Association of red‐mark syndrome with a Rickettsia‐like organism and its connection with strawberry disease in the USA

Red‐mark syndrome (RMS), a disease seen mostly in rainbow trout, Oncorhynchus mykiss, is of unknown aetiology. The research presented here indicates the presence of an intracellular bacterium in RMS‐affected fish. A positive reaction was observed using immunohistochemistry (IHC) with skin lesions, liver, kidney and spleen of affected fish sampled from several locations within the United Kingdom using two different polyclonal antisera raised against Piscirickettsia salmonis. The same reaction was also seen with a number of different anti‐P. salmonis monoclonal antibodies (MAbs). A disease with similar clinical signs to RMS, referred to as strawberry disease (SD), has been reported in the USA. A Rickettsia‐like organism (RLO) has recently been associated with SD based on analysis of 16S rDNA sequences. Using the same panel of anti‐P. salmonis antibodies used to screen the RMS samples, similar staining was obtained in tissue of SD‐affected fish by IHC. A polymerase chain reaction (PCR) using RLO‐specific primers was also performed on RMS‐affected fish from the United Kingdom, and the samples were positive for the RLO 16S rRNA sequence. These findings suggest that the same aetiological agent may be responsible for RMS in the United Kingdom and SD in the USA.     

New Zealand rickettsia‐like organism (NZ‐RLO) and Tenacibaculum maritimum: Distribution and phylogeny in farmed Chinook salmon (Oncorhynchus tshawytscha)

A total of 777 fish from three growing regions of New Zealand Chinook salmon farms comprising of five sites were tested. Quantitative PCR was used to determine the distribution of New Zealand rickettsia‐like organism and Tenacibaculum maritimum. Genetic information from these bacteria were then compared with strains reported worldwide. Using this information, suggested associations of pathogens with clinically affected fish were made. NZ‐RLO was detected in two of the three regions, and T. maritimum was detected in all regions. Three strains of NZ‐RLO were identified during this study. Based on analysis of the ITS rRNA gene, NZ‐RLO1 appears to be part of an Australasian grouping sharing high similarity with the Tasmanian RLO, NZ‐RLO2 was shown to be the same as an Irish strain, and NZ‐RLO3 was shown be closely related to two strains from Chile. Based on multi‐locus sequence typing, the New Zealand T. maritimum was the same as Australian strains. NZ‐RLOs were detected more frequently in fish with skin ulcers than fish without skin ulcers. While additional research is required to investigate the pathogenicity of these organisms, this is the first time that NZ‐RLOs have been associated with the development of clinical infections in farmed Chinook salmon.     

In vivo growth and genomic characterization of rickettsia‐like organisms isolated from farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand

A rickettsia‐like organism, designated NZ‐RLO2, was isolated from Chinook salmon (Oncorhynchus tshawytscha) farmed in the South Island, New Zealand. In vivo growth showed NZ‐RLO2 was able to grow in CHSE‐214, EPC, BHK‐21, C6/36 and Sf21 cell lines, while Piscirickettsia salmonis LF‐89^T grew in all but BHK‐21 and Sf21. NZ‐RLO2 grew optimally in EPC at 15°C, CHSE‐214 and EPC at 18°C. The growth of LF‐89 ^T was optimal at 15°C, 18°C and 22°C in CHSE‐24, but appeared less efficient in EPC cells at all temperatures. Pan‐genome comparison of predicted proteomes shows that available Chilean strains of P. salmonis grouped into two clusters (p‐value = 94%). NZ‐RLO2 was genetically different from previously described NZ‐RLO1, and both strains grouped separately from the Chilean strains in one of the two clusters (p‐value = 88%), but were closely related to each other. TaqMan and Sybr Green real‐time PCR targeting RNA polymerase (rpoB) and DNA primase (dnaG), respectively, were developed to detect NZ‐RLO2. This study indicates that the New Zealand strains showed a closer genetic relationship to one of the Chilean P. salmonis clusters; however, more Piscirickettsia genomes from wider geographical regions and diverse hosts are needed to better understand the classification within this genus.     

Quantitative PCR demonstrates a positive correlation between a Rickettsia-like organism and severity of strawberry disease lesions in rainbow trout, Oncorhynchus mykiss (Walbaum)

Strawberry disease (SD) is an inflammatory skin disorder in rainbow trout, Oncorhynchus mykiss (Walbaum). The aetiology of SD is unknown although the 16S rDNA sequence of a Rickettsia-like organism (RLO) has been associated with SD lesions using a nested PCR assay. In this study, we developed a Taqman quantitative PCR assay (qPCR) that targeted the RLO 16S rDNA sequence to examine the distribution of RLO relative to lesion status. We compared 18 lesion samples from 13 fish representing high or low lesion severity as judged by gross examination. QPCR results showed that there was a higher number of RLO sequences in high severity lesions (mean of 12,068 copies) compared with fewer copies of RLO sequence in low severity lesions (mean of 3287 copies, P = 0.012). Grossly normal skin samples (n = 13) from SD-affected fish were all negative by qPCR except two samples (121 and 139 copies). The qPCR assay described herein is a useful tool to investigate the role of RLO in SD in the absence of a culture system for RLO. Our results demonstrate a positive correlation between copy number and lesion severity consistent with the hypothesis that the RLO is the aetiologic agent of SD.     

Mortality from scale drop disease in farmed Lates calcarifer in Southeast Asia

In Southeast Asia, a new disease called scale drop disease (SDD) caused by a novel Megalocytivirus (SDDV) has emerged in farmed Asian sea bass (Lates calcarifer) in Singapore, Malaysia and Indonesia. We received samples from an Eastern Thai province that also showed gross signs of SDD (loss of scales). Clinical samples of 0.2–1.1 kg L. calcarifer collected between 2016 and 2018 were examined for evidence of SDDV infection. Histopathology was similar to that in the first report of SDDV from Singapore including necrosis, inflammation and nuclear pyknosis and karyorrhexis in the multiple organs. Intracytoplasmic inclusion bodies were also observed in the muscle tissue. In a density‐gradient fraction from muscle extracts, TEM revealed enveloped, hexagonal megalocytiviral‐like particles (~100–180 nm). By PCR using primers derived from the Singaporean SDDV genome sequence, four different genes were amplified and sequenced from the Thai isolate revealing 98.7%–99.9% identity between the two isolates. Since viral inclusions were rarely observed, clinical signs and histopathology could not be used to easily distinguish between SDD caused by bacteria or SDDV. We therefore recommend that PCR screening be used to monitor broodstock, fry and grow‐out fish to estimate the current impact of SDDV in Southeast Asia and to prevent its spread.     

Acute dermatitis in farmed trout: an emerging disease

A new skin condition, known as puffy skin disease (PSD), emerged in farmed rainbow trout Oncorhynchus mykiss (Walbaum) in 2002. The number of new cases increased considerably from 2006. Clinical signs include white or grey skin patches, which become raised and red with excessive mucous production and scale loss. Fish are inappetant and lose condition. Histologically, the key feature is epithelial hyperplasia. We undertook a questionnaire study of trout farmers in England and Wales to investigate prevalence and risk factors. PSD was reported on 37% (n = 49) of rainbow trout sites, located in 28 river catchments. The increase in cases from 2006 onwards was mirrored by the increase in red mark syndrome (RMS). Prevalence and severity of PSD were highest in the summer months. The presence of PSD was associated with RMS (OR = 9.7, P < 0.001). Sites receiving live rainbow trout in the previous 12 months were considerably more likely to have PSD (OR = 5.3. P < 0.01), which suggests an infectious aetiology. The size of affected fish and prevalence varied between farms, indicating that farm‐level factors are important. Future research should further investigate the aetiology of PSD and practices to manage the disease.     

First identification and characterization of Streptococcus iniae obtained from tilapia (Oreochromis aureus) farmed in Mexico

This is the first study to isolate, identify and characterize Streptococcus iniae as the causative disease agent in two tilapia (Oreochromis aureus) populations. The populations were geographically isolated, of distinct origins, and did not share water sources. Affected fish showed various external (e.g., exophthalmia and cachexia, among others) and internal (e.g., granulomatous septicaemia and interstitial nephritis, among others) signs. All internal organ samples produced pure cultures, two of which (one from each farm, termed S‐1 and S‐2) were subjected to biochemical, PCR and 16S rRNA sequencing (99.5% similarity) analyses, confirming S. iniae identification. The two isolates presented genetic homogeneity regardless of technique (i.e., RAPD, REP‐PCR and ERIC‐PCR analyses). Pathogenic potentials were assessed through intraperitoneal injection challenges in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio). Rainbow trout mortalities were respectively 40% and 70% at 10⁴ and 10⁶ CFU per fish with the S‐1 isolate, while 100% mortality rates were recorded in zebrafish at 10² and 10⁴ CFU per fish with the S‐2 isolate. The obtained data clearly indicate a relationship between intensified aquaculture activities in Mexico and new disease appearances. Future studies should establish clinical significances for the tilapia industry.     

Development of piscirickettsiosis in Atlantic salmon (Salmo salar L.) smolts after intraperitoneal and cohabitant challenge using an EM90‐like isolate: A comparative study

Piscirickettsiosis, caused by the intracellular Gram‐negative bacteria Piscirickettsia salmonis, is at present the most devastating disease in the Chilean salmon industry. The aim of this study was to analyse disease development after challenge with a P. salmonis strain (EM90‐like) under a controlled environment by comparing intraperitoneal challenge with cohabitation challenge. The P. salmonis EM90‐like isolate was cultured in a liquid medium for the challenge of 400 Atlantic salmon (Salmo salar) smolts. Cumulative mortality was registered, necropsy was performed, and bacterial distribution in the tissues and histopathological changes were analysed. The results revealed a similar progression of the disease for the two different challenge models. Pathological and histopathological changes became more visible during the development of the clinical phase of the disease. Bacterial DNA was identified in all the analysed tissues indicating a systemic infection. Bacterial tropism to visceral organs was demonstrated by real‐time quantitative PCR and immunohistochemistry. Better knowledge of disease development during P. salmonis infection may contribute to further development of challenge models that mimic the field situation during piscirickettsiosis outbreaks. The models can be used to develop and test future preventive measures against the disease.     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

A comparative molecular study of the presence of “Candidatus arthromitus” in the digestive system of rainbow trout,Oncorhynchus mykiss (Walbaum), healthy and affected with rainbow trout gastroenteritis

Observations were made using histopathological techniques in conjunction with a nested polymerase chain reaction (PCR) protocol for the specific detection of “Candidatus arthromitus” on DNA extracted from wax‐embedded tissues and fresh digestive contents of rainbow trout. Samples positive for “Candidatus arthromitus” DNA included fish with rainbow trout gastroenteritis (RTGE), clinically normal cohabiting fish, and apparently healthy controls from RTGE positive and RTGE negative sites. The results obtained from the PCR were confirmed by nucleotide sequencing. “Candidatus arthromitus” DNA was found in distal intestine as well as in sections of pyloric caeca, suggesting that both these locations are appropriate for molecular detection of “Candidatus arthromitus” DNA in trout. Furthermore, rainbow trout fry distal intestinal samples from two different hatcheries where RTGE had not been reported were also positive. Differences in “Candidatus arthromitus” DNA detection between paraffin wax‐embedded and fresh digestive content samples from the same fish suggested that it may be predominantly epithelium‐associated in healthy trout. Parallel histopathological observations indicated that pyloric caeca are the preferred site for visualizing segmented filamentous bacteria (SFB) in trout with RTGE. The results of this study showed that the presence of SFB was not invariably associated with clinical disease and that more information is required to understand the role of these organisms.     

Effect of quorum quenching bacteria on growth,virulence factors and biofilm formation of Yersinia ruckeri in vitro and an in vivo evaluation of their probiotic effect in rainbow trout

Five N‐acyl homoserine lactone‐degrading bacteria (quorum quenching (QQ) strains) were selected to evaluate their impacts on growth, virulence factors and biofilm formation in Yersinia ruckeri in vitro. No difference was observed among the growth pattern of Y. ruckeri in monoculture and coculture with the QQ strains. To investigate the regulation of virulence factors by quorum sensing in Y. ruckeri, cultures were supplemented with 3oxo‐C8‐HSL. The results indicated that swimming motility and biofilm formation are positively regulated by QS (p < 0.05), whereas caseinase, phospholipase and haemolysin productions are not influenced by 3oxo‐C8‐HSL (p > 0.05). The QQs were able to decrease swimming motility and biofilm formation in Y. ruckeri. QQ bacteria were supplemented to trout feed at 10⁸ CFU/g (for 40 days). Their probiotic effect was verified by Y. ruckeri challenge either by immersion or injection in trout. All strains could significantly increase fish survival with Bacillus thuringiensis and Citrobacter gillenii showing the highest and lowest relative percentage survival (RPS) values (respectively, 85% and 38%). Besides, there was no difference between the RPS values by either immersion or injection challenge expect for B. thuringiensis. The putative involvement of the QQ capacity in the protection against Yersinia is discussed.     

Isolation of bacterial probiotic candidates from the gastrointestinal tract of rainbow trout,Oncorhynchus mykiss (Walbaum), and screening for inhibitory activity against Flavobacterium psychrophilum

In this study, 318 bacterial strains were isolated from the gastrointestinal (GI) tracts of 29 rainbow trout, Oncorhynchus mykiss (Walbaum). These bacteria were screened in vitro for their ability to inhibit growth of Flavobacterium psychrophilum, the causative agent of coldwater disease. Bacteria observed to inhibit F. psychrophilum growth were further screened against rainbow trout bile, as an indicator of their ability to survive in the GI tract. This screening resulted in narrowing the pool to 24 bacterial isolates. Those 24 isolates were then tested for pathogenicity in rainbow trout by intraperitoneal injection. Following a 28‐day challenge, eight isolates were shown to cause direct mortality and were eliminated from further study. As a result, 16 bacterial isolates were identified as probiotic candidates with the potential to control or reduce disease caused by F. psychrophilum.     

Probiotic characteristics and aflatoxin B1 binding ability of Debaryomyces hansenii and Kazaschtania exigua from rainbow trout environment

The aim of this study was to evaluate probiotic properties and the aflatoxin B₁ adsorption ability of yeasts isolated from rainbow trout intestine and fish feed to assess their use in the formulation of feed additives. Growth at pH 2, bacterial pathogens inhibition, bacterial pathogens co‐aggregation, autoaggregation, homologous and heterologous inhibition against lactic acid bacteria were evaluated. Moreover, aflatoxin B₁ adsorption was tested. All strains were able to maintain viable (10⁷ cells/ml) at pH 2. All strains isolated from intestine were identified as Kazaschtania exigua, while strains isolated from feed were all identified as Debaryomyces hansenii. Kazaschtania exigua RC035 and RC037 showed the strongest antimicrobial activity while K. exigua RC037 and RC038 were the most efficient co‐aggregating bacterial pathogens. All strains exhibited strong autoaggregation. None of the tested yeast strains showed homologous inhibition towards other yeasts and heterologous inhibition towards lactic acid bacteria strains. Debaryomyces hansenii RC031 demonstrated aflatoxin B₁ adsorption capacity (21%). The results of the present study indicate that select strains of Kazaschtania exigua and D. hansenii showed potential to improve the health of rainbow trout by inhibiting pathogens and binding AFB₁ and their use as probiotics may improve the production of rainbow trout in aquaculture systems.     

The isolation of a rickettsia-like organism causing disease and mortality in Chilean salmonids and its confirmation by Koch's postulate

Abstract. A rickettsia-like organism (RLO) was isolated from infected coho salmon, Oncorhynchus kisutch (Walbaum), in cultures from six fish cell lines, but could not be cultured on 41 artificial media. The organism was confirmed by Koch's postulates as the aetiological agent of a systemic disease causing signifieant mortality among coho in Chile. The organism was a Gram-negative, obligate intracellular pathogen frequently observed within intracyto-plasmic vacuoles or inclusions of host cells, and appears to belong to the order Rickettsiales, family Rickettsiaceae and possibly the tribe Ehrlichiae. The organism, as well as the clinical signs and pathology it produces, is described. The disease, originally observed only in coho raised in seawater net pens, was reproduced experimentally in fish in both freshwater and seawater aquaria. Horizontal transmission without parasite vectors was observed in fish in both aquaria. The organism was shown, for the first time, to cause disease and mortality in Atlantic, Salmo salar L., and Chinook, O. tshawytscha (Walbaum), salmon and rainbow trout, O. mykiss (Walbaum). Because of the systemic nature of the disease, it is proposed that it be called 'salmonid rickettsial septicaemia'.     

Purified deoxynivalenol or feed restriction reduces mortality in rainbow trout,Oncorhynchus mykiss (Walbaum), with experimental bacterial coldwater disease but biologically relevant concentrations of deoxynivalenol do not impair the growth of Flavobacterium psychrophilum

Diets containing deoxynivalenol (DON) were fed to rainbow trout Oncorhynchus mykiss (Walbaum) for 4 weeks followed by experimental infection (intraperitoneal) with Flavobacterium psychrophilum (4.1 × 10⁶ colony‐forming units [CFU] mL⁻¹). Mortality of rainbow trout fed either 6.4 mg kg⁻¹ DON or trout pair‐fed the control diet was significantly reduced (P < 0.05) in comparison with trout fed the control diet to apparent satiation (<0.1 mg kg⁻¹ DON). In a second experiment, trout were fed one of three experimental diets; a control diet, a diet produced with corn naturally contaminated with DON (3.3 mg kg⁻¹ DON) or a diet containing purified DON (3.8 mg kg⁻¹); however, these fish were not experimentally infected. The presence of DON resulted in significant reduction (P < 0.0001) in feed intake as well as weight gain after 4 weeks. Respiratory burst of head‐kidney leucocytes isolated from rainbow trout fed diets containing purified DON (3.8 mg kg⁻¹) was significantly higher (P < 0.05) at 35 day post‐exposure compared with controls. The antimicrobial activity of DON was examined by subjecting F. psychrophilum in vitro to serial dilutions of the chemical. Complete inhibition occurred at a concentration of 75 mg L⁻¹ DON, but no effect was observed below this concentration (0–30 mg L⁻¹).     

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 10⁸ cfu/fish and after mortality had begun, fish were given erythromycin‐ and florfenicol‐medicated feed at a rate of 75 mg kg^?1 day^?1 and 10 mg kg^?1 day^?1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30‐day trial. Relative to antibiotic‐free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.     

Identification of RAPD‐SCAR marker linked to white spot syndrome virus resistance in populations of giant black tiger shrimp,Penaeus monodon Fabricius

White spot disease (WSD) caused by white spot syndrome virus (WSSV) creates severe epizootics in shrimp aquaculture industry worldwide. Despite several efforts, no such permanent remedy was yet developed. Selective breeding using DNA markers would be a cost‐effective strategy for long‐term solution of this problem. In the present investigation, out of 30 random primers, only one primer produced a statistically significant (P < 0.01) randomly amplified polymorphic DNA (RAPD) marker of 502 bp, which provided a good discrimination between disease resistant and disease susceptible populations of Penaeus monodon from three geographical locations along the East coast of India. Because RAPD markers are dominant, a sequence characterized amplified region (SCAR) marker was developed by cloning and sequencing of 502 bp RAPD fragment, which generates a single 457 bp DNA fragment after PCR amplification only in the disease resistant shrimps. Challenge experiment was also conducted to validate this 457 bp SCAR marker, and the results suggested that the WSSV loads were 2.25 × 10³ fold higher in disease susceptible than that in disease resistant shrimps using real‐time PCR. Therefore, this 457 bp DNA SCAR marker will be very valuable towards the development of disease‐free shrimp aquaculture industry.     

Determination and transferability of plasmid‐mediated antibiotic resistance genes of the bacteria isolated from rainbow trout

Antibiotic resistance and the presence of resistance genes (ARGs) were investigated in the bacteria isolated from rainbow trout (Oncorhynchus mykiss) from different trout farms located in Turkey. The most frequent types of antibiotic resistance were towards β‐lactams (cephalothin [70% of bacterial isolates], amoxicillin [63%], ampicillin [62%], ticarcillin [56%], aztreonam [51%]), macrolide [erythromycin, 68%] and sulphonamide [sulphamethoxazole, 51%]. Of bacterial isolates, 51% were multiple drug resistant (MDR), while 35% of the isolates were extensively drug resistant (XDR). None of isolates were pandrug resistant (PDR). The most common ARGs were ampC (36%) and sul1 (24%). The class 1 integron gene cassette was detected in 51% of the bacteria. There was a strong positive correlation between the antibiotic resistance rate and the presence of ARGs (r² = .932). Gene encodes bla_CTX‐M1, one of the extended spectrum beta‐lactamase enzymes, was first described in Aeromonas caviae, Photobacterium damselae, Pseudomonas luteola and Burkholderia cepacia. It was determined that 35% of the bacteria harboured at least one plasmid. Plasmid‐mediated ARGs were identified to be tetracyclines (tetA, tetB, tetC, tetD), sulphonamides (sul1, sul3) and β lactams (ampC, bla_pse). Thus, results suggest that ARG contamination situation deliberates resistance to tetracycline, aminoglycoside, chloramphenicol and sulphonamide. Therefore, the presence and activity of ARGs in fish and in environmental bacteria may play an important role in the spread of resistance genes among bacteria by transposition or integron gene cassettes.     

Biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum) using two bacterial isolates (LE89 and LE141) of Pseudomonas fluorescens

The probiotic activity of 15 bacterial isolates that inhibit Saprolegnia parasitica in vitro was tested for the biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum), adding the bacteria to tank water for 14 days at a concentration of 10⁶ bacteria ml^?1 water. Pseudomonas fluorescens LE89 and Pseudomonas fluorescens LE141 were effective in controlling experimental infection with S. parasitica since of the fish treated with LE89, 24.5% ± 16.27% (p < 0.05) became infected, as did 42.8% ± 8.41% (p < 0.05) of those treated with LE141. Given their protective effect when administered in water, their effect was also studied when administered in feed before and after experimental infection. Both bacterial isolates survived low pH levels and the action of bile, grew in skin and intestinal mucus, were resistant to several antibiotics and survived in feed; however, neither of the two isolates prevented S. parasitica infection when administered in feed.