Ciliate parasite Ichthyophthirius multifiliis causing acute mortality in tiger barb (Puntigrus tetrazona, Bleeker, 1855) and widow tetra (Gymnocorymbus ternetzi, Boulenger, 1895)

Abstract

A mixed group of tiger barb (Puntigrus tetrazona) and different colored glo widow tetra (Gymnocorymbus ternetzi) were procured from Galiff street market of Kolkata, India and stocked in a glass aquarium with 250L water capacity for ornamental fish decorative purpose. After a few days of stocking, fishes were (mean weight of 1.8 ± 0.5 g and mean length 5.0 ± 0.3 cm) found severely infected with white spot disease. Initially, the manifestation was clearly observed in Tiger barb followed by widow tetra with clinical signs of white spot, loss of fin, complete loss of gill filaments, and anorexia. The causative agent was identified as protozoan ciliate parasite Ichthyophthirius multifiliis, by wet mount preparation. The histopathological observation of infected fish skin tissue depicts that epidermis were severely damaged and presence of trophozoite parasite in dermis near the blood vessels. Molecular characterization of the parasitic pathogen was carried out by partial18s rDNA sequencing and the obtained sequences were showing 100% similarity with previously reported Indian I. multifiliis sequence.

Introduction

The Ichthyophthirius multifiliis is a broad host range holotrichous ciliate parasite of freshwater fish, ubiquitously found all over the world. The parasite has a complex life cycle including a host-specific trophont stage which eventually comes out of the host and encysted into tomont and after binary fission, hundreds of infective theront released free swimming to infect the susceptible host. It is one of the parasites associated with mass mortalities in wild as well as cultured fish (Wurstbaugh and Tapia 1988; Jessop 1995). The parasite is commonly encountered in ornamental fish but many food fish including carp, tilapia, rainbow trout, eel, and striped catfish are also reported to be susceptible. Among ornamental fish barbs, zebrafish, platy, angel, fighter fish, gourami, and goldfish were reported varyingly susceptible to this parasite (Price and Bone 1985; Cherry 2003; Maceda-Veiga et al. 2009; Wei et al. 2013; Liu and Lu 2004; Mecha and Gonzales-Plasus 2023). The severity of infection depends on water temperature, size, variety of fish, and secondary infection (Mikkelsen et al. 2006; Cherry 2003; Price and Bone 1985; Liu and Lu 2004). There are no commercial vaccines available for the prevention of I. multifiliis and repeated chemical treatment, and elevations in water temperature are found to be successful disease control strategies. However, a certain degree of immunity may be acquired in case of exposure to survival from natural infection.

I. multifiliis infection has been reported in several places of India and some of them are associated with mass mortalities (Chanda et al. 2011; Mallik et al. 2015; Kumar et al. 2018 & Mamun et al. 2020; Kumar et al. 2022). However, molecular analysis of the Indian strain has been relatively sporadic to date. In this paper mortality of two ornamental fish namely tiger barb (Puntigrus tetrazona) and different colored glo widow tetra (Gymnocorymbus ternetzi) due to I. multifiliis infestation were first time reported and 18 s rDNA sequencing was carried out for molecular characterization.

Materials and Methods

Sampling details

Total hundred fishes (50 tiger barb and 50 glo tetra) of mean weight (g) of 2.5 ± 0.3 and mean length (cm) 5.5 ± 0.1 were procured from Kolkata Galiff street and maintained in a glass tank with 250L water capacity during the month of November. After a week of stocking, tiger barb displayed early manifestations of infection and consecutively after two days tetras. The clinical signs include white spot throughout the body, loss of fin, complete loss of gill filaments, and anorexia (Fig. 1a). The infected fish were dark in color, remain isolated, slow-moving, and stop feeding. Mortality was within 48 h unset of symptom. The water temperature at the time of mortality was 23 °C and pH 7.5.

Fig. 1.

a The infected Tiger barb and neon widow tetra showing clinical manifestation numerous white spots throughout the body. b Wet mount of skin mucus showing ich parasite of 250 um width having horse shoe shaped macronucleus

Microscopic observation

A fresh wet mount of skin scrap was prepared on clean glass slide with a drop of filtered water and observed under light microscope (RXLr-5). The individual parasites were separated and measured under microscope. Rest of the mucus scrap (0.5 ml) was collected aseptically in 1.5 ml eppendorf tube for molecular identification.

Molecular analysis

The total genomic DNA was purified using DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer’s instructions. The product was then quantified in a Nanodrop (Thermo Scientific, Wilmington, USA) spectrophotometer at 160 ng/μl. The reported primer (Jousson et al. 2005) forward primerIMRf1 (5’-AGTGACAAGAAATAGCAAGCCAGGAG-3’) and reverse primer IMRr1 (5’-ACCCAGCTAAATAGGCAGAAGTTCAA-3’) located at positions 455 and 647 of I. multifiliis 18S sequence were used for polymerase chain reaction amplification. The PCR amplification was carried out in a 25 μL reaction volume containing 100 ng of template DNA, 200 μM of each dNTP, 10 pmol of each primer, 1 U of Taq DNA polymerase, and 1 × Taq buffer with 15 mM MgCl₂ (Thermo Fisher Scientific, USA). The thermal condition was set as initial denaturation at 94 °C for 5 min, followed by 35 cycles (1 min denaturation at 94 °C, 1 min primer annealing at 58 °C, and 1 min primer extension at 72 °C) then the final extension step of 7 min at 72 °C. The gel electrophoresis was performed with 2% agarose. The amplicons were sequenced (Barcodebiosciences, Bangalore) in both direction and the obtained sequence were submitted in NCBI GenBank database. The obtained sequence with accession number were analysed with Basic Local Alignment Search Tool (BLAST).

Histopathology

The small pieces from skin of moribund fish were preserved in neutral buffered formalin for histological preparation. After 72 h fixation, the tissue were washed under tap water, dehydrated and cleaned with xylene prior to paraffin embedding. Sections of 5 micromieter thick were prepared and stained in H & E and mounted with DPX solution. The stained slides were observed under light microscope (RXLr-5).

Result and discussion

The wet mount of infected fish mucus was observed under a light microscope and multiple spherical parasites of more than 100-µm diameter and with horseshoe-shaped macronucleus were clearly observed (Fig. 1b). These are identified to be tissue-specific trophont stages of ciliate parasite I. multifiliis. The parasitic trophonts were only observed in moribund conditions or when the host was near death, whereas it gradually absorbed into a dense spherical body with indistinct internal structure in the case of dead fish. Compiled information on I. multifiliis outbreaks reported from different countries is mentioned in Table 1. It indicates that the preferred temperature for the outbreak remain < 30°C and higher than 10°C (12–23°C) as mentioned by Mallik et al. (2015). The size of the trophont remains in a range of 0.25–0.66 mm.

Table 1.

Comparative statement of data on I. multifiliis reported from different countries

Countries	Host Fish species	Prevalence (%)	Temperature (0c)	Size of parasite	References
India, Uttarakhand	Schizothorax richardsonii	84.80 ± 1.83	21.4 ± 0.120	0.8 mm	Mallik et al. (2015)
India, Mangalore	Pangasianodon hypophthalmus	100	18	0.9 mm	Mamun et al. (2020)
India, Mangalore	Pterophyllum scalare & Trichopodus trichopterus	No data	23	1 mm	Mamun et al. (2020)
India, West Bengal	Puntigrus tetrazona & Gymnocorymbus ternetzi	100 in both	23	0.25 mm	Present study
China, Tibet	Schizothorax macropogon & Tachysurus fulvidraco	No data	12.5 (S. macropogon) (T. fulvidraco)	0.28–0.49 mm (S. macropogon) 0.33–0.66 mm (T. fulvidraco)	Li et al. (2022)
Brazil	Steindachneridium parahybae	100	20	No data	Corrêa et al. (2019)
Canada, Nova Scotia	Anguilla rostrata	23–12	14	No data	Jessop (1995)

The PCR amplification was showing clear amplicon band visible at 200 bp size. The amplicons were sequenced in both direction and obtained sequence were submitted in NCBI GenBank database with accession number OM370900, OM370901, OM370902 and OM370903 (Fig. 2). All of the obtained sequence with accession number were analysed with Basic Local Alignment Search Tool (BLAST) and showed 100% similarity with previously reported I. multifiliis sequences. The parasite I. multifiliis is classified under suborder Ophryoglenina of order Hymenostomatida which carry a “watch glass organelle” near the oral region (Lynn et al. 1991). Similar to other taxa of ciliates these are histophagous, polymorphic lifestyle and reproduce by palintomy. A phylogenetic tree was made to compare the closeness with other ciliates (U17355, X56533, X56532, M98015) by selecting 18srDNA (Fig. 3). Other than previously reported I. multifiliis sequence, the nearest match is with Ophryoglena catenula, which is another ciliate of same suborder and result corroborates previous report by Wright and Lynn (1995).

Fig. 2.

H & E staining of infected fish skin depicting: darkly stained spherical I. Multifiliis with distinct macronucleus underneath dermal layer and epidermis has greatly reduced

Fig. 3.

Neighbour-joining tree without distance correction by clustal omega predicting closeness with other ciliates

In histological observation of skin tissue slides, a picture of late stage of infection was detected (Fig. 2). The parasite with typical macronucleus was clearly located within dermis near the blood vessels. The epidermis has greatly reduced and the vascularisation of spogiose dermis has engorged and more prominent. The parasite could reach the extreme deep of epidermal layer within 24 h of post exposure as previously reported (Hines and Spira 1974). Initial hyperplasia of skin epithelial layer, excess mucus cell production is similar phenomenon as in case of other parasites Trichodina, Argulus, Macrogyrodactylus congolensis (Adly et al. 2015; Kabata 1970; Maduenyane et al. 2022) infecting fish skin.

Funding

The authors received no specific funding for this work.

Declarations

Conflict of interest

The authors declare that they have no conflict of interests.