Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single‐sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.     

Effect of repeated exposure to AQUI‐S® on the viability and growth of Neoparamoeba perurans

There have been recent efforts amongst immunologists to develop approaches for following individual fish during challenges with viral and bacterial pathogens. This study contributes to assessing the feasibility of using such approaches to study amoebic gill disease (AGD). Neoparamoeba perurans, agent of AGD, has been responsible for widespread economic and fish loss in salmonid aquaculture. With the emergence of AGD in Europe, research into infection dynamics and host response has increased. This study investigated the effect of repeat exposure to anaesthesia, a necessary requirement when following disease progression in individual fish, on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28‐day period to AQUI‐S^® (isoeugenol), a popular anaesthetic choice for AGD challenges, at a concentration and duration required to sedate post‐smolt salmonids. Population growth was measured by sequential counts of amoeba over the period, while viability of non‐attached amoeba in the culture was assessed with a vital stain. AQUI‐S^® was found to be a suitable choice for in vivo ectoparasitic challenges with N. perurans during which repetitive anaesthesia is required for analysis of disease progression.     

Evaluation of sodium percarbonate as a bath treatment for amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a major health challenge for Atlantic salmon aquaculture globally. While freshwater bathing for 2 hr is effective in reducing infection severity, there is need for more rapid and lower cost alternatives. To this end, a combination of sodium percarbonate (SPC) in freshwater was examined for its treatment efficacy. Initial in vitro studies showed a reduction in amoeba viability when exposed for 30 min to freshwater containing >500 mg/L SPC. Subsequently, AGD‐affected salmon were bathed for 30 min in 16°C freshwater containing 100, 500 or 1,000 mg/L SPC, or for 2 hr in 16°C freshwater to mimic industry practice. Treatment at the highest SPC concentration caused extensive gill damage and substantial mortality. Neither occurred to a significant extent at lower SPC concentrations. Gill pathology of surviving fish 10 days post‐treatment (dpt) was comparable to or more severe than pre‐treatment, and significantly (p < .001) more severe than in 2 hr freshwater bathed fish. N. perurans DNA was confirmed by qPCR in all treatment groups at 10 dpt. The data indicate that a 30‐min exposure to SPC in freshwater is not a suitable alternative to existing freshwater treatment of AGD.     

PCR survey for Paramoeba perurans in fauna,environmental samples and fish associated with marine farming sites for Atlantic salmon (Salmo salar L.)

Amoebic gill disease (AGD) caused by the amoeba Paramoeba perurans is an increasing problem in Atlantic salmon aquaculture. In the present PCR survey, the focus was to identify reservoir species or environmental samples where P. perurans could be present throughout the year, regardless of the infection status in farmed Atlantic salmon. A total of 1200 samples were collected at or in the proximity to farming sites with AGD, or with history of AGD, and analysed for the presence of P. perurans. No results supported biofouling organisms, salmon lice, biofilm or sediment to maintain P. perurans. However, during clinical AGD in Atlantic salmon, the amoeba were detected in several samples, including water, biofilm, plankton, several filter feeders and wild fish. It is likely that some of these samples were positive as a result of the continuous exposure through water. Positive wild fish may contribute to the spread of P. perurans. Cleaner fish tested positive for P. perurans when salmon tested negative, indicating that they may withhold the amoeba longer than salmon. The results demonstrate the high infection pressure produced from an AGD‐afflicted Atlantic salmon population and thus the importance of early intervention to reduce infection pressure and horizontal spread of P. perurans within farms.     

Identification and pathogenicity study of emerging fish pathogens Acinetobacter junii and Acinetobacter pittii recovered from a disease outbreak in Labeo catla (Hamilton, 1822) and Hypophthalmichthys molitrix (Valenciennes, 1844) of freshwater wetland in West Bengal,India

The disease outbreaks in aquaculture system of wetlands are the major cause of fish mortality. Among various bacterial septicaemic diseases, fish mortality caused by Acinetobacter spp. is recently reported in different fish species. Fish disease outbreak was investigated in a wetland of West Bengal, India to identify the aetiological factors involved. The moribund fish were examined and subjected to bacterial isolation. Two bacterial causative agents were identified as Acinetobacter junii and Acinetobacter pittii by biochemical characterization and 16S rRNA gene amplification. Both the isolates were oxidase‐negative, nitrate‐negative, catalase‐positive and indole‐negative. The molecular identification using 16S rRNA gene sequencing and phylogenetic tree analysis further confirmed the two Acinetobacter spp. with 97%–99% similarity. The antibiotic resistance patterns of these two bacteria revealed that both of them were resistant to β‐lactam, cefalexin, cephalothin, amoxyclav, cefuroxime, cefadroxil, clindamycin, vancomycin and penicillin. In addition, A. pittii was also resistant to other antibiotics of cephams group such as ceftazidime and cefotaxime. In the challenge experiment, both A. junii and A. pittii were found to be pathogenic with LD₅₀ of 1.24 × 10⁵ and 1.88 × 10⁷ cfu/fish respectively. Histopathological examination of gill, liver and kidney revealed prominent changes supporting bacterial septicaemia. The investigation reports for the first time on concurrent infection by A. junii and multidrug‐resistant (MDR)‐A. pittii as emerging fish pathogens to cause severe mortality in Labeo catla and Hypophthalmichthys molitrix in a freshwater wetland.     

Efficacy of single and multiple doses of fenbendazole against gill parasites (Dactylogyrus sp.) of Labeo rohita (Hamilton, 1822) and its physio‐metabolic effects on the fish

The study was conducted to investigate the efficacy and physio‐metabolic responses of single and multiple doses of fenbendazole (FBZ) in Labeo rohita against gill parasite (Dacylogyrus sp.). Two Isoenergetic (395 Kcal DE/100 g) and isonitrogenous (35% CP) purified diets viz. control (without FBZ) and treatment (0.2% FBZ) were prepared. The efficacy of FBZ was measured after administration of single dose (20 mg/kg b. wt.) on 0th day and multiple doses of FBZ, that is, 20 mg/kg b. wt. on 0th, 3rd and 7th day through diet. Although oral administration of FBZ at single dose could significantly reduce the parasite in fish in comparison with the control group, 100% efficacy of the drug was not achieved up to 15th day of treatment. On the other hand, multiple doses of FBZ could show almost 100% efficacy on 15th day in terms of elimination of parasites from gill. The drug induced oxidative stress in examined fish. There was no significant change in activities of hepatic and muscle aspartate aminotransferase and alanine aminotransferase of L. rohita up to 15th day after oral administration of FBZ at single dose. But both single and multiple doses of FBZ could significantly enhance the hepatic superoxide dismutase, catalase and glutathione‐S‐transferase activities in fish up to 7th day of drug administration and significantly decreased thereafter up to 15th and/or 30th day. It is concluded that oral administration of FBZ at multiple doses (20 mg/kg b. wt.) on 1st, 3rd and 7th day could be effective against Dactylogyrus sp. infection in L. rohita.     

Ca. Branchiomonas cysticola,Ca. Piscichlamydia salmonis and Salmon Gill Pox Virus transmit horizontally in Atlantic salmon held in fresh water

Elucidation of the role of infectious agents putatively involved in gill disease is commonly hampered by the lack of culture systems for these organisms. In this study, a farmed population of Atlantic salmon pre‐smolts, displaying proliferative gill disease with associated Candidatus Branchiomonas cysticola, Ca. Piscichlamydia salmonis and Atlantic salmon gill pox virus (SGPV) infections, was identified. A subpopulation of the diseased fish was used as a source of waterborne infection towards a population of naïve Atlantic salmon pre‐smolts. Ca. B. cysticola infection became established in exposed naïve fish at high prevalence within the first month of exposure and the bacterial load increased over the study period. Ca. P. salmonis and SGPV infections were identified only at low prevalence in exposed fish during the trial. Although clinically healthy, at termination of the trial the exposed, naïve fish displayed histologically visible pathological changes typified by epithelial hyperplasia and subepithelial inflammation with associated bacterial inclusions, confirmed by fluorescent in situ hybridization to contain Ca. B. cysticola. The results strongly suggest that Ca. B. cysticola infections transmit directly from fish to fish and that the bacterium is directly associated with the pathological changes observed in the exposed, previously naïve fish.     

Effects of temperature on amoebic gill disease development: Does it play a role?

A relationship between increasing water temperature and amoebic gill disease (AGD) prevalence in Atlantic salmon (Salmo salar) has been noted at fish farms in numerous countries. In Scotland (UK), temperatures above 12°C are considered to be an important risk factor for AGD outbreaks. Thus, the purpose of this study was to test for the presence of an association between temperature and variation in the severity of AGD in Atlantic salmon at 10 and 15°C. The results showed an association between temperature and variation in AGD severity in salmon from analysis of histopathology and Paramoeba perurans load, reflecting an earlier and stronger infection post‐amoebae exposure at the higher temperature. While no significant difference between the two temperature treatment groups was found in plasma cortisol levels, both glucose and lactate levels increased when gill pathology was evident at both temperatures. Expression analysis of immune‐ and stress‐related genes showed more modulation in gills than in head kidney, revealing an organ‐specific response and an interplay between temperature and infection. In conclusion, temperature may not only affect the host response, but perhaps also favour higher attachment/growth capacity of the amoebae as seen with the earlier and stronger P. perurans infection at 15°C.     

Clinical assessment of chloramine-T and freshwater as treatments for the control of gill amoebae in Atlantic salmon, Salmo salar L.

Infections of gill amoebae that manifest as amoebic gill disease (AGD) occur in Atlantic salmon in Tasmania. The treatment of choice is freshwater bathing; however, the effectiveness of this treatment has declined over time. In this experiment, cage trials of chloramine‐T (Cl‐T) to treat AGD in Atlantic salmon were conducted over 3 months, and involved an initial bath in either freshwater or seawater with Cl‐T, followed by a second bath 6 weeks later. Amoeba densities were reduced to 50–80% of original values for both treatments. Neoparamoeba sp. density was not affected by bathing, and was not significantly different over the course of the experiment. Lesion prevalence was higher for Cl‐T‐treated fish than for freshwater‐treated fish, with overall prevalence levels of 14.30±1.00% and 8.03±0.57% respectively. This was also seen for gross gill scores. In the fortnight after each of the two baths, Cl‐T‐treated fish had significantly higher lesion levels, although this difference was then resolved by 4 weeks post bathing. The use of Cl‐T in seawater is at least as effective as freshwater at reducing amoebae density, and may be a more practical alternative when freshwater is in short supply.     

The induction of laboratory-based amoebic gill disease revisited

Previous work in our laboratory defined a method of inducing laboratory‐based amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L. Gills of AGD‐affected fish were scraped and the debris placed into fish‐holding systems, eliciting AGD in naïve Atlantic salmon. While this method is consistently successful in inducing AGD, variability in the kinetics and severity of infections has been observed. It is believed that the infections are influenced by inherently variable viability of post‐harvest amoeba trophozoites. Here, a new method of experimental induction of AGD is presented that redefines the infection model including the minimum infective dose. Amoebae were partially purified from the gills of AGD‐affected Atlantic salmon. Trophozoites were characterized by light microscopy and immunocytochemistry and designated Neoparamoeba sp., possibly Neoparamoeba pemaquidensis. Cells were placed into experimental infection systems ranging in concentration from 0 to 500 cells L^?1. AGD was detected by gross and histological examination in fish held in all systems inoculated with amoebae. The number of gross and histological AGD lesions per gill was proportional to the inoculating concentration of amoebae indicating that the severity of disease is a function of amoeba density in the water column. The implications of these observations are discussed in the context of the existing AGD literature base as well as Atlantic salmon farming in south‐eastern Tasmania.     

Recovery of Bacillus mycoides,B. pseudomycoides and Aeromonas hydrophila from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) with gill disease

During a 3‐month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.     

Isolation,molecular characterization and antimicrobial susceptibility of Aeromonas spp. obtained from Tiger Grouper (Epinephelus fuscoguttatus) and Marble Goby (Oxyeleotris marmoratus) fish in Sabah,Malaysia

Aeromonads are ubiquitous in aquatic environments and have been implicated in fish and human infections. In this study, we isolated, studied antimicrobial susceptibility patterns and screened the existence of 15 virulence genes in aeromonads from two famously consumed fish species—seven marine Tiger Grouper (Epinephelus fuscoguttatus) and eight freshwater Marble Goby (Oxyeleotris marmoratus) from the aquaculture hatchery in Sabah, Malaysia. A total of 30 aeromonads (17 A. caviae, 9 A. rivuli, 4 A. dhakensis) were identified using PCR targeting GCAT gene, rpoD‐restriction fragment length polymorphism and multi‐locus phylogenetic analysis. All 30 strains were resistant to amoxicillin and cephalothin and five strains were multidrug‐resistant. Nine virulence genes (lip, ela, eno, fla, aerA, hylA, dam, alt and ser) present in A. dhakensis, suggesting the virulence potential of this species as a fish pathogen. This study offers as a baseline for future studies in monitoring and managing these two fish in aquaculture industry.     

Experimental Lactococcus garvieae infection in rainbow trout,Oncorhynchus mykiss,Walbaum 1792: a comparative histopathological and immunohistochemical study

The aim of this study was to induce Lactococcus garvieae infection in young and adult fish through different routes [intraperitoneal (IP) and immersion (IM)] and to investigate the pathogenesis and histopathological and immunohistochemical findings comparatively. For this purpose, a total of 180 rainbow trout (90 young, 20 ± 5 g and 90 adult, 80 ± 10 g) obtained from a commercial fish farm were used. The fish were divided into eight groups, four experimental groups (Young‐Adult IP groups and Young‐Adult IM groups, each contain 30 fish) and four control groups (Young‐Adult IP Control groups and Young‐Adult IM control groups, each contain 15 fishes). The experimental study was conducted using L. garvieae, and confirmatory identification was performed by PCR. The sequence result of the PCR amplicon of 16S rDNA from isolate L. garvieae LAC1 was determined and deposited in the GenBank database under accession number KC883976 . Fish in the IP groups were intraperitoneally administered an inoculate containing 10⁶ cfu mL^?1 bacteria 0.1 mL. In the IM groups, fish were kept in inoculated water containing 10⁸ cfu mL^?1 bacteria for 20 min. Mortality as well as clinical and pathological findings was recorded daily, and significant differences in macroscopic and microscopic results were observed between the IP and IM administration groups. All tissue samples were immunohistochemically stained by the avidin‐biotin‐peroxidase complex and immunofluorescence (IF) methods using polyclonal antibody to detect L. garvieae antigens. In immunoperoxidase staining in the IP groups, positive reactions to bacterial antigens were most commonly seen in the spleen, kidney, heart, liver, peritoneum and swim bladder. In the IM groups, bacterial antigens were most commonly found in the eye, gill, spleen and kidney. In the IF method, the distribution of antigens in tissue and organs was similar to the reactions with immunoperoxidase staining. Finally, in this experimental study, an important correlation was seen between the distribution of L. garvieae antigens and lesions developing in many organ and tissues.     

Occurrence of trypanosomiasis in net‐cage cultured groupers (Cromileptes altivelis and Epinephelus fuscoguttatus) in Nanshan port of Sanya,Hainan province,China

In the present study, reported was an unidentified trypanosome that caused massive mortalities of cultured humpback grouper, Cromileptes altivelis (Valenciennes, 1828) and brown‐marbled grouper, Epinephelus fuscoguttatus (Forsskål, 1775) in the net‐cages in Sanya, China, and some general pathological features of E. fuscoguttatus after experimental infection of the trypanosome. Before dying in the net cages of diseased fish, listlessness and anorexia were the observable symptoms. The blood of the diseased fish was light‐coloured in comparison with that of healthy one. E. fuscoguttatus experimentally infected with this trypanosome are with different degrees of engorgement in gill lamella, liver, spleen and kidney. Tissue lesion and necrosis were observed in liver and spleen of diseased fish.     

Impact of sublethal di‐n‐butyl phthalate on the aquaculture fish species Nile tilapia (Oreochromis niloticus): histopathology and oxidative stress assessment

Phthalates such as di‐n‐butyl phthalate (DBP) and their esters are widely used plasticizers, their ubiquitous presence in daily life, inevitably leads to their restricted use due to important environmental pollution and health impacts and endocrine disruption potential. The aim of this study was to examine the effects of a sublethal concentration of 10 mg L^?1 DBP on haematocrit (HCT) values, gills and liver histology, malondialdehyde (MDA, 2‐thiobarbituric acid‐TBA reactivity) and reduced glutathione (GSH) levels in gills and liver tissue as oxidative stress biomarkers in the aquaculture fish species Nile tilapia (Oreochromis niloticus) after 24 (DBP‐24) and 96 (DBP‐96) h exposure. No differences were found between per cent HCT values in the 24 h exposure groups (P > 0.05). Response of antioxidant defence systems in liver and gill tissues of the fish were dependent on exposure duration and changed to a higher extent during 96 h. MDA levels in liver tissue increased in DBP treated fish in comparison to the control fish. However, the differences between the exposure and control groups were not significant (P > 0.05). A statistically significant decrease (P > 0.05) was recorded in gill MDA levels in the DBP‐96 group when compared to the control and DBP‐24 groups. The liver GSH levels were unchanged in the DBP treated fish. However, GSH levels were significantly lower in the gill tissue of the DBP‐96 group. Exposure to DBP caused several degenerative changes in the histology of gill and liver tissue. Gills displayed hyperaemia, epithelial lifting, oedema, talengiectasia, epithelial hyperplasia and fusion of secondary lamellae, whereas in liver several circulatory anomalies (hyperaemia, blood congestion and sinosoid dilatation) and vacuolization of hepatocytes were observed. Histopathological results demonstrated that the gills were more affected than the liver perhaps due to their direct contact with DBP.     

Stimulatory effect of dietary clove,Eugenia caryophyllata,bud extract on growth performance,nutrient utilization,antioxidant capacity,and tolerance of African catfish,Clarias gariepinus (B.), to Aeromonas hydrophila infection

The occurrence of pathogenic bacteria, Aeromonas hydrophila, in farm‐raised fish requires urgent attention. Continuous and indiscriminate use of antibiotics as growth promoters and disease control agents in aquaculture have been discouraged because of the risk of development of antibiotic‐resistant bacterial strains. There is steady interest in the use of botanicals, such as clove, Eugenia caryophyllata, buds extract (ECBE), as alternatives. Hence, the present study evaluated the effect of dietary ECBE supplementation on the growth performance, physiological, antioxidant, and immunity biomarkers of African catfish, Clarias gariepinus. Fish (11.7 ± 0.5 g) were fed diets containing 0.0 (control), 5.0, 10.0, or 15.0 g ECBE/kg diet up to apparent satiation twice daily for 12 weeks. After the feeding trial, fish from each treatment were challenged with A. hydrophila infection by intraperitoneal injection and kept under observation for 14 days to record any abnormal clinical signs and daily mortality. The results demonstrated that fish performance and feed intake were significantly enhanced with increasing ECBE levels, and its optimum level is 15 g/kg diet. Further, the dietary ECBE increased significantly the intestinal villi length/width and absorption area in a dose‐dependent manner. There are significant progressive increases in the values of red blood cells, hemoglobin, hematocrit, white blood cells, platelets, lymphocytes, and heterocytes, while monocytes, eosinophil, and basophils decreased significantly due to dietary ECBE in a dose‐dependent manner. Highest glucose, cholesterol, total protein, globulin, and albumin‐globulin ratios, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea, and creatinine values were found in fish fed 15 g ECBE/kg diets, while lowest values were recorded in fish fed the control diet. Despite the high AST and ALT values, no visible lesions or damage were observed in the liver cells of fish fed ECBE‐enriched diets. In addition, the inclusion of ECBE in fish diets enhanced the antioxidant and immunity capacity. Fish mortality after the bacterial challenge was higher in fish fed the control diet (82.3%) than those fed ECBE‐enriched diets. The lowest fish mortality was observed in fish fed the 15 ECBE/kg diet (4.7%) [Correction added on 16 November 2018: this section has been revised for clarity.].     

Visceral granulomas in farmed large yellow croaker,Larimichthys crocea (Richardson), caused by a bacterial pathogen,Pseudomonas plecoglossicida

An enzootic disease characterized by granulomas in internal organs occurred in cage‐farmed large yellow croaker, Larimichthys crocea (Richardson), in April and November 2010, in Ningbo, Zhejiang Province. One bacterial strain, named XSDHY‐P, was isolated from the diseased fish and identified by biochemical characterization, fatty acid methyl ester (FAME) analysis and multilocus sequence analysis (MLSA). According to the results obtained from the biochemical tests, FAME analysis and phylogenetic analysis derived from 16S ribosomal RNA, gyrB, oprF, oprI, oprL and rpoD gene sequencing, the bacterial isolate, XSDHY‐P, was identified as Pseudomonas plecoglossicida. Moreover, lethal dose, 50% trials were carried out to demonstrate the virulence of XSDHY‐P in large yellow croaker when administered at 2.13 × 10⁵ colony‐forming units per fish. Visceral granulomas were found in the experimentally infected fish as well as in the naturally infected fish, indicating that P. plecoglossicida is another bacterial pathogen that causes granulomatosis in L. crocea.     

Gill pathology in Scottish farmed Atlantic salmon,Salmo salar L., associated with the microsporidian Desmozoon lepeophtherii Freeman et Sommerville, 2009

Gill disorders have emerged in recent years as a significant problem in the production of marine‐stage Atlantic salmon Salmo salar L. The multi‐aetiological condition ‘proliferative gill inflammation’ (PGI) has been reported to cause heavy losses in western Norway, yet reports of Scottish cases of the disease have remained anecdotal. In the present study, histopathological material from a marine production site in the Scottish Highlands experiencing mortalities due to a seasonal gill disease with proliferative‐type pathology was examined using light microscopy, special staining techniques and transmission electron microscopy (TEM). The microsporidian Desmozoon lepeophtherii Freeman et Sommerville, 2009 (syn. Paranucleospora theridion) was identified by staining using a Gram Twort method and TEM associated with distinctive proliferative and necrotic pathology confined to the interlamellar Malpighian cell areas of the primary filaments. Epitheliocystis was not a feature of the gill pathology observed. It is believed this is the first report of D. lepeophtherii being identified associated with pathology in a Scottish gill disease case, and supports anecdotal reports that a disease at least partly synonymous with PGI as described by Norwegian researchers is present in Scottish aquaculture.