areas with similar preferences¹. Many of these conditions are found in aquaculture situations, especially within older more highly organic systems, with brown or blackwater species of fish.

Vertical Transmission²

Some work investigating the epidemiology of vertical transmission has been done in a few species. In Mexican platies (Xiphophorus maculatus), vertical transmission via the ovaries has been demonstrated. In salmon, however, transmission via the ovaries is believed not to occur.

Very Preliminary Studies

Mycobacteriosis is a common disease of older broodstock, and once entrenched within a population or system, is difficult to eliminate short of depopulation and disinfection. Fish from a number of farms experiencing chronic morbidity and mortality as well as poor fry survival from 1997-1998 were diagnosed with mycobacteriosis at our laboratory. Affected broodstock species included Blue Rams(Papiliochromis ramirezi), Gold Severums (Heros severus), Spiketail Paradisefish (Pseudosphromenus sp.), Red and Blue Colombian Tetras (undescribed species), and Neon Rainbowfish (Melanotaenia praecox). In one case involving Blue Rams, broodstock were heavily infected with mycobacterial organisms. Although offspring demonstrated poor survival, no acid fast bacteria were seen in fry examined histologically, and other factors including water quality could not be completely ruled out. In another case involving Spiketail Paradise fish, after a positive diagnosis was made, several generations were allowed to self propagate and were periodically examined and culled in an outdoor pond. After approximately three generations, fish were still positive. In all instances, depopulation and disinfection were recommended and where possible, carried out.

At five farms to date, water and sediment samples were collected from various locations, including areas holding affected populations. All five farms had a history of mycobacterial infection in broodstock. These samples were sent to Quest Diagnostics for processing and culture, to obtain an initial environmental prevalence of mycobacteria. Mycobacterium fortuitum was the common isolate from most of the locations sampled, including water and sediment from sumps and indoor holding areas of broodfish and juveniles, pond water, and biological sponge media "filtrate."

The second phase of this study is currently in progress, and is a bit more delicate, since valuable broodfish are involved. Broodfish from affected populations at several farms will be sampled from one or more of the following sites: gills, ulceration scrape/scale, blood, fecal/intestinal tract, kidney, liver, spleen, ovaries, testes. Where possible, mature eggs and milt will be stripped from fish for sampling. Samples will be processed for culture and/or submitted for histopathology. External signs, culture results, and pathology will be correlated.

In addition, offspring of affected broodstock populations will be sampled and evaluated similarly, to identify any potential carriers or diseased fish. Offspring and their growout environment will be assessed periodically. Preliminary findings and future directions will be discussed.

References

1.  Falkinham, personal communication.

2.  Inglis V, Roberts RJ, Bromage NR. 1993. Bacterial Diseases of Fish. Chapter 14, Mycobacteriosis: Nocardiosis, p.224. Halsted Press, New York.

3.  Kirschner Jr, RA, Parker BC, Falkinham III, JO 1992. Epidemiology of Infection by Nontuberculous Mycobacteria: Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in Acid, Brown-Water Swamps of the Southeastern United States and Their Association with Environmental Variables. Am. Rev. Respir. Dis.; 145: 271-275.

4.  Thornton CG. 1997. Methods for processing mycobacteria. U.S. 5,658, 749.

5.  Thornton CG, MacLellan KM, Brink Jr, TL, et al. In press. A novel method for processing respiratory specimens using C₁₈ Carboxypropylbetaine (CB-18) for the detection of mycobacteria in a blinded study.