Protozoan infections in farmed fish from Brazil: diagnosis and pathogenesis

Infecções por protozoários em peixes cultivados no Brasil: diagnóstico e patogênese

Mauricio Laterça Martins Lucas Cardoso Natalia Marchiori Santiago Benites de Pádua About the authors

Abstracts

The Phylum Protozoa brings together several organisms evolutionarily different that may act as ecto or endoparasites of fishes over the world being responsible for diseases, which, in turn, may lead to economical and social impacts in different countries. Apart from the recent advances for the diagnosis of fish diseases in Brazil, little is known on the protozoan parasites and their relationship with environment and host. This revision presents the most important protozoan parasites found in farmed fish from Brazil, not only with emphasis on its diagnosis, biology, transmission and host-parasite relationship, but also on some information that may be useful to researchers in determining the correct diagnosis in fish farms.

Fish parasites; disease; ciliate; dinoflagellate; pathogenicity


O filo Protozoa reúne diversos organismos evolutivamente distintos que podem atuar como ecto ou endoparasitos de peixes em todo o mundo, sendo responsáveis por doenças as quais, por sua vez, podem ocasionar impactos econômico e social nos diferentes países. Apesar dos recentes avanços no campo de diagnóstico de doenças em peixes no Brasil, ainda pouco se conhece sobre a fauna de protozoários parasitos de peixes e suas relações com o ambiente e hospedeiro. Esta revisão apresenta os mais importantes protozoários parasitos encontrados em peixes cultivados no Brasil, não apenas com ênfase no seu diagnóstico, biologia, transmissão e relação hospedeiro-parasito, mas também algumas informações que podem ser úteis para pesquisadores para o correto diagnóstico em pisciculturas.

Parasitos de peixes; enfermidade; ciliado; dinoflagelado; patogenicidade


Introduction

The Phylum Protozoa gathers several organisms evolutionarily different that may act as ecto and endoparasites in fish (LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.), as well as in other vertebrates and also invertebrates. They are the causative agents of diseases in the global aquaculture causing, among other things, damage and reduced growth of the host fish (MORAES & MARTINS, 2004Moraes FR, Martins ML. Condições predisponentes e principais enfermidades de teleósteos em piscicultura intensiva. In: Cyrino JEP, Urbinati EC, Fracalossi DM, Castagnolli N, editors. Tópicos especiais em piscicultura de água doce tropical intensiva. São Paulo: TecArt; 2004, p. 343-383.), favoring secondary bacterial infections (XU et al., 2012Xu D-H, Shoemaker CA, Martins ML, Pridgeon JW, Klesius PH. Enhanced susceptibility of channel catfish to the bacterium after parasitism by . Edwardsiella ictaluriIchthyophthirius multifiliisVet Microbiol 2012; 158(1-2): 216-219. http://dx.doi.org/10.1016/j.vetmic.2012.02.007. PMid:22397934
http://dx.doi.org/10.1016/j.vetmic.2012....
) and mortality, all leading to constraints in global aquaculture production.

In this way, parasitic protozoan diseases are responsible not only for great losses to the commercial fishing industry, but also for a negative social impact in developing countries where aquaculture activities contribute to food production of high nutritional value to the needy population (BONDAD-REANTASO et al., 2005Bondad-Reantaso MG, Subasinghe RP, Arthur JR, Ogawa K, Chinabut S, Adlard R et al. Disease and health management in Asian aquaculture. Vet Parasitol 2005; 132(3-4): 249-272. http://dx.doi.org/10.1016/j.vetpar.2005.07.005. PMid:16099592
http://dx.doi.org/10.1016/j.vetpar.2005....
).

A number of prophylactic and curative measures have been suggested, although many of the recommended chemicals may be over-used or misused by aquacultural workers, leading to parasite's potential drug resistance and negative impact on the aquatic environment. In this way, it must be highlighted the importance of disease impact in order to elaborate efficient strategies for early diagnosis and fast intervention in management practices in fish farms so as to obtain a stable and sustainable production (PÁDUA & CRUZ, 2014Pádua SB, Cruz C. Health challenges in tilapia culture in Brazil. Aquaculture Asia Pacific 2014; 10(2): 37-39.).

This revision presents the most important protozoan parasites found in farmed fish in Brazil, with emphasis on its diagnosis, distribution, biology, transmission routes and host-parasite relationship that may be useful to researchers in determining the correct diagnosis of fish pathogens.

Ciliophora

These unicellular protozoans possess mobile cilia involving the external body surface in some stage of their life cycle. Cytostome, macronucleous and micronucleous present. Reproduction by binary fission and conjugation. Apiosoma, Balantidium, Chilodonella, Epistylis, Ichthyophthirius multifiliis, Nyctotherus, Rhynchodinium paradoxum, Tetrahymena and Trichodinidae are the main representatives.

Apiosoma Blanchard, 1885

It comprises sessile peritrichid ciliated protozoans in the adult stage, with a conical body shape provided by contractile and nutritive vacuoles, infundibulum (oral cavity), scopula (from which the parasite attaches to host surface), peristomial disc, macronucleous and micronucleous (LI et al., 2008Li M, Wang J, Zhu D, Gu Z, Zhang J, Gong X. Study of (Blanchard 1885) occurring on fry of freshwater fishes in Hongze, China with consideration of the genus . Apiosoma piscicolaApiosomaParasitol Res 2008; 102(5): 931-937. http://dx.doi.org/10.1007/s00436-007-0856-5. PMid:18247054
http://dx.doi.org/10.1007/s00436-007-085...
; EL-TANTAWY et al., 2013El-Tantawy SAM, Reda ESA, Abdel-Aziz A, Abou El-Nour MF, Rady I. spp. and Scopulata epibranchialis infesting Nile perch fish in Dakahlia Province, Egypt. ApiosomaLates niloticusN Y Sci J 2013; 6(6): 111-118.).

Like other sessile peritrichids such as Epistylis and Heteropolaria Foissner et Schubert, 1977, they use fish only for attachment and do not invade the epithelial cells, thus feeding by filtration of suspended material in the water. This phenomenon is termed epibiosis, in which the ciliate acts as epibiont and the host as basibiont (substrate organism) (PÁDUA et al., 2012bPádua SD, Ishikawa MM, Kasai RYD, Jerônimo GT, Carrijo-Mauad JR. Parasitic infestations in hybrid surubim catfish fry ( x . Pseudoplatystoma reticulatumP. corruscans)Rev Bras Med Vet 2012b; 34(3): 235-240.).

Life cycle

Apiosoma reproduces by binary fission and conjugation (LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.). Apart from these strategies, they can develop non-sessile forms denominated telotroch (free-swimming migratory stage which detaches from the parent colony in order to search for new hosts for attachment) in the water.

Transmission

It is transmitted by the free-swimming infective telotrochs.

Diagnosis

Microscopic examination of fresh-mounted scraps of fish's skin, fins and gills is the main technique for diagnosis of Apiosoma (Figure 1a,b). As the parasite presents a robust and long (40-70 µm) body shape, it can be therefore easily diagnosed even in infections with low parasite densities. For a detailed view of the morphological features used for specific identification, the following staining techniques can be used: silver nitrate impregnation (Figure 1c), protargol and Giemsa (Figure 1d), Heidenhain, Ehrlich or Harris haematoxylin, as well as neutral red (LI et al., 2008Li M, Wang J, Zhu D, Gu Z, Zhang J, Gong X. Study of (Blanchard 1885) occurring on fry of freshwater fishes in Hongze, China with consideration of the genus . Apiosoma piscicolaApiosomaParasitol Res 2008; 102(5): 931-937. http://dx.doi.org/10.1007/s00436-007-0856-5. PMid:18247054
http://dx.doi.org/10.1007/s00436-007-085...
). The main characteristics used for identification are body length and width; presence of scopula; body shape; position, length and width of macronucleus; micronucleus diameter; position of contractile vacuole; peristomial disc diameter and peduncle width (LI et al., 2008Li M, Wang J, Zhu D, Gu Z, Zhang J, Gong X. Study of (Blanchard 1885) occurring on fry of freshwater fishes in Hongze, China with consideration of the genus . Apiosoma piscicolaApiosomaParasitol Res 2008; 102(5): 931-937. http://dx.doi.org/10.1007/s00436-007-0856-5. PMid:18247054
http://dx.doi.org/10.1007/s00436-007-085...
; EL-TANTAWY et al., 2013El-Tantawy SAM, Reda ESA, Abdel-Aziz A, Abou El-Nour MF, Rady I. spp. and Scopulata epibranchialis infesting Nile perch fish in Dakahlia Province, Egypt. ApiosomaLates niloticusN Y Sci J 2013; 6(6): 111-118.).

Figure 1
Apiosoma attached on the epithelium (a) and scale (b) of Nile tilapia larva Oreochromis niloticus in fresh-mounted slide. Silver nitrate impregnated specimen to observe the ciliary (c) and stained with Giemsa (d) showing the nuclear apparatus.

Pathogenesis and clinical signs

As the parasite attaches on the host by scopula and do not invade epithelial cells, the pathological alterations are discrete or even less evident. When heavily infested on the fish gills, however, these parasites cause reduced breathing surface to gas exchanges on the gills. In this case, fish will experience respiratory distress and may be seen near the surface of the water, gasping for air (DURBOROW, 2003Durborow RM. Protozoan parasites. Southern Regional Aquaculture Center; 2003. vol. 4701.). Differently, it was observed displacement of scales possibly due to secondary infections on tilapia fingerlings highly infested on the skin (PÁDUA, S.B. personal communication).

Clinical signs may be seen in diseased fish but are not specific and are frequently associated with respiratory difficulty and hyperventilation. Skin color alterations like darkness can also be observed. Li et al. (2008)Li M, Wang J, Zhu D, Gu Z, Zhang J, Gong X. Study of (Blanchard 1885) occurring on fry of freshwater fishes in Hongze, China with consideration of the genus . Apiosoma piscicolaApiosomaParasitol Res 2008; 102(5): 931-937. http://dx.doi.org/10.1007/s00436-007-0856-5. PMid:18247054
http://dx.doi.org/10.1007/s00436-007-085...
detected loss of equilibrium, low swimming on the water surface and anorexia. As with most protozoans, environmental debasement and crowded conditions cause them to become more damaging (DURBOROW, 2003Durborow RM. Protozoan parasites. Southern Regional Aquaculture Center; 2003. vol. 4701.).

Chilodonella Strand, 1926

Leaf-shaped ciliated protozoans, oval, dorsoventrally flattened, slightly asymmetric (Figure 2a-c) and mobiles. Macro and micronucleus well evidenced (Figure 2c); the ventral surface has two longitudinal rows of ciliary kineties (Figure 2b,d) (PÁDUA et al., 2013aPádua SB, Martins ML, Carrijo-Mauad JR, Ishikawa MM, Jerônimo GT, Dias-Neto J, et al. First record of Chilodonella hexasticha (Ciliophora: Chilodonellidae) in Brazilian cultured fish: a morphological and pathological assessment. Vet Parasitol 2013a; 191(1-2): 154-160. http://dx.doi.org/10.1016/j.vetpar.2012.07.030. PMid:22902259
http://dx.doi.org/10.1016/j.vetpar.2012....
). Chilodonella species are free-living but some of them parasitize the skin, gills and fins of both freshwater, marine and estuary fish (PÁDUA et al., 2013aPádua SB, Martins ML, Carrijo-Mauad JR, Ishikawa MM, Jerônimo GT, Dias-Neto J, et al. First record of Chilodonella hexasticha (Ciliophora: Chilodonellidae) in Brazilian cultured fish: a morphological and pathological assessment. Vet Parasitol 2013a; 191(1-2): 154-160. http://dx.doi.org/10.1016/j.vetpar.2012.07.030. PMid:22902259
http://dx.doi.org/10.1016/j.vetpar.2012....
). Only two species have been observed causing damage in fish: Chilodonella hexasticha Kiernik, 1909, mainly found in tropical fishes and C. piscicola (Zacharias, 1894) Jankowski, 1980 (syn. C. cyprini Moroff, 1902) mainly parasitizing fishes from subtropical and temperate waters. In Brazil, Pádua et al. (2013a)Pádua SB, Martins ML, Carrijo-Mauad JR, Ishikawa MM, Jerônimo GT, Dias-Neto J, et al. First record of Chilodonella hexasticha (Ciliophora: Chilodonellidae) in Brazilian cultured fish: a morphological and pathological assessment. Vet Parasitol 2013a; 191(1-2): 154-160. http://dx.doi.org/10.1016/j.vetpar.2012.07.030. PMid:22902259
http://dx.doi.org/10.1016/j.vetpar.2012....
have reported for the first time C. hexasticha causing outbreak mortality in Nile tilapia (Oreochromis niloticus), pacu (Piaractus mesopotamicus), and tuvira (Gymnotus aff. inaequilabiatus).

Figure 2
Chilodonella hexasticha observed in fresh-mounted slide from the gills of Nile tilapia Oreochromis niloticus (a), with detail of ciliary kineties in silver nitrate impregnation (b), nuclear apparatus and great amount of bacteria when stained by Giemsa (c), transversal fission of the parasite after silver nitrate impregnation (d). Bar: 10 µm (b,d).

Life cycle

Chilodonellids show monoxen life cycle with transversal division on the host (Figure 2d), besides sexual reproduction by conjugation (PYNE et al., 1974Pyne CK, Ruch F, Leemann U, Schneider S. Development of the macronuclear anlage in the ciliate . I. Morphological and cytophotometric studies on the evolution of DNA. ChilodonellauncinateChromosoma 1974; 48(3): 225-238. http://dx.doi.org/10.1007/BF00326506. PMid:4217265
http://dx.doi.org/10.1007/BF00326506...
; PÁDUA et al., 2013aPádua SB, Martins ML, Carrijo-Mauad JR, Ishikawa MM, Jerônimo GT, Dias-Neto J, et al. First record of Chilodonella hexasticha (Ciliophora: Chilodonellidae) in Brazilian cultured fish: a morphological and pathological assessment. Vet Parasitol 2013a; 191(1-2): 154-160. http://dx.doi.org/10.1016/j.vetpar.2012.07.030. PMid:22902259
http://dx.doi.org/10.1016/j.vetpar.2012....
). Some researchers suggest that chilodonellids have one life stage capable of forming resistant cysts for parasite maintenance, but this strategy is not yet fully understood.

Transmission

The transmission of chilodonellosis occurs especially by direct contact among infested and healthy fish. The parasite disseminates in fish farming via routine utensils and water in fish transporting, which can be considered as the most important dispersion factors.

Diagnosis

Diagnosis can be made from microscopic examination of fresh-mounted scraps of the skin, fins and gills of suspected fish. In fresh-mounted slides the rapid movement of the parasite, generally to a single direction can be easily detected. Silver nitrate impregnation technique allied to Giemsa or haematoxylin staining procedures are fundamental to observe the main taxonomic characters for specific identification (PÁDUA et al., 2013aPádua SB, Martins ML, Carrijo-Mauad JR, Ishikawa MM, Jerônimo GT, Dias-Neto J, et al. First record of Chilodonella hexasticha (Ciliophora: Chilodonellidae) in Brazilian cultured fish: a morphological and pathological assessment. Vet Parasitol 2013a; 191(1-2): 154-160. http://dx.doi.org/10.1016/j.vetpar.2012.07.030. PMid:22902259
http://dx.doi.org/10.1016/j.vetpar.2012....
). The numbers of ciliary kineties constitute the most important taxonomic character that distinguishes C. hexasticha from C. piscicola. In this way, C. piscicola has more numerous and less spaced ciliary kineties (KAZUBSKI & MIGALA, 1974Kazubski SL, Migala K. Studies on the distinctness of Chilodonella cyprini (Moroff) and Ch. Hexasticha (Kiernik) (), ciliate parasite of fishes. Chlamydodontidae, GymnostomatidaActa Protozool 1974; 13: 9-40.).

Histological techniques also allow the researcher to find out the definitive diagnosis. However, not all details can be observed in infested tissue from histological sections, allowing only its determination at the generic level.

Pathogenesis and clinical signs

Infestations by Chilodonella cause severe lesions when compared to other ciliates with similar lifestyle, as for example trichodinids. Pathological changes caused by Chilodonella are related to its abrasive action on the host epithelium, being the gill filaments the most sensitive organ to the parasite attack. In acute cases, an increase in the mucus production with consequent congestion of the gills maybe found. Consequently in severely infested fish, epithelial proliferation, necrosis and desquamation culminating in blood capillary rupture and mononuclear inflammatory cell infiltrate can be found (PÁDUA et al., 2013aPádua SB, Martins ML, Carrijo-Mauad JR, Ishikawa MM, Jerônimo GT, Dias-Neto J, et al. First record of Chilodonella hexasticha (Ciliophora: Chilodonellidae) in Brazilian cultured fish: a morphological and pathological assessment. Vet Parasitol 2013a; 191(1-2): 154-160. http://dx.doi.org/10.1016/j.vetpar.2012.07.030. PMid:22902259
http://dx.doi.org/10.1016/j.vetpar.2012....
). Since host health and defense system are strongly affected, the disease is frequently accompanied by bacterial opportunistic secondary infections (Figure 1c) that provoke systemic infection and host death.

Clinical manifestation comprises non-specific signs such as respiratory difficulty, loss of equilibrium and appetite. Apart from these alterations, whitish lesions on the gills, scaleness, darkened skin, skin and fins ulcers, and haemorrhagic areas are common in mixed infection with bacteria.

Epistylis Ehrenberg, 1830

Representatives of Epistylis are colonial ciliates with bell-shaped body, provided by a long peduncle (not contractile) in which, in the apex, is located the zooid cell with a nucleus, contractile vacuoles and cilia (LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.). Similar to Apiosoma, this sessile peritrichid uses fish as a substrate for attachment (epibiosis) and feeds on suspended particles in the water (PÁDUA et al., 2012aPádua SB, Menezes Filho RN, Dias Neto J, Ishikawa MM, Jerônimo GT, Martins ML, et al. Epistilíase: uma doença emergente no Brasil. Panor. Aquic. 2012a; 22(129): 22-31.). When present in a population under high stocking densities associated with bacteria that colonizes its peduncle, this ciliate might cause host damage and then be named as parasite. In Brazil, epistyliasis has been characterized as an emergent disease with greater impact in farmed catfish (ISHIKAWA et al., 2012Ishikawa MM, Pádua SB, Ventura AS, Jerônimo GT, Russo MR, Carrijo-Mauad JR, et al. Biologia e estratégias na sanidade de alevinos de bagres carnívoros. Embrapa Agropecuária Oeste; 2012. p. 1-35. Documentos 115.; PÁDUA et al., 2012aPádua SB, Menezes Filho RN, Dias Neto J, Ishikawa MM, Jerônimo GT, Martins ML, et al. Epistilíase: uma doença emergente no Brasil. Panor. Aquic. 2012a; 22(129): 22-31.,b, 2013b).

Life cycle

These ciliates rely on binary fission of the zooids (Figure 3b) for asexual propagation, but they may reproduce by conjugation on its sexual reproduction (ISHIKAWA et al., 2012Ishikawa MM, Pádua SB, Ventura AS, Jerônimo GT, Russo MR, Carrijo-Mauad JR, et al. Biologia e estratégias na sanidade de alevinos de bagres carnívoros. Embrapa Agropecuária Oeste; 2012. p. 1-35. Documentos 115.). Similar to Apiosoma, it occurs as non-sessile telotrochs in the water column (ISHIKAWA et al., 2012Ishikawa MM, Pádua SB, Ventura AS, Jerônimo GT, Russo MR, Carrijo-Mauad JR, et al. Biologia e estratégias na sanidade de alevinos de bagres carnívoros. Embrapa Agropecuária Oeste; 2012. p. 1-35. Documentos 115.).

Figure 3
Epistylis sp. (a) in fresh-mounted slide under light microscope. Zooid in binary fission (b) and detail of bacteria (arrow heads) attached on the peduncle of the ciliate. Nile tilapia Oreochromis niloticus (c) with fin erosion and loss of scale (arrowheads) associated with Epistylis colonization, and hybrid surubim Pseudoplatystoma sp. (d) showing total erosion of dorsal fin and colonies of Epistylis sp. on the dorsal fin and head (arrow heads).

Transmission

Epistyliasis is transmitted from infective telotrochs searching for new hosts for attachment to develop new colonies. In addition, Pádua et al. (2013b)Pádua SB, Ishikawa MM, Ventura AS, Jerônimo GT, Martins ML, Tavares LER. Brazilian catfish parasitized by sp. (Ciliophora, Epistylididae), with description of parasite intensity score. EpistylisParasitol Res 2013b; 112(1): 443-446. http://dx.doi.org/10.1007/s00436-012-3069-5. PMid:22890898
http://dx.doi.org/10.1007/s00436-012-306...
suggested that zooplanctonic microcrustaceans may act as vectors or reservoir hosts for Epistylis in farming conditions. In fact, it is common to find these ciliates fixed on the body surface of those organisms increasing thus disease dissemination (VISSE, 2007Visse M. Detrimental effect of peritrich ciliates ( sp.) as epibionts on the survival of the copepod EpistylisAcartia bifilosa.Proc Estonian Acad Sci Biol Ecol 2007; 56(3): 173-178.). Besides, it is a routine practice for fish farming to add manure to the ponds in order to improve the primary productivity of the water resulting in increased phytoplankton and zooplancton production for fish feeding. Nevertheless, as a result of the addition of nutrients into the water, the sanitary risk of an increase in the number of disease-vectors protozoans is imminent.

Diagnosis

Macroscopical observation of epistyliasis from whitish to yellowish colonies on the fish surface, fins, operculum, head and mouth should be accompanied by fresh-mounted slides observed in microscope (Figure 3a). This is especially true because observation of parasite colonies at first view (naked eye) can lead to misleading or confusing diagnosis since it can be easily confused with fungal infection. From microscopic observation, contraction movements of the zooids afford the definitive diagnosis. As complementary material, the histopathological analysis provides a more comprehensive view of the disease and its effect on tissues but do not preserve the important characteristics found in fresh-mounted material. In early infestation it is not possible to detect the colonies by the naked eye (PÁDUA et al., 2013bPádua SB, Ishikawa MM, Ventura AS, Jerônimo GT, Martins ML, Tavares LER. Brazilian catfish parasitized by sp. (Ciliophora, Epistylididae), with description of parasite intensity score. EpistylisParasitol Res 2013b; 112(1): 443-446. http://dx.doi.org/10.1007/s00436-012-3069-5. PMid:22890898
http://dx.doi.org/10.1007/s00436-012-306...
), being necessary to examine scraps from skin, fins and gills.

The identification at specific level from in vivo analysis must be complemented in samples impregnated with silver proteinate (protargol). Length and width of zooid, cell and colony shape, peristomial lips diameter, arrangement of buccal ciliary, nucleus shape and legth and width of peduncle are important characteristics to be measured (LI et al., 2012Li M, Li W, Ge X, Wang C, Zhang L, Huang F, et al. First report of Epistylis unioi Gong 1986 (Sessilida: Epistylididae) infecting fry of in Hubei, China. Pelteobagrus fulvidracoZootaxa 2012; 3556: 80-88.).

Differential diagnosis

To confirm Epistylis on fish, microscopical analysis is fundamental, once the fungus Saprolegnia sp. can also develop colonies visible to the naked eye. It can be differentiated from the other sessile peritrichids, Apiosoma and Ambiphrya, by the formation of branched colonies. On the other hand, other ciliates not commonly found to be parasitizing fish such as Zoothamnium and Carchesium also develop branched colonies similar to Epistylis. Nevertheless, they differ from the latter in their peduncle contraction movement.

Pathogenesis and clinical signs

Its pathology is strongly associated with the presence of bacteria on the peduncle (Figure 3b, arrow heads). As they do not invade directly the host cells in the attachment process, neither pathological alterations in infested tissue nor substances are released on the host surface (HAZEN et al., 1978Hazen TC, Raker ML, Esch GW, Fliermans CB. Ultrastruct of red-sore lesions on largemouth bass (Micropterussalmoides): associattion of the ciliate sp. and the bacterium . EpistylisAeromonas hydrophilaJ Protozool 1978; 25(3 Pt 2): 351-355. http://dx.doi.org/10.1111/j.1550-7408.1978.tb03901.x. PMid:102785
http://dx.doi.org/10.1111/j.1550-7408.19...
). Moreover, these authors confirmed the involvement of Aeromonas hydrophila as the bacterium responsible in colonizing the parasite and secreting lytic enzimes that degrade the adjacent tissue. In this case, haemorrhagic alterations associated with peritrichid colonies in channel catfish can determine the red-sore disease. The colonization of parasite depends on the fish species and developmental stage of fish as well as the production system conditions.

In highly infested fish by Epistylis sp. it can be seen fin erosion and skin ulcers related to bacterial enzymes activity (Figure 3c,d). On histological sections, degeneration and epithelial necrosis adjacent to the colonies, intense desquamation, as well as increased mucus production and inflammatory infiltrate are also related (PÁDUA et al., 2012aPádua SB, Menezes Filho RN, Dias Neto J, Ishikawa MM, Jerônimo GT, Martins ML, et al. Epistilíase: uma doença emergente no Brasil. Panor. Aquic. 2012a; 22(129): 22-31.). The fish death occurs generally after secondary invasion by opportunistic bacteria thus causing systemic infection.

Ichthyophthirius multifiliis Fouquet, 1876

The causative agent of ichthyophthiriasis or white spot disease is one of the most important fish parasites of worldwide distribution compromising skin, fins, gills and eyes of farmed fish. This parasite is not host specific and any freshwater fish can potentially transmit the parasite (EIRAS, 2013aEiras JC. Ciliophora. In: Pavanelli GC, Takemoto RM, Eiras JC, editors. Parasitologia de peixes de água doce do Brasil. Maringá: Eduem; 2013a. p. 233-245.). Similar to that found for channel catfish (Ictalurus punctatus) in the United States (XU &KLESIUS, 2004Xu D-H, Klesius PH. Two year study on the infectivity of Ichthyophthirius multifiliis in channel catfish Ictalurus . punctatusDis Aquat Organ 2004; 59(2): 131-134. http://dx.doi.org/10.3354/dao059131. PMid:15212279
http://dx.doi.org/10.3354/dao059131...
), in Brazil it is considered an important parasitic disease in farmed hybrid surubim catfish (Pseudoplatystoma sp.) (ISHIKAWA et al., 2012Ishikawa MM, Pádua SB, Ventura AS, Jerônimo GT, Russo MR, Carrijo-Mauad JR, et al. Biologia e estratégias na sanidade de alevinos de bagres carnívoros. Embrapa Agropecuária Oeste; 2012. p. 1-35. Documentos 115.), including the Central-North region of Brazil where water temperature variations are less frequent.

During a parasitological survey of ornamental fishes from North Brazil, Tavares-Dias et al. (2009)Tavares-Dias M, Lemos JRG, Martins ML, Jerônimo GT. Metazoan and protozoan parasites of freshwater ornamental fish from Brazil. In: Tavares-Dias M, editor. Manejo e sanidade de peixes em cultivo. Macapá: Embrapa Amapá; 2009. p. 469-494. cap. 19. identified I. multifiliis in the gills of Paracheirodon axelrodi, Hyphessobrycon copelandi and Dianema urostriatum. Several fish species are susceptible to I. multifiliis including the silver catfish jundiá (Rhamdia quelen) (MARTINS et al., 2013Martins ML, Marchiori NC, Garcia P. Doenças parasitárias do jundiá. In: Barcellos LJG, Fagundes M, Ferreira D. Workshop sobre jundiá: história e perspectivas. Passo Fundo: UPF; 2013. p. 184-221.) and the amazonian hybrid pintado (Pseudoplatystoma sp. x Leiarius marmoratus) are susceptible to I. multifiliis.

Life cycle

The life cycle of I. multifiliis is monoxenic and involves only a fish to be completed. Similar to Amyloodinium and Piscinoodinium, its life cycle has three stages as follows:

  1. I

    Theront: infective and mobile form measuring 30 to 50 µm; it needs to find a host otherwise it will die. Theronts are provided by a structure named perforatorium used in fish cell invasion.

  2. II

    Trophont: adult mobile stage found in fish epithelium, can reach 800 to 1,000 µm in diameter.

  3. III

    Tomont: free form of the parasite provided by a cyst for protection. Attaches to plants and substrate to divide asexually and originate 500 to 1000 daughter cells named tomites that will differentiate in infective theronts and so search for a new host.

Transmission

Its transmission occurs by co-habitation (XU et al., 2007Xu D-H, Klesius PH, Shoemaker CA. Evaluation of a cohabitation challenge model in immunization trials for channel catfish Ictalurus punctatus against . Ichthyophthirius multifiliisDis Aquat Organ 2007; 74(1): 49-55. http://dx.doi.org/10.3354/dao074049. PMid:17425263
http://dx.doi.org/10.3354/dao074049...
) with infested fish or directly from the theronts. Fishery utensils used in fish farms and water transport are potential vectors of ichthyophthiriasis. The release of theronts into the water is strongly associated with temperature. At water temperatures above 24°C, the life cycle is favored and completed rapidly. Differently, temperatures below 10°C or above 28°C can inhibit the parasite life cycle (ISHIKAWA et al., 2012Ishikawa MM, Pádua SB, Ventura AS, Jerônimo GT, Russo MR, Carrijo-Mauad JR, et al. Biologia e estratégias na sanidade de alevinos de bagres carnívoros. Embrapa Agropecuária Oeste; 2012. p. 1-35. Documentos 115.). An exception, however, must be commented when it was observed mortalities of up to 50% in fingerlings of tambaqui Colossoma macropomum infected with I. multifiliis at Central-North and North regions of Brazil, when water temperatures ranged from 29.5 to 31.5 °C (PÁDUA, S.B. personal communication). In this way, studies on different strains of I. multifiliis to verify its susceptibility under uncommon conditions must be encouraged.

Diagnosis

Ichthyophthiriasis diagnosis is made based on macroscopical observation of trophonts within the host's skin and microscopical analysis of fresh-mounted material (skin, fins and gills scraps) between a slide and a coverslip (Figure 4a). Under an optical microscope, the observation of the mobile pear-shaped theronts and mature trophonts uniformly covered by a layer of external cilia and with a horse-shoe shaped nucleus confirms the diagnosis (Figure 4b). Histopathological analysis can also reveals the parasitosis (Figure 4c,d).

Figure 4
Hybrid surubim Pseudoplatystoma sp. presenting white spots on the skin and fins (a). Ichthyophthirius multifiliis observed in fresh-mounted slide from the skin: mature trophont and its horse-shoe shaped macronucleus (b - arrowhead) and several imature trophonts in developmental stages (b - continued arrow). Histological section of the gill lamellae (c), with inflammatory infiltrate (c - asterisk), and the parasite inserted in the skin of infected fish (d - arrow head). Proliferation of mucous cells next to the parasite attachment (d - dotted arrows). Bar: 150 µm (c) and 100 µm (d). Figure 4 a was obtained from Pádua et al. (2012b)Pádua SD, Ishikawa MM, Kasai RYD, Jerônimo GT, Carrijo-Mauad JR. Parasitic infestations in hybrid surubim catfish fry ( x . Pseudoplatystoma reticulatumP. corruscans)Rev Bras Med Vet 2012b; 34(3): 235-240. and Figure 4 d from Pádua et al. (2014)Pádua SB, Jerônimo GT, Ishikawa MM, Belo MAA, Martins ML, Pelisari T, et al. Parasitological assessment and host-parasite relationship in farmed cachara catfish fingerlings Pseudoplatystoma reticulatum (Eigenmann & Eigenmann 1889). Neotrop Helminthol 2014; 8(1): 37-45..

Differential diagnosis

Because of its similarity with the dinoflagellate Piscinoodinium pillulare, definitive diagnosis ought to be careful. This dinoflagellate shows similar color and shape to I. multifiliis, but does not swim and has no cilia around the body. Moreover, P. pillulare has rounded nucleus instead of the horse-shoe shaped nucleus in I. multifiliis.

Pathogenesis and clinical signs

Pathological alterations associated with ichthyophthiriasis are related to theront invasion on the epithelium layers with posterior histophagia stage of trophont. This process provokes inflammatory reaction of the host with intense epithelial proliferation by goblet cells, and in severe cases it can be observed fusion of secondary lamellae, degeneration and epithelial cell necrosis forming several ulcers on the epithelium after the releasing of mature trophonts (PÁDUA et al., 2014Pádua SB, Jerônimo GT, Ishikawa MM, Belo MAA, Martins ML, Pelisari T, et al. Parasitological assessment and host-parasite relationship in farmed cachara catfish fingerlings Pseudoplatystoma reticulatum (Eigenmann & Eigenmann 1889). Neotrop Helminthol 2014; 8(1): 37-45.).

The main clinical sign is the presence of white spots on the fish surface including skin, fins, eyes, buccal cavity and gills. It is common to observe fish with respiratory difficulty, flashing behavior in ponds and aquaria, and in cage-reared fish flashing on the sieve cages, leading to muddy water in earth ponds (MARTINS et al., 2000Martins ML, Morais FR, Fujimoto RY, Onaka EM, Nomura DT, Silva CAH, et al. Parasitic infections in cultivated freshwater fishes a survey of diagnosticated cases from 1993 to 1998. Rev Bras Parasitol Vet 2000; 9(1): 23-28. PMid:24728357.; ISHIKAWA et al., 2012Ishikawa MM, Pádua SB, Ventura AS, Jerônimo GT, Russo MR, Carrijo-Mauad JR, et al. Biologia e estratégias na sanidade de alevinos de bagres carnívoros. Embrapa Agropecuária Oeste; 2012. p. 1-35. Documentos 115.).

Trichodinidae Claus, 1874

Trichodinids are mobile ciliates characterized by the presence of a body covered by a slender membrane surrounded by an adoral ciliary spiral, a horse-shoe shaped macronucleus and an adhesive disc provided with a denticulate ring in which the denticles are found (Figure 5c) (BASSON & VAN AS, 2006Basson L, Van As JG. Trichodinidae and other ciliophorans (Phylum Ciliophora). In: Woo PTK. Fish diseases and disorders. 2nd ed. Cab International; 2006. p. 154-182. vol. 1. Protozoan and Metazoan infections.). In Brazil, the genera Trichodina, Paratrichodina, Tripartiella and Trichodinella have been found parasitizing aquatic animals (PÁDUA et al., 2011aPádua SB, Martins ML, Varandas DN, Dias Neto J, Ishikawa MM, Pilarski F. Tricodinídeos: quem são e o que eles podem causar nos peixes. Panorama da Aquicultura 2011a; 121(127): 22-29.), including zooplanctonic microcrustacean (SILVA et al., 2009Silva WM, Roche KF, Vicente FS, Delben AAST. First record of the peritrich Basson and Van As, 1991 (Protozoa: Ciliophora) on a South American calanoid . Trichodina diaptomiNotodiaptomus deitersi (Poppe, 1890) (Crustacea: Copepoda)J Eukaryot Microbiol 2009; 56(4): 385. http://dx.doi.org/10.1111/j.1550-7408.2009.00414.x. PMid:19602084
http://dx.doi.org/10.1111/j.1550-7408.20...
), gastropod mollusc (PINTO et al., 2006Pinto HA, Wieloch AH, Melo AL. Uma nova espécie de Trichodina Ehrenberg, 1838 (Ciliophora: Trichodinidae) em (Crosse, 1864) (Mollusca: Planorbidae). Biomphalaria schrammiLundiana 2006; 7(2): 121-124.), bivalve mollusc from mangrove (SABRY et al., 2013Sabry RC, Gesteira TCV, Magalhães ARM, Barracco MA, Guertler C, Ferreira LP, et al. Parasitological survey of mangrove oyster, , in the Pacoti River Estuary, Ceará State, Brazil. Crassostrea rhizophoraeJ Invertebr Pathol 2013; 112(1): 24-32. http://dx.doi.org/10.1016/j.jip.2012.10.004. PMid:23147104
http://dx.doi.org/10.1016/j.jip.2012.10....
), ornamental fishes (MARTINS et al., 2012Martins ML, Marchiori N, Roumbedakis K, Lami F. Trichodina nobilis Chen, 1963 and Hirschmann et Partsch, 1955 from ornamental freshwater fishes in Brazil. Trichodina reticulataBraz J Biol 2012; 72(2): 281-286. http://dx.doi.org/10.1590/S1519-69842012000200008. PMid:22735135
http://dx.doi.org/10.1590/S1519-69842012...
), wild fishes (BITTENCOURT et al., 2014Bittencourt LS, Pinheiro DA, Cárdenas MQ, Fernandes BM, Tavares-Dias M. Parasites of native Cichlidae populations and invasive (Linnaeus, 1758) in tributary of Amazonas River (Brazil). Oreochromis niloticusRev Bras Parasitol Vet 2014; 23(1): 44-54. http://dx.doi.org/10.1590/S1984-29612014006. PMid:24728360
http://dx.doi.org/10.1590/S1984-29612014...
), as well as farmed fish (VALLADÃO et al., 2013Valladão GMR, Pádua SB, Gallani SU, Menezes-Filho RN, Dias-Neto J, Martins ML, et al. Paratrichodina africana (Ciliophora): a pathogenic gill parasite in farmed Nile tilapia. Vet Parasitol 2013; 197(3-4): 705-710. http://dx.doi.org/10.1016/j.vetpar.2013.04.043. PMid:23731856
http://dx.doi.org/10.1016/j.vetpar.2013....
) and amphibians (FERNANDES et al., 2011Fernandes NM, Sartini B, Dias RJ, D'Agosto M. Quantitative study of Trichodina heterodentata (Ciliophora: Mobilia) infrapopulations infesting tadpoles of a Brazilian endemic toad Rhinella pombali (Anura: Bufonidae). Zoologia 2011; 28(6): 777-783. http://dx.doi.org/10.1590/S1984-46702011000600011.
http://dx.doi.org/10.1590/S1984-46702011...
). Until 2006 trichodinids from farmed fishes in Brazil were recognized only at their generic level. From that year on, several studies have been performed in order to identify trichodinids at specific level (GHIRALDELLI et al., 2006Ghiraldelli L, Martins ML, Adamante WB, Yamashita MM. First record of Van as and Basson, 1989 (Protozoa: Ciliophora) from cultured Nile tilapia in the state of Santa Catarina, Brazil. Trichodina compactaInt J Zool Res 2006; 2(4): 369-375. http://dx.doi.org/10.3923/ijzr.2006.369.375.
http://dx.doi.org/10.3923/ijzr.2006.369....
; PINTO et al., 2009Pinto E, Garcia AM, Figueiredo HCP, Rodrigues MP, Martins ML. First record of . (Ciliophora: Peritrichia) in . Tripartiella spPseudoplatystoma corruscans (Osteichthyes: Pimelodidae) cultured in the state of Mato Grosso do Sul, Brazil, with the description of a new speciesBol Inst Pesca 2009; 35(1): 91-97.; JERÔNIMO et al., 2011Jerônimo GT, Speck GM, Cechinel MM, Gonçalves ELT, Martins ML. Seasonal variation on the ectoparasitic communities of Nile tilapia cultured in three regions in southern Brazil. Braz J Biol 2011; 71(2): 365-373. http://dx.doi.org/10.1590/S1519-69842011000300005. PMid:21755153
http://dx.doi.org/10.1590/S1519-69842011...
; MARTINS et al., 2010aMartins ML, Marchiori N, Nunes G, Rodrigues MP. First record of Trichodina heterodentata (Ciliophora: Trichodinidae) from channel catfish, cultivated in Brazil. Ictalurus punctatusBraz J Biol 2010a; 70(3): 637-644. http://dx.doi.org/10.1590/S1519-69842010000300022. PMid:20730352
http://dx.doi.org/10.1590/S1519-69842010...
; MARTINS et al., 2012Martins ML, Marchiori N, Roumbedakis K, Lami F. Trichodina nobilis Chen, 1963 and Hirschmann et Partsch, 1955 from ornamental freshwater fishes in Brazil. Trichodina reticulataBraz J Biol 2012; 72(2): 281-286. http://dx.doi.org/10.1590/S1519-69842012000200008. PMid:22735135
http://dx.doi.org/10.1590/S1519-69842012...
; MIRANDA et al., 2012Miranda LH, Marchiori N, Alfaro CR, Martins ML. First record of (Ciliophora: Trichodinidae) from cultivated in Peru. Trichodina heterodentataArapaima gigasActa Amazonica 2012; 42(3): 433-438. http://dx.doi.org/10.1590/S0044-59672012000300016.
http://dx.doi.org/10.1590/S0044-59672012...
; PÁDUA et al., 2012cPádua SB, Martins ML, Carraschi SP, Cruz C, Ishikawa MM. Trichodina heterodentata (Ciliophora: Trichodinidae): a new parasite for (Pisces: Characidae). Piaractus mesopotamicusZootaxa 2012c; 3422: 62-68.).

Figure 5
Trichodinids in fresh-mounted smear from the skin (a), Trichodina centrostrigeata in differential interference contrast microscope (b), adhesive disc of Trichodina magna in silver nitrate impregnation (c) and an adhesive disc of a trichodinid during binary fission in silver nitrate impregnation (d). Bar: 50 µm (b) and 10 µm (c,d).

Trichodinids can be found parasitizing both freshwater and marine fishes on the body surface, buccal cavity and gills. Nevertheless, relatively few of them have become endoparasites in the intestine, kidney and urinary bladder of their hosts (LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.). Proliferation of the group in the environment seems to be associated with bad water quality, total number of bacteria and ecological aspects of the fish species. In this way, the use of trichodinids as an indicator for eutrophication in brackish-water environments was suggested (PALM & DOBBERSTEIN, 1999Palm HW, Dobberstein RC. Occurrence of trichodinid ciliates (Peritricha: Urceolariidae) in the Kiel Fjord, Baltic Sea, and its possible use as a biological indicator. Parasitol Res 1999; 85(8-9): 726-732. http://dx.doi.org/10.1007/s004360050622. PMid:10431740
http://dx.doi.org/10.1007/s004360050622...
).

Their reproduction in fish farms has been related to high stocking density, high organic matter contents and increased water temperature (BASSON & VAN AS, 2006Basson L, Van As JG. Trichodinidae and other ciliophorans (Phylum Ciliophora). In: Woo PTK. Fish diseases and disorders. 2nd ed. Cab International; 2006. p. 154-182. vol. 1. Protozoan and Metazoan infections.; MARTINS et al., 2010bMartins ML, Azevedo TMO, Ghiraldelli L, Bernardi N. Can the parasitic fauna on Nile tilapias be affected by different production systems? An Acad Bras Cienc 2010b; 82(2): 493-500. http://dx.doi.org/10.1590/S0001-37652010000200024. PMid:20563429
http://dx.doi.org/10.1590/S0001-37652010...
). Differently, some trichodinid species were found to be suppressed with increased water temperature (YEMMEN et al., 2011Yemmen C, Ktari MH, Bahri S. Seasonality and histopathology of Trichodina puytoraci Lom, 1962, a parasite of flathead mullet () from Tunisia. Mugil cephalusActa Adriat 2011; 52(1): 15-20.).

Life cycle

Trichodinids have monoxenic life cycle and reproduce mainly by binary fission (conjugation is also possible) on the host (Figure 5d). In a short period of time they can reproduce rapidly and reach 100% prevalence and up to 299,100 parasites per host as observed by Martins et al. (2010b)Martins ML, Azevedo TMO, Ghiraldelli L, Bernardi N. Can the parasitic fauna on Nile tilapias be affected by different production systems? An Acad Bras Cienc 2010b; 82(2): 493-500. http://dx.doi.org/10.1590/S0001-37652010000200024. PMid:20563429
http://dx.doi.org/10.1590/S0001-37652010...
.

Transmission

Trichodinids can be horizontally transmitted by direct contact or by contaminated water in which the parasite searches for new hosts. Contaminated fish farming utensils are also another important source of trichodinids transmission.

Diagnosis

Trichodinid diagnosis can be made from scraps of skin, fins and gills of diseased fish observed under a stereomicroscope by an expert researcher or under a microscope (Figure 5a,b). In fresh-mounted material between a slide and a coverslip the parasites move rapidly in circle. The most important techniques for specific diagnosis are silver nitrate impregnation (Figure 5c) and staining by Giemsa or haematoxylin (LOM, 1958Lom J. A contribution to the systematics and morphology of endoparasitic trichodinids from amphibians, with a proposal of uniform specific characteristics. J Protozool 1958; 5(4): 251-263. http://dx.doi.org/10.1111/j.1550-7408.1958.tb02563.x.
http://dx.doi.org/10.1111/j.1550-7408.19...
; VAN AS & BASSON, 1989Van As JG, Basson L. A further contribution to the taxonomy of the Trichodinidae (Ciliophora: Peritrichia) and a review of the taxonomic status of some fish ectoparasitic trichodinids. Syst Parasitol 1989; 14(3): 157-179. http://dx.doi.org/10.1007/BF02187051.
http://dx.doi.org/10.1007/BF02187051...
).

Histological sections can also allow identifying the parasites only at the family level, because the shape of the denticles varies within and between the genera.

Pathogenesis and clinical signs

According to Basson and Van As (2006)Basson L, Van As JG. Trichodinidae and other ciliophorans (Phylum Ciliophora). In: Woo PTK. Fish diseases and disorders. 2nd ed. Cab International; 2006. p. 154-182. vol. 1. Protozoan and Metazoan infections., in a “firmly attached Trichodina, the rim of the border membrane “bites” into the surfaces of the fish's epithelial cells, and the surface it encircles is forcibly vaulted as by a sucker”; these activities (attachment and rotating movements) may cause serious irritation and damage to the epithelial or epidermal cells of fish.

The disease may occur in acute form mainly in larvae and fingerlings causing ulcers, subepithelial oedema, displacement of the secondary lamellae in the gill filaments, hyperplasia and mononuclear inflammatory infiltration (VALLADÃO et al., 2014Valladão GMR, Gallani SU, Pádua SB, Martins ML, Pilarski F. (Ciliophora) infestation on . Trichodina heterodentataProchilodus lineatus larvae: a host-parasite relationship studyParasitology 2014; 141(5): 662-669. http://dx.doi.org/10.1017/S0031182013001480. PMid:24476792
http://dx.doi.org/10.1017/S0031182013001...
). On the other hand, in chronic infestations trichodinids may induce an increase in the mucus cells of the epithelium and gill filaments, discrete hyperplasia with partial or total fusion of the secondary lamellae, inflammatory infiltrate and gill necrosis (VALLADÃO et al., 2013Valladão GMR, Pádua SB, Gallani SU, Menezes-Filho RN, Dias-Neto J, Martins ML, et al. Paratrichodina africana (Ciliophora): a pathogenic gill parasite in farmed Nile tilapia. Vet Parasitol 2013; 197(3-4): 705-710. http://dx.doi.org/10.1016/j.vetpar.2013.04.043. PMid:23731856
http://dx.doi.org/10.1016/j.vetpar.2013....
). The micro-lesions caused by the presence of these parasites are next colonized by bacteria (VALLADÃO et al., 2014Valladão GMR, Gallani SU, Pádua SB, Martins ML, Pilarski F. (Ciliophora) infestation on . Trichodina heterodentataProchilodus lineatus larvae: a host-parasite relationship studyParasitology 2014; 141(5): 662-669. http://dx.doi.org/10.1017/S0031182013001480. PMid:24476792
http://dx.doi.org/10.1017/S0031182013001...
), which, in turn, are responsible for opportunistic infections and accelerate the progression of the disease thus causing outbreaks of mortality.

Clinical signs are not specific and diseased fish may present darkness of the skin, whitish areas in the gills, hypoxia and flashing on the ponds or aquaria surface (PÁDUA et al., 2011aPádua SB, Martins ML, Varandas DN, Dias Neto J, Ishikawa MM, Pilarski F. Tricodinídeos: quem são e o que eles podem causar nos peixes. Panorama da Aquicultura 2011a; 121(127): 22-29.). Lethargy and erratic swimming on the body surface are frequently found in diseased fish larvae (VALLADÃO et al., 2013Valladão GMR, Pádua SB, Gallani SU, Menezes-Filho RN, Dias-Neto J, Martins ML, et al. Paratrichodina africana (Ciliophora): a pathogenic gill parasite in farmed Nile tilapia. Vet Parasitol 2013; 197(3-4): 705-710. http://dx.doi.org/10.1016/j.vetpar.2013.04.043. PMid:23731856
http://dx.doi.org/10.1016/j.vetpar.2013....
).

Flagellata

Flagellate protozoans are mainly characterized by the presence of one or more flagella for movement. The majority of them are ectoparasites while others can be found parasitizing internal organs (EIRAS, 1994Eiras JC. Elementos de ictioparasitologia. Porto: Fundação Engenheiro Antônio de Almeida; 1994.). They reproduce by longitudinal binary fission as for kinetoplastids or present a three-phased life cycle such as dinoflagellates. Amyloodinium ocellatum, Piscinoodinium pillulare, Trypanosoma, Cryptobia and Ichthyobodo are the main representatives.

Amyloodinium ocellatum Brown et Hovasse, 1946

Amyloodinium comprises dinoflagellates of varied shape depending on the life stage. The causative agent of velvet disease A. ocellatum is ubiquitous, affects marine farmed fish and may provoke important outbreak mortalities and economical losses in aquaculture systems (LEVY et al., 2007Levy MG, Poore MF, Colorni A, Noga EJ, Vandersea MW, Litaker RW. A highly specific PCR assay for detecting the fish ectoparasite . Amyloodinium ocellatumDis Aquat Organ 2007; 73(3): 219-226. http://dx.doi.org/10.3354/dao073219. PMid:17330741
http://dx.doi.org/10.3354/dao073219...
; PEREIRA et al., 2011Pereira JC, Abrantes I, Martins I, Barata J, Frias P, Pereira I. Ecological and morphological features of occurrences in cultivated gilthead seabream L.: a case study. Amyloodinium ocellatumSparus aurataAquaculture 2011; 310(3): 289-297. http://dx.doi.org/10.1016/j.aquaculture.2010.11.011.
http://dx.doi.org/10.1016/j.aquaculture....
; MOREIRA et al., 2013Moreira CB, Hashimoto GSO, Rombenso AN, Candiotto FB, Martins ML, Tsuzuki MY. Outbreak of mortality among cage-reared cobia (Rachycentron canadum) associated with parasitism. Rev Bras Parasitol Vet 2013; 22(4): 588-591. http://dx.doi.org/10.1590/S1984-29612013000400021. PMid:24473886
http://dx.doi.org/10.1590/S1984-29612013...
). The trophont stage is pear or rounded-shaped, has golden to brownish color, presents chloroplasts and is capable of feeding when adhered on the fish surface or gills by its rhizocysts. The tomont is the reproductive form (it encysts on the substrate) and the dinospore corresponds to the infective free-swimming stage with a longitudinal and transverse flagellum (NOGA, 1987Noga EJ. Propagation in cell culture of the dinoflagellate Amyloodinium, an ectoparasite of marine fishes. Science 1987; 236(4806): 1302-1304. http://dx.doi.org/10.1126/science.236.4806.1302. PMid:17770332
http://dx.doi.org/10.1126/science.236.48...
; GUERRA-SANTOS et al., 2012Guerra-Santos B, Albinati RC, Moreira ELT, Lima FW, Azevedo TM, Costa DSP, et al. Parâmetros hematológicos e alterações histopatológicas em bijupirá ( Linnaeus, 1766) com amyloodiniose. Rachycentron canadumPesq Vet Bras 2012; 32(11): 1184-1190. http://dx.doi.org/10.1590/S0100-736X2012001100019.
http://dx.doi.org/10.1590/S0100-736X2012...
; WOO & ARDELLI, 2014Woo PTK, Ardelli BF. Immunity against selected piscine flagellates. Dev Comp Immunol 2014; 43(2): 268-279. http://dx.doi.org/10.1016/j.dci.2013.07.006. PMid:23872230
http://dx.doi.org/10.1016/j.dci.2013.07....
).

Life cycle

The life cycle has no intermediate hosts and presents the following three stages: dinospore, the infective form; trophont, that develops on the host; and tomont, the reproductive stage with consecutive divisions developed on the ponds or aquaria substrates (Figure 6). Reproduction by binary fission occurs when tomonts divide repeatedly producing the free-swimming infective dinospores (FRANCIS-FLOYD & FLOYD, 2011Francis-Floyd R, Floyd MR. : an important parasite of cultured marine fish [Amyloodinium ocellatumonline]. Florida: Southern Regional Aquaculture Center (SRAC), Institute of Food and Agricultural Sciences, University of Florida; 2011 [cited 2014 Jan 30]. Available from: http://agrilifecdn.tamu.edu/fisheries/files/2013/09/SRAC-Publication-No.-4705-Amyloodinium-ocellatum-an-Important-Parasite-of-Cultured-Marine-Fish.pdf.; WOO & ARDELLI, 2014Woo PTK, Ardelli BF. Immunity against selected piscine flagellates. Dev Comp Immunol 2014; 43(2): 268-279. http://dx.doi.org/10.1016/j.dci.2013.07.006. PMid:23872230
http://dx.doi.org/10.1016/j.dci.2013.07....
).

Figure 6
Life-cycle of Amyloodinium ocellatum. Modified from Pereira et al. (2011)Pereira JC, Abrantes I, Martins I, Barata J, Frias P, Pereira I. Ecological and morphological features of occurrences in cultivated gilthead seabream L.: a case study. Amyloodinium ocellatumSparus aurataAquaculture 2011; 310(3): 289-297. http://dx.doi.org/10.1016/j.aquaculture.2010.11.011.
http://dx.doi.org/10.1016/j.aquaculture....
.

Transmission

Obligatory parasite of monoxenic life cycle, Amyloodinium has low host specificity parasitizing mainly the gills followed by skin of farmed and wild marine and estuary fishes (REED & FRANCIS-FLOYD, 1994Reed P, Francis-Floyd R. Amyloodinium infections of Marine Fish [online]. Florida: Institute of Food and Agricultural Sciences, University of Florida; 1994 [cited 2014 Jan 30]. VM90. Available from: http://fisheries.tamu.edu/files/2013/09/Amyloodinium-Infections-of-Marine-1195 Fish.pdf.; ABREU et al., 2005Abreu PC, Robaldo RB, Sampaio LA, Bianchini A, Odebrecht C. Recurrent amyloodiniosis on broodstock of the Brazilian flounder : dinospore monitoring and prophylactic measures. Paralichthys orbignyanusJ World Aquac Soc 2005; 36(1): 42-50. http://dx.doi.org/10.1111/j.1749-7345.2005.tb00129.x.
http://dx.doi.org/10.1111/j.1749-7345.20...
; WOO, 2007Woo PTK. Protective immunity in fish against protozoan diseases. Parassitologia 2007; 49(3): 185-191. PMid:18410078.). It can be transmitted by direct contact with infective dinospores present in the water. In open aquaculture systems, such as cage farming, the parasite can be easily transmitted to wild fish. In this way, Roberts-Thomson et al. (2006)Roberts-Thomson A, Barnes A, Fielder DS, Lester RJG, Adlard RD. Aerosol dispersal of the fish pathogen, . Amyloodinium ocellatumAquaculture 2006; 257(1-4): 118-123. http://dx.doi.org/10.1016/j.aquaculture.2006.02.058.
http://dx.doi.org/10.1016/j.aquaculture....
related tomonts of A. ocellatum transported by dynamic airflow system until 2 meters from the source of infection. The capacity of transport or dissemination of tomonts was proved by those authors and constitute one more possibility of infection in indoor systems.

Diagnosis

Diagnosis of A. ocellatum is made from scrapings of body surface and gills between a glass slide and a coverslip to be observed in stereomicroscope or light microscope (MONTGOMERY-BROCK et al., 2001Montgomery-Brock D, Sato VT, Brock JA, Tamaru CS. The application of hydrogen peroxide as a treatment for the ectoparasite Amyloodinium ocellatum (Brown 1931) on the Pacific threadfin Polydactylus sexfilis.J World Aquac Soc 2001; 32(2): 250-254. http://dx.doi.org/10.1111/j.1749-7345.2001.tb01103.x.
http://dx.doi.org/10.1111/j.1749-7345.20...
; ABREU et al., 2005Abreu PC, Robaldo RB, Sampaio LA, Bianchini A, Odebrecht C. Recurrent amyloodiniosis on broodstock of the Brazilian flounder : dinospore monitoring and prophylactic measures. Paralichthys orbignyanusJ World Aquac Soc 2005; 36(1): 42-50. http://dx.doi.org/10.1111/j.1749-7345.2005.tb00129.x.
http://dx.doi.org/10.1111/j.1749-7345.20...
; GUERRA-SANTOS et al., 2012Guerra-Santos B, Albinati RC, Moreira ELT, Lima FW, Azevedo TM, Costa DSP, et al. Parâmetros hematológicos e alterações histopatológicas em bijupirá ( Linnaeus, 1766) com amyloodiniose. Rachycentron canadumPesq Vet Bras 2012; 32(11): 1184-1190. http://dx.doi.org/10.1590/S0100-736X2012001100019.
http://dx.doi.org/10.1590/S0100-736X2012...
). In addition, microscopic examination of histological sections of parasitized tissue also reveals the presence of the parasite (GUERRA-SANTOS et al., 2012Guerra-Santos B, Albinati RC, Moreira ELT, Lima FW, Azevedo TM, Costa DSP, et al. Parâmetros hematológicos e alterações histopatológicas em bijupirá ( Linnaeus, 1766) com amyloodiniose. Rachycentron canadumPesq Vet Bras 2012; 32(11): 1184-1190. http://dx.doi.org/10.1590/S0100-736X2012001100019.
http://dx.doi.org/10.1590/S0100-736X2012...
), while the serological exam with the presence of specific antibodies is yet little explored (CECCHINI et al., 2001Cecchini S, Saroglia M, Terova G, Albanesi F. Detection of antibody response against Amyloodinium ocellatum (Brown, 1931) is serum of naturally infected European sea bass by an enzyme-linked immunosorbent assay (ELISA). Bull Eur Assoc Fish Pathol 2001; 21(3): 104-108.). Its fixation is made directly in the slides with 5% formalin solution or in a flask with 10% buffered formaline solution for histopathology. The most applied staining techniques are May-grunwald and Giemsa or haematoxylin and eosin (GUERRA-SANTOS, 2011Guerra-Santos B. Uso do medicamento homeopático sulphur no controle do Amyloodinium sp. em bijupirá (Rachycentron canadum Linnaeus, 1766) [Tese]. Salvador: Universidade Federal da Bahia; 2011.), as well as iodine to evidence the amyloid granules.

In dinospores, the presence of longitudinal and transverse flagella, length and width of cellular body, distribution pattern of epitheca and hypotheca plates and observation of a peduncle, rhizocysts, pores and nodules are the main characteristics for parasite identification (LANDSBERG et al., 1994Landsberg JH, Steldlnger KA, Blakesley BA, Zondervan RL. Scanning electron microscope study of dinospores of cf. , a pathogenic dinoflagellate parasite of marine fish, and comments on its relationship to the Peridiniales. AmyloodiniumocellatumDis Aquat Organ 1994; 20(1): 23-32. http://dx.doi.org/10.3354/dao020023.
http://dx.doi.org/10.3354/dao020023...
), as well as genetic information and trophont measurements (ABREU et al., 2005Abreu PC, Robaldo RB, Sampaio LA, Bianchini A, Odebrecht C. Recurrent amyloodiniosis on broodstock of the Brazilian flounder : dinospore monitoring and prophylactic measures. Paralichthys orbignyanusJ World Aquac Soc 2005; 36(1): 42-50. http://dx.doi.org/10.1111/j.1749-7345.2005.tb00129.x.
http://dx.doi.org/10.1111/j.1749-7345.20...
).

Pathogenesis and clinical signs

Tissue alteration on the host is a result of the mechanical action of trophonts during the attachment process by the ryzoids on the epithelial cells. Among the acute pathological changes an increase in mucus production and gill congestion is common (GUERRA-SANTOS et al., 2012Guerra-Santos B, Albinati RC, Moreira ELT, Lima FW, Azevedo TM, Costa DSP, et al. Parâmetros hematológicos e alterações histopatológicas em bijupirá ( Linnaeus, 1766) com amyloodiniose. Rachycentron canadumPesq Vet Bras 2012; 32(11): 1184-1190. http://dx.doi.org/10.1590/S0100-736X2012001100019.
http://dx.doi.org/10.1590/S0100-736X2012...
), frequently leading to vacuolar degeneration, epithelial rupture, hyperplasia and fusion of the secondary lamellae and necrosis in severely affected fish (CRUZ-LACIERDA et al., 2004Cruz-Lacierda ER, Maeno Y, Pineda AJT, Matey VE. Mass mortality of hatchery-reared milkfish (Chanos chanos) and mangrove red snapper (Lutjanus argentimaculatus) caused by Amyloodinium ocellatum (Dinoflagellida). Aquaculture 2004; 236(1-4): 85-94. http://dx.doi.org/10.1016/j.aquaculture.2004.02.012.
http://dx.doi.org/10.1016/j.aquaculture....
; SARAIVA et al., 2011Saraiva A, Jerónimo D, Cruz C. Amyloodinium ocellatum (Chromalveolata: Dinoflagellata) in farmed turbot. Aquaculture 2011; 320(1-2): 34-36. http://dx.doi.org/10.1016/j.aquaculture.2011.07.034.
http://dx.doi.org/10.1016/j.aquaculture....
).

Anorexia, erratic swimming, lethargy, “flashing” onto the substrates, skin depigmentation, emaciation and branchial hyperventilation are the main clinical signs (ABREU et al., 2005Abreu PC, Robaldo RB, Sampaio LA, Bianchini A, Odebrecht C. Recurrent amyloodiniosis on broodstock of the Brazilian flounder : dinospore monitoring and prophylactic measures. Paralichthys orbignyanusJ World Aquac Soc 2005; 36(1): 42-50. http://dx.doi.org/10.1111/j.1749-7345.2005.tb00129.x.
http://dx.doi.org/10.1111/j.1749-7345.20...
; FRANCIS-FLOYD & FLOYD, 2011Francis-Floyd R, Floyd MR. : an important parasite of cultured marine fish [Amyloodinium ocellatumonline]. Florida: Southern Regional Aquaculture Center (SRAC), Institute of Food and Agricultural Sciences, University of Florida; 2011 [cited 2014 Jan 30]. Available from: http://agrilifecdn.tamu.edu/fisheries/files/2013/09/SRAC-Publication-No.-4705-Amyloodinium-ocellatum-an-Important-Parasite-of-Cultured-Marine-Fish.pdf.; GUERRA-SANTOS et al., 2012Guerra-Santos B, Albinati RC, Moreira ELT, Lima FW, Azevedo TM, Costa DSP, et al. Parâmetros hematológicos e alterações histopatológicas em bijupirá ( Linnaeus, 1766) com amyloodiniose. Rachycentron canadumPesq Vet Bras 2012; 32(11): 1184-1190. http://dx.doi.org/10.1590/S0100-736X2012001100019.
http://dx.doi.org/10.1590/S0100-736X2012...
).

Piscinoodinium pillulare Lom, 1981

Causative agent of piscinoodiniasis or velvet disease has similar morphological and developmental characteristics of A. ocellatum but is considered an important parasite in freshwater fish (NOGA & LEVY, 2006Noga EJ, Levy MG. Phylum Dinoflagellata. In: Woo PTK, editor. Fish disease: diagnosis and treatment. 2nd ed. CABI International; 2006. p. 16.). With worldwide distribution, this parasite is not host-specific (MARTINS et al., 2001Martins ML, Moraes JRE, Andrade PM, Schalch SHC, Moraes FR. Piscinoodinium pillulare (Schäperclaus, 1954) Lom, 1981 (Dinoflagellida) infection in cultivated freshwater fish from the northeast region of Sao Paulo State, Brazil. Parasitological and pathological aspects. Braz J Biol 2001; 61(4): 639-644. http://dx.doi.org/10.1590/S1519-69842001000400013. PMid:12071320
http://dx.doi.org/10.1590/S1519-69842001...
) and is responsible for important sanitary problems in Brazil in native farmed fishes such as tambaqui (Colossoma macropomum), pacu (Piaractus mesopotamicus), pirapitinga (Piaractus brachypomus) besides their hybrids, piauçu (Leporinus macrocephalus) and curimbatá (Prochilodus lineatus), in hatchery of Amazonian pintado (SILVA, W.C. personal communication), in exotic channel catfish (Ictalurus punctatus) and less frequently in Nile tilapia (Oreochromis niloticus). This dinoflagellate was also reported in five ornamental freshwater fishes from an exporter´s holding facility in the Amazonas State (TAVARES-DIAS et al., 2009Tavares-Dias M, Lemos JRG, Martins ML, Jerônimo GT. Metazoan and protozoan parasites of freshwater ornamental fish from Brazil. In: Tavares-Dias M, editor. Manejo e sanidade de peixes em cultivo. Macapá: Embrapa Amapá; 2009. p. 469-494. cap. 19.). Under a microscope, it can be observed three forms of the dinoflagellate from the scraps of fish: pear-shaped, banana-shaped and the mature rounded parasite of brownish color (MARTINS et al., 2001Martins ML, Moraes JRE, Andrade PM, Schalch SHC, Moraes FR. Piscinoodinium pillulare (Schäperclaus, 1954) Lom, 1981 (Dinoflagellida) infection in cultivated freshwater fish from the northeast region of Sao Paulo State, Brazil. Parasitological and pathological aspects. Braz J Biol 2001; 61(4): 639-644. http://dx.doi.org/10.1590/S1519-69842001000400013. PMid:12071320
http://dx.doi.org/10.1590/S1519-69842001...
; FOIN, 2005Foin AA. Parasites et parasitoses des poissons d'ornement d'eau douce: aide au diagnostic et propositions de traitement [Tese]. Maisons-Alfort: École Nationale Veterinaire d' Alfort; 2005.), although in high infestations different developmental stages can be found. Special care must be taken on the mature trophonts that could be confused with I. multifiliis by an inexperienced person.

Life cycle

Its life cycle is similar to A. ocellatum (three-phased cycle), with trophont, tomont and dinospore on the host or aquatic environment. Imobile trophonts adhere to hosts by their rhizocysts (KLINGER & FRANCIS-FLOYD, 1998Klinger RE, Francis-Floyd R. Introduction to freshwater fish parasites [online]. Florida: Institute of Food and Agricultural Sciences, University of Florida; 1998. CIR716. Available from: http://edis.ifas.ufl.edu/pdffiles/FA/FA04100.pdf.; FOIN, 2005Foin AA. Parasites et parasitoses des poissons d'ornement d'eau douce: aide au diagnostic et propositions de traitement [Tese]. Maisons-Alfort: École Nationale Veterinaire d' Alfort; 2005.). Tomonts on the substrate undergo sucessive divisions in order to originate free-swimming dinospores provided with flagella.

Transmission

Piscinoodiniasis can be transmitted by contact with infested fish and dinospores from contaminated water (FOIN, 2005Foin AA. Parasites et parasitoses des poissons d'ornement d'eau douce: aide au diagnostic et propositions de traitement [Tese]. Maisons-Alfort: École Nationale Veterinaire d' Alfort; 2005.) and also by fish farming utensils with no disinfection after using them. The transport of live fish among fish farms represents an efficient way of transmission of infectious-parasitic diseases once the water can load infective or resistant stages of parasites, bacteria and viruses of sanitary importance.

Diagnosis

Traditional evaluation by scraps from the body surface and gills observed in stereomicroscope or microscope is used to diagnose piscinoodiniasis in fish. The detection of trophonts stage allows the definitive diagnosis in posterior slides fixed in methylic alcohol for 10 min and stained with diluted Giemsa (one drop per 1 mL of distilled water for 120 to 180 min) (Figure 7) or iodine staining to view the amyloid granules (MARTINS et al., 2001Martins ML, Moraes JRE, Andrade PM, Schalch SHC, Moraes FR. Piscinoodinium pillulare (Schäperclaus, 1954) Lom, 1981 (Dinoflagellida) infection in cultivated freshwater fish from the northeast region of Sao Paulo State, Brazil. Parasitological and pathological aspects. Braz J Biol 2001; 61(4): 639-644. http://dx.doi.org/10.1590/S1519-69842001000400013. PMid:12071320
http://dx.doi.org/10.1590/S1519-69842001...
). Routine histopathological analysis may also provide definitive diagnosis by viewing the trophonts attached on the gill filaments. As low parasitic infestations do not cause clinical signs and disease in fish, prophylactic measures must adopted.

Figure 7
Piscinoodinium pillulare from gill scraps of tambaqui Colossoma macropomum. Pear-shaped trophonts, rounded (arrowheads) and one dinospore (arrow). In detail, two parasites stained in Giemsa exhibiting oval to rounded nucleus with the absence of micronucleus.

Pathogenesis and clinical signs

Pathological alterations are associated with the mechanical action of the parasite after attachment to the host. In this stage trophonts insert their rhizocysts from the fixing disc and invade the cytoplasm of epithelial cells of the host (LOM & SCHUBERT, 1983Lom J, Schubert G. Ultrastructural study of (Schäperclaus, 1954) Lom, 1981 with special emphasis on its attachment to the fish host. Piscinoodinium pillulareJ Fish Dis 1983; 6(5): 411-428. http://dx.doi.org/10.1111/j.1365-2761.1983.tb00096.x.
http://dx.doi.org/10.1111/j.1365-2761.19...
).

In low parasitic levels is common to observe an increase in the mucus production and gill congestion. On the other hand, in severe infections where the disease leads to a chronical form it has been observed proliferative alterations, including fusion of the secondary lamellae, mucus cell proliferation, inflammatory cell infiltration associated with degeneration and necrosis of the epithelial cells, subepithelial oedema, haemorhages and ulcers (MARTINS et al., 2001Martins ML, Moraes JRE, Andrade PM, Schalch SHC, Moraes FR. Piscinoodinium pillulare (Schäperclaus, 1954) Lom, 1981 (Dinoflagellida) infection in cultivated freshwater fish from the northeast region of Sao Paulo State, Brazil. Parasitological and pathological aspects. Braz J Biol 2001; 61(4): 639-644. http://dx.doi.org/10.1590/S1519-69842001000400013. PMid:12071320
http://dx.doi.org/10.1590/S1519-69842001...
). Recently, a case of mixed parasitism by P. pillulare and a myxozoan parasite in farmed fish was reported by Sant'Ana et al. (2012)Sant'Ana FJF, Oliveira SL, Rabelo RE, Vulcani VAS, Silva SMG, Ferreira Junior JA. Surtos de infecção por Piscinoodinium pillulare e Henneguya spp. em pacus () criados intensivamente no Sudoeste de Goiás. Piaractus mesopotamicusPesq Vet Bras 2012; 32(2): 121-125. http://dx.doi.org/10.1590/S0100-736X2012000200005.
http://dx.doi.org/10.1590/S0100-736X2012...
, when 90% of diseased fish died in 15 days.

Proliferative disturbs in the gills associated with the interlamellar presence of parasites are responsible for hypoxia, loss of equilibrium and erratic movements (SANT'ANA et al., 2012Sant'Ana FJF, Oliveira SL, Rabelo RE, Vulcani VAS, Silva SMG, Ferreira Junior JA. Surtos de infecção por Piscinoodinium pillulare e Henneguya spp. em pacus () criados intensivamente no Sudoeste de Goiás. Piaractus mesopotamicusPesq Vet Bras 2012; 32(2): 121-125. http://dx.doi.org/10.1590/S0100-736X2012000200005.
http://dx.doi.org/10.1590/S0100-736X2012...
). Consequently, fish search for for more oxygen on the water surface or water inlet in ponds. Heavily infested fish present “flashing” on the substrate as a response of parasite's irritant action. Macroscopical observation of gills heavily infested by P. pillulare may reveal brownish coloration (Figure 8a,b). Trophonts of P. pillulare attach mainly on the primary lamellae filling all the interlamellar space between the secondary lamellae (Figure 8c), where the rhizocysts can be found attached to the host epithelial cells (Figure 8d).

Figure 8
Pathological changes in fish with piscinoodiniasis. Brownish gill of tambaqui Colossoma macropomum (a), hundreds of parasites adhered in gill filaments observed in stereomicroscope (b), as well as from histological section (c), rhizocysts penetrating host's skin (d - arrow). Bar: 100 µm (c) and 25 µm (d).

Trypanosoma Gruby, 1843

This haemoflagellate parasite is ubiquitous throughout freshwater and marine environments and may cause problems in aquaculture. Members of Trypanosoma present a slender body, elongated, cylinder-shaped with more or less thin extremities, free flagellum, undulating membrane besides the nucleus and kinetoplast (Figure 9a) and volutin granules disposed generally in the middle of the body (HUSSEIN et al., 2010Hussein ANA, Rabie SA, Mohammed NE, Hussein NM. Light and scanning electron microscopic studies of trypanosomes parasites infecting freshwater fishes in Qena Governorate, Egypt. Egypt Acad J Biolog Sci 2010; 2(1): 17-31.).

Figure 9
Trypanosoma sp. (a) observed from blood extension of tuvira Gymnotus aff. inaequilabiatus; Cryptobia sp. (b) from gill scraps of hybrid tambacu (C. macropomum x P. mesopotamicus) observed in a phase-contrast microscope and Ichthyobodo sp. (c) from skin of Nile tilapia Oreochromis niloticus previously stained with Giemsa. Figure 9 a was obtained from Pádua et al. (2011b)Pádua SB, Ishikawa MM, Satake F, Jerônimo GT, Pilarski F. First record of Trypanosoma sp. (Protozoa: Kinetoplastida) in tuvira (Gymnotus aff. inaequilabiatus) in the Pantanal wetland, Mato Grosso do Sul State, Brazil. Rev Bras Parasitol Vet 2011b; 20(1): 85-87. http://dx.doi.org/10.1590/S1984-29612011000100019. PMid:21439241
http://dx.doi.org/10.1590/S1984-29612011...
.

Life cycle

These protozoans have a heteroxenic life cycle involving an annelid Hirudinea as an intermediate host and vertebrates as definitive hosts. However, little is known about the distinct stages of their life cycle infecting South American fishes and, additionally, they are rarely found in farmed fish. During the infectious process on the host, asexual reproduction by cellular division was described in infected knifefish Gymnotus aff. inaequilabiatus (PÁDUA et al., 2011bPádua SB, Ishikawa MM, Satake F, Jerônimo GT, Pilarski F. First record of Trypanosoma sp. (Protozoa: Kinetoplastida) in tuvira (Gymnotus aff. inaequilabiatus) in the Pantanal wetland, Mato Grosso do Sul State, Brazil. Rev Bras Parasitol Vet 2011b; 20(1): 85-87. http://dx.doi.org/10.1590/S1984-29612011000100019. PMid:21439241
http://dx.doi.org/10.1590/S1984-29612011...
). Tripomastigote, epimastigote, amastigote and several dividing flagellate forms were observed in the stomach of leech Batracobdella gemmata, vector of trypanosomiasis to the loricariid catfish Hypostomus punctatus (D'AGOSTO & SERRA-FREIRE, 1993D'agosto M, Serra-Freire NM. Estádios evolutivos de tripanossomas de Hipostomus punctatus Valenciennes (Osteichthyes, Loricariidae) em infecção natural de Batracobdella gemmata Blanchard (Hirudinea, Glossiphoniidae). Rev Bras Zool 1993; 10(3): 417-426.).

Transmission

Transmission occurs during the leech parasitism on fishes (BRUNO et al., 2006Bruno DW, Nowak B, Elliott DG. Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections. Dis Aquat Organ 2006; 70(1-2): 1-36. http://dx.doi.org/10.3354/dao070001. PMid:16875388
http://dx.doi.org/10.3354/dao070001...
) and in the case of loricariids, they are found attached to the regions of the body not covered by plates or on the gill filaments inoculating the infective form (D'AGOSTO & SERRA-FREIRE, 1993D'agosto M, Serra-Freire NM. Estádios evolutivos de tripanossomas de Hipostomus punctatus Valenciennes (Osteichthyes, Loricariidae) em infecção natural de Batracobdella gemmata Blanchard (Hirudinea, Glossiphoniidae). Rev Bras Zool 1993; 10(3): 417-426.).

Diagnosis

Blood smears stained with a combination of May Grünwald-Giemsa or May Grünwald-Giemsa-Wright or only Giemsa are simple techniques for morphological characterization of the haemoflagellates. Nevertheless, this is a low-sensitive method and because of that, it is recommended to prepare fresh-mounted slides with a drop of blood between a glass and a coverslip as well as centrifuge the blood in a microhaematocrit tube for parasite concentration in order to provide high-sensitive method for diagnosis (WOO, 1969Woo PTK. The haematocrit centrifuge for the detection of trypanosomes in blood. Can J Zool 1969; 47(5): 921-923. http://dx.doi.org/10.1139/z69-150. PMid:5343381
http://dx.doi.org/10.1139/z69-150...
).

Histological sections of the liver, spleen and kidney stained with Ehrlich haematoxylin and eosin can improve the diagnosis (HUSSEIN et al., 2010Hussein ANA, Rabie SA, Mohammed NE, Hussein NM. Light and scanning electron microscopic studies of trypanosomes parasites infecting freshwater fishes in Qena Governorate, Egypt. Egypt Acad J Biolog Sci 2010; 2(1): 17-31.). Parasite identification at species level should be performed by analysis of morphological and morphometric characteristics (total length, width, nucleus length and width, distance from the nucleus centre to the posterior and anterior extremities, flagellum length and number of undulating membrane), besides application of molecular phylogenetic methods (MASLOV et al., 2001Maslov DA, Podlipaev SA, Lukes J. Phylogeny of the kinetoplastida: taxonomic problems and insights into the evolution of parasitism. Mem Inst Oswaldo Cruz 2001; 96(3): 397-402. http://dx.doi.org/10.1590/S0074-02762001000300021. PMid:11313652
http://dx.doi.org/10.1590/S0074-02762001...
; HUSSEIN et al., 2010Hussein ANA, Rabie SA, Mohammed NE, Hussein NM. Light and scanning electron microscopic studies of trypanosomes parasites infecting freshwater fishes in Qena Governorate, Egypt. Egypt Acad J Biolog Sci 2010; 2(1): 17-31.; FERREIRA & AVENANT-OLDEWAGE, 2013Ferreira ML, Avenant-Oldewage A. Notes on the occurrence of sp. (Kinetoplastida: Trypanosomatidae) in freshwater fishes from South Africa. TrypanosomaOnderstepoort J Vet Res 2013; 80(1): 529. http://dx.doi.org/10.4102/ojvr.v80i1.529. PMid:23718161
http://dx.doi.org/10.4102/ojvr.v80i1.529...
). Molecular tools for DNA detection are poorly explored in diagnosis of tryponosomiasis in fish from Brazil despite their recognized importance.

Pathogenesis and clinical signs

In case of cultured fish condition high levels of parasitism could be responsible for lethargy, gill paleness, splenomegaly, nephromegaly and altered color of the liver.

Cryptobia Leidy, 1846

These biflagellates have a triangulate to elongated body shape, a kinetoplast and a nucleus at the anterior end. Two flagella arise at the anterior end; the anterior flagellum is free while the recurrent one is attached to the body and extends beyond it as a free flagellum responsable for the movement (Figure 9b) (KUPERMAN et al., 2002Kuperman BI, Matey VE, Barlow SB. Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea. Hydrobiologia 2002; 473(1-3): 93-102. http://dx.doi.org/10.1023/A:1016577516848.
http://dx.doi.org/10.1023/A:101657751684...
; BRUNO et al., 2006Bruno DW, Nowak B, Elliott DG. Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections. Dis Aquat Organ 2006; 70(1-2): 1-36. http://dx.doi.org/10.3354/dao070001. PMid:16875388
http://dx.doi.org/10.3354/dao070001...
). It can be found parasitizing either the gills or skin of marine and freshwater fishes around the world and the majority of them are ectocommensals. However, some species are pathogenic for young fish (KUPERMAN et al., 2002Kuperman BI, Matey VE, Barlow SB. Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea. Hydrobiologia 2002; 473(1-3): 93-102. http://dx.doi.org/10.1023/A:1016577516848.
http://dx.doi.org/10.1023/A:101657751684...
; RANZANI-PAIVA et al., 2005Ranzani-Paiva MJ, Felizardo NN, Luque JL. Parasitological and hematological analysis of Nile tilapia Oreochromis niloticus Linnaeus, 1757 from Guarapiranga reservoir, São Paulo State, Brazil. Acta Sci Biol Sci 2005; 27: 231-237. http://dx.doi.org/10.4025/actascibiolsci.v27i3.1334.
http://dx.doi.org/10.4025/actascibiolsci...
).

Cryptobia branchialis Nie (Chen, 1956) has been registered in different continents (KUPERMAN et al., 2002Kuperman BI, Matey VE, Barlow SB. Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea. Hydrobiologia 2002; 473(1-3): 93-102. http://dx.doi.org/10.1023/A:1016577516848.
http://dx.doi.org/10.1023/A:101657751684...
). In Brazil, several occurrences of Cryptobia sp. exist (EIRAS et al., 2012Eiras JC, Takemoto RM, Pavanelli GC, Luque JL. Checklist of protozoan parasites of fishes from Brazil. Zootaxa 2012; 3221: 1-25.), but so far little is known about the parasite species involved. High infestations by this flagellate are frequently diagnosed on pacu, tambaqui, pirapitinga and their hybrids, as well as Nile tilapia, the hybrid surubim (Pseudoplatystoma spp.) and hybrid Amazonian pintado.

Life cycle

Cryptobia presents monoxenic life cycle, with longitudinal fission occuring on the host to posterior dettachment from the gills of the host to be free in water column (KUPERMAN et al., 2002Kuperman BI, Matey VE, Barlow SB. Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea. Hydrobiologia 2002; 473(1-3): 93-102. http://dx.doi.org/10.1023/A:1016577516848.
http://dx.doi.org/10.1023/A:101657751684...
).

Transmission

It presents horizontal transmission either by direct contact (host to host) or contaminated water with free-swimming infective forms. Fishery utensils as well as the water used in fish transport might be the source of parasitism in aquaculture facilities.

Diagnosis

Cryptobiosis can be diagnosed by observation of fresh-mounted smears of skin and gills under microscope. Later, infected smears can be stained with, for example, silver albumose (protargol) impregnation in order to provide a correct diagnosis. For fixation prior to impregnation, Hollande's fluid can be used (KOZLOFF, 2004Kozloff EN. Redescription of Leidy, 1846 (Kinetoplasta: Bodonea: Cryptobiidae), disposition of flagellates mistakenly assigned to this species, and description of a new species from a North American pulmonate snail. Cryptobia helicisActa Protozool 2004; 43: 123-132.). Observation of flagella, length and body width, nucleus diameter, cell shape and the aciculum (a bunch of microtubules to reinforce the parasite pharynx) are some of the most important diagnostic features for Cryptobia. Differently, the observation of flagella, disposition and contraction movement are fundamental information to the correct diagnosis. After that, the smears containing the parasites must be stained for posterior analysis under a light microscope (for example, protargol impregnation as recommended by Kozloff (2004)Kozloff EN. Redescription of Leidy, 1846 (Kinetoplasta: Bodonea: Cryptobiidae), disposition of flagellates mistakenly assigned to this species, and description of a new species from a North American pulmonate snail. Cryptobia helicisActa Protozool 2004; 43: 123-132.), in order to evidence the flagelli, nucleus, cell chape and the presence of aciculum.Bruno et al. (2006)Bruno DW, Nowak B, Elliott DG. Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections. Dis Aquat Organ 2006; 70(1-2): 1-36. http://dx.doi.org/10.3354/dao070001. PMid:16875388
http://dx.doi.org/10.3354/dao070001...
used the techniques of Feulgen or Giemsa staining for observation of the kinetoplast.

A combination of fixative solution containing methylic alcohol for 10 min and later staining with diluted Giemsa (one drop per 1 mL of distilled water, for 120 to 180 min) affords a detailed observation of parasite morphology, although flagella are poorly evidenced. The length and body width, flagelli length, nucleus diameter and position as well as the length and position of kinetoplast, and the cell shape are used for identification at specific level (PUTZ, 1972Putz RE. Crybtobia cataractae sp. n. (Kinetoplastida: Cryptobiidae), a hemoflagellate of some cyprinid fishes of West Virginia. J Helminthol Soc Wash 1972; 39(1): 18-22.; KOZLOFF, 2004Kozloff EN. Redescription of Leidy, 1846 (Kinetoplasta: Bodonea: Cryptobiidae), disposition of flagellates mistakenly assigned to this species, and description of a new species from a North American pulmonate snail. Cryptobia helicisActa Protozool 2004; 43: 123-132.).

Differential diagnosis

The diagnosis of Cryptobia sp. in the gills must be made carefully due to its similarity with Ichthyobodo spp. (both have two flagella). Nevertheless, Ichthyobodo is mainly found on the skin. In order to differentiate both parasites, fresh-mounted microscope slides with special aware of flagella's arrangement are useful. Cryptobia sp. presents flagella with rapid movements while in Ichthyobodo the flagella move in circles.

Pathogenesis and clinical signs

Its pathogenicity is controverted and the findings of Kuperman et al. (2002)Kuperman BI, Matey VE, Barlow SB. Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea. Hydrobiologia 2002; 473(1-3): 93-102. http://dx.doi.org/10.1023/A:1016577516848.
http://dx.doi.org/10.1023/A:101657751684...
suggest that the parasite neither invades the host cells during the attachment process nor cause pathological alterations. However, observations from young tilapia highly infested by the parasite showed a direct relation with an increase in the gill mucus production, gill filaments oedema and reduced respiratory lamellae (KUPERMAN et al., 2002Kuperman BI, Matey VE, Barlow SB. Flagellate Cryptobia branchialis (Bodonida: Kinetoplastida), ectoparasite of tilapia from the Salton Sea. Hydrobiologia 2002; 473(1-3): 93-102. http://dx.doi.org/10.1023/A:1016577516848.
http://dx.doi.org/10.1023/A:101657751684...
).

Ichthyobodo Pinto, 1928

Causative agent of ichthyobodiasis, these obligatory parasites are small biflagellated kinetoplastids found in skin, fins and gills of wild and farmed marine and freshwater fishes from temperate and tropical waters (ROBERTSON, 1985Robertson DA. A review of . In: Ichthyobodo necator (Henneguy, 1883) an important and damaging fish parasiteMuir JF, Roberts RJ, editors. Recent advances in aquaculture. Springer; 1985. p. 1-30.. http://dx.doi.org/10.1007/978-1-4684-8736-7_1.
http://dx.doi.org/10.1007/978-1-4684-873...
; LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.; TODAL et al., 2004Todal JA, Karlsbakk E, Isaksen TE, Plarre H, Urawa S, Mouton A, et al. Ichthyobodo necator (Kinetoplastida) - a complex of sibling species. Dis Aquat Organ 2004; 58(1): 9-16. http://dx.doi.org/10.3354/dao058009. PMid:15038446
http://dx.doi.org/10.3354/dao058009...
). Widely distributed, Ichthyobodo necator (Henneguy, 1883) was registered in different fish species from Brazil (EIRAS et al., 2012Eiras JC, Takemoto RM, Pavanelli GC, Luque JL. Checklist of protozoan parasites of fishes from Brazil. Zootaxa 2012; 3221: 1-25.). Similar to Cryptobia, it is pear-shaped (Figure 9c) and when fixed on the host it shows circling or zigzag movements (LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.).

Life cycle

Ichthyobodo presents monoxenic life cycle, asexual reproduction in which occurs the longitudinal fission of the parasite cell (ROBERTSON, 1985Robertson DA. A review of . In: Ichthyobodo necator (Henneguy, 1883) an important and damaging fish parasiteMuir JF, Roberts RJ, editors. Recent advances in aquaculture. Springer; 1985. p. 1-30.. http://dx.doi.org/10.1007/978-1-4684-8736-7_1.
http://dx.doi.org/10.1007/978-1-4684-873...
). Cells containing two pairs of flagella can be found moments before binary fission (LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.).

Transmission

The parasitosis is horizontally transmitted by direct contact among diseased and healthy fish. Free-swimming infective parasites are responsible for disease dissemination as well as contaminated fishery utensils.

Diagnosis

Fresh-mounted smears of skin and gills viewed under microscope constitute the most employed technique for routine diagnosis. Cryptobia is more or less pyriform and has two unequal flagella extending posterior-laterally, while Ichthyobodo is more elongate-shaped and has two flagella, one posterior and one recurrent which is attached to the body forming the posterior free flagellum.

Smears previously fixed in methylic alcohol and stained with haematoxylin, Feulgen or Giemsa can be used for microscopical analysis (LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.; TODAL et al., 2004Todal JA, Karlsbakk E, Isaksen TE, Plarre H, Urawa S, Mouton A, et al. Ichthyobodo necator (Kinetoplastida) - a complex of sibling species. Dis Aquat Organ 2004; 58(1): 9-16. http://dx.doi.org/10.3354/dao058009. PMid:15038446
http://dx.doi.org/10.3354/dao058009...
; ISAKSEN, 2013Isaksen TE. infections on farmed and wild fish [IchthyobodoTese]. Bergen: University of Bergen; 2013.). The distribution pattern and kinetoplast morphology are useful information for specific identification level (MOREIRA et al., 2004Moreira D, López-García P, Vickerman K. An updated view of kinetoplastid phylogeny using environmental sequences and a closer outgroup: proposal for a new classification of the class Kinetoplastea. Int J Syst Evol Microbiol 2004; 54(Pt 5): 1861-1875. http://dx.doi.org/10.1099/ijs.0.63081-0. PMid:15388756
http://dx.doi.org/10.1099/ijs.0.63081-0...
).

Histopathological analysis also presents an efficient tool for diagnosis of ichthyobodiasis (URAWA et al., 1991Urawa S, Ueki N, Nakai T, Yamasaki H. High mortality of cultured juvenile Japanese flounder, (Temminek & Sehlegel), caused by the parasitic flagellate sp. Paralichthys olivaceusIchthyobodoJ Fish Dis 1991; 14(4): 489-494. http://dx.doi.org/10.1111/j.1365-2761.1991.tb00602.x.
http://dx.doi.org/10.1111/j.1365-2761.19...
; BRUNO et al., 2006Bruno DW, Nowak B, Elliott DG. Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections. Dis Aquat Organ 2006; 70(1-2): 1-36. http://dx.doi.org/10.3354/dao070001. PMid:16875388
http://dx.doi.org/10.3354/dao070001...
), but constitutes a non-sensitive technique in low parasitism intensity. Molecular methods of DNA extraction are more efficient (ISAKSEN et al., 2012Isaksen TE, Karlsbakk E, Repstad O, Nylund A. Molecular tools for the detection and identification of spp. (Kinetoplastida), important fish parasites. IchthyobodoParasitol Int 2012; 61(4): 675-683. http://dx.doi.org/10.1016/j.parint.2012.07.006. PMid:22824336
http://dx.doi.org/10.1016/j.parint.2012....
) but less used and unpractical as a routine diagnostic method.

Pathogenesis and clinical signs

In Brazil this parasite does not cause significant mortality and in most cases low intensity infections were recorded in larvae and young fishes. Among the pathological alterations, it has been observed destruction of the epithelial cells due to parasite attachment (LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.), as well as degenerative and proliferative epithelial alterations with reduced mucus production (URAWA et al., 1991Urawa S, Ueki N, Nakai T, Yamasaki H. High mortality of cultured juvenile Japanese flounder, (Temminek & Sehlegel), caused by the parasitic flagellate sp. Paralichthys olivaceusIchthyobodoJ Fish Dis 1991; 14(4): 489-494. http://dx.doi.org/10.1111/j.1365-2761.1991.tb00602.x.
http://dx.doi.org/10.1111/j.1365-2761.19...
). Clinical signs are non-specific indicating respiratory difficulty and altered skin and gill color (URAWA et al., 1991Urawa S, Ueki N, Nakai T, Yamasaki H. High mortality of cultured juvenile Japanese flounder, (Temminek & Sehlegel), caused by the parasitic flagellate sp. Paralichthys olivaceusIchthyobodoJ Fish Dis 1991; 14(4): 489-494. http://dx.doi.org/10.1111/j.1365-2761.1991.tb00602.x.
http://dx.doi.org/10.1111/j.1365-2761.19...
; LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.).

Other Parasites

Entamoeba Casagrandi et Barbagallo, 1897

Entamoeba belongs to the amoebae group, moves and feeds by using extensions of its cytoplasm (pseudopods) and reproduces by binary fission. According to Ponce-Gordo and Martínez-Díaz (2010)Ponce-Gordo F, Martínez-Díaz RA. Taxonomía y filogenia del género .: una revisión histórica. EntamoebaRev Ibero-Latinoam Parasitol 2010; 69(1): 5-37., the main morphological characteristic, which distinguishes Entamoeba from other genera, is the morphology of the nucleus. When cysts are mature, the different species of Entamoeba present a characteristic number of nuclei, which can be 1 belonging to bovis group, 4 to histolytica group, 8 to coli group and as gingivalis group when the number of cysts is not known. Analysis of Entamoeba can be made with histological sections of the affected organ, after fixing in 10% formalin solution and staining with iron haematoxylin or Heidenhain-Mallory Azan (ORIAS & NOBLE, 1971Orias JD, Noble ER. sp. n. and other parasites from a North Atlantic fish. Entamoeba nezumiaJ Parasitol 1971; 57(5): 945-947. http://dx.doi.org/10.2307/3277840. PMid:5133900
http://dx.doi.org/10.2307/3277840...
). Smears of the intestine viewed under microscope with a drop of saline solution or iodine (BULLOCK, 1966Bullock WL. Entamoeba gadi sp. n. from the rectum of the pollock, (L., 1758), with some observations on its cytochemistry. Pollachius virensJ Parasitol 1966; 52(4): 679-684. http://dx.doi.org/10.2307/3276429. PMid:4226197
http://dx.doi.org/10.2307/3276429...
) are used. Until the present, only Entamoeba pimelodi found in the intestine of yellow catfish Pimelodus clarias was observed parasitizing freshwater fishes in Brazil (CUNHA & PENIDO, 1926Cunha AM, Penido JCN. n. sp., parasite d'un poisson d'eau douce. Entamoeba pimelodiC R Soc Biol Paris 1926; 95: 1010-1011.).

Balantidium Clarapède et Lachmann, 1858

Members of Balantidium are unicellular and endocommensal parasites in the intestine of their hosts. Some of these ciliates appear to be host-specific, whereas others are generalists (BRUNO et al., 2006Bruno DW, Nowak B, Elliott DG. Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections. Dis Aquat Organ 2006; 70(1-2): 1-36. http://dx.doi.org/10.3354/dao070001. PMid:16875388
http://dx.doi.org/10.3354/dao070001...
). Two developmental stages are present: the trophozoyte, flexible and covered by a row of longitudinal cilia reaching 40 to 140 µm and the cyst, ovoid and measuring 40 to 60 µm in diameter (BRUNO et al., 2006Bruno DW, Nowak B, Elliott DG. Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections. Dis Aquat Organ 2006; 70(1-2): 1-36. http://dx.doi.org/10.3354/dao070001. PMid:16875388
http://dx.doi.org/10.3354/dao070001...
). Its horizontal transmission is made by oral and fecal via. In Brazil, Balantidium piscicola Geza, 1913 was found parasitizing caranha Piaractus brachypomus and mandi catfish Synodontis clarias (JUNIOR, 1913Junior GE. Ueber Organisationsverhältnisse von (Daday). Nyctotherus piscicolaArch Protistenkd 1913; 29: 364-386.; PINTO, 1928Pinto C. Myxosporideos e outros protozoários intestinais de peixes observados na América do Sul. Arch Inst Biol 1928; 1: 102-136.).

Theileria Bettencourt, Franco et Borges, 1907

Members of Theileria spp. undergo exoerythrocytic merogony in the lymphocytes, erythroblasts and other cells of the internal organs, and this is followed by invasion of the erythrocytes by the merozoites, which may or may not reproduce (LAINSON, 2007Lainson R. Theileria electrophori n.sp., a parasite of the electric eel (Osteichthyes: Cypriniformes: Gymnotidae) from Amazonian Brazil. Electrophorus electricusMem Inst Oswaldo Cruz 2007; 102(2): 155-157. http://dx.doi.org/10.1590/S0074-02762007000200005. PMid:17426878
http://dx.doi.org/10.1590/S0074-02762007...
). When division takes place, the parasite produces no more than four daughter cells (LEVINE, 1988 apudLAINSON, 2007Lainson R. Theileria electrophori n.sp., a parasite of the electric eel (Osteichthyes: Cypriniformes: Gymnotidae) from Amazonian Brazil. Electrophorus electricusMem Inst Oswaldo Cruz 2007; 102(2): 155-157. http://dx.doi.org/10.1590/S0074-02762007000200005. PMid:17426878
http://dx.doi.org/10.1590/S0074-02762007...
). The elongated bacillary or bayonet forms are characteristic for Theileria (BARNETT, 2012Barnett SF. . In: TheileriaKreir J (Ed.). Parasitic Protozoa: Babesia, Theileria, Myxosporida, Microsporida, Bartonellaceae, Anaplasmataceae, Ehrlichia, and Pneumocystis. Elsevier; 2012. p. 77-92. vol. 4.). In Brazil, Theileria electrophori is, so far, the only fish Theileria species reported from the viscera of a single juvenile of the electric eel Electrophorus electricus from the State of Pará, North Brazil. Air-dried smears of infected organs are fixed in absolute methyl alcohol and stained by Giemsa's method may be useful for accurate diagnosis of Theileria.

Calyptospora Overstreet, Hawkins et Fournie, 1984

These intracellular protozoan parasites are found in the liver and intestine of their hosts and have a heteroxenic life cycle transmitted by an infected crustacean ingested by a fish. In fish host, the oocysts (elliptical, ovoid or pear-shaped) present 4 sporocysts covered by a thin veil fixed by the presence of wall projections named sporopodia. Moreover, it presents a suture on the wall that do not divide the cell into two valves (EIRAS, 2013bEiras JC. Apicomplexa. In: Pavanelli GC, Takemoto RM, Eiras JC, editors. Parasitologia de peixes de água doce do Brasil. Maringá: Eduem; 2013b. p. 217-231.).

Three species of Calyptospora were described parasitizing hepatocytes of Brazilian fishes: C. serrasalmi Cheung, Nigrelli and Ruggieri, 1986 from piranha Serrasalmus niger (CHEUNG et al., 1986Cheung PJ, Nigrelli RF, Ruggieri GD. Calyptospora serrasalmi sp. nov. (Coccidia: Calyptosporidae) from liver of the black piranha, Serrasalmus niger Schomburgk. J Aquaricult Aquat Sci 1986; 4(3): 54-57.); C. tucunarensis Békési and Molnár, 1991 from tucunaré Cichla ocellaris (BÉKÉSI & MOLNÁR, 1991Békési L, Molnár K. n. sp. (Apicomplexa: Sporozoea) from liver of tucunare in Brazil. Calyptospora tucunarensisCichla ocellarisSyst Parasitol 1991; 18(2): 127-132. http://dx.doi.org/10.1007/BF00017665.
http://dx.doi.org/10.1007/BF00017665...
) and C. spinosa Azevedo, Matos and Matos, 1993 from joaninha Crenicichla lepidota (AZEVEDO et al. 1993Azevedo C, Matos P, Matos E. Morphological data of spinosa n. sp. (Apicomplexa, Calyptosporidae) parasite of . CalyptosporaCrenicichla lepidota HECKEL, 1840 (Teleostei) from Amazon riverEur J Protistol 1993; 29(2): 171-175. http://dx.doi.org/10.1016/S0932-4739(11)80270-1. PMid:23195539
http://dx.doi.org/10.1016/S0932-4739(11)...
). Coccidiosis by Calyptospora was found by Bonar et al. (2006)Bonar CJ, Poynton SL, Schulman FY, Rietcheck RL, Garner MM. Hepatic Calyptospora sp. (Apicomplexa) infection in a wild-born, aquarium-held clutch of juvenile arapaima (Osteoglossidae). Arapaima gigasDis Aquat Organ 2006; 70(1-2): 81-92. http://dx.doi.org/10.3354/dao070081. PMid:16875394
http://dx.doi.org/10.3354/dao070081...
in Arapaima gigas exported to the United States. Albuquerque & Carvalho Brasil-Sato (2010)Albuquerque MC, Carvalho Brasil-Sato M. First report of sp. (Apicomplexa, Calyptosporidae) in forage characid fish from the Três Marias Reservoir, São Francisco Basin, Brazil. CalyptosporaEur J Protistol 2010; 46(2): 150-152. http://dx.doi.org/10.1016/j.ejop.2009.12.005. PMid:20163938
http://dx.doi.org/10.1016/j.ejop.2009.12...
registered Calyptospora sp. in the liver and intestines of piaba-facão Triportheus guentheri and in the intestines of piaba Tetragonopterus chalceus. Additionally, Silva et al. (2012)Silva MC, São Clemente SC, Picanço Júnior JA, Silva MVO, Matos ER. . Calyptospora sp. in Brachyplatystomavaillantii trapped at the Vigia, State of Pará, BrazilRev Bras Parasitol Vet 2012; 21(2): 176-178. PMid:22832762. have reported in piramutaba Brachyplatystoma vaillantii and Santiago et al. (2012)Santiago H, Corrêa JL, Tortelly R, Menezes RC, Matos P, Matos E. Morphology and histopathology of Calyptospora sp. (Apicomplexa: Calyptosporidae) in speckled peacock bass, Humboldt, 1821 (Perciformes: Cichlidae), from the Marajó-Açu River, Marajó Island, Brazil. Cichla temensisParasitol Res 2012; 110(6): 2569-2572. http://dx.doi.org/10.1007/s00436-011-2770-0. PMid:22200958
http://dx.doi.org/10.1007/s00436-011-277...
in tucunaré Cichla temensis. Fresh-mounted smears can be observed in light microscope. For histological analysis the infected organs are fixed in Davidson or 10% formalin solution and stained with hematoxylina and eosin (BÉKÉSI & MOLNÁR, 1991Békési L, Molnár K. n. sp. (Apicomplexa: Sporozoea) from liver of tucunare in Brazil. Calyptospora tucunarensisCichla ocellarisSyst Parasitol 1991; 18(2): 127-132. http://dx.doi.org/10.1007/BF00017665.
http://dx.doi.org/10.1007/BF00017665...
; SANTIAGO et al., 2012Santiago H, Corrêa JL, Tortelly R, Menezes RC, Matos P, Matos E. Morphology and histopathology of Calyptospora sp. (Apicomplexa: Calyptosporidae) in speckled peacock bass, Humboldt, 1821 (Perciformes: Cichlidae), from the Marajó-Açu River, Marajó Island, Brazil. Cichla temensisParasitol Res 2012; 110(6): 2569-2572. http://dx.doi.org/10.1007/s00436-011-2770-0. PMid:22200958
http://dx.doi.org/10.1007/s00436-011-277...
). The oocysts can be studied by transmission electronic microscopy (AZEVEDO et al., 1993Azevedo C, Matos P, Matos E. Morphological data of spinosa n. sp. (Apicomplexa, Calyptosporidae) parasite of . CalyptosporaCrenicichla lepidota HECKEL, 1840 (Teleostei) from Amazon riverEur J Protistol 1993; 29(2): 171-175. http://dx.doi.org/10.1016/S0932-4739(11)80270-1. PMid:23195539
http://dx.doi.org/10.1016/S0932-4739(11)...
).

Haemogregarina Danylewsky, 1885, Cyrilia Lainson, 1981 and Desseria Siddall, 1995

These groups comprise several blood protozoan parasites (DINIZ et al., 2002Diniz JA, Silva EO, Souza W, Lainson R. Some observations on the fine structure of trophozoites of the haemogregarine (Adeleina: Haemogregarinidae) in erythrocytes of the fish (Synbranchidae). Cyrilia lignieresiSynbranchus marmoratusParasitol Res 2002; 88(7): 593-597. http://dx.doi.org/10.1007/s00436-002-0603-x. PMid:12107449
http://dx.doi.org/10.1007/s00436-002-060...
; EIRAS, 2013bEiras JC. Apicomplexa. In: Pavanelli GC, Takemoto RM, Eiras JC, editors. Parasitologia de peixes de água doce do Brasil. Maringá: Eduem; 2013b. p. 217-231.). They are commonly found in both erythrocytes and leukocytes of marine fishes (DAVIES, 1995Davies AJ. The biology of fish haemogregarines. Adv Parasitol 1995; 36: 117-203. http://dx.doi.org/10.1016/S0065-308X(08)60491-1.
http://dx.doi.org/10.1016/S0065-308X(08)...
), except for Cyrilia spp., which parasitize only the erythrocytes of freshwater fish. According to Davies et al. (2008)Davies AJ, Amado LL, Cook RT, Bianchini A, Eiras JC. Potential environmental and host gender influences on prevalence of (Adeleorina:Haemogregarinidae) and suspected . Haemogregarina platessaeHaemohormidium terraenovae (incertae sedis) in Brazilian flounder from the Patos Lagoon Estuary, southern BrazilFolia Parasitol 2008; 55(3): 161-170. http://dx.doi.org/10.14411/fp.2008.023. PMid:19202674
http://dx.doi.org/10.14411/fp.2008.023...
, although most fish haemogregarine life cycles are unknown, fishes are likely to act as intermediate hosts, while leeches or gnathiid isopods are probably the definitive ones.

Cyrilia and Haemogregarina are both characterized by the presence of an intra-erythrocytic merogony phase in fish host, while in Desseria spp. this stage is not found (DAVIES et al., 2004Davies AJ, Smit NJ, Hayes PM, Seddon AM, Wertheim D. Haemogregarina bigemina (Protozoa: Apicomplexa: Adeleorina) - past, present and future. Folia Parasitol 2004; 51(2-3): 99-108. http://dx.doi.org/10.14411/fp.2004.015. PMid:15357389
http://dx.doi.org/10.14411/fp.2004.015...
). In Brazil, Haemogregarina was found parasitizing lungfish piramboia Lepidosiren paradoxa, mullet Mugil liza and sole Paralichthys orbignyanus (JEPPS, 1927Jepps MW. Note on a haemogregarine in . Lepidosiren paradoxaParasitology 1927; 19(3): 285-287. http://dx.doi.org/10.1017/S0031182000005722.
http://dx.doi.org/10.1017/S0031182000005...
; EIRAS et al., 1995Eiras JC, Ranzani-Paiva MJT, Davies AJ. Observations on Haemogregarina mugili (Apicomplexa) and Trypanosoma froesi (Sarcomastigophora) from the blood of Günther, 1880 (Pisces: Mugilidae) in Brazil. Mugil platanusRes Rev Parasitol 1995; 55(3): 173-176.; DAVIES et al., 2008Davies AJ, Amado LL, Cook RT, Bianchini A, Eiras JC. Potential environmental and host gender influences on prevalence of (Adeleorina:Haemogregarinidae) and suspected . Haemogregarina platessaeHaemohormidium terraenovae (incertae sedis) in Brazilian flounder from the Patos Lagoon Estuary, southern BrazilFolia Parasitol 2008; 55(3): 161-170. http://dx.doi.org/10.14411/fp.2008.023. PMid:19202674
http://dx.doi.org/10.14411/fp.2008.023...
), while Cyrilia lignieresi Laveran, 1906 was found in the blood of marbled swamp eel Synbranchus marmoratus (LAINSON 1981Lainson R. On (Neiva and Pinto, 1926) gen. nov. (Haemogregarinidae) and Trypanosoma bourouli Neiva and Pinto, in the fish . In: Cyrilia gomesiSynbranchus marmoratus: simultaneous transmission by the leech HaementerialutziCanning EU, editor. Parasitological topics. London: Society of Protozoologists; 1981. p. 150-158. Special Publication vol. 1., 1992Lainson R. A protozoologist in Amazonia: neglected parasites, with particular reference to members of the Coccidia (Protozoa, Apicomplexa). Cienc Cult 1992; 44(2-3): 81-93.; DINIZ et al. 2002Diniz JA, Silva EO, Souza W, Lainson R. Some observations on the fine structure of trophozoites of the haemogregarine (Adeleina: Haemogregarinidae) in erythrocytes of the fish (Synbranchidae). Cyrilia lignieresiSynbranchus marmoratusParasitol Res 2002; 88(7): 593-597. http://dx.doi.org/10.1007/s00436-002-0603-x. PMid:12107449
http://dx.doi.org/10.1007/s00436-002-060...
).

Eimeria Schneider, 1875

Frequently found parasitizing the intestinal wall of vertebrates including fish (MOLNÁR et al., 2012Molnár K, Ostoros G, Dunams-Morel D, Rosenthal BM. that infect fish are diverse and are related to, but distinct from, those that infect terrestrial vertebrates. EimeriaInfect Genet Evol 2012; 12(8): 1810-1815. http://dx.doi.org/10.1016/j.meegid.2012.06.017. PMid:22824419
http://dx.doi.org/10.1016/j.meegid.2012....
). This group was originally confused with Calyptospora but according to Molnár (2006)Molnár K. Phylum Apicomplexa. In: Woo PTK, editor. Fish diseases and disorders: protozoan and metazoan infections. Cambridge: CABI Publishing; 2006. p. 183-204., differently from the latter, Eimeria does not have intermediate hosts in its life cycle. Sporocysts can be recognized by the presence of a “stieda” body (the opening of the sporocyst from where sporozoites goes out) (MOLNÁR et al., 2012Molnár K, Ostoros G, Dunams-Morel D, Rosenthal BM. that infect fish are diverse and are related to, but distinct from, those that infect terrestrial vertebrates. EimeriaInfect Genet Evol 2012; 12(8): 1810-1815. http://dx.doi.org/10.1016/j.meegid.2012.06.017. PMid:22824419
http://dx.doi.org/10.1016/j.meegid.2012....
). According to Eiras (2013b)Eiras JC. Apicomplexa. In: Pavanelli GC, Takemoto RM, Eiras JC, editors. Parasitologia de peixes de água doce do Brasil. Maringá: Eduem; 2013b. p. 217-231. the degree of the lesions are variable. As for example, E. sardinae cause testis deformation and castration in Sardina pilchardus. Up to now, Eimeria sp. was only observed in the intestine of lungfish L. paradoxa in the State of Pará, North Brazil by Lainson & Ribeiro (2006)Lainson R, Ribeiro L. n.sp. (Apicomplexa:Eimeriidae) of the South American lungfish Lepidosiren paradoxa (Osteichthyes:Dipnoi) from Amazonian Brazil. Eimeria lepidosirenisMem Inst Oswaldo Cruz 2006; 101(3): 327-329. http://dx.doi.org/10.1590/S0074-02762006000300018. PMid:16862331
http://dx.doi.org/10.1590/S0074-02762006...
.

Nyctotherus Leidy, 1849 and Rhynchodinium paradoxum Cunha et Penido, 1927

Ciliated Nyctotherus is present in digestive tract of insects, amphibians and fishes. The trophozoite has around 200 µm, is oval and provided with cilia arranged in longitudinal rows and a cytostome located in the middle of the body (THATCHER, 2006Thatcher VE. Amazon fish parasites. Sofia: Pensoft Publishers; 2006.; EIRAS, 2013aEiras JC. Ciliophora. In: Pavanelli GC, Takemoto RM, Eiras JC, editors. Parasitologia de peixes de água doce do Brasil. Maringá: Eduem; 2013a. p. 233-245.); cytopharynx - a duct that communicates cytostome with the interiorof the parasite- with undulating membrane. Great macronucleus, triangular and located at the anterior end of the body.

According to Thatcher (2006)Thatcher VE. Amazon fish parasites. Sofia: Pensoft Publishers; 2006., these protozoans do not cause severe pathology and can be considered as endocommensals (EIRAS, 2013aEiras JC. Ciliophora. In: Pavanelli GC, Takemoto RM, Eiras JC, editors. Parasitologia de peixes de água doce do Brasil. Maringá: Eduem; 2013a. p. 233-245.). In Brazil, it was observed in P. brachypomus, Acestrorhampus sp. and P. clarias (PINTO, 1928Pinto C. Myxosporideos e outros protozoários intestinais de peixes observados na América do Sul. Arch Inst Biol 1928; 1: 102-136.).

Rhynchodinium paradoxum shows elongated and cylinder-shaped body, bean-shaped macronucleus located at the anterior end of the body. Cilia are long but absent on the posterior end of the body (THATCHER, 2006Thatcher VE. Amazon fish parasites. Sofia: Pensoft Publishers; 2006.). This protozoan was found in the intestine of granulated catfish abotoado Pterodoras granulosus (CUNHA & PENIDO, 1927Cunha AM, Penido JCN. n. gen. protozoaire parasite dúm poissson déau douce. Rynchodinium paradoxumC R Soc Biol Paris 1927; 97(36): 1793-1794.).

Tetrahymena Furgason, 1940

Tetrahymena is considered an important pathogenic agent to ornamental freshwater fish where it may cause severe mortalities (BRUNO et al., 2006Bruno DW, Nowak B, Elliott DG. Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections. Dis Aquat Organ 2006; 70(1-2): 1-36. http://dx.doi.org/10.3354/dao070001. PMid:16875388
http://dx.doi.org/10.3354/dao070001...
) (Figure 10). This protozoan has a pear-shaped body covered by a row of cilia, besides macronucleus and micronucleus. According to Bruno et al. (2006)Bruno DW, Nowak B, Elliott DG. Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections. Dis Aquat Organ 2006; 70(1-2): 1-36. http://dx.doi.org/10.3354/dao070001. PMid:16875388
http://dx.doi.org/10.3354/dao070001...
, these ciliates do not form cysts and studies suggest that Tetrahymena penetrates the host epithelium (especially where there are wounds) reaching the blood and parasitizing the gills, kidney, eyes and brain (EIRAS, 2013aEiras JC. Ciliophora. In: Pavanelli GC, Takemoto RM, Eiras JC, editors. Parasitologia de peixes de água doce do Brasil. Maringá: Eduem; 2013a. p. 233-245.).

Figure 10
Tetrahymena sp. from the skin of guppy (Poecilia sp.) observed in light microscope at 40 x magnification.

In Brazil, this parasite was found parasitizing the butterfly fish Carnegiella strigata (TAVARES-DIAS et al., 2010Tavares-Dias M, Lemos JRG, Martins ML. Parasitic fauna of eight species of ornamental freshwater fish species from the middle Negro River in the Brazilian Amazon Region. Rev Bras Parasitol Vet 2010; 19(2): 103-107. http://dx.doi.org/10.4322/rbpv.01902007. PMid:20624347
http://dx.doi.org/10.4322/rbpv.01902007...
), and can be commonly found parasitizing guppies. Cutaneous infestation by Tetrahymena causes scaleness, whitish lesions on the skin and opportunistic secondary infection by bacteria.

Hexamita Dujardin, 1838

Members of Hexamita possess oval body, bilateral symmetry, provided by four pairs of flagella, three anterior for locomotion and one posterior, besides two spherical nuclei located at the anterior end (ROTHENBACHER & BOHL, 1975Rothenbacher HJ, Bohl M. Hexamita: pathogenicity and therapy. Fisch und Umwelt 1975; 1: 13-23. [cited 2014 Jan 23]. Available from: http://www.dfo-mpo.gc.ca/Library/115785.pdf.; FOIN, 2005Foin AA. Parasites et parasitoses des poissons d'ornement d'eau douce: aide au diagnostic et propositions de traitement [Tese]. Maisons-Alfort: École Nationale Veterinaire d' Alfort; 2005.). They are opportunistic endoparasites of the intestinal tract of wild and farmed marine, freshwater and ornamental fishes in tropical and temperate waters (ROTHENBACHER & BOHL, 1975Rothenbacher HJ, Bohl M. Hexamita: pathogenicity and therapy. Fisch und Umwelt 1975; 1: 13-23. [cited 2014 Jan 23]. Available from: http://www.dfo-mpo.gc.ca/Library/115785.pdf.; EIRAS, 1994Eiras JC. Elementos de ictioparasitologia. Porto: Fundação Engenheiro Antônio de Almeida; 1994.; FRANCIS-FLOYD & REED, 1994Francis-Floyd R, Reed P. Management of in ornamental cichlids [Hexamitaonline]. Florida: Institute of Food and Agricultural Sciences, University of Florida; 1994 [cited 2014 Jan 23]. Available from: http://www.extension.org/mediawiki/files/0/02/Management_of_hexamita_in_ornamental_cichlids.pdf.; FOIN, 2005Foin AA. Parasites et parasitoses des poissons d'ornement d'eau douce: aide au diagnostic et propositions de traitement [Tese]. Maisons-Alfort: École Nationale Veterinaire d' Alfort; 2005.). It can also be found in the swimbladder, liver, spleen, blood, kidney, and heart of hosts (FOIN, 2005Foin AA. Parasites et parasitoses des poissons d'ornement d'eau douce: aide au diagnostic et propositions de traitement [Tese]. Maisons-Alfort: École Nationale Veterinaire d' Alfort; 2005.). After analysis of 7,139 fish specimens between the years 1987 and 1990, Békési (1992)Békési L. Evaluation of data on ichthyopathological analyses in the Brazilian Northeast. Cienc Cult 1992; 44(6): 400-403. registered the presence of Hexamita in Brazil from the intestine of Prochilodus brevis (syn. P. cearensis). These parasites present a monoxenic life cycle; the pear-shaped trophozoites change to spherical before cellular division (WOO, 2006Woo PTK. Diplomonadida (Phylum Parabasalia) and Kinetoplastea (Phylum Euglenozoa). In: Woo PTK, editor. Fish diseases and disorders. 2nd ed. Wallingford: CABI Publishing, 2006. p. 116-153. cap. 4. vol. 1. Protozoan and Metazoan infections. http://dx.doi.org/10.1079/9780851990156.0046.
http://dx.doi.org/10.1079/9780851990156....
). They can be horizontaly transmitted by the releasing of trophozoites and oocysts into the water from the fish feces that will be ingested by other hosts (LOM & DYKOVÁ, 1992Lom J, Dyková I. Protozoan parasites of fishes. Amsterdam: Elsevier Science; 1992. 315 p. vol. 26. Developments in Aquaculture and Fisheries Science.; FOIN, 2005Foin AA. Parasites et parasitoses des poissons d'ornement d'eau douce: aide au diagnostic et propositions de traitement [Tese]. Maisons-Alfort: École Nationale Veterinaire d' Alfort; 2005.). The diagnosis can be made by analysing the feces of infected fish under high magnification (KLINGER & FRANCIS-FLOYD, 1998Klinger RE, Francis-Floyd R. Introduction to freshwater fish parasites [online]. Florida: Institute of Food and Agricultural Sciences, University of Florida; 1998. CIR716. Available from: http://edis.ifas.ufl.edu/pdffiles/FA/FA04100.pdf.; FOIN, 2005Foin AA. Parasites et parasitoses des poissons d'ornement d'eau douce: aide au diagnostic et propositions de traitement [Tese]. Maisons-Alfort: École Nationale Veterinaire d' Alfort; 2005.).

Distribution of Parasites in Brazil

Several protozoan parasites of fishes have been reported in national territory, as a result of efforts of Brazilian researchers from different localities. Nevertheless, unpublished data of the present authors indicate that much more observations are yet to be reported as shown in Figure 11.

Figure 11
Geographical distribution of (a) ciliated protozoan and (b) other protozoan parasite groups of fishes from Brazil. Symbols indicate the register of the below-mentioned species: ☉ Entamoeba pimelodi;Amyloodinium;Cryptobia;Hexamita;Ichthyobodo; ⓿ Piscinoodinium;Trypanosoma;Calyptospora;Cyrilia lignieresi;Eimeria lepidosirensis;Haemogregarina;Apiosoma;Balantidium;Chilodonella;Epistylis;Ichthyophthirius multifiliis;Nyctotherus piscicola;  Rynchodinium paradoxum;Tetrahymena and Trichodinidae. Colour black indicates previous reports; colour red indicates unpublished data from the present authors.

Attention on the Control Strategies

The use of chemicals in order to control protozoan parasites can be, sometimes, difficult to administer, costly, not completely efficient and even environmentally hazardous. Dozens of protocols used in many other countries for tropical and temperate fishes have been adapted to fish farms in Brazil with no scientific criteria. In fact, if applied erroneously, it may cause environmental degradation such as impairment of water quality (pH and dissolved oxygen alterations can lead to great fish mortality), besides the fact that several chemoterapeutics employed are corrosive, carcinogenic or even explosive. On this view, they must be manipulated with PPE´s (Personel Protection Equipment) and special care. Nevertheless, in field practices it is usually observed low knowledge when employing these techniques. Prophylactic measurements or immunoprophylaxis adopted in the fish farms and ornamental industry are important tools to minimize the effects of parasitism and to estimulate the fish immune system (MARTINS et al., 2011Martins ML, Shoemaker CA, Xu D-H, Klesius PH. Effect of parasitism on vaccine efficacy against in Nile tilapia. Streptococcus iniaeAquaculture 2011; 314(1-4): 18-23. http://dx.doi.org/10.1016/j.aquaculture.2011.01.022.
http://dx.doi.org/10.1016/j.aquaculture....
).

In order to control outbreaks of diseases in fish farms it is fundamental to first characterize carefully the hygienic-sanitary state of the facility. This includes being aware of disease diagnosis history, recognition of pathogen dispersal and/or transmission modes involved. The use of chemotherapeutics cannot be linked only when an outbreak of mortality occurs, in which treatment is employed as a rescue measure to reduce the economic losses. Such products must be applied strategically in each rearing phases in order to have the best fish response, with lowest environmental impact and safety to the operating person.

Disease control in hatcheries that produce fingerlings is a key point to reach the high sanitary quality and regular production (PÁDUA & CRUZ, 2014Pádua SB, Cruz C. Health challenges in tilapia culture in Brazil. Aquaculture Asia Pacific 2014; 10(2): 37-39.). In these farms, transmission of diseases from the broodstock to eggs, larvae and fingerlings deserves special attention. Efforts to control diseases must be concentrated on these units, which represent the beginning of the production cycle.

In tilapia hatcheries where the eggs are collected from the mouth, it may be necessary to disinfect them before storage in incubators. The use of Chloramine-T is an efficient measure for disinfecting eggs against viruses, bacteria, fungi and parasites found in this process. In order to proceed, the use of small capacity circulating systems (200 – 1,000 L) should be implemented so as to supply 2 to 6 incubators that will recirculate water with 30 mg/L of Chloramine-T, during 10 to 20 min of exposition. It is important to assure that water will circulate inside a mechanical filtration system with the aid of an acrylic filter mat. In this case, it is worth mentioning that the use of activated carbon elements or other substances with adsorption effects is not recommended, since they could kidnap the molecule of the drug used for disinfection.

Adequate management include not handling fish when abiotic factors such as water temperature and pH are exceeding limits generally considered safe for the fish species, avoid translocation of fingerlings from one place to another without proper care, always use good quality feeds, monitor regularly the quality of the water entering the farm, avoid water sharing among ponds and disinfect fish handling equipment. After that, it is necessary to adopt other measures of biosecurity to ensure larvae remain free of parasites during subsequent rearing phases. For such, it is important to sectorize fish farm facilities, thus delimitating areas for equipments and for passage of employees. Other important measures to avoid disease dissemination include preventing entry of wild fish in the farm (this can be done using screens in the water supply canal), implementing sanitary barriers such as cleaning and disinfecting ponds, using a quarantine tank, depurating fish before transport and using wetlands in the water supply canal.

Wetlands macrophytes such as Eichhornia spp. (Figure 12) can be considered an efficient tool to contain dispersion of resistant forms of parasites, such as eggs and resistent cysts into the farm, as well as to provide a huge surface area for attached microbial growth. However, one must establish a routine for renewal of these aquatic plants so that the saturation capacity of retention of particles in its submerged roots does not occurs. To this end, it must be performed the removal of adult plants, or under the senescent stage, every 15 to 45 days, varying according to the season and the amount of suspended material in the water.

Figure 12
Constructed wetland in freshwater fish farm for use as a sanitary barrier and water cleaning.

After storage of fingerlings in a farm destinated to fish fattening, the use of sanitary measures may be expensive and sometimes operationally difficult. On the other hand, the implementation of sanitary measures in hatcheries is more economic and efficient in suppressing or erradicating the disease (PÁDUA & CRUZ, 2014Pádua SB, Cruz C. Health challenges in tilapia culture in Brazil. Aquaculture Asia Pacific 2014; 10(2): 37-39.). Similar to that found in industrial pig and poultry farming, young fish should be sent to growing units in a good health status and possibly vaccinated, minimizing thus parasite hazard to an acceptable level. It must be emphasized that it is complex to achieve fish completely negative for parasites and so prophylaxis and best management practices are the best ways of achieving adequate health status of farmed fish (BOYD et al., 2008Boyd CEB, Lim C, Queiroz J, Salie K, De Wet L, McNevin A. Best management practices for responsible aquaculture. Washington: Aquaculture Collaborative Research Support Program; 2008. 47 p.).

Acknowledgements

The authors thank Fundect (Process nº 23/200.202/2010 and 10/14490-1) for financial support and CNPq (National Council of Scientific and Technological Development) for grant to M.L. Martins (302493/2010-7) and financial support (472598/2011-2); Roney Nogueira de Menezes Filho (Aquivet Saúde Aquática) for collaboration on diagnostic information from the field and control approach used in fingerlings hatchery-farming; Wellington Correa Silva and Fernando Franceschini Macedo (Rações VB) for valorous information on the protozoan occurrence in native fish from the Central-North region of Brazil; Dra. Ana Paula de Araújo (Acquapiscis) for information of parasitic protozoan in marine fish; Dra. Márcia Mayumi Ishikawa (Embrapa Agropecuária Oeste) for information on native fish protozoosis inluding bait fish.

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Publication Dates

  • Publication in this collection
    Jan-Mar 2015

History

  • Received
    19 Jan 2015
  • Accepted
    02 Feb 2015
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