Implementation and analysis of diagnostic techniques for Mycobacterium spp. and Francisella spp. in granulomatous disease of fish in breeding and wild aquaculture of São Paulo/Brazil

There are several etiological agents of acute, chronic, granulomatous, systemic, or focal diseases in animals worldwide. In aquaculture, in fish, reptiles, amphibians and crustaceans, bacteria have caused great losses in production, due to the death of the infected animals or the bad aspect of the sick ones, that makes commercialization unfeasible [1]. In addition, according to Miller and Neely [1], and Wang et al. [2], many of the bacteria pathogenic to aquatic animals are zoonotic, affecting the public health. The growing expansion of consumption in national and international markets since 1990 for various fish, especially tilapia, led to new cultivation strategies, focused especially on the increase of the stocking density and the use of formulated feeds, characterized by a marked reduction in natural food. Therefore, it is necessary to know the bacterial agents, their pathogenesis, which includes the parasite-host-environment relationship, so that new effective protocols can be used, to avoid the dispersion and circulation of these bacterial agents [3].

When tracking the first responses to bacterial infection, the immediate immune response, the knowledge comes that the macrophages residing in the tissue are the cells that first contact the infecting bacteria. In the case of Mycobacteria [4], the macrophages residing in the first response, phagocytize and eradicate mycobacteria, suggesting that, in order to establish a successful infection, the mycobacteria must escape from the initially infected resident macrophages, or cause macrophage apoptotic death and go to the monocytes thus permitting growth. Some cytosolic pathogens, to prevent antimicrobial autophagy, invoke specific mechanisms, such as altering its surface when recruiting proteins from the host, as is the case of Francisella, a cytosolic pathogen that rapidly breaks down its phagosome and resides and successfully proliferates in the cytosol, for an extended period of time, without triggering an autophagy response [5]. 1.1 Mycobacterium Spp.

Mycobacterium spp. is a pathogen capable of causing serious and costly diseases in many invertebrates such as crustaceans [6] and vertebrates, such as humans (tuberculosis, leprosy, Buruli ulcer), livestock (bovine tuberculosis) and ectothermic animals (reptiles, amphibians and fish) [7, 8, 9, 10, 11, 12, 13]. In recent years, due to the decrease in fishing activities, there has been an increasing interest in fish farming, and this increase in farms has favored the development of diseases such as mycobacteriosis and franciselosis [14].

Mycobacterium marinum, M. fortuitum and M. chelonae are the main agents of the disease, called mycobacteriosis or tuberculosis of wild or captive fish, and they are apart of 120 or more species of mycobacteria [15]. In marine ornamental fish and inland water fish these diseases are relatively common [3, 16, 17, 18]. It is a zoonosis because some species offish mycobacteria are potentially capable of infecting humans [19, 20].

In fish, the severity of the infection varies from chronic, with no major tissue changes, in which a few fish die, to conditions of severe and acute infections, with high mortality [5].

In humans, M. marinum causes skin lesions such as pool granulomas [21, 22]. This disease is associated with aquatic activities such as swimming, fishing, managing aquariums, sailing, fish bites, fin wounds, and cleaning tanks and aquariums [21]. M. fortuitum can cause severe injuries such as lung diseases [23, 24].

Microscopically, we generally observe the formation of granulomas. Epithelioid granulomas, or immune granulomas, are characteristic of insoluble particles, typically microorganisms that can induce an immune response. Their center can be filled with caseous necrosis. Macrophages phagocytize such agents and present antigens toT lymphocytes. Their role is to prevent the spread of these agents, and they reveal them to the giant Langhans cell. Macroscopically we have a variety of lesions such as external ulcers, exophthalmos, weight loss or can even occur without symptoms, and this happens when the infection is acute. They mainly appear in the spleen, kidney and liver, in the form of whitish gray areas that can coalesce. As a result of all that, the granuloma is composed of macrophages, epithelioid cells, giant cells, and it is surrounded by T lymphocytes and, in some cases, plasmocytes. The older ones develop a fibroblast capsule and connective tissue. The mechanism of granuloma formation has not been fully clarified [4]. 1.2 Francisella Spp.

Recently, the bacterium of the genus Francisella spp, an emerging pathogen and infectious agent extremely virulent for several animal species, has been found in marine and freshwater fish, amphibians, reptiles and even mollusks. It has been associated with massive tilapia mortalities in commercial farms in Taiwan, Hawaii and Costa Rica [25, 26, 27, 28], and, with breeding casualties between 5% and 80%, with an average of 50%. In 2005, this bacteriosis, initially confused with the disease caused by Piscirikettsia bacteria (common cause of septicemia in salmonids), decimated the tilapia stocks of one of the main producers and exporters of fresh fillets to the United States, Aqua Corporation in Costa Rica [27]. The Francisella genus, of the Francisellaceae family, is composed of non-mobile, gram-negative, strictly aerobic bacteria, and facultative intracellular coccobacilli [29] and comprises three widely known species, F. tularensis, F philomiragia, and F. novicida. Some authors consider the species F. novicida as a subspecies of F. tularensis, since it is being divided into three subspecies, F. tularensis spp tularensis, holorctica and mediasiatica [30, 31, 32]. The species F. philomiragia is divided into F. noatunensis spp noatunensis and F. noatunensis spp orientalis [33, 34]. Some authors do not consider it as a zoonotic factor and, therefore, apparently without the risk of people becoming contaminated [31]. Others consider F. tularensis spp tularensis, found mainly in North America, as the most virulent for animals and humans [30, 35]. Considering the apparent link between F. tularensis and aquatic environments, fish and amphibians have been considered likely reservoirs [36].

Clinical signs, not specific to this bacteriosis, include loss of appetite, pallor, lethargic behavior, and erratic swimming. Focal hemorrhagic areas, loss of scales, erosion of the epidermis, exophthalmos, renomegaly and splenomegaly can be observed [28]. Internally, a more specific sign of the disease is observed, which is the presence of many white nodules in the gills, with epithelial hyperplasia and whitish nodules in the spleen, kidney, and gonads. These nodules have occasionally been noticed also in the liver and the heart. The lesions contain a large number of cocobastonetes which accumulate in cellular cytoplasm and, therefore, the presence of focal and diffuse necrotizing vasculitis, particularly in the spleen and kidney, is common, resulting in chronic inflammation and granuloma formation [25].

The primary target cells for Francisella spp invertebrates are phagocytes [37], epithelial cells and dendritic cells [38, 39]. Phagocytes are important in the initial control of infections by internalizing pathogens and in the formation of phagolysosomes that eventually degrade this content. Meanwhile, intracellular bacteria, including members of the genus Francisella, have developed resistance to this phagolysosome degradation. Golovliov et al. [20], Clemens et al. [5], Santic et al. [40] and Birkbeck et al. [41], noticed that the tilapia affected in fish farms in South America, showed intramuscular lesions, significantly affecting the processing of the carcasses. Up to 30% of the fish fillets from the affected stocks showed dark granulomatous lesions.

Other granulomatous diseases in fish can be caused by Nocardia spp, Rhodococcus spp., Renibacterium salmoninarum, Citrobacter freundii, Photobacterium damselae, Vibrio spp., Seriola liquifaciens, Edwardsiella tarda, Piscirickettsia spp, and Flavobacterium spp. [42].

The aim of this study was to investigate these diseases offish and to evaluate the best diagnostic technique.