First report of Kudoa sp. in the palate and pharyngeal musculature of Gobioides grahamae Palmer and Wheeler, 1955 (Perciformes, Gobiidae) from Marajó Island, Brazil

[Primeiro registro de Kudoa sp. na musculatura do palato e faringeal de Gobioides grahamae Palmer e Wheeler, 1955 (Perciformes, Gobiidae) da Ilha do Marajó, Brasil]

ABSTRACT

The aim of this study was to describe the first occurrence ofKudoasp. inGobioides grahamae, contributing to the understanding of this group of parasites in the Amazonian ichthyofauna. Forty specimens ofG. grahamaecollected from the natural environment were analyzed. Cysts ofKudoasp. were diffusely distributed through the striated skeletal muscle fibers with severe edema and inflammatory infiltrate composed of lymphocytes were observed in 30% of the specimens. Edema and marked coagulation necrosis of the muscle fibers was associated with infection byKudoasp. spores, which had accumulated inside the skeletal muscle fibers. Although there are no records of foodborne outbreaks caused by Kudoa spp. in Brazil, it is of paramount importance that we evaluate its occurrence, since the consumption of fish, especially raw fish, has increased because of the adoption of Japanese cuisine. To minimize the economic impacts on the fisheries market and the risk of this parasite to public health, it is necessary to initiate a program to monitor the presence of this likely underdiagnosed, emerging parasite.

Keywords:
muscle fibers; parasite; Myxozoa; Multivalvulida

RESUMO

O objetivo deste estudo foi descrever a primeira ocorrência de Kudoa sp. em Gobioides grahamae, contribuindo, assim, para a compreensão desse grupo de parasitas na ictiofauna amazônica. Foram analisados 40 espécimes de G. grahamae coletados de ambiente natural. Cistos de Kudoa sp. foram distribuídos difusamente através das fibras musculares esqueléticas estriadas com presença de edema grave e infiltrado inflamatório composto de linfócitos, que foram observados em 30% dos espécimes. Edema e necrose de coagulação acentuada das fibras musculares foram associados com a infecção por esporos de Kudoa sp., acumulados no interior das fibras musculares da faringe. Apesar de não haver registros de surtos de origem alimentar causada por Kudoa spp. no Brasil, é de suma importância a avaliação de sua ocorrência, uma vez que o consumo de peixe, especialmente peixe cru, aumentou por causa da adoção da culinária japonesa. Para minimizar os impactos econômicos no mercado da pesca e o risco desse parasita para a saúde pública, é necessário iniciar um programa para monitorar a presença desse parasita emergente, possivelmente subdiagnosticada.

Palavras-chave:
fibra muscular; parasita; Myxozoa; Multivalvulida

INTRODUCTION

The host range of parasites belonging to the phylum Myxozoa comprises mainly fish. The taxonomy of this phylum is based primarily on spore morphology and structural characteristics, such as the number of valves, number of polar capsules, and size of spores, which provide data for the classification of these organisms (Whipps et al., 2004).

The family Kudoidae (Myxozoa: Multivalvulida) includes the genus Kudoa (Meglitsch, 1947) whose principle characteristic is the presence of four valves (in most cases), each one containing one polar capsule (Moran et al., 1999; Casal et al., 2008). This genus has a worldwide distribution and is composed of approximately 95 reported species that parastitize marine and estuarine fishes (Eiras et al., 2014a). Most of these species are histozoic parasites that infect skeletal muscle and form macroscopic cysts, although they are reported to be present in other organs such as the brain, pericardium, digestive tract, kidneys, and ovaries (Matsukane et al., 2010; Casal et al., 2008; Mansour et al., 2013).

The identification and study of these parasites is highly important in fisheries and aquaculture because infection with this pathogen reduces the market value of products and generates economic losses for the industry (Shirakashieret al., 2014). In addition, the effects on public health should be taken into account, because Kudoaparasitism can cause food poisoning and allergic reactions, as previously described forKudoa septempunctata(Kawai et al., 2012;Iwashita et al., 2013). The aim of this study was to describe the first occurrence ofKudoasp. inGobioides grahamae, contributing to the understanding of this group of parasites in the Amazonian ichthyofauna.

MATERIALS AND METHODS

Forty specimens ofG. grahamaewere collected from the natural environment of an area adjacent to the town of Salvaterra (00 of 45'21"S 48°30' 54" W) on the Island of Marajó, in the state of Pará, Brazil. Live specimens were brought to the Laboratório de Pesquisa Carlos Azevedo (LPCA) at Universidade Federal Rural da Amazônia (UFRA) (Belém/PA) and kept in aquariums at a temperature of 28-30ºC. The animals were anesthetized with tricaine methanesulfonate (MS-222) at a concentration of 50mg/L and dissected using a stereoscopic microscope. This study was approved by the Ethics Committee for Experiments with Animals/UFRA (report number 013/2014).

The parasitized tissues were cut into 0.5cm-thick sections, fixed in Davidson solution (formaldehyde PA, acetic acid PA, alcohol 95%, distilled water) for 24h, dehydrated in an increasing series of absolute ethanol, diaphanized in xylol, embedded in paraffin, and then stained with hematoxylin-eosin, Ziehl-Neelsen, and Masson's trichrome. These sections were used for histopathological analyses and results were documented. The measurements of spores were made according to Lom and Dyková (1992) under 1000X magnification in a light microscope with a Zeiss Primo Star photographic camera AxioCam ERC 5s using the AxioVision 5.1 software.

RESULTS AND DISCUSSION

The analysis of the specimens revealed the presence of filamentous cysts in the pharyngeal muscles. Cysts were diffusely distributed through the striated skeletal muscle fibers of 30% (12/40) of the specimens (Figure 1). Similar to that described by Eiras et al. (2016) and Oliveira et al. (2015), the parasites were located inside and between the muscle fibers. The spores were identified by light microscopies belonging to the genusKudoa, based on characteristics including the quadrangular or pseudoquadrangular format, with the presence of four equal and pyriform polar capsules located side-by-side and the apex converging on the apical pole of spores, as described by Casal et al. (2008) and Azevedo et al. (2015) (Figure 2).

Microscopic analysis showed a degenerated epithelium of the palate and multifocal areas of necrosis. Cysts ofKudoasp. with severe edema and inflammatory infiltrate composed of lymphocytes were observed in the submucosa. Edema and marked coagulation necrosis of the muscle fibers was associated with infection byKudoasp. spores (Figure 3 and 4).

Figures 1-4
Kudoa sp. in musculature of Gobioides grahamae. 1. Region palate pharyngeal (*) with filamentous cysts (arrows). Kudoa sp. fresh spores observed (Objective 2X). 2. Cyst (C) and spores (arrows) of Kudoa sp. in fresh muscle (Objective 40X). 3. Kudoa sp. spores (*) located between muscle fibers (m=skeletal muscles, C=conjuntive tissue) (Masson's Trichrome Stain), scale bar: 100µm. 4. Kudoa sp. spores (*) located inside the skeletal muscles fibers of the palate. spores (*) fibers muscles (m) (Staining in Ziehl-Neelsen). Scale bar: 100µm.

Similar histopathological aspects as described here were observed inMorone americana(Bunton and Poynton, 1991), inTrichiurus lepturus(Andrada et al., 2005), and inSebastes paucispinis(Heckmann and Jensen, 1978). Casal et al. (2008) described the disintegration of the myofibrils inAequidens plagiozonatusassociated with parasitism byKudoa aequidens, without observing an inflammatory reaction. In addition, muscular necrosis has been described as a result of Kudoaparasitism in two other species of the Gobiidae family (Gobius paganellusandPomatoschistus micros), in a study carried out by Davies et al. (1998).

Barja and Toranzo (1993) and Henning et al. (2013) suggested that the myoliquefaction of skeletal muscle is related to the fact that these parasites produce proteolytic enzymes that, after the death of the host, act to break down muscle. On the other hand, Eiras (2016b) describes the absence of myoliquefaction in the species Lutjanus analis, Bagre marinus, Aspistor luniscutis and Lutjanus jocu, parasitized by Kudoa sp.

According to Lom and Dyková (1992), myxosporidia infection in a particular host is a result of the interaction of physiological (host resistance) and ecological (environment, nutrition) factors. They report that prevalence of myxospordia can be very high, reaching up to 100%, despite the fact that only a few fish within a stock may be infected initially. They report prevalence of two species, Kudoa paniformisandKudoa thyrsites, up to 90% in Merluccius productus.Bunton and Poynton (1991) also observed a high prevalence (85%) of Kudoa spp. inMorone americana, Vaz (2000) reported prevalence of 84.7% in Trachurus trachurus and Oliveira et al. (2015) reported prevalence of 100% by Kudoa sp in Plagioscion squamosissimus.

This reported prevalence is higher than that found in this study. Values closer to ours were reported by Casal et al. (2008) forK. aequidens(35.7%) and by Menezeset al. (1989) forSardina pilchardus(30%). Andrada et al. (2005) reported a lower prevalence (19%) inTrichiurus lepturus. The majority of Kudoaspecies have specific hosts. However,K. thyrsitesseems to have less host specificity, with reports of this parasite in at least 23 species in several families of fish (Moran et al., 1999).

In Brazil, this genus is recorded in 14 species of fish, in the North, Northeast, South and Southeast (Table 1). There is no record of parasitism of the Kudoa genus in Gobiidae family fish in Brazil. Velasco et al. (2012) described necrotizing myositis in the muscles of the palate ofGobioides broussonetii, due to parasitism byMyxobolussp. captured in the same location as the hosts of this study. Many authors describe infections byKudoaas subclinical, and cysts may not be detected by the naked eye. Therefore, a fish infected with thousands of spores in its muscles can go unnoticed and be consumed (Cruz et al. 2003).

Table 1
Comparative data of occurrence of Kudoa genus in brazilian fishes

Host	Parasite	Locality	Prevalence	Autor
Mugil platanus	Kudoa sp.	Rio de Janeiro-RJ	-	Knoff and Serra-Freire (1993)
Trichiurus lepturus	Kudoa sp.	Niterói-RJ	19,15%	Andrada et al. (2005)
Aequidens plagiozonatus	Kudoa aequidens	Peixe Boi-PA	35,7%	Casal et al. (2008)
Scomberomorus brasiliensis	Kudoa sp.	Mossoró-RN	80%	Eiras et al. (2014b)
Plagioscion squamosissimus	Kudoa sp.	Outeiro-PA	100%	Oliveira et al. (2015)
Chaetobranchopsis orbicularis	Kudoa orbicularis	Cachoeira do Arari (Marajó Island)-PA	47,6%	Azevedo et al.(2016)
Mugil liza, Odontesthesbonariensis and Micropogonias furnieri	Kudoa sp.	Rio Grande-RS	-	Eiras et al. (2016a)
Lutjanus analis, Bagre marinus, Aspistor luniscutis and Lutjanus jocu	Kudoa spp.	Aracaju-SE	-	Eiras et al. (2016b)
Cathorops spixii and Cathorops agassizii	Kudoa sp.	Vigia de Nazaré-PA		Santos et al. (2019)
Gobioides grahamae	Kudoa sp.	Salvaterra (Marajó Island)-PA	30%	Present study

Martinez de Velasco et al. (2008) reported thatKudoaspp. can induce an allergic reaction in some people, and through epidemiological analyses and animal tests, Kawai et al. (2012) reportedKudoa septempunctataas a causative agent in outbreaks of food poisoning. Jeon et al. (2014) suggested that, owing to the outbreaks that have been described, it is necessary to develop effective, feasible methods to detectK. septempunctatato ensure food safety and to facilitate marketing. According to Lee (2017), in 2015, a total of 11 outbreaks caused by K. septempunctata were described associated with the consumption of sushi and sashimi. Diarrhea and vomiting were the most common symptoms observed in these outbreaks.

Although there are no records of foodborne outbreaks caused by Kudoa spp. in Brazil, it is of paramount importance that we evaluate its occurrence, since the consumption of fish, especially raw fish, has increased because of the adoption of Japanese cuisine. To minimize the economic impacts on the fisheries market and the risk of this parasite to public health, it is necessary to initiate a program to monitor the presence of this likely underdiagnosed, emerging parasite. The present study was approved by the Ethics Committee for Animal Experimentation of the Universidade Federal Rural da Amazônia (UFRA) (CEUA approval no. 013/2014).

ACKNOWLEDGEMENTS

The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Edital Universal 2011) and Sistema de Autorização e Informação em Biodiversidade (SISBIO) / Instituto Chico Mendes de Conservação e Biodiversidade (ICMBIO) / Instituto Brasileiro do meio ambiente e dos Recursos Naturais Renováveis (IBAMA) - Licença 27119.

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Publication Dates

• Publication in this collection
  08 May 2020
• Date of issue
  Mar-Apr 2020

History

• Received
  01 Oct 2018
• Accepted
  01 Oct 2019