The characteristic of myxosporean infection in several organs of nile tilapia cultivation

The myxosporean is a parasite that frequently infects several fish organs, therefore it can be identified by histopathologically assessing the character of the infection in the infected organs. The present study was carried out to assess the infection of myxosporean (Myxobolus sp.) in nile tilapia cultivation by observing the histomorphological characteristic of several organs. Live Nile tilapia sample from Gresik cultivations immediately was done necropsy and observation on gill, intestine and liver. Histological preparation was made for the gills, liver and intestines and observed with HE staining. In the gills of nile tilapia, variable numbers of active spore of Myxobolus sp. can displace the gill lamellae, inflammatory and incite hyperplastic responses, lamellar fusion, and gill distortion in several infections. The spore infiltrates the surface of the liver of the host causing serious damage to the liver tissue. Fish infected with the parasite exhibit necrosis and cysts in the gut. Parasites were detected within multiple segments of the intestine. While occurrences within the intestinal lumen were infrequent, conspicuous clinical indications were notably absent upon inspection of the fish’s external body surface. The findings unequivocally establish that Myxobolus sp. infection in tilapia induces substantial damage to the crucial organs of the fish.


Introduction
Nile Tilapia (Oreochromis niloticus, O. niloticus) is a freshwater fish that is widely known throughout the world, including in Indonesia.Nile Tilapia has many advantages including its delicious taste, relatively high nutritional content, fast growth, easy to cultivate because it easily adapts to the environment [1].Tilapia possesses robust disease resistance and a diminished reliance on respiration, enabling it to withstand unfavorable conditions characterized by low oxygen levels and elevated ammonia concentrations.These attributes underscore its adaptability to challenging environmental circumstances [2].In most areas of the province of East Java nile tilapia is widely cultivated as a commodity that has high economic potential.So far, various diseases and water quality problems have disrupted tilapia cultivation.
Myxosporeiasis such as myxobolusis caused by Myxobolus sp. is a disease that often interferes with tilapia cultivation but has not been widely reported by the cultivator community.It is suspected that infection by Myxobolus sp.occurs frequently in nile tilapia cultivated including in several areas of cultivation of East Java.The life cycle of myxosporea is complex, encompassing vegetative stages within two distinct hosts: an aquatic invertebrate (typically an annelid, occasionally a bryozoan) and an ectothermic vertebrate, commonly a fish.Due to this parasite can infect several organs of fish, its identification can be done by assessing the character and damage of the infection in the infected organs.Given these considerations, a pathological examination of Myxobolus infections becomes imperative in order to ascertain the extent of damage inflicted upon the affected tissues.Histopathological analysis can serve as a valuable biomarker for assessing the health status of fish, including those afflicted by diseases, enabling comprehensive health monitoring.
Vital organs pivotal for metabolic processes offer an avenue for early diagnosis of fish health issues through observation and analysis [3].Similarly, within Indonesia, there has been limited exploration of myxoboliasis in freshwater fish, particularly tilapia, resulting in a relatively unexplored realm of myxosporean species diversity.This research aims to assess the characteristic and damage of several organs such as gills, intestines and liver histopathologically due to infection of Myxobolus sp.

Sample collection and examination
All experiments related to animals were conducted based on the Guideline of Animal Ethics and Welfare procedure and approved by the Faculty Veterinary Medicine of Universitas Airlangga.Tilapia samples were collected from five tilapia aquaculture locations in Gresik, one of the districts in East Java Province, Indonesia.Twenty tilapia fish were selected at random from each cultivation site, with no consideration given to the presence or absence of observable disease symptoms.Nile tilapia samples harvested directly from Gresik Nile tilapia cultivation underwent immediate necropsy, followed by meticulous observation of various internal organs including the gills, liver, and intestines.These examinations were conducted at the Parasitology Laboratory of the Faculty of Veterinary Medicine, Universitas Airlangga, and were completed within a 24-hour timeframe.Before conducting the necropsy, a thorough external examination was performed on the fish to ascertain the presence of any anomalies.

Histopathological preparation
Subsequently, a necropsy was conducted, during which various organs, including the gills and internal structures such as the liver and intestines, were meticulously isolated for subsequent histopathological examination.The organs of the fish comprising gills, liver, and intestines were meticulously prepared for histological analysis, following the Stoskopf method [4].Subsequently, the tissue specimens were thoroughly rinsed with normal saline and then meticulously fixed in a 10% buffered formalin solution, allowing for a 24-hour fixation period.Following the fixation process, the tissue underwent gradual dehydration through a succession of ascending alcohol concentrations.Subsequently, the tissue was embedded in paraffin wax and skillfully sectioned at a thickness of 5 μm.Tissue sections were subjected to staining using the hematoxylin-eosin (H&E) method.

Microscopic examination and parasite assessment
The ensuing histopathological analysis was conducted in accordance with the guidelines outlined in the book by Genten et al. [5].Every slide was meticulously examined utilizing a high-powered compound light microscope set at a magnification of 1000x.Visual documentation was facilitated by capturing images using a Nikon DS-Fi2 HTC-one camera, mounted on a Nikon H600L microscope.Each tested slide was subjected to thorough examination across five distinct fields of view, allowing for a comprehensive histomorphological assessment to determine the presence or absence of myxosporean parasites.The quantification of parasites was meticulously performed to gauge the extent of infection, all under a magnification of 1000x.

Results and discussion
All samples from several Gresik tilapia aquaculture had no clinical signs observed.Samples from several Gresik tilapia aquaculture had no clinical signs observed.As a low subclinical (enzootic) infection and as a cystic tomont, this parasite survives in the body of the fish.Such conditions persist in the environment, becoming clinically epizootic infections when fish are stressed due to poor management practices (e.g.poor feeding, overcrowding and poor sanitation).The pathogen is not host specific and recovery from disease confers resistance to reinfection [6].Despite the absence of clinical indications 3 of myxosporean infection in the tilapia samples, the parasites were detected and identified in the present study through histopathological analysis.Three possibilities can be occurred in that condition, firstly the infection status is still at the subclinical level and secondly the host is immune to reinfection because it has been infected before.The third is that O. niloticus can survive in adverse environmental conditions due to its strong disease resistance and light respiratory needs so that it can tolerate low oxygen and high ammonia levels which are predisposing factors for myxosporean (Myxobolus sp.) infections.After necropsy and histomorphological observation of gill, liver and intestine were found group of myxosporean parasites that to be target in this study, in the intestines were found intestine myxosporean (Myxobolus sp.) in several aquacultured nile tilapia in Gresik (Fig. 1).

Figure 1.
Histopathological alterations indicated considerable harm to the gill lamellar tissue, encompassing conditions including congestion, oedema, hyperplasia, and lamellar fusion, all attributable to the infection caused by the myxosporean (Myxobolus sp.) (A).Despite these changes, it is notable that no nodules were detected within the afflicted fish gills., there was epithelial proliferation (B), the observation revealed intestinal edema accompanied by epithelial erosion, with sporadic presence of myxosporean (Myxobolus sp.) organisms observed on certain sections of the mucosal surface of Nile tilapia intestines.Furthermore, it was observed that these parasites had progressed to the extent of infiltrating the deeper layers of the intestinal tissue.(C & D), liver of nile tilapia infected with Myxobolus sp showed hepatic cells experiencing necrosis (E & F).A-F, 1000x magnification.
The primary respiratory organs of fish are their gills, functioning through the vital process of respiratory gas diffusion, which facilitates the exchange of oxygen and carbon dioxide between the bloodstream and the surrounding water [7].Comprising filamentous structures, gills possess an expansive surface area, with delicate filaments and gill lamellae (respiratory epithelium) of a remarkably thin nature, functioning as the focal points for the exchange of respiratory gases [8].Histological examinations conducted under a microscope revealed distinguishable disparities between non-infected carps and their infected counterparts, with the former exhibiting notably lesser tissue damage within their bodies compared to the latter.The observations unveiled damage to the gill lamellar tissue, characterized by evident indicators such as oedema, congestion, hyperplasia, and fusion of the lamellae.Although healthy fish gills exhibited an absence of nodules, certain alterations were still detected in fish devoid of Myxobolus sp.infection.Both infected and non-infected fish exhibited the presence of gill lamellae, along with indications of oedema and congestion.Oedema denotes cellular inflammation stemming from an accumulation of surplus fluid within a tissue, causing the tissue to swell and impede its proper functionality.
Oedematous conditions can notably compromise the efficiency of gas diffusion due to the considerably reduced absorption surface area of the lamellae.Elevated hydrostatic pressure, often linked to liquid infiltration into the body, can precipitate oedema [7].Invariably, oedema ushers in the detachment of epithelial layers from secondary lamellae, coupled with the subsequent demise of epithelial cells.This cascade of events disrupts the epithelium's role as a receptacle for dissolved gases [9].Notably, gill swelling or oedema can ensue from direct contact between myxosporeans and the epithelial cells lining the gill surface, inducing irritation.The aquatic environment of nile tilapia farming commonly hosts parasites such as Trichodina, Trematoda, and Myxosporidia.These organisms might contribute to some instances of primary-level epithelial hyperplasia.It's worth noting, however, that the extent of hyperplasia instigated by parasites is seldom extensive [9].This hyperplastic cell network is believed to proliferate from the primary epithelium, resulting in the differentiation of cells necessary to adapt to environmental shifts, particularly in cases of injury or inflammation.Notably, Lovy et al.'s research uncovered that damaged gill filament cartilage often aligns with instances of epithelial hyperplasia and lamellar fusion [10].In the Table 1, the prevalence of myxosporean infection (Myxobolus sp.) in several sampled tilapia aquaculture in Gresik district where fish sampling was carried out not based on visible clinical symptoms that lead to myxobolus infection.Each cultivation was taken as many as 20 heads at five cultivation locations.Table 1 showed the prevalence of myxosporean infection (Myxobolus sp.) in several sampled tilapia aquaculture in Gresik district where fish sampling was carried out not based on visible clinical symptoms that lead to myxobolus.Each cultivation was taken as many as 20 heads at five cultivation locations.After necropsy was carried out on all the fish taken, then histopathological examination was carried out on several internal organs, the average prevalence rate was 29%.This condition indicated that infection with myxosporean (Myxobolus sp.) has attacked almost all nile tilapia farming in Gresik district, although most of them did not show clinical signs of myxosporeasis (Myxobolusis).In general, Myxobolus sp.infection in tilapia takes place without clinical symptoms.This is due to the fact that tilapia is a fish that can survive in poor environmental conditions due to its high disease resistance and low respiratory demands.As a result, tilapia can tolerate low oxygen and high ammonia levels, as well as numerous water quality issues and various diseases.[2].
The data obtained was very interesting where the results of histopathological examination of the intestinal organs showed a tendency for parasites seen in the small intestine to be less than in the large intestine, this occurs in almost all samples of tilapia (Table 2 and Fig. 2).Similar to natural infection of Unicauda fimbrethilae n. sp.(Cnidaria: Myxosporea: Myxobolidae), in the small intestine frequently is found lower than the large intestine in asymptomatic fish [11].The previous study on freshwater giltheads for observation of myxosporean (Enteromyxum leei) in intestine reported the large intestine is the parasite main target in the infection process [12].Therefore, lower level of infection was found in the proximal portions than in the distal bowel.) was recognized through GI Tract histomorphological observation of several farmed fish in some farms in Gresik.At the evaluation of every five microscopic fields of view, it was found that the parasites in the large intestine were higher than in the small intestine.nucleus shrinking (pyknosis) and enlarged cells, the morphology is not clear or absent karyolysis.Some parts of the cell gradually disappear from the tissue causes a decrease in tissue activity which indicates the occurrence of necrosis [13].Therefore, cell death is imminent.Myxobolus sp.cysts (in circles) have been found in the intestine.In the mucus lining the intestinal wall, Myxobolus sp.spores are usually detected.It can occur as a solitary spore intervening or as a small group of invading spores and attaching to the intestinal cell wall [14].Myolytic caused by cyst infection Myxobolus sp. in the intestine can undermine epithel of mucosa and villi.Myolytic occurs in the intestinal wall due to intestinal infection [15].Figures 1E and 1F showed liver damage in tilapia infected with Myxobolus sp.Necrosis and inflammation were hallmarks of damage found in fish infected with Myxobolus sp (Fig. 1E & 1F).Although the area of necrosis was not extensive, Myxobolus infection clearly caused necrosis of liver cells (Fig. 1E & 1F).The liver tissue necrosis makes the staining of hematoxylin non-absorbable.Several bio-agents such as bacteria, parasites fungi dan viruses, as well as blood transport disturbance factors that affect blood supply to certain tissues can cause necrosis in fish livers.Triggers of specific damage to these cells are usually caused by all of these disturbances [16].Red blood spots were found due to excessive erythrocytes coming out of the vessels on liver cell analysis.The previous study reported that inflammation is recognized through existing blood clots due to many erythrocytes are discovered coming out of the blood vessels [17].Response of this inflammatory purposed to restore tissue and depress necrosis-causing agents.In parasitic infections, generally changes of cell such as coccidiosis of the liver can also happen.The symptom is a lumpy enlarged oocyst with development of granulomas from a fibrotic connective tissue or capsule that partially replaces the parenchyma of host's liver.So that there is a decrease in weight and even death due to this condition due to damage to the functional activity of the liver [18].

Conclusion
Myxosporean (Myxobolus sp.) infects gills, liver and intestine tract of fish, but nothing clinical signs obviously based on inspection of the fish body surface carried out, however histopathologically of those organs were clearly seen the characteristic and damage of infection of Myxobolus sp.

Figure 2 .
Figure 2. The existence of Myxosporean (Myxobolus sp.) was recognized through GI Tract histomorphological observation of several farmed fish in some farms in Gresik.At the evaluation of every five microscopic fields of view, it was found that the parasites in the large intestine were higher than in the small intestine.

Figure
Figure IC and 1D showed changes in intestinal cells of infected tilapia.In healthy fish intestine, lamina propria, villi structure, epithelial cells of mucosa and goblet cells are still observable and the structure of the intestinal tissue can be completely stained.While damage to cells of intestine in the necrosis form occurs in suffered fish.Cells of necrosis can be known from the characteristics of the cell

Table 1 .
Prevalence of myxosporean (Myxobolus sp.) in nile tilapia Gresik cultivation by histomorphological examination of the internal organs

Table 2 .
The presence of myxosporean (Myxobolus sp.) was assessed by examining five microscopic fields within both the large and small intestines of each Nile tilapia cultivation group from GresikThe parasites per five fields of view (Mean ± SD)