Cryptosporidium Species in Fish: An Update †

: Cryptosporidium species have been detected in fish, in a wide range of geographical regions. Currently, three species are genetically characterized as piscine-host-specific: C. molnari , C. scophthalmi , C. huwi . One potential novel species, 23 additional piscine genotypes, other non-piscine-host-specific Cryptosporidium species ( C. parvum , C. hominis , C. scrofarum , C. xiaoi ) and the rat genotype III have been genetically characterized in fish. The pathology of cryptosporidiosis is very important for the aquaculture industry, causing mortalities in farmed fish, while the presence of Cryptosporidium zoonotic subtypes in edible fish increases the fish-borne zoonotic potential risk, which is of major importance from a public health point of view.


Introduction
Cryptosporidium spp. are included in the "top ten" list of foodborne parasites of greatest global concern, which affect people's health, infecting tissues and causing clinical symptoms [1]. Cryptosporidium is identified as the second most common cause of diarrhoea worldwide and death in children, after rotavirus [2]. Symptoms of cryptosporidiosis include diarrhoea, vomiting, nausea, lack of appetite and cramps. There is no effective treatment or vaccine available. It is usually a self-limiting disease in the case of immunocompetent individuals but can be chronic in immunocompromised people and young children. It is caused by the enteric protozoan parasite of the genus Cryptosporidium, which infects a huge range of domestic animals, wildlife population and humans [3].
The current knowledge on Cryptosporidium species infecting piscine hosts is steadily increasing, since Cryptosporidium species have been detected in marine and freshwater, cultured, captive and ornamental fish, in a wide range of geographical regions. The presence of zoonotic Cryptosporidium subtypes in fish samples has been described in various studies [4][5][6][7]. Fish-borne zoonotic potential risk from Cryptosporidium species is of major importance from a public health point of view. The presence of these parasites in edible fish may be the etiological agent responsible for outbreaks of fish-borne cryptosporidiosis. It may also produce a variety of clinical signs in infected fish, which are more obvious in the case of heavy infections. The pathology of cryptosporidiosis in cultured fish is important, followed by poor growth rates and increased mortality [8,9] causing an economic impact in the aquaculture industry, which is expanding worldwide.

Cryptosporidium Species in Piscine Hosts
Cryptosporidium species have been detected in marine and freshwater, cultured, captive and ornamental fish in a wide range of geographical regions ( Figure 1). Most commonly, the parasite has been reported in Australia, where 58% of worldwide reports have been recorded. The first piscine host of Cryptosporidium was the tropical fish Naso lituratus, in which C. nasorum was identified [10]. To date, three species are genetically characterized as piscine-host-specific: C. molnari, C. scophthalmi, C. huwi, which are the main three recorded piscine species (Table 1). C. molnari has been genetically characterized in gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) [11,12]. C. huwi has been identified in the ornamental guppy (Poecilia reticulata) [13], the golden tiger barb (Puntigrus tetrazona) [14] and the Neon tetra (Paracheirodon innesi) [15]. C. scophthalmi was firstly reported in turbot [8], but no molecular data are available. However, molecular characterization has identified a C. scophthalmi-like organism in Scophthalmus maximus [16]. These main three recorded piscine species have been reported mainly in Spain, France, Australia and North East Atlantic, while C. molnari and similar genotypes is the species with the widest distribution ( Figure 2). One potential novel species has been isolated in angelfish [17] in Washington State, USA.   According to molecular data, piscine genotypes 2-8 (PG2-PG8) [5,13,14,19], genotype 9 (G9) [7] and novel genotypes (NG, NGC1-5,7), one novel genotype similar to C. molnari in koi carp causing disseminated infection [20] and 14 un-named novel genotypes [4,14,15,21] are additional piscine genotypes (Table 2), which have been reported worldwide, as described in Figure 3.

Cryptosporidium Zoonotic Species in Fish
Humans are susceptible to a wide range of Cryptosporidium spp. and genotypes, with C. hominis and C. parvum as the main species infecting humans globally [22][23][24]. According to a recent evolutionary genomic survey of anthroponotic Cryptosporidium species, C. hominis and C. p. anthroponosum are the main parasites that primarily infect humans, as C. parvum can be further divided into two branches with distinct host associations: the C. parvum anthroponosum (anthroponotic) and the C. parvum parvum (zoonotic) [25].
Several Cryptosporidium species have been detected in fish. The presence of zoonotic subtypes in fish is probably related to water contamination by animal and human wastes. C. parvum zoonotic subtypes are of major significance to public health, as they represent the most common source of zoonotic infection [3]. This has been described in various studies [4][5][6][7] including a great range of hosts and geographic regions. During the last decade, it has been detected in 18 freshwater and marine fish species including edible fish, both wild and farmed. Prevalence of infection was low in most cases. Trout species are recorded as the most sensitive piscine host, as the highest prevalence has been recorded in case of Brown trout [26] and European trout in NW Spain [7].
C. parvum developmental stages are mainly detected in the digestive track (stomach, pyloric caeca, intestine). However, the isolated presence of other non-pathogenic Cryptosporidium species in fish fillets clearly highlights the risk of infection to humans [6]. This fact indicates the potential zoonotic risk of transmission from the fish digestive tract to the fish fillet, in case of C. parvum infected edible fish. Cryptosporidium spp. can be transmitted either by consumption of undercooked fish, or by contact with fish during preparation and handling, especially in immunosuppressed patients [27,28].

Pathology
Cryptosporidium is an obligate intracellular protozoan parasite that mainly infects the microvillus border of the gastrointestinal epithelium. In contrast with the epicellular location of Cryptosporidium species from other vertebrates, in case of piscine Cryptosporidium species sporulation takes place deep within the epithelium [11][12][13].
The pathology of cryptosporidiosis is related to host sensitivity and its response against the parasite. In some cases, the prevalence and the intensity of infection is low and the pathological impact is absent [6,29] or very mild, while oocysts appear degenerated and surrounded by host cellular reaction, as in the case of seabass infected with C. molnari [11].
When severe parasitosis occurs, infected fish are in poor physical condition, characterized by listlessness, muscle wasting and atrophy. Poor growth rates and increased mortality are recorded, while anorexia, severe emaciation or swollen coelomic cavities and abdominal swelling ascites have been observed. During necropsy, indigestion, intestinal distension arising from mucous intestinal contents, gas and whitish faeces are the main findings [8,10,17,18].
Piscine cryptosporidiosis may produce a variety of clinical signs in infected fish, which are more obvious in case of heavy infections. The histopathological effect of the parasite is similar in most cases, as necrosis, vacuolisation, degradation, disaggregation and sloughing of epithelial cells is observed in infected tissues [30]. In the case of gastric cryptosporidiosis, organisms are distributed among the gastric mucosa. Gastritis is present in infected fish, varying from mild to moderate, depending on the intensity of the parasitosis [31]. When inflammation is severe, necrosis of gastric mucosal epithelial cells, mucosal glandular atrophy and glandular loss may be present. Vacuolation appears surrounding oocysts engulfed by hypertrophied cells, probably macrophages. Massive vacuolation and abundant debris appear within the cells and in the lumen in treated fish, while in severely affected fish, clusters of oocysts occupy wide vacuolated areas of the intestinal epithelium [31]. Infected intestines also show different degrees of damage, usually correlated with the infection intensity. Displacement of the microvilli and focal indentation at sites of attachment of the parasites on the mucosal epithelium is recorded [10]. In moderate infections, mild intestinal lesions are observed, while in massive infections severe intestinal lesions are mentioned and most of the epithelial surface is covered by oocysts. In such advanced infections, normal mucosal architecture is distorted, leading to desquamation of the intestinal mucosa, and epithelial cells are distended by large vacuoles containing clusters of oocysts. In these cases, the inflammatory response may be severe and exocytosis of some lymphocytes into the epithelium is common. The abundance of rodlet cells in infected epithelia is also evident and numerous goblet cells are also observed [8].
Cryptosporidium spp. sporogonial stages, apart from gastrointestinal track, have been detected in other tissues. The parasite has been recorded in bile duct epithelial cells of turbot, causing the fibrosis of the bile ducts and necrosis of adjacent hepatic cells [32]. The liver was also a target organ in the case of Koi carp, being pale brown with severe granulomatous inflammatory lesions [20]. In this case, superficial haemorrhages in the skin and hyperaemia of the gills were present, while the spleen was swollen and irregularly granular. Extensive inflammation was evident on histological analysis, with many granulomas present in the kidneys, the spleen, the base of the secondary lamellae and the cartilage of the gills and between the skeletal muscles. The intestines were empty and pale with a thickened mucosa and haemorrhages, while many granulomas were present in the tip of the villi in the lamina propria and near the crypts [20].

Conclusions
Cryptosporidium species have been recorded in a wide range of piscine hosts worldwide. The presence of Cryptosporidium zoonotic subtypes in edible fish, which are commonly consumed raw or slightly processed, increases the fish-borne zoonotic potential risk. Furthermore, the pathology of cryptosporidiosis in fish is very important. Intensive aquaculture practices, stressful conditions, high host density, water supply and temperature, in combination with the immunological immaturity of the host, increase the prevalence, the mortality rate and interactions with other pathogens in farmed fish, having a significant economic impact on the aquaculture industry.