Infected Fish Tissues Research Articles

Inclusion of cricket (Gryllus bimaculatus) meal in African catfish (Clarias gariepinus) feed influences disease resistance

In our present study, we observed the effect of cricket meal (CM) on disease resistance of African catfish. Fish were fed diets containing 350 g kg–1 and 400 g kg–1 of CM and 350 g kg–1 of fishmeal as control. The fish were divided into triplicates treatments of ten fish per replicate, weighed 22.5±0.6 g and fed with experimental diets for 40 days before being challenged against Aeromonas hydrophila. Relative percentage of survival (RPS) was recorded over 12 days post-challenge. White blood cell count, total protein, globulin and lysozyme showed significantly increasing levels in those fed with cricket meal diet compared to the control group. Mortalities at 12-day post-challenge significantly decreased to 30% (RPS: 66.7%) and 27% (RPS: 70%) for 35% and 40% CM respectively compared to 90% mortality in control group. Intestines and liver tissues of infected fish were dissected for pathogenic confirmation. The intestines of control diet showed the highest bacterial load (58.2×107 cfu g–1) compared to CM diets. The current study indicates that dietary CM could enhance the innate immune system and disease resistance of African catfish.

Sclerocollum saudii Al-Jahdali, 2010 (Acanthocephala: Cavisomidae) as a sentinel for heavy-metal pollution in the Red Sea.

Currently, fish helminth parasites, especially cestodes and acanthocephalans, are regarded as sentinel organisms to elucidate metal pollution in aquatic ecosystems. Here, 34 specimens of the fish Siganus rivulatus were collected in the Red Sea, from a seriously polluted, small lagoon named Sharm-Elmaya Bay, at Sharm El-Sheikh, South Sinai, Egypt; 22 (64.7%) were infected by Sclerocollum saudii (Acanthocephala: Cavisomidae). Thus, 22 natural infrapopulations (26-245 individuals) of this parasite were collected from infected fish. Samples of water and sediments from the bay, samples of muscle, intestine and liver from each fish, and samples from the parasite were taken for analysis of heavy metals (cadmium (Cd) and lead (Pb)). Both Cd and Pb concentrations in sediments were higher than those in water. The concentration of these metals were significantly higher in tissues (intestine, liver and muscle) of non-infected fish than those in infected fish, with Pb concentrations consistently higher than those of Cd, and both were drastically decreased in the order: liver > intestine > muscle. Metal concentrations in this acanthocephalan were much higher than those in its fish host. There were strong negative relationships between metal concentrations in tissues (intestine, liver and muscle) of infected fish and infrapopulation size, and between metal concentrations in the acanthocephalan and its infrapopulation size. These relationships strongly suggest competition for these metals between the fish host and its acanthocephalan parasite, and intraspecific competition among acanthocephalan individuals for available metals in the fish intestine. Bioconcentration factors were relatively high, since the mean Cd concentration in S. saudii was 239, 68 and 329 times higher than those in intestine, liver and muscle tissues, respectively, of its fish host. Also, mean Pb concentration was 55, 13 and 289 times higher than those in these tissues, respectively. The host-parasite system described here seems to be promising for biomonitoring of metal pollution in the Red Sea.

Pathogenicity of snakehead vesiculovirus in rice field eels (Monopterus albus)

Snakehead vesiculovirus (SHVV) has caused mass mortality to cultured snakehead fish in China, resulting in enormous economic losses in snakehead fish culture. In this report, the whole genome of SHVV was sequenced. Interestingly, it shared more than 94% nucleotide sequence identity with Monopterus albus rhabdovirus (MoARV), which has caused great economic loss to cultured rice field eel (Monopterus albus). Therefore, the concern of cross-species infection of these viruses prompted us to investigate the susceptibility of rice field eel to SHVV infection. The results showed that rice field eel was susceptible to SHVV in both intracoelomical injection and immersion routes. Severe hemorrhage was observed on the skin and visceral organs of SHVV-infected rice field eels. Histopathological examination showed vacuoles in the tissues of infected liver, kidney and heart. Viral RNA or protein was detected in the tissues of infected fish by reverse transcription polymerization chain reaction (RT-PCR), in situ hybridization (ISH), or immunohistochemistry assay (IHC). Investigation of the epidemic of vesiculovirus in rice field eel as well as other co-cultured fish is invaluable for the prevention of vesiculovirus infection.

Detecting Aphanomyces Invadans in Pure Cultures and EUS-infected Fish Lesions by Applying PCR

Aphanomyces invadans is an oomycete fungus which causes Epizootic Ulcerative Syndrome (EUS) disease in wide range of fresh and brackish water fish worldwide imposing serious economic losses. A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of fish with the A.invadans. A set of primers (1APM 1F and 1APM 6R) was used to specifically amplify A. invadans DNA. The PCR amplifies a 400 bp amplicon. A protocol for the extraction of A. invadans DNA from infected fish tissue and pure fungal cultures was developed. The method was tested on seven EUS-susceptible fish species (snakehead, snakeskin gourami, moonlight gourami, koi carp, catfish, gold fish, climbing perch) and one EUS-resistant fish (tilapia), artificially infected with A. invadans and pure cultures of Aphanomyces spp., Saprolegnia spp., Achlya spp., and Allomyces sp. Detection of A. invadans was possible at the early stage of sampling, which was 24 hours post injection in both EUS-susceptible and resistant fish. Resistant fish was found to be PCR-negative after 6 days of inoculation but in susceptible fish PCR-positive results obtained even after day 28 or in dead fish. Therefore PCR may be a useful method for detection EUS infection in fish from early stage of disease onset.

Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections.

Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp, Cyprinus carpio L. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×107. The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3.

Isolation of a Ranavirus-type grouper iridovirus in mainland China and comparison of its pathogenicity with that of a Megalocytivirus-type grouper iridovirus

Megalocytivirus- and Ranavirus-type grouper iridoviruses (GIV-M and GIV-R, respectively) are causative agents in cultured groupers. GIV-R is recoded in the grouper industry in Chinese Taiwan and Singapore but rarely found in other regions or countries, including mainland China. In this study, a novel GIV-R, designated as GIV-R-SY1301, was isolated and identified from diseased juvenile hybrid groupers (tiger grouper Epinephelus fuscoguttatus ♀×giant grouper Epinephelus lanceolatus ♂) in Sanya, Hainan Island of China through PCR detection, cell culture, and transmission electron microscopy observation. Multiple alignment analysis based on the full-length major capsid protein (MCP) genes (1392bp) showed that twenty-three and four nucleotide substitutions were observed among GIV-R-SY1301 and two early GIV-R isolates from Taiwan and Singapore, respectively. At the protein level, there are one or two amino acid substitutions in MCP among these isolates. Artificial challenge with GIV-R-SY1301 and a GIV-M isolate (OSGIV-HN11) indicated that both OSGIV-NH11 and GIV-R-SY1301 were highly virulent to infected tiger groupers. Cumulative mortalities of 97.5% (39/40) and 85% (34/40) were obtained in OSGIV-HN11- and GIV-R-SY1301-challenged fish, respectively. Histopathological analysis showed numerous abnormally enlarged cells in OSGIV-HN11-infected grouper spleen and kidney tissues but not in GIV-R-SY1301-infected grouper tissues. Abnormally enlarged cells in infected fish tissues could be a notable histopathological marker to distinguish GIV-M infection from GIV-R infection. Taken together, this study is the first to isolate and characterize GIV-R iridovirus in mainland China. GIV-R would be an alarming agent to grouper industry in mainland China, as well as GIV-M has been influencing. Statements of relevance1.First isolation and characterization of a Ranavirus-type grouper iridovirus in mainland China;2.Comparative pathogenicity of Ranavirus- and Megalocytivirus-type GIV was firstly studied; both Ranavirus- and Megalocytivirus-type GIV showed highly virulent to infected tiger groupers under artificial infection of the same viral dosages. By contrast, GIV-M showed a higher virulence than GIV-R, but the latter showed a more rapid lethality;3.The differential histopathology caused by GIV-R and GIV-M was also studied. Abnormally enlarged cells in infected fish tissues could be a notable histopathological marker to distinguish GIV-M infection from GIV-R infection. All these findings contributed important information for grouper aquaculture worldwide.

Rapid detection of Streptococcus iniae in red tilapia tissue (Oreochromis sp.) by polymerase chain reaction

Streptococcus iniae, as a cause of Streptococcosis outbreak of cultured tilapia, is a hemolytic, Gram-positive bacterium. In this study, a one-step PCR method was developed for the detection of S. iniae from fish kidney. The oligonucleotide primers, assigned as LOX-1 and LOX-2, are designed from lactate oxidase gene of S. iniae. Using extracted DNA from bacterial cells or head kidney of S. iniae infected fish, the PCR reaction yields a 870 bp fragment specific to the S. iniae sequence. Primers LOX-1 and LOX-2 did not amplify DNA from other common bacteria in cultured fish (Streptococcus agalactiae, Aeromonas hydrophila, Edwardsiella ictaluri, Vibrio harveyi). The detection limit of this primer pair is 102 CFU/ml of S. iniae. The improved one-step PCR method provides a diagnostic tool for: (i) the detection of S. iniae directly from infected fish tissue; (ii) and the identification of S. iniae from bacteria cell.

Molecular and Immunohistochemical Diagnosis of Photobacterium damselae Subspecies piscicida During Naturally Occurring Disease in Egypt

AbstractIn the marine aquaculture industry, photobacteriosis caused by the Photobacterium damselae subspecies piscicida, Phdp is a globally significant disease. A number of clinical photobacteriosis outbreaks among yearling cultured and broodstocks of gilthead sea bream were sampled and submitted to our laboratory during the summer and autumn of 2013. The tissues of infected fish were subjected to an ordinary bacteriological identification and were analyzed using the polymerase chain reaction (PCR) and immunohistochemistry techniques. The results indicated that the selective primers that have been designed for detecting the gene encoding the apoptotic‐induced protein, AIP56, represent a powerful tool for sensitive and specific detection of virulent strains of Phdp. AIP56 toxin triggers apoptosis of host macrophages and neutrophils, contributing to the lesions observed during the pathological investigation. Immunohistochemistry allows bacterial identification and antigen expression to be directly correlated to the disease; the immune‐positive bacteria were detected in gills, liver, kidneys, spleen, and brain tissues in acute photobacteriosis. These also appeared in the necrotic areas of the granulomas of chronically infected fish. Molecular and immunohistochemical methods were useful as research and diagnostic tools in different stages of the disease; moreover, they appear to have enormous potential in retrospective epidemiological investigations.

Infecting Pacific Herring with Ichthyophonus sp. in the Laboratory.

The protistan parasite Ichthyophonus sp. occurs in coastal populations of Pacific Herring Clupea pallasii throughout the northeast Pacific region, but the route(s) by which these planktivorous fish become infected is unknown. Several methods for establishing Ichthyophonus infections in laboratory challenges were examined. Infections were most effectively established after intraperitoneal (IP) injections with suspended parasite isolates from culture or after repeated feedings with infected fish tissues. Among groups that were offered the infected tissues, infection prevalence was greater after multiple feedings (65%) than after a single feeding (5%). Additionally, among groups that were exposed to parasite suspensions prepared from culture isolates, infection prevalence was greater after exposure by IP injection (74%) than after exposure via gastric intubation (12%); the flushing of parasite suspensions over the gills did not lead to infections in any of the experimental fish. Although the consumption of infected fish tissues is unlikely to be the primary route of Ichthyophonus sp. transmission in wild populations of Pacific Herring, this route may contribute to abnormally high infection prevalence in areas where juveniles have access to infected offal.

Zebrafish as a useful model for zoonotic Vibrio parahaemolyticus pathogenicity in fish and human

Vibrio parahaemolyticus is an important aquatic zoonotic pathogen worldwide that causes vibriosis in many marine fish, and sepsis, gastroenteritis and wound infection in humans. However, the pathogenesis of different sources of V. parahaemolyticus is not fully understood. Here, we examined the pathogenicity and histopathology of fish (V. parahaemolyticus 1.2164) and human (V. parahaemolyticus 17) strains in a zebrafish (Danio rerio). We found that different infection routes resulted in different mortality in zebrafish. Moreover, death due to V. parahaemolyticus 1.2164 infection occurred quicker than that caused by V. parahaemolyticus 17 infection. Hematoxylin-eosin staining of liver, kidney and intestine sections showed histological lesions in all three organs after infection with either strain. V. parahaemolyticus 1.2164 caused more severe damage than V. parahaemolyticus 17. In particular, V. parahaemolyticus 1.2164 treatment induced more serious hydropic degeneration and venous sinus necrosis in the liver than V. parahaemolyticus 17 treatment. The expression levels of three proinflammatory cytokines, interleukin 1β (il1β), interferon phi 1 (ifnϕ1) and tumor necrosis factor α (tnfα), as determined by quantitative real-time PCR, were upregulated in all examined tissues of infected fish. Notably, the peak levels of tnfα were significantly higher than those of il1β and ifnϕ1, suggesting, together with pathological results, that tnfα and il1β play an important role in acute sepsis. High amounts of tnfα may be related to acute liver necrosis, while ifnϕ1 may respond to V. parahaemolyticus and play an antibacterial role for chronically infected adult zebrafish. Taken together, our results suggest that the zebrafish model of V. parahaemolyticus infection is useful for studying strain differences in V. parahaemolyticus pathogenesis.

Isolation and Identification of Tenacibaculum maritimum, the Causative Agent of Tenacibaculosis in Farmed Sea Bass (Dicentrarchus labrax) on the Aegean Sea Coast of Turkey

In this paper we describe the isolation and identification of Tenacibaculum maritimum from infected sea bass by using bacteriological, histopathological, and molecular methods. Fish were reared in five different floating net cage farms and two hatcheries located in several regions on the Aegean Sea coast of Turkey. We observed external erythemic and erosive jaw and operculum superficial or deep ulcerative skin lesions on the head and body surfaces, gill rot, hemorrhagic and erythemic fins in infected individuals, and slight exophthalmia was also detected. In addition, internal hemorrhages and hyperemia in the visceral organs and bloody fluid in the peritoneal cavity were observed. Bacteria isolated from the kidney and skin lesions of the infected fish produced flat, pale colored colonies on Flexibacter maritimus medium (FMM), marine enriched Cytophaga agar (MECA) and Marine Agar (MA) and were identified as Tenacibaculum maritimum, Vibrio sp. and motile or non-motile Aeromonas species. Histopathologically, degeneration and liquefactive necrosis in liver, kidney, and spleen was observed in the individuals infected with Tenacibaculum maritimum. The identification of T. maritimum was performed directly from the infected fish tissue using nested polymerase chain reaction. In this study, T. maritimum induced mostly mixed infection with Vibrio sp. and motile or non-motile aeromonad species in the four fish farms resulting in 25% mortality. However, this bacterium produced an unmixed infection with the same mortality in young sea bass from only one farm.

A Comparative Study of the Health Status of Tilapia Fish Through Various Environmental Changes

A number of 360 apparently healthy and naturally infected Nile tilapia with body weight range (40-80) grams were obtained from Abassa fish farms, Sharkia Governorate, Egypt, random collected from pond with uncovered cement ponds (A) and greenhouse cement ponds (B) (180 from each) during winter season of 2013 /2014 ( December and January). Water parameters were recorded at the collection time namely temperature, ammonia and dissolved oxygen. At the present work tilapia fish were subjected to clinical and post mortem examination. All Fish were examined for isolation and identification of bacterial, mycotic and parasitic pathogens and histopathological examinations were performed on infected fish tissue. The isolated bacteria were Pseudomonas aeruginosa , Pseudomonas fluorescens , Proteus vulgaris , Escherichia coli , Enterobacter intermedius , Enterobacter aerogenes , Enterobacter cloacae and Aeromonas hydrophila . The bacterial prevalence was higher in pond B (57.8%) than in pond A (33.3%). Mycological examination revealed that fungal isolation of four genera were isolated from pond (A) ( Penicillium spp, Aspergillus niger , Blastomyces spp and Scopulariopsis brevicaulis ) but in pond (B) were positive to fiver genera ( Penicillium spp, Aspergillus niger , Blastomyces spp and Scopulariopsis brevicaulis ). The fungal isolates were higher in pond (B) 77.8% than in pond (A) 55.6%. The examination of tilapia fish revealed the presence of four different parasitic agents which were identified, as Myxobolus tilapiae , Trichodina heterodentata and two encysted metacercariae (EMC) Centrocestus formosanus in gills and Heterophyes sp in muscles .The parasitic agents was higher in pond (B) 77.8% than in pond 55.6%(A). The results of the histopathological examination of naturally infected fish were also discussed. It was concluded that it be good to using greenhouse in fish culture to avoid the decrease in water temperature during winter season but other factors must be taken in consideration like water sources to avoid spread of diseases.

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for the detection of the fathead minnow nidovirus

Fathead minnow nidovirus (FHMNV) is a serious baitfish-pathogenic virus in North America. Studies to trace the spread of the virus and determine its host range are hampered by the absence of reliable diagnostic assays. In this study, a one-step, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed that targets a region in the FHMNV spike protein gene. The assay was optimized, and the best results were obtained at 8mM of Mg2+ with an incubation time of 40min at 63°C in the presence of calcein. The analytical sensitivity of the RT-LAMP method was estimated to be as low as 5 viral copies and was 1000-fold more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) method. The diagnostic sensitivity and specificity of the developed RT-LAMP assay versus the RT-PCR assay was 100% and 95.7%, respectively. A quantitative RT-LAMP of FHMNV with a high correlation coefficient (r2=0.9926) was also developed and the result of quantitation of viral copies in tissue samples of infected fish showed that the viral loads of the infected fish tissue samples reached up to 4.7×1010 copies per mg. It is anticipated that the developed RT-LAMP and quantitative RT-LAMP methods will be instrumental for diagnosis and surveillance of FHMNV.

Simultaneous Detection of Streptococcus spp. and Aeromonas spp. from Diseased Tilapia (Oreochromis niloticus) using Multiplex-Polymerase Chain Reaction

A multiplex Polymerase Chain Reaction (m-PCR) assay was developed for simultaneous detection of two major pathogens, Streptococcal and Aeromonad bacteria, in farmed tilapia. DNA fragments of Streptococcus spp. and Aeromonas spp. were amplified using genus specific primers, C1/C2 and AERF/AERR, which produced PCR of 207 bp and 953 bp, respectively. The lowest concentration of each Streptococcus and Aeromonas spp. extracted genomic DNA from a colony detected by m-PCR was 2 ng. The m-PCR assay was proven applicable for detection of bacterial genomic DNA in tissues (brain and posterior kidney) of infected fish. Specificity of the assay tested with other Gram-positive (Staphylococcus aureus) and Gram-negative water borne bacteria (Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhimurium, Vibrio harveyi and Vibrio parahemolyticus) showed no amplification. As Streptococcal and Aeromonad infections are common concurrent bacterial diseases, the m-PCR assay established in this study enabled effective simultaneous detection of these two major bacterial infections responsible for current economic losses in tilapia farming in Thailand.

First Detection of Spring Viremia Of Carp Virus in Cyprinus Carpio, In Lake Shkodra/Scadar Albania

Spring viremia of carp is a highly contagious viral disease caused by Rhabdovirus carpio which is a single strand RNA virus. A combination of one step RT-PCR (reverse transcription) and Semi-nested PCR was used to detect spring viremia of carp virus in infected cell cultures (EPC) and fish tissues. All the fish samples were collected from Lake Shkodra and from artificial ponds in different region of Albania. Total RNA was extracted from 500 μl of fish tissue extract or from 100 μl of supernatant from infected EPC cell cultures. One step RT-PCR is performed using a modification of the methods of Stone et al.(1) in a 15 μl reaction mix volume. We used SVCV F1 (primer forward) 5’- TCT-TGG-AGC-CAA-ATA-GCT-CAR*-R*TC-3’ and SVCV R2 ( primer reverse) 5’-AGA-TGG-TAT-GGACCC- CAA-TAC-ATH*-CAN*-CAY*-3 primers for the amplification of 714 bp fragment of SVCV cDNA (2). The reaction mix was subjected to thermal cycles (2). Amplified cDNA was analyzed in agarose gel electrophoresis. For the second round of amplification we used a semi-nested PCR with SVCV F1 5’-TCT-TGG-AGC-CAA-ATA-GCT-CAR*- R*TC-3’ and SVCV R4 5’-CTG-GGG-TTT-CCN*-CCT-CAA-AGY*-TGY*-3* primers (1). Amplified fragment cDNA (606 bp) is analyzed by agarose gel electrophoreses (2).The results of gel electrophoreses was positive for three fish samples (Cyprinus carpio) collected from Lake Shkodra. This is the first report of the detection of spring viremia of carp virus in Albania in a natural habitat like Lake Shkodra.

Pharmacokinetics of streptomycin sulfate in Staphylococcus aureus-infected Clarias gariepinus (Burchell 1822)

Most bacteria causing fish diseases in Nigeria have been observed to be susceptible to aminoglycosides like streptomycin. However, route of administration for effective therapy has been a challenge due to the reported poor intestinal absorption, hence a need to study its kinetics.Clarias gariepinus were divided into four groups of 14 fish each. Fish in A were Staphylococcus aureus-infected while those in B were uninfected and both received streptomycin by bath at 70mg/l, while it was 70mg/kg via gastric gavage for groups C which were Staphylococcus aureus-infected and D which were uninfected. Blood and tissue collection was done between 1 and 120h post exposure to streptomycin. Samples were analyzed by ELISA, while some portions of tissues of infected fish were fixed for histopathology.Sum of area under curve values for serum and kidney (AUC0–8h) were 5876.5μgh/l and 399,287μgh/kg; 4519.3μgh/l and 183,474μgh/kg; 27,771.2μgh/l and 82,539μgh/kg; as well as 18,768.4μgh/l and 33,252.5μgh/kg respectively for groups A, B, C and D. Microscopically the lesions were localized and extensive hepatic coagulative necrosis with diffuse tubular necrosis.Possible systemic therapeutic value is suggested, depending on increased distribution of streptomycin and levels of streptomycin in kidneys of diseased fish at corresponding times being higher than in sera.

Comparison of three methods for detection of Mycobacterium marinum in goldfish (Carassius auratus)

Abstract Mycobacterium marinum is a well-recognised mycobacterial species which produces granulomatous reactions in a variety of aquatic organisms. Detection of this organism primarily relies on histological and bacteriological examination of infected fish tissues but recently a number of molecular methods have also been employed. In this study a comparison of three such methods in the detection of M. marinum in goldfish ( Carassius auratus ) was carried out. Histological analysis revealed that fish infected with M. marinum often developed granulomas in the absence of external lesions and clinical signs of mycobacteriosis. Approximately 40% of all infected fish had greater than/equal to 10 4 mycobacteria per gram of splenic tissue. Approximately 89% of samples (8/9) with 4 CFU/g of spleen were positive by PCR. Statistical analysis revealed significant differences between the 3 methods of comparison for the entire sample ( N = 15), but very significant differences between PCR and other detection methods ( p = 0.0202). Overall, this study demonstrates that PCR is a rapid, specific, and sensitive method for detecting M. marinum in goldfish.

Bacterial distribution and tissue targets following experimental Edwardsiella ictaluri infection in Nile tilapia Oreochromis niloticus

Edwardsiella ictaluri, a Gram-negative enteric bacterium, is the known etiological agent of enteric septicemia of catfish. In the last few years, different strains have been implicated as the causative agent of mortality events in cultured fish, including Nile tilapia Oreochromis niloticus L. Due to the emergent nature of edwardsiellosis in non-ictalurid fish, little is known about the dynamics of E. ictaluri infection in tilapia. The purpose of this study was to gain a better understanding of the pathogenesis of edwardsiellosis in tilapia by determining the median lethal and infective doses, tissue targets of infection, rate of bacterial dissemination, and the specific tissue response to E. ictaluri following an immersion challenge with bacterial strains recovered from outbreak events in tilapia. In addition to histopathology assessment, the bacterial burdens in several tissues of infected fish were determined over a 2 wk course of infection using quantitative real-time PCR (qPCR). The collected data suggest the cutaneous and oral routes as the main ports of entry for the organism, which later spreads hematogenously throughout the body. Even though histopathological assessment of infected fish revealed involvement of a wide range of tissues, the severity of the necrotizing and granulomatous lesions in the spleen and head kidney, with concomitant high levels of bacterial DNA in these organs determined by qPCR, identifies them as the main targets of infection.

Fluorescent In Situ Hybridization: A New Tool for the Direct Identification and Detection of F. psychrophilum

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium (“Pan-Flavo”) and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum.

Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena crocea)

In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.