Determination of Co-infection in Diseased Seven khramulya (Capoeta capoeta)

This study was conducted identify the cause of mortality in diseased Seven khramulya (Capoeta capoeta) and to determine the damage to the fish tissue caused by the agents identified. While hemorrhage on the fins and abdominal region of diseased fish, necrosis in gills, darkening color and loss of scales were externally determined, the presence of bloody and smelling liquid in the abdominal cavity, necrosis of visceral organs, splenomegaly, hyperemia and hemorrhage in the visceral organs were internally observed. As a result of the parasitological examination, Gyrodactylus sp. were found on the gills of the diseased fish, and Trichodina sp. were found on their skin. Bacteriologically, isolated bacteria were identified as Aeromonas hydrophila, Vibrio fluvialis, Staphylococcus warneri and S. capitis. Histopathologically, thickening of epicardium in heart tissue, myopathy, periglomerular and tubular edema melanomacrophage and hemosiderin foci, the infiltration of inflammatory cells in the kidney, hyperemia, infiltration of inflammatory cells in the liver were observed. A. hydrophila, V. fluvialis, S. warneri, and S. capitis were first isolated and identified as disease agents from the co-infection in diseased Seven khyramulya, and furthermore, pathological changes in tissues caused by these pathogenic bacteria were investigated in detail. Research Article


INTRODUCTION
The Cyprinidae family constitutes the most important family among the fish species distributed over the world (Blanc et al., 1971;Howes, 1991). The genus Capoeta, a member of this family, is distributed from West Asia to Central Asia, and the fish species in this genus live in fast or slow flowing lakes and rivers (Geldiay and Balik, 1996). It was reported that this genus has five species and 7 subspecies in fresh waters of Turkey (Geldiay and Balik, 1996;Demirsoy, 1997). It was also reported that the most important species that are cultured among these species were common carps (Pullin, 1986).
Seven khramulya (Capoeta capoeta) is a fish species with economic importance, which is consumed as food by local people in the Southeastern Anatolia Region (Geldiay and Balık, 1996;Şen and Canpolat, 2011). It was reported that the total fishing of Seven khramulya species in Turkey was 695 tons in 2015 and reached 708 tons in 2016 (TUIK, 2016). However, according to the data for 2016, it is observed that it decreased by 1.6% compared to 2015. Therefore, it was indicated that studies should be conducted on the farming of this fish (Demir, 2017).
Despite the reports indicating that bacterial pathogens such as A.hydrophila, A.sobria, V.fluvialis, A. salmonicida, Flavobacterium sp. cause disease in common carps in previous studies (Guz and Kozinska, 2004;Monette et al., 2006;Adanır and Turutoğlu, 2007), there is no study on the bacterial diseases of this fish, which is considered to be cultured and is a Cyprinid species.
This study was conducted to identify the agents causing mortality in Seven khramulya through parasitological and bacteriological methods and to determine the histopathological damage to tissues caused by the agents identified.

Fish Samples
Diseased Seven khramulya (150-200 g), which is found in freshwater in the Euphrates Region and where deaths are observed, was used as a material in this study. The temperature of the water where diseased fish found was 20-21°C.

Bacteriological Examination
For microbiological analysis, samples were taken from liver, spleen and kidney from all moribund Seven khramulya. They were inoculated onto Tryptic Soy Agar (TSA) and Brain Heart Infusion Agar (BHIA). Petri plates were incubated at 24-25 °C for 24-48 h. The isolates recovered from diseased fish were identified by using conventional bacteriological method. In addition, all isolates were determined together with their biochemical characteristics using rapid identification kits such as API20E and API STAPH (Buller, 2004;Austin and Austin, 2016).

Parasitological Examination
The presence of external parasites was investigated by examining the fresh samples from the gills and skin of diseased Seven khramulya under a light microscope. Furthermore, the samples taken from the intestine, stomach, and gallbladder of the same fish after autopsy were examined for internal parasites (Bullock, 1978;Timur and Timur, 2003).

Histological Examination
Samples of tissues from liver, kidney, spleen, gut, gills, heart and gill immediately fixed in 10% buffered formalin and processed for paraffin embedding. Histological sections (4-5µm) were stained with haematoxylin and eosin (H&E) and examined by light microscopy (Culling, 1963).

Clinical Findings
While clinical findings such as weakness, slight loss of scales, necrosis in gills, cloud like thin white film layer on the body surface and hemorrhage in the dorsal and tail fin are remarkable in the external examination of diseased Seven khramulya (Fig. 1a,b), accumulation of fluid in the abdominal cavity, hemorrhage and hyperemia in internal organs, and splenomegaly were observed in the internal examination (Fig. 1c).

Parasitological Findings
As a result of the parasitological examination, Gyrodactylus sp., one of the external monogenean parasites, was found on the gills and Trichodina sp., one of the protozoan ciliates, was found on the skin of the diseased Seven khramulya.

Bacteriological Findings
Bacteria with 4 different colony morphologies reproduced in the medium at the end of the incubation period. A total of 20 bacterial isolates were obtained from these colonies, and these isolates were examined in 4 different groups based on their morphological, physiological and biochemical characteristics. The bacteria of the first isolate (n=5) were identified as Aeromonas sp. since they were Gram-negative, motile bacilli, gave a positive reaction to cytochrome oxidase and catalase tests and also were sensitive to O/129 test. Since isolate number 2 (n=5) was Gram-negative motility bacilli, gave a positive reaction to cytochrome and oxidase test and was sensitive to O/129 test, the isolated bacteria were identified as Vibrio sp. It was determined that isolates number 3 (n=5) and 4 (n=5) were Gram-positive, cluster forming, cocci shaped bacteria and also gave a negative reaction to cytochrome oxidase and a positive reaction to catalase. Therefore, these isolated bacteria were identified as Staphylococcus spp. According to the results of all biochemical tests, the isolated bacteria were identified as A. hydrophila (isolate 1), V. fluvialis (isolate 2), S. warneri (isolate3), and S. capitis (isolate 4). All results obtained were confirmed by the API 20E and API STAPH kit. The morphological, physiological and biochemical characteristics of the isolated bacteria and their API profiles are presented in Table 1.

Histopathological Findings
Histopathologically, thickening of epicardium in the heart tissue of the diseased fish (Fig. 2a), myopathy and inflammatory cell infiltration (Fig. 2b), periglomerular and tubular edema in the renal tissue of the kidney, melanomacrophage and hemosiderin foci, necrosis and inflammation reaction in the hematopoietic tissue were detected (Fig. 2c). Furthermore, it was also remarkable that there was hyperemia in the liver tissue of infected fish, intense infiltration of inflammatory cells, and necrosis in hepatic cells (Fig. 2d). As a result of the parasitological examination, hyperplasia at the ends of the primary filaments of the gills in which Gyrodactylus sp. was detected (Fig. 2f), and edema in secondary lamellae were observed (Fig. 2e).

DISCUSSION
Stress factors, such as the increase in organic load occurring in the habitat of fish that are found in nature and cultured, oxygen deficiency and pollutants, cause bacterial diseases and therefore mortality in fish populations (Roberts, 2001; Austin and Austin, 2016). Aeromonas infections are an important bacterial disease observed in many freshwater fish (Roberts, 2001;Austin and Austin, 2016). The pathogens that cause this infection include bacterial species such as A. hydrophila, A. caviae, A. sobira, A. veronii, A. salmonicida, and A. bestiarum (Timur and Timur, 2003;Buller, 2004;Austin and Austin, 2016). It was reported that mobile Aeromonas species that cause diseases in nature, aquaculture medium and even in aquarium fish production exist in large amounts in places where there is organic pollution. In the studies conducted so far, A. hydrophila was reported to be the pathogen bacteria, which was defined as an opportunistic pathogen and was the most predominantly isolated pathogen among these species (Roberts, 2001;Nielsen et al., 2001;Timur and Timur, 2003). While some of the bacteria examined in this study are Gram-negative and motile bacilli and identified as A. hydrophila due to their morphological, physiological and biochemical characteristics, as previously reported (Buller, 2004;Austin and Austin, 2016).
It was reported that A. hydrophila causes primary or co-infections in cyprinid fish and causes mass deaths as a primary agent in common carps cultured in Northern Greece (Sioutas et al., 1991), and as a coinfection with Flavobacterium sp. in common carps in the habitat in the St. Lawrence River in Canada (Monette et al., 2006). As reported by Bohai et al. (1993) in various carp species, A. hydrophila and V. fluvialis were isolated and identified in this study. As stated previously by Monette et al (2006), we think that the reason for observing this pathogen, which was also reported in common carps in nature, in the coinfection in diseased fish examined is the physical and environmental factors.
Vibriosis disease caused by different Vibrio species was reported in many naturally grown or cultured freshwater and sea fish species (Roberts, 2001;Austin and Austin, 2016). Vibrio species causing disease in freshwater fish mostly involve pathogens such as V. anguillarum, V. fluvialis (Han-Chang, 2009). In this study, V. fluvialis was isolated and identified from diseased Seven khramulya, as it was reported in various diseased common carp by Bohai et al (1993).
In freshwater fish, Gram-positive bacteria such as S. aureus, S. epidermidis, S. cohnii subsp cohnii, and S. warneri (Varvarigos, 2001;Akaylı et al., 2011;Austin and Austin, 2016) mainly cause stapylococcosis. There was a report indicating that S. warneri isolated from diseased Seven khramulya causes disease in Oncorhynchus mykiss cultured in our country (Metin et al., 2014). There are no studies indicating that isolated S. capitis causes disease in fish we found.
Protozoan parasites that cause disease in fish are defined as opportunistic or compulsory pathogens. Protozoan parasites found on fish skins include species such as Ichthyobodo necator, Ichthyophthirius multifilis, Trichodina spp. (Roberts, 2001;Durborow, 2003). Although Trichodina sp. is a parasite that is usually and intensely found in freshwater, it is a type of parasite that causes disease in addition to primary pathogens during stress. In this study, Trichodina sp.
was found in the skin of diseased Seven khramulya, as it was also reported by previous researchers (Sanmartin et al., 1991;Nilsen, 1995;Asmat, 2001).
Gram-negative and Gram-positive pathogen bacteria causing hemorrhagic septicemia in fish cause serious disorders in the structure, function, and morphology of the tissues (Roberts, 2001). Histologically, it was determined in this study that bacterial agents cause co-infection and degeneration in the hepatocytes of the liver, tubular and glomerular destruction in the renal tissue, as it was stated by Yu et al (2010). As reported by Dar et al (2016), hypertrophy in the lamellae and degenerative changes in the gill epithelium in the gill tissue of silver carp (Hypophthalmichthys molitrix) infected with A. sobria were also detected in diseased Seven khramulya.
As a result, the disease observed in Seven khramulya in nature was mostly accompanied by A. hydrophila along with the pathogens such as V. fluvialis, S. warneri, and S. capitis. S. capitis was isolated and identified as a disease agent from diseased Seven khramulya for the first time in this study. Furthermore, we report for the first time in this comprehensive study that these pathogenic bacteria isolated and identified from co-infection may cause disease in Seven khramulya. We think that the external parasites observed in diseased fish examined act as secondary factors causing disease with bacterial pathogens.

CONCLUSION
Common carps are among the most important freshwater fish cultured in the world and Turkey. Seven khramulya is an important fish species that is found in natural water in our country and aimed to be raised. The data in this study shed light on future intense cultivation of Seven khramulya in Turkey, as contributes to pre-identification of bacterial and parasitic diseases that may pose a risk to fish.