Quantitative detection of parasitic ciliate Cryptocaryon irritans in seawater with an optimized sample processing method.

The protozoan Cryptocaryon irritans is one of the most important ectoparasites of marine fish, causing 'white spot disease' and mass mortality in aquaculture. To accurately predict disease outbreaks and develop prevention strategies, improved detection methods are required that are sensitive, convenient and rapid. In this study, a pair of specific primers based on the C. irritans 18S rRNA gene was developed and used in a real-time PCR (qPCR) assay. This assay was able to detect five theronts in 1 L of natural seawater. Furthermore, a linear model was established to analyse the log of Ct value and parasite abundance in seawater (y = -2.9623x + 24.2930), and the coefficient of determination (R2 ) value was 0.979. A lysis buffer was optimized for theront DNA extraction and used for storage sample. This method was superior to the commercial water DNA kit, and there was no significant degradation of DNA at room temperature for 24-96 hr. A dilution method was developed to manage qPCR inhibitors and used to investigate natural seawater samples in a net cage farm with diseased fish, and the findings were consistent with the actual situation. This study provides a valuable tool for assisting in the early monitoring and control of cryptocaryoniasis in aquaculture.

Updating specific PCR primer for detection of Cryptocaryon irritans from reared Larimichthys polyactis.

Artificial breeding of small yellow croaker (Larimichthys polyactis) was recently achieved, providing a bright future for its commercial farming. In May 2019, a disease outbreak occurred among small yellow croakers in an aquaculture farm near Xiangshan Bay, charactering by white spots spotted on the surface of fish skin, gills and fins. The parasite was preliminarily identified as Cryptocaryon irritans based on morphological feature of the parasite and the symptoms on fish. However, the previously published specific primer pairs failed to confirm the existence of C. iriitans. Six nucleotides mismatches were discovered after mapping specific forward primer back to targeted gene. Therefore, an updated PCR specific primer was developed within the 9th highly variable region of 18S rRNA gene and conserved in all C. irritans sequences available in GenBank database. The specificity was verified in silico by Primer-BLAST against GenBank nucleotide. Laboratory cultured ciliates (Mesanophrys, Pseudokeronopsis and Uronema) as well as natural microbial community samples collected from sea water and river water was used as negative control to verify the specificity of the primer in situ. Besides, tank transfer method was used to evaluate the treatment of the parasite infection. By tank transfer method, 2.00 ± 0.61 out of 10 fish that already sever infected were successfully survived after 8 days treatment, meanwhile the control group died out at d 6. More loss to the treatment group during first five days was observed and may attribute to the combined effect from infection and stress the recent domesticated fish suffered during rotation. Therefore, tank transfer method was also effective to prevent small yellow croaker from further infection, however the loss of the small yellow croaker suffered from stress during rotation also needs to be carefully concerned. In conclusion, this study reported the first diagnose of C. irritans infection on small yellow croaker, provided updated specific primer to detect C. irritans infection on fish body and reported the effect of tank transfer on small yellow croaker treatment.

Current status of parasitic ciliates Chilodonella spp. (Phyllopharyngea: Chilodonellidae) in freshwater fish aquaculture.

Freshwater fish farming contributes to more than two-thirds of global aquaculture production. Parasitic ciliates are one of the largest causes of production loss in freshwater farmed fishes, with species from the genus Chilodonella being particularly problematic. While Chilodonella spp. include 'free-living' fauna, some species are involved in mortality events of fish, particularly in high-density aquaculture. Indeed, chilodonellosis causes major productivity losses in over 16 species of farmed freshwater fishes in more than 14 countries. Traditionally, Chilodonella species are identified based on morphological features; however, the genus comprises yet uncharacterized cryptic species, which indicates the necessity for molecular diagnostic methods. This review synthesizes current knowledge on the biology, ecology and geographic distribution of harmful Chilodonella spp. and examines pathological signs, diagnostic methods and treatments. Recent advances in molecular diagnostics and the ability to culture Chilodonella spp. in vitro will enable the development of preventative management practices and sustained freshwater fish aquaculture production.

Cryptocaryon irritans recombinant proteins as potential antigens for sero-surveillance of cryptocaryonosis.

Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.

Protozoan infections in farmed fish from Brazil: diagnosis and pathogenesis.

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.

Protozoan infections in farmed fish from Brazil: diagnosis and pathogenesis / Infecções por protozoários em peixes cultivados no Brasil: diagnóstico e patogênese

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.

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.

Validation of FLOTAC for the detection and quantification of Troglodytella abrassarti and Neobalantidium coli in chimpanzees and pigs.

We carried out a calibration of FLOTAC for ciliates Troglodytella abrassarti and Neobalantidium coli based on the selection of a most appropriate flotation solutions, and we also tested its accuracy (i.e., number of detected stages out of known added number of stages to fecal samples) and sensitivity for trophozoites of both ciliates in chimpanzee feces and N. coli cysts in pig feces, compared the detection threshold of FLOTAC with MIF-based sedimentation, and, subsequently, tested the losses of ciliate stages during sample preparation. Nine flotation solutions were evaluated, and ZnSO4 solution (specific gravity [s.g.] 1.2) showed to be the most suitable for trophozoite detection, while Sheather's solution (s.g. 1.33) was selected as most suitable for cysts. The FLOTAC sensitivity in detection of both stages varied: for trophozoites, we found all samples were positive when the intensity of infection 10 trophozoites per gram and higher, whereas for cysts the sensitivity was lower. The accuracy of FLOTAC negatively correlated with infection intensity, and the merthiolate-iodine-formaldehyde sedimentation-based quantification had a lower detection threshold. We demonstrated additional losses of stages of T. abrassarti and N. coli due to their retention in the sediment, which is probably a major reason for discrepancies in the numbers of countable ciliates between both methods. In conclusion, the FLOTAC should not be considered as a gold standard for quantification of intestinal ciliates in primates; instead, we recommend the modified MIF method.

Identification of the pathogenic ciliate Pseudocohnilembus persalinus (Oligohymenophorea: Scuticociliatia) by fluorescence in situ hybridization.

Many scuticociliatid ciliates are regarded as devastating pathogens in aquaculture. Among these, Pseudocohnilembus persalinus is a facultative pathogen that often results in refractory diseases of mariculture fish. Although traditional silver staining methods have been successfully used to identify these ciliates, their identification is hampered by their small size and their morphological similarity to closely related species. We designed an alternative method of identification of P. persalinus using an SSU-rDNA targeted oligonucleotide probe labeled with a fluorochrome, and optimized in a fluorescence in situ hybridization (FISH) protocol. The assay results in a clear identification by strong fluorescence signals from the oligonucleotide probe. The method can be used for quick and early detection of P. persalinus infections on host fish, as well as other susceptible organisms in aquiculture water. It may also be used in studies of the geographical distribution of this scuticociliate.

Ichthyophthiriasis: emphases on the epizootiology.

Ichthyophthiriasis (white spot disease) is an economically important protozoosis caused by Ichthyophthirius multifiliis in freshwater fish. Medication prevention and curation are the main methods to control this disease with vaccines in laboratory, but the efficacy of drugs practically acts on the free-living (nonparasitic) stage of I. multifiliis and can be easily impaired by a variety of environmental factors. Thus, study on the biological properties of I. multifiliis and the complicated ecological relationships between I. multifiliis and other biotic or abiotic factors that influence epidemicity of ichthyophthiriasis will contribute to integrated control of ichthyophthiriasis. In this article, some I. multifiliis biological properties, such as systematic position and life cycle of I. multifiliis, are briefly reviewed, and the seemingly abnormal phenomenon associated with in vitro cultures is specially discussed; then, the epizootiology of ichthyophthiriasis is emphasized, which involves various biotic or abiotic factors that impact the life and action of I. multifiliis. The susceptibility and stress reaction of fish to I. multifiliis infection are stated. Also, the pathogenicity and diagnosis of ichthyophthiriasis were covered, and an overall assessment is finally made on ichthyophthiriasis control.

First record of Chilodonella hexasticha (Ciliophora: Chilodonellidae) in Brazilian cultured fish: a morphological and pathological assessment.

Chilodonelids are small ciliated protozoans found worldwide and can be dangerous in culture conditions. This study presents morphometric data on the ciliate Chilodonella that is found in cultured Nile tilapia (Oreochromis niloticus), native bait fish tuvira (Gymnotus aff. inaequilabiatus) and native pacu (Piaractus mesopotamicus) and includes a histopathological assessment of the changes that occur in the pacu. For parasitic diagnosis, skin and gill samples were scraped onto slides, dried at room temperature, stained with Giemsa or impregnated with silver nitrate, and the measurements were obtained from photomicrographs. In the diseased pacu, the first gill arch was collected and fixed in a 10% buffered formalin solution for histopathological analysis. Parasite specimens from the different collection sites were identified morphologically as C. hexasticha Kiernik (1909). Diseased fish exhibited depigmentation, skin ulceration, scale loss, excessive mucus production and gill lesions. Histopathological analysis of pacu gills displayed epithelial proliferation with mononuclear inflammatory infiltrate, hemorrhages, and scattering necrosis. In Brazilian-farmed fish this is the first record of C. hexasticha, which has great pathogenic potential in cultured freshwater species. In addition, two new hosts are presented.

Trichodina colisae (Ciliophora: Trichodinidae): new parasite records for two freshwater fish species farmed in Brazil / Trichodina colisae (Ciliophora: Trichodinidae): novo registro de parasito para duas espécies de peixes de água doce cultivadas no Brasil

Family Trichodinidae comprises ciliate protozoa distributed worldwide; they are considered some of the main parasitological agents infecting cultivated fish. However, the trichodinidae parasitizing important fish species cultured in Brazil are unknown, and more taxonomic studies on this group of parasites are required. This research morphologically characterizes Trichodina colisae Asmat & Sultana, (2005) of pacu (Piaractus mesopotamicus) and patinga hybrid (P. mesopotamicus × P. brachypomus) cultivated in the central and southeast regions of the country. Fresh assemblies were made from mucus scraped from the skin, fins and gills, fixed with methanol and, subsequently, impregnated with silver nitrate and stained with Giemsa for assessment under light microscopy. This research reports not only the second occurrence of T. colisae in the world, but also its first occurrence in South America.(AU)

Tricodinídeos são protozoários ciliados móveis com ampla distribuição mundial; são considerados um dos agentes parasitários que mais acometem peixes cultivados. No Brasil, a maioria dos tricodinídeos que parasitam importantes espécies de peixes cultivados são desconhecidos, o que requer mais estudos taxonômicos com esse grupo de parasitos. Este estudo caracteriza morfologicamente Trichodina colisae Asmat & Sultana, 2005 de pacu (Piaractus mesopotamicus) e do híbrido patinga (P. mesopotamicus × P. brachypomus) cultivados, respectivamente, no Centro-Oeste e Sudeste do Brasil. Foram feitas montagens a fresco do raspado de muco da pele, nadadeiras e brânquias, fixados com metanol e, posteriormente, impregnados com nitrato de prata e coradas com Giemsa para avaliação em microscopia óptica. O presente estudo relata não só a segunda ocorrência de T. colisae no mundo, mas também a primeira ocorrência na América do Sul.(AU)

Trichodina nobilis Chen, 1963 and Trichodina reticulata Hirschmann et Partsch, 1955 from ornamental freshwater fishes in Brazil.

In the present work Trichodina reticulata and T. nobilis (Ciliophora: Trichodinidae) are morphologically characterised from ornamental freshwater fish culture in the State of Santa Catarina, Brazil. The prevalence of infection and a list of comparative measurements are discussed. We examined "southern platyfish" Xiphophorus maculatus (n = 35), "goldfish" Carassius auratus (n = 31), "guppy" Poecilia reticulata (n = 20), "sailfin molly" Poecilia latipinna (n = 6), "beta" Betta splendens (n = 2) and "spotted headstander" Chilodus punctatus (n = 1). After being anesthetised in a benzocaine solution, fishes were examined for parasitological evaluation. A total of 51.57% fishes were parasitised by Trichodina spp. Carassius auratus was the most parasitised species, followed by X. maculatus and P. reticulata. Beta splendens, C. punctatus and P. latipinna were not parasitised by any trichodinid species. Two species of Trichodina were collected from the skin of fish: T. nobilis was found in C. auratus, P. reticulata and X. maculatus and T. reticulata was only observed in C. auratus. The importance of adequate handling in ornamental fish culture are also discussed.

Trichodina nobilis Chen, 1963 and Trichodina reticulata Hirschmann et Partsch, 1955 from ornamental freshwater fishes in Brazil / Trichodina nobilis Chen, 1963 e Trichodina reticulata Hirschmann et Partsch, 1955 de peixes ornamentais de água doce no Brasil

In the present work Trichodina reticulata and T. nobilis (Ciliophora: Trichodinidae) are morphologically characterised from ornamental freshwater fish culture in the State of Santa Catarina, Brazil. The prevalence of infection and a list of comparative measurements are discussed. We examined "southern platyfish" Xiphophorus maculatus (n = 35), "goldfish" Carassius auratus (n = 31), "guppy" Poecilia reticulata (n = 20), "sailfin molly" Poecilia latipinna (n = 6), "beta" Betta splendens (n = 2) and "spotted headstander" Chilodus punctatus (n = 1). After being anesthetised in a benzocaine solution, fishes were examined for parasitological evaluation. A total of 51.57% fishes were parasitised by Trichodina spp. Carassius auratus was the most parasitised species, followed by X. maculatus and P. reticulata. Beta splendens, C. punctatus and P. latipinna were not parasitised by any trichodinid species. Two species of Trichodina were collected from the skin of fish: T. nobilis was found in C. auratus, P. reticulata and X. maculatus and T. reticulata was only observed in C. auratus. The importance of adequate handling in ornamental fish culture are also discussed.

No presente trabalho Trichodina reticulata e T. nobilis (Ciliophora: Trichodinidae) de peixes ornamentais de água doce cultivados no estado de Santa Catarina, Brasil são caracterizadas morfologicamente. A prevalência de infecção e uma lista comparative de medidas são discutidas. Foram examinados "platis" Xiphophorus maculatus (n = 35), "kinguios" Carassius auratus (n = 31), "guppys" Poecilia reticulata (n = 20), "molinésias" Poecilia latipinna (n = 6), "betas" Betta splendens (n = 2) e "espada" Chilodus punctatus (n = 1). Após a anestesia com solução de benzocaína, os peixes foram submetidos à avaliação parasitológca. Um total de 51,57% peixes estavam parasitados por Trichodina spp. Carassius auratus foi a espécie mais parasitada, seguida por X. maculatus e P. reticulata. Beta splendens, C. punctatus e P. latipinna não estavam parasitados por tricodinídeos. Duas species de Trichodina foram coletadas da superfície corporal dos peixes: T. nobilis foi encontrada em C. auratus, P. reticulata e X. maculatus e T. reticulata foi observada apenas em C. auratus. A importância do manejo adequado em cultivos de peixes ornamentais também foi discutida.

Development of specific PCR assays for the detection of Cryptocaryon irritans.

Cryptocaryon irritans is one of the most important protozoan pathogens of marine fish, causing the "white spot" disease and posing a significant problem to marine aquaculture. In the present study, a C. irritans-specific reverse primer (S15) was designed based on the published sequence of the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) of C. irritans and used together with the conserved forward primer P1 to develop a specific polymerase chain reaction (PCR) assay for direct, rapid, and specific detection of C. irritans. The specificity of these primers was tested with both closely and distantly related ciliates (Pseudokeroronpsis rubra, Pseudokeroronpsis carnae, Euplotes sp. 1, Ichthyophthirius multifiliis, Pseudourostyla cristata, and Paramecium caudaium), and only C. irritans was detected and no product was amplified from any other ciliates examined in this study using the specific primer set P1-S15. The specific PCR assay was able to detect as low as 45 pg of C. irritans DNA and a nested PCR assay using two primer sets (P1/NC2, P1/S15) increased the sensitivity, allowing the detection of a single C. irritans. The species-specific PCR assays should provide useful tools for the diagnosis, prevention, and molecular epidemiological investigations of C. irritans infection in marine fish.