Parasitological diagnosis in ornamental freshwater fish from different fish farmers of five Brazilian states

Abstract This study aimed to search parasites in 333 ornamental fish from five Brazilian states (Ceará, Minas Gerais, São Paulo, Paraná and Santa Catarina). Fish were sent from eight farms located in the municipalities of Fortaleza, Patrocínio do Muriaé, São Francisco do Glória, Cascavel, Timbó, Iguape, Jacareí and Mairinque. All fish received anesthesia earlier to euthanasia procedures. After the search for parasites, it was verified that 70.6% (235/333) of fishes were infected by at least one type of parasite, being 12 types of parasites identified: monogeneans, digenean metacercariae, cestodes, nematodes, Lernaea cyprinacea, trichodinids, Piscinoodinium pillulare, Ichthyophthirius multifiliis, diplomonad flagellates, Ichthyobodo sp., Chilodonella sp., and Tetrahymena sp. The proportion of infected fishes among the farms is compared through statistical tests, besides, animal handling adopted in each farm is also discussed. The importance of ensuring fish health in order to make the ornamental freshwater fish industry economically viable and reduce losses in production is highlighted.


Introduction
The ornamental freshwater fish industry has grown substantially over the past decades worldwide (FAO, 2020).In Brazil, this activity has become more common, with fish being the fourth most prevalent pet in Brazilian residences, present in approximately 20.8 million houses (ABINPET, 2022).The Amazon is a critical center for the extraction of ornamental fish from the northern region of Brazil, whose main market is exportation to Europe, Asian and United States (Anjos et al., 2009), while Minas Gerais is the main producer hub, supplying animals to different Brazilian regions (Magalhães and Jacobi, 2013).
Animals were transported alive in plastic bags with oxygen to a wholesale shop in the city of São Paulo, where the parasitological analysis were performed.For this study, animals were randomly collected from the plastic bags at the moment of arrival, and fish species were identified and euthanized.

Water quality and evaluation of biosecurity practices in fish farms
During technical visits at the farms, the water quality and biosecurity practices were verified through the presence or absence of methods for disease and parasite prevention (filters, biofilters, and UV radiation devices), staff routines towards fish care were observed, along with feeding practices, fallowing and quarantine routines, equipment to control water quality (mechanical aerators), and water quality testing routines (e.g., measurement of pH, ammonia, and dissolved oxygen).In addition, contact with wild animals, such as birds and crustaceans, was evaluated.

Clinical evaluation, collection and parasite analysis
Prior to necropsy procedures, fish were clinically examined and classified as either 'alert' or 'prostrated'.After that, one of the following clinical signs were identified and recorded: difficulty to breathe properly, skin wounds or ulcers, irregular, clamped or damaged fins, excessive mucus, and white spots.
For parasite analysis, skin scraping was performed first, followed by a sedation procedure with Eugenol (100 mg L -1 ) diluted in a 4 L container (Roubach et al., 2005).Animals were sedated after being in contact with the solution for approximately 5 min or less, depending on the species, followed by spinal cord section euthanasia (Noga, 2010).Following euthanasia, gill and viscera wet mounts were performed through optic microscopic evaluation on 4 x, 10 x, 20 x and 40 x magnification.The parasites were identified based on their structure, morphology and movement pattern (Woo, 2006;Noga, 2010;Martins et al., 2015;Cardoso et al., 2018).For a better examination of tissues by wet mount, samples were mounted between a glass slide and coverslip with a drop of saline solution (Martins et al., 2015).

Statistical analysis
We evaluated whether there was a difference in the proportion of infected animals between each municipality.A Chi-square test was performed to compare the proportion of infected animals in different municipalities and the alpha value established to reject the null hypothesis was stressful situations may occur, causing an imbalance of this triad.This imbalance may be due to excessive amounts of ammonia, nitrite and nitrate, low dissolved oxygen, water eutrophication, lack of control of the water temperature, frequent pH imbalance and inadequate handling (Moraes and Martins, 2004), causing stress and making fish susceptible to infectious and parasitic diseases.Biosecurity, internal and external barriers, disinfection and antisepsis of equipment and aquaria, quarantining of fish prior to entry into the system, among other prophylactics measures, must be taken in order to preserve the physiological conditions and health of the animals to avoid parasite proliferation.The lack of correct hygiene handling practices and failure of proper water treatment equipment installation, cause severe sanitary problems because of inefficient pathogen control (Cardoso et al., 2017(Cardoso et al., , 2018(Cardoso et al., , 2020a;;Relvas et al., 2020).
The monitoring of fish health status must be one of the most important activities in farms, wholesalers, stores, and in importation and exportation holding facilities.Transport of live animals contributes to parasite dissemination not only in ornamental fish, but also in native fish and farmed fish (Tavares- Dias et al., 2009), endangering native populations and posing a threat to the aquaculture industry.The introduction of a new pathogen may have devastating effects on the health and economic viability of aquatic animal populations if they do not evolve appropriate defense mechanisms against pathogens (Cardoso and Balian, 2018).The most common diseases affecting cultured fish are caused by exotic pathogens, which are inadvertently introduced into a region via infected fish from another geographic area.The implementation of methods to prevent or manage the transmission of infectious diseases is important in preventing the escape of pathogens from a farm, so that it does not affect wild populations or adjacent farms (Whittington and Chong, 2007;Becker et al., 2014;Trujillo-González et al., 2018).
Parasitic infections represent an important challenge for commercial suppliers of ornamental fish, and only an effective biosecurity program will maintain healthy animals, reducing the risk of acquiring disease in a facility.In this context, knowing the parasite groups that can affect ornamental fish is essential for aquaculture development.However, in Brazil, there is still a lack of data and robust research being conducted.Additionally, research is not being conducted by official laboratories, making the present research important in order to initiate an epidemiological mapping of sanitary conditions of fish grown in national territory.Therefore, the aim of this study was to identify the parasite groups of ornamental freshwater fish from some fish producing regions that had different animal handling characteristics.All statistical analyses were computed using R Core Team (2020) software.
In the farms from Fortaleza and Mairinque, fish were raised in fiberglass tanks; the facilities had internal and external barriers, preventing fish from having contact with other animals; there was frequent monitoring and adjustment of pH levels, ammonia, nitrite and nitrate; before introducing new individuals to the system, fish stayed in quarantine; and there was a vet technician responsible for sanitary aspects and animal health in both municipalities.In Cascavel, Jacareí, Patrocínio do Muriaé and Timbó, fish were pound-raised and had contact with outside animals because there were no internal or exterior barriers; there was also no control of water temperature; farms had a poor sanitation system, with frequent low dissolved oxygen and high levels of occurrence of water eutrophication; equipment or aquaria did not have any type of disinfection or antisepsis; there was an inefficient pathogen control.In Iguape and São Francisco do Glória, fish were also pond-raised and had contact with outside animals; the farms did not have any control over water temperature; nor did they implement frequent monitoring and control of pH, ammonia, nitrite and nitrate.

Discussion
Our results demonstrate that a great diversity of parasites currently affects ornamental fish from the main  Parasites in ornamental fish in Brazil producing regions in Brazil.Fortaleza had the lowest proportion of infected fish (23.7% -9/38) in this study, and thus it was used to compare the proportion of infected animals with the other municipalities.
When compared to Fortaleza, the proportion of infected fish was equal in São Francisco do Glória (57.9% -33/57), Iguape (66.7% -14/21) and Mairinque (50% -22/44).In Fortaleza and Mairinque: both farms employ proper measures for raising their fish, resulting in fewer infected animals in these farms.Although São Francisco do Glória and Iguape raised fish in pound-raised systems, which meant that fish had contact with outside animals and that there was not proper water quality control, the proportion of infected animals was equal to Fortaleza.This result could be explained by the small number of evaluated specimens in Iguape: only 21 analyzed fish belonging to three species (Corydora paleatus, Xiphophorus maculatus and Misgurnus anguillicaudatus), of which 57.1% (12/21) were infect by digenean metacercariae.In addition, in São Francisco do Glória, even though fish were raised in pond-raised tanks, animal handling was better in this county, which could explain the proportion of infected animals being similar to that of Fortaleza.
The proportion of infected fish in Timbó (100% -55/55), Jacareí (96.8% -30/31), Patrocício do Muriaé (88% -44/50), and Cascavel (75.7% -28/37) was different from Fortaleza.In these four counties, a greater proportion of infections were observed.Pond-raised fish farms are normally characterized by a lack of proper water quality control or biosecurity measures.These characteristics enable pathogens reproduction and spread, and as a result, more animals were infected in these farms in relation to Fortaleza.
All fish analyzed from Timbó were Goldfish, Carassius auratus, from a new producer in the region, and 100% (55/55) of the fish were infected with trichodinids.Trichodinids have been previously described to infect ornamental freshwater fish from different Brazilian regions (Piazza et al., 2006;Tavares-Dias et al., 2009, 2010;Martins et al., 2012;Florindo et al., 2017b;Santos et al., 2017;Cardoso et al., 2018).In natural habitats, these ectoparasites cause no clinical effects and have also been reported in cultured fish (Martins and Ghiraldelli, 2008).However, under highly intensive aquaculture systems with inadequate handling conditions, these ciliates may proliferate extensively and become pathogenic parasites responsible for, not only for serious host inhibited growth, but also losses up to 50% of fish stocks (Madsen et al., 2000;Guiraldelli, 2006).When starting a new farm, it is important to follow biosecurity rules and prophylactic measures to preserve animal health, avoid parasite proliferation, and ensure that animals without health issues are commercialized.
The proportion of infected fish in Timbó did not differ from that in Jacareí (p=1).In Jacareí and Timbó, 87.1% (27/31) and 88% (44/55) of fish were infected with monogeneans, respectively.These flatworms have already been reported in different ornamental freshwater fish from Brazil (Tavares- Dias et al., 2009Dias et al., , 2010;;Florindo et al., 2017a;Santos et al., 2017;Cardoso et al., 2018;Hoshino et al., 2018).They have a direct life cycle and can cause respiratory disease when infecting gills, or scale loss may occur if the parasite is attached to the skin.Large numbers of monogeneans on either the skin or gills may result in significant damage and mortality (Cardoso et al., 2017;Francis-Floyd et al., 2019).In this study, only 4.8% (16/333) of the infected fish presented difficulty in breathing properly, and 2.7% (9/333) showed scale irritation or damage during clinical assessment.Herein, it is also important to highlight that scale damage might have been caused during the transport of animals.Despite these findings, these results demonstrate that few fish presented sings of infection when commercialized.Heavy monogenean infestations are usually indicators of poor sanitation and deteriorating water quality (Chen et al., 2020), a scenario found in producers from the counties of Jacareí and Timbó.If left untreated, monogeneans can lead to serious economic loss for fish farmers.
In Jacareí, 25.8% (8/31) of the fish were infected by I. multifiliis.This protozoan is a widespread parasite with no host specificity and has been described in a range of ornamental fish in Brazil (Piazza et al., 2006;Tavares-Dias et al., 2009, 2010;Eiras et al., 2012;Santos et al., 2017;Cardoso et al., 2018;Hoshino et al., 2018).Fishery utensils used in fish farms and stressful situations, such as overcrowding, low dissolved oxygen, and high temperatures, favor the I. multifiliis life cycle and disease outbreaks (von Gersdorff Jørgensen, 2016).In this study, none of the fish infected with I. multifiliis presented the telltale clinical sing of I. multifiliis infestation during clinical assessment, white spots.This demonstrates that infected fish may have the parasite but may not yet have developed common clinical signs of the disease prior to removal from the system.Although I. multifiliis showed low prevalence rates in the analyzed fish, sanitary handling must be considered to avoid the introduction of diseased or asymptomatic fish.
In Iguape, fish were pond-raised and had contact with animals outside of the pounds, including birds, which are common hosts for of adult digeneans, from which metacercariae are part of their life cycle.An important finding was yellow spot disease in Corydora paleatus.The morphology matched the genus Clinostomum that have zoonotic potential, but this was the only parasite that we had the opportunity to attempt molecular identification and there was no amplification of the PCR reaction.A total of 57.1% (12/21) of fishes from Iguape were infected with digenean metacercariae.This digenean life stages commonly found in ornamental fish raised in tanks or lakes with access to other hosts, such as crustaceans, mollusks, birds and mammals (Alves et al., 2001;Piazza et al., 2006;Santos et al., 2017;Cardoso et al., 2018;Hoshino et al., 2018).Except for the counties of Mairinque and Fortaleza, all farms evaluated had poundraised systems that enabled the direct contact of fish with birds, mammals, mollusks and crustaceans.Trematode cysts can infect gills, skin, muscles and viscera, and are generally innocuous; however, high infestations can be lethal (Dias et al., 2003).When in metacercaria form, these worms cannot be treated; therefore, precautions must be taken to prevent the parasitosis.
In Patrocínio do Muiaré and Cascavel, the proportion of infected animals was not significantly different (p=1).
Of the fish from Patrocínio do Muriaé, 44% (22/50) were infected with Chilodonella sp., while, in Cascavel, 21.6% (8/37) and 18.9% (7/37) were infected with P. pillulare and Tetrahymena sp., respectively.These ectoparasites have been frequently reported in studies from different Brazilian regions (Piazza et al., 2006;Tavares-Dias et al., 2009, 2010;Eiras et al., 2012;Florindo et al., 2017b;Cardoso et al., 2018) and their transmission occurs especially through direct contact between infested and healthy fish.Elevated intensities of these protozoan parasites are common in cultured fish because the parasite disseminates in fish population via the use of routine utensils and water used during fish transport (Martins et al., 2002(Martins et al., , 2015;;Tavares-Dias et al., 2010).Pathological changes caused by these agents are related to their abrasive action on the host epithelium, with the gill filaments being the most sensitive organ for parasites to attack.Clinical signs are non-specific, indicating respiratory difficulty and skin irritation, resulting in excessive mucus (Martins et al., 2015).In this study, only 0.9% (3/333) of fish had excessive mucus during clinical assessment, demonstrating that few infected fish present signs of infection when commercialized, even though they are infected.
Lernaea cyprinacea was found just in Patrocínio do Muriaé.This parasite is a non-native species that was first cataloged in Brazil in the 1960s, being disseminated from Brazilian regions, like São Paulo, southwest Brazil (Cardoso et al., 2018), Sergipe northeastern Brazil (Assis et al., 2014), andPará, north Brazil (Barros et al., 2024), corroborating the data obtained from the present study.This invasive parasite can cause severe damage on fish populations and spread rapidly in a farming environment (Assis et al., 2014), so the adoption of quarantine and not disposing of waste water in common sewage are actions that prevent the spread and establishment of the parasite.
All fish that came from Mairinque were angelfish, Pterophyllum scalare, which were raised in polyethylene tanks, a material that is easy cleaned and disinfected.Only two types of parasites were found in this farm: 15.9% (7/44) of fish from Mairinque were infected by nematodes and 40.9% (18/44) by diplomonad flagellates.Like metacercariae, nematodes also present a complex life cycle that requires two or more intermediate hosts, and because of the absence of other hosts during the life cycle, they are relatively uncommon in cultured fish.However, fish fed live and wild-caught arthropods can become infected, enabling them to be either intermediate or final hosts for nematodes (Woo, 2006;Noga, 2010).Crustaceans and arthropods were part of the diet of fish from Mairinque, which most likely explains the infection.Diplomonad flagellates are comprised of various Spironucleus species that are mostly found in the intestine and are directly transmitted from fish to fish.Many cases of diplomonad flagellates in aquarium fish are mixed infections that involve other bacterial opportunists (Gallani et al., 2016) or parasites (e.g., Capillaria nematode infections in angelfish) (Noga, 2010).
Host-parasite interactions can be affected by inadequate handling, suboptimal water quality, inadequate nutrition, and stress (Piazza et al., 2006;Martins et al., 2011;Hoshino et al., 2018).The prevalence rates found in this study might have been influenced by the lack of control of the fish populations, water quality, high stocking density, inadequate water temperature, and a lack of quarantine measures for introduced fish.The farms from the municipalities of Fortaleza and Mairinque had a veterinarian to assist fish management and health, and raised fish in fiberglass tanks, a material that facilitated cleaning and disinfection of water environment.Conversely, the counties of Cascavel, Iguape, Jacareí, Patrocínio do Muriaé, São Francisco do Glória and Timbó did not had a technician present to ensure an effective biosecurity program to maintain healthy animals, thus reducing the risk of infectious disease in the facility.Additionally, they had pond-raised tanks that not only enabled the contact of fish with hosts that could disseminate parasites, but also made cleaning and disinfection more complicated.
The absence of clinical signs in fish on farms allows for the trading of infected fish and the spreading of parasites, or other harmful organisms, along the aquatic trade chain.Parasites may be normally present on fish skin, fins, and gills, or in internal organs without putting the host at risk.However, when fish are exposed to environmental or behavioral stressors, the potential damage from those organisms increases, enabling host susceptibility, causing imbalance between host, parasite and environment (Martins et al., 2002).Therefore, the manifestation of these diseases only occurs when the parasites encounter favorable situations for their proliferation.
Cestodes were found in fish from only two farms: São Franscisco do Glória 8% (5/57) and Cascavel 2,7% (1/37).Although we didn't identify genus and species of the parasite, the only Ciclassoma salvini analyzed from Cascavel presented a cestode very similar to the Asian fish tapeworm Schyzocotyle acheilognathi commonly found in native freshwater fishes from Asia.This cestode has low host specificity and for that reason it has been registered parasitizing more than 200 cultured and wild fish species, besides amphibians, reptiles and birds from different regions of the world.With a high pathogenic potential, S. acheilognathi may cause mortalities in highly infected fish.This cestode has already been found in Brazil (Souza et al., 2018).
Apart from host damage, depigmentation, skin ulceration, scale loss, excessive mucus production, gill lesions and reduced growth of fish, parasite infection also increases the mortality ratio by favoring secondary bacterial infections (Xu et al., 2012).Thus, prophylactic measurements adopted in farms and the ornamental fish industry are important tools to minimize the effects of parasitism (Martins et al., 2011;Cardoso et al., 2020b).

Conclusions
Ornamental fish commercialized in Brazil have high infection ratios and in order to provide the market with better and healthier fish, it is necessary to improve handling and maintenance of fish in the field in wholesaler facilities and pet stores.As showed in this study, few fish presented signs of infection when they were in the farms.Even though they had parasites, they might have not Brazilian Journal of Biology, 2023, vol.83, e270067 9/10 Parasites in ornamental fish in Brazil yet developed clinical signs.Therefore, monitoring and treatment of diseases in fish farms is essential to prevent the spread of diseases in the aquatic trade chain and to prevent aquarists from abandoning this hobby due to a decrease om the availability of healthy animals.More detailed research, including the use of molecular biology to identify parasites that can infect a range of ornamental freshwater fish, is necessary to enrich this initial study.In addition, this study can serve as a basis for new research that has a more adjusted design.

Figure 3 .
Figure 3. Proportion of infected fish in each municipality analyzed in the study.Note: a,b,c,d,e,f: Equal letters correspond to equal proportions by the pairwise comparisons for proportions with Bonferroni's correction.
Total 0.05 (p<0.05).After the comparison of proportions, if p<0.05, pairwise comparisons were calculated through multiple comparisons with Bonferroni's method to adjust the p-value.