Abstract: Background and Objective: Sessiline ciliates live as eco commensals (low numbers) and parasites (high numbers) on different hosts, like mollusks copepods, mysids and fish. Riboscyphidia ecto-protozoan is moderately pathogenic but high numbers of it on the gills can physically prevent gas exchange. The present study aimed to describe the epizoic ciliates Riboscyphidia found on the Red Sea cultured Asian sea bass and obtain more information on the Epidemiology of the parasite with special references to control and histopathological examination of naturally infected sea bass. Materials and Methods: The occurrence of epizoic ciliates on the adult Asian Sea bass. About 100 Asian sea bass were collected by the fishing net at a private marine fish farm at Ismailia governorate and transferred to the hydrobiology laboratory at National Research Centre. A parasitological and histopathological study of epizoic sessile ciliate species was done. ANOVA test was used for Statistical analysis. Results: Riboscyphidia sp. was found and isolated after parasitological examination of investigated adult’s Asian sea bass. The prevalence of Riboscyphidiosis was 64%. Sessile ciliates were found on gills, skin and fins. The clinical signs of Riboscyphidiosis were respiratory distress, flashing and off food. Histopathological alterations in naturally infested Asian sea bass were investigated. Conclusion: The treatment of choice of Riboscyphidiosis was prolonged immersion by Copper citrate with a dose of 0.56 mg mL–1 for 7 days.
INTRODUCTION
The intensive fish breeding leads to the recognition of several serious infections of which ectoparasites are striking by their rapid development spread by direct dissemination among all living in close contact.
One of the largest and most widely distributed mobile ciliate genera is Trichodina present on aquatic animal’s hosts1. Sessiline ciliates live as commensals (low numbers) and parasites (high numbers) on different hosts, like mollusks2, copepods, mysids3,4 and fish5. At high numbers, these ciliates have been reported as causing mortality in juvenile and adult cultured fish, causing severe economic losses in different localities of the world6.
The ectocommensal ciliates Riboscyphidia attach to the gills or skin with a holdfast7. Attachment causes superficial destruction to the cells8. Like the sessile, colonial commensals, reproduce by simple binary division and use the host primarily for nutrition and attachment. They derive little, if any, nutrition directly from the host. They feed also on bacteria and suspended organic debris, which is prevalent in food. Thus, they are a good indication of bad water quality9.
Riboscyphidia ciliates parasites for fish, when presented on the fish with low numbers but when the fish stressed (Infected with bacteria, parasites or protozoa or change in the water parameters) act as ectocommensal. Literature about Riboscyphidiosis was scanty and a lot of data was missed about non-motile (Sessiline) ciliates. Thus, the present study aimed to investigate the epizoic of ciliates infect the Red Sea cultured Asian sea bass and obtain more information on epidemiology of the parasite depending on host characteristics and environmental factors and searching for the method to control infection of Riboscyphidia in such fish.
MATERIALS AND METHODS
Studying area: The study was carried out at the Department of Hydrobiology, National Research Centre, Dokki, Giza, Egypt, from September-December, 2019.
Naturally infected fish: Total 100 (1-2 kg) the Red Sea cultured Asian sea bass was collected from private marine fish farm cultured in circular cement ponds at Ismailia province, collected in plastic bags with oxygen and transferred to the Department of Hydrobiology, National Research Centre, Dokki, Giza. Clinical, post mortem and parasitological examinations were done.
Parasitological examination: Scraping from skin, fins and gills were taken, spread on glass slides, dried, fixed with methanol and stained with potassium permanganate and covered with cover slides. Gill filaments were cut and placed in a Petri dish filled with filtered Seawater and examined with a compound microscope (X40)10.
Isolation and identification of the parasite: Wet mounts from skin or gills with parasites were prepared and examination was done through the light electric compound microscope using high power (X40)9. Prevalence of infection of cultured Asian sea bass with Riboscyphidia sp. was also determined.
Treatment design: Naturally infected 120 Asian sea bass were divided into four groups each group 10 with three replicates, 1st group subjected for treatment with formalin bath 150 ppm for 60 min and the 2nd group subjected for treatment with formalin bath with 250 ppm for 30 min. While, the 3rd group was subjected to treatment with chelated copper-like copper citrate (2.23 g+5 H2O+1.5 g citric acid) 0.56 mg mL–1 for 7 days (prolonged immersion), while 4th group was set as control group without treatment (Table 1).
Histopathological studies: Histopathological samples were taken from the skin, fins, gills and musculature of naturally infected investigated fish, Asian sea bass fixed in 10% formol saline for 24 hrs. Samples were prepared and examined according to Bancroft and Stevens11.
Statistical analysis: Data were analyzed for significant differences using the ANOVA test at p<0.05 using SPSS program version 9.0.
| Table 1: Treatment of Riboscyphidia infection in the Red Sea cultured Asian sea bass | |||||
| Groups | Drug | Number of fish |
Treatment type | Dosage of drug | Duration |
| 1st | Formalin | 10 |
Bath | 150 ppm | 60 min |
| 2nd | Formalin | 10 |
Bath | 250 ppm | 30 min |
| 3rd | Copper citrate | 10 |
Prolonged immersion | 0.56 mg mL–1 | 7 days |
| 4th | Control | 10 |
No treatment | ----- | ------ |
RESULTS
Clinical signs and postmortem lesions: Infested Asian sea bass showed, lethargic, flashing, swimming upside, surfacing, gulping the atmospheric air and even jumped outside water, sloughed scales especially at the region of the caudal surface and caudal peduncle and tail fins also at the region of the dorsal musculature. Ulcers also may found on the lesion or not with the growth of green algae on the exposed skin. Post mortem lesions were focused on gills that appeared severely congested stuck and slimmed (Fig. 1a, b). Riboscyphidia sp. attached to primary gill filaments surface epithelium resulting in blocking of gas exchange leading to respiratory distress and suffocation of infected fishes (Fig. 2a-d).
| Table 2: Efficacy of treatment of Riboscyphidia infection in the Red Sea cultured Asian sea bass | |||||||
| Groups | Drug | Number of fish |
Treatment | Dosage of drug | Duration | Treated fish | Treatment (%) |
| 1st | Formalin | 10 |
Bath | 150 ppm | 60 min | 9 | 90* |
| 2nd | Formalin | 10 |
Bath | 250 ppm | 30 min | 7 | 70* |
| 3rd | Copper citrate | 10 |
Prolonged immersion | 0.56 mg mL–1 | 7 days | 10 | 100* |
| 4th | Control | 10 |
No treatment | ----- | ------ | ---- | ----- |
| *Significance n = 10 | |||||||
| Fig. 1(a-b): | Post mortem lesions of gills (a) Infected Asian sea bass congested and stuck gills filaments and (b) Eroded dorsal fins with a shallow ulcer at the dorsal musculature of infected fishes |
| Fig. 2(a-d): | Riboscyphidia sp. attached to surface epithelium of primary gill filaments (a) Primary gill filaments with a high density of Riboscyphidia attached with the epithelial lining of the gill filament (arrow), (b-c) Magnified non-mobile ciliate Riboscyphidia (Arrows) and (d) Stained Riboscyphidia with potassium permanganate (arrow) |
| Fig. 3: | Riboscyphidia parasite (ciliate) with horseshoe nucleus stained with potassium permanganate |
| Fig. 4(a-d): | Histopathological examination of gills, skin and musculature of Asian sea bass Gill suffered from telangiectasis and severe hyperplasia and sticking of secondary gill filaments and infiltration of inflammatory cells (a-b), Skeletal muscle bundles suffered from Zenker's necrosis with infiltration of inflammatory cells, (c) Skin layer suffered from the focal deposition of melanin pigments (d) |
Identification of the parasite: After microscopical examination and from morphological features of the isolated parasite it was identified as Riboscyphidia (Ciliate) sp. (Fig. 3).
Prevalence of Riboscyphidia: From 100 cultured Asian sea bass, about 64 fish was infected with Riboscyphidia sp. with a prevalence of about 64%.
Results of Riboscyphidia infection treatment: The present study displayed that the treatment of 1st group with a formalin bath 150 ppm for 60 min resulted in the treatment of 90% of fish, while the 2nd group displayed treatment of 70% only on the other hand 3rd group which was treated with chelated copper resulted that 100% of fish treated with no mortality of fish Table 2.
Histopathological studies: Samples from gills, skin and musculature were collected and subjected for histopathological examination and revealed that primary gill filament suffered from telangiectasia and severe hyperplasia and sticking of secondary gill filaments and infiltration of inflammatory cells, (Fig. 4a, b) Skeletal musculature suffered from Zenker's necrosis and hyalinization with infiltration of inflammatory cells (Fig. 4c) and skin layer of infected fish suffered from focal melanin pigment deposition (Fig. 4d).
DISCUSSION
The present study revealed the epidemiology and pathogenesis and control of Riboscyphidia ciliate on cultured Asian sea bass. Riboscyphidia ectoprotozoan are moderately pathogenic but with high numbers of it on the gills can physically prevent gas exchange. They may also act as a portal for bacterial colonization12. The goal of the study was to determine epizoic ciliates Riboscyphidia infecting the Red Sea cultured Asian sea bass and obtain more information on the epidemiology of the parasite depending on host characteristics and environmental factors and searching for a method to control infection of Riboscyphidia in such fish.
Regarding the clinical signs and post mortem, the present study revealed that infested Asian sea bass was showed, lethargic, flashing, swimming upside, surfacing and sloughed scales. Post mortem lesions were focused on gills that appeared severely congested and slimmed, present results nearly agree with that obtained by Fernandez-Leborans et al.4 and Noga9 most infections were concentrated in gills thus all observed sings were respiratory distress like surfacing.
Concerning the identification of infected parasite present study displayed that from morphological features of the isolated parasite it was identified as Riboscyphidia (Ciliate) sp. according to Lom7.
Regarding the result of the treatment of infected fishes with Riboscyphidia, the present study revealed that the treatment of choice was chelated copper citrate, the result nearly agrees with the results recorded by Van and Basson1 and Noga9.
Regarding the results of the histopathological examination of the present study revealed that gills, skin and musculature were subjected for histopathological examination and revealed that primary gill filament suffered from telangiectasis and severe hyperplasia and the skeletal musculature suffered from Zenker's necrosis and hyalinization. The results confirmed with the results obtained by Noga9, Bancroft and Stevens11 and Lee et al.12
Riboscyphidia ciliate infection is ectocommensal when found on fishes with low numbers or as secondary infection accompanied by bacterial or mycotic infection, Riboscyphidia is easily treated but the treatment of choice was copper citrate bath treatment “prolonged immersion”. It is moderately pathogenic for fishes causing death for young fish.
CONCLUSION
From the present study, it was concluded that Riboscyphidia infection mainly infects fishes as a secondary infection after bacterial or mycotic disease. And prolonged immersion of copper citrate at the dose 0.56 mg L–1 was recorded as the best treatment for Riboscyphidia infection in the Red Sea cultured Asian sea bass.
SIGNIFICANCE STATEMENT
This study discovers the methods of control and treatment of Riboscyphidia when infecting Asian sea bass that can be beneficial for Veterinarians and people who work in fish industries. This study will help the researcher to uncover the critical areas of pathogenesis and epizootics of Riboscyphidia on infected the Asian sea bass with the determination of treatment of choice of Riboscyphidia that many researchers were not able to explore. Thus, a new theory on control of the Riboscyphidia may be arrived at.