Communications: Isolation of Several Mycobacterium Species from Fish

Abstract

Several species of marine fish caught in the wild and of freshwater ornamental fish were used in this study. Infected organs (liver, spleen, and kidney) were sampled for mycobacteria. Decontaminated tissue samples were plated onto selective media for mycobacterial recovery. After initial isolation, fluorescent and acid-fast staining techniques identified bacterial colonies to genus. Profiles of biochemical growth characteristics were used to further identify the isolates to species. Five species of Mycobacterium were identified: M. simiae, M. scrofulaceum, M. marinum, M. chelonae, and M. fortuitum. Of these, M. simiae and M. scrofulaceum have not been previously reported from fish. Tissue samples containing focal granulomatous lesions were prepared for electron microscopic examination.     

Phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. in Malaysia

Mycobacteriosis due to mycobacteria is one of the most common bacterial diseases in ornamental fish. We describe here the phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. using ATCC Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium chelonae as references. A total of four isolates (M1, M2, M3, M4) were obtained from four out of 106 fish samples using selective agar, and identified to Mycobacterium genus using acid-fast staining and 16s rRNA gene-based genus specific polymerase chain reaction. DNA sequencing and NCBI-BLAST analysis further identified isolate M1 as M. marinum and isolates M2, M3, M4 as M. fortuitum. Morphological, physiological and biochemical tests were carried out for phenotypic characterizations. Universal M13 and wild-type phage M13 RAPD dendogram was generated to illustrate the genetic relationship of the isolates and reference strains.     

Multitiered Health Assessment of Atlantic Menhaden in the Pamlico River,North Carolina

Abstract

During the fall of 2001 and 2002, Atlantic menhaden Brevoortia tyrannus were collected from several creeks in the Pamlico River, North Carolina, to investigate recent fish kills and ulcerative skin lesions. High skin lesion prevalence (>50%) was associated with the Atlantic menhaden kills in fall 2001, whereas there were no fish kills in fall 2002 and skin lesion prevalence was lower (≤50%). Indicators of tissue damage (histopathological analyses of gills, heart, liver, intestine, and anterior kidney), body condition (liver somatic index), and immune status (transforming growth factor-β (TGF-β) messenger RNA (mRNA) production, hematology, plasma chemistry, and splenosomatic index) were compared between Atlantic menhaden with and without ulcerative skin lesions in fall. Atlantic menhaden with ulcerative skin lesions had significantly higher liver somatic indices, neutrophil and monocyte percentages, and splenic mononuclear cell TGF-β mRNA levels than did fish without lesions. Hematocrit values, plasma protein, and Ca concentrations were significantly lower in fish with ulcerative skin lesions than in those without. The indicators used in this study at multiple levels of biological organization have provided valuable baseline data for understanding the health status of lesioned and nonlesioned Atlantic menhaden in the Pamlico River.     

Isolation,identification and associated risk factors of non‐tuberculous mycobacteria infection in humans and dromedary camels in Samburu County,Kenya

Non‐tuberculous mycobacteria are of public health significance, and zoonotic infection is attributed to the sociocultural practice of consumption of raw milk and the close human–livestock contact in pastoral communities. This study aimed at isolation, identification of mycobacteria from human sputum and camel milk and risk factors assessment in Samburu East, Kenya. Six hundred and twelve camels and 48 people presumed to have tuberculosis (TB) from 86 households in Wamba and Waso regions were screened. Camels were categorized into Somali, Turkana and Rendile breeds. Single intradermal comparative tuberculin test (SICTT) was used as a herd‐screening test on lactating camels and a milk sample collected from reactive camels. Sputum samples were collected from eligible members of participating households. A standard questionnaire on possible risk factors for both humans and camels was administered to respective household heads or their representatives. Total camel skin test reactors were 238/612 (38.9%). Milk and sputum samples were analysed at KEMRI/TB research laboratory for microscopy, GeneXpert^®, culture and identification. Isolates were identified using 16S rRNA gene sequencing at Inqaba biotec in South Africa. Sixty‐four isolates were acid‐fast bacilli (AFB) positive of which M. fortuitum (3), M. szulgai (20), M. monacense (5), M. lehmanni (4), M. litorale (4), M. elephantis (3), M. duvalii (3), M. brasiliensis (1), M. arcueilense (1) and M. lentiflavum (1) were from milk; M. fortuitum (1), M. szulgai (2) and M. litorale (1) were from humans. Risk factors included the following: Turkana breed (OR = 3.4; 95% CI: 1.2–9.3), replacements from outside the County (OR = 2.1; 95% CI: 0.3–12.3), presence of other domestic species (small stock; OR = 4.6) and replacement from within the herd (OR = 3.2; 95% CI: 0.7–14.7). Zoonotic risk practices included raw milk consumption, shared housing and handling camels. Monitoring of zoonotic NTM through surveillance and notification systems is required.     

Amyloodiniosis in Cultured Hybrid Striped Bass (Morone saxatilis × M. chrysops) in North Carolina

Abstract

An outbreak of amyloodiniosis in pondcultured hybrid striped bass × white bass (female Morone saxatilis × male M. chrysops) is the northernmost documented outbreak caused by an endemic source in fish of the western Atlantic. Dinospores produced by this isolate of Amyloodinium ocellatum were anteroposteriorly flattened, as contrasted with the gymnodinoid dinospores of the A. ocellatum-type species, suggesting that more than one strain or species of A. ocellatum may exist.     

Multitiered health assessment of Atlantic menhaden in the Pamlico River, North Carolina

During the fall of 2001 and 2002, Atlantic menhaden Brevoortia tyrannus were collected from several creeks in the Pamlico River, North Carolina, to investigate recent fish kills and ulcerative skin lesions. High skin lesion prevalence (>50%) was associated with the Atlantic menhaden kills in fall 2001, whereas there were no fish kills in fall 2002 and skin lesion prevalence was lower (< or =50%). Indicators of tissue damage (histopathological analyses of gills, heart, liver, intestine, and anterior kidney), body condition (liver somatic index), and immune status (transforming growth factor-beta [TGF-beta] messenger RNA [mRNA] production, hematology, plasma chemistry, and splenosomatic index) were compared between Atlantic menhaden with and without ulcerative skin lesions in fall. Atlantic menhaden with ulcerative skin lesions had significantly higher liver somatic indices, neutrophil and monocyte percentages, and splenic mononuclear cell TGF-beta mRNA levels than did fish without lesions. Hematocrit values, plasma protein, and Ca concentrations were significantly lower in fish with ulcerative skin lesions than in those without. The indicators used in this study at multiple levels of biological organization have provided valuable baseline data for understanding the health status of lesioned and nonlesioned Atlantic menhaden in the Pamlico River.     

Primary Isolation and Characterization of Tenacibaculum maritimum from Chilean Atlantic Salmon Mortalities Associated with a Pseudochattonella spp. Algal Bloom

Abstract

This study presents the first isolation of Tenacibaculum maritimum from farmed Atlantic Salmon Salmo salar in Chile. The isolate, designated T. maritimum Ch-2402, was isolated from gills of Atlantic Salmon at a farm located in region X, Los Lagos, Chile, during the harmful algal bloom caused by Pseudochattonella spp. in February 2016. The algal bloom is reported to have caused 40,000 metric tons of mortality in this salmon farming area. The bacterium T. maritimum, which causes tenacibaculosis, is recognized as an important pathogen of marine fish worldwide. Genetic, phylogenetic, and phenotypic characterizations were used to describe the T. maritimum Ch-2402 isolate. The isolate was similar to the type strain of T. maritimum but was genetically unique. Tenacibaculum dicentrarchi isolates were also recovered during sampling from the same farm. Based on the fact that T. maritimum has been shown to cause disease in Atlantic Salmon in other regions, the presence of this bacterium poses a potential risk of disease to fish in the Chilean aquaculture industry.

Received November 6, 2016; accepted May 29, 2017 Published online July 26, 2017     

Isolation and Classification of a Nocardia species from Diseased Giant Gourami Osphronemus goramy

Abstract

An enzootic caused by bacteria occurred among cultured giant gourami in two freshwater fish farms in Kagoshima, Japan, in 1982. Typical external characteristics of the diseased fish were sloughed scales, exophthalmia, and opaque eyes. Internally, numerous creamy white nodules were found in the gills, spleen, kidney, and liver and a lesser number in the bladder and stomach. Nocardia sp. was isolated from spleen and kidney tissues of all diseased fish examined. To clarify the taxonomic position of the isolates, 164 tests were performed, and results were compared to reference strains of six species of Nocardia and one species of Rhodococcus. The isolates formed a homogenous group in a single taxon at a similarity of 98.7% based on an average linkage analysis and were clearly separated from the reference strains. The isolated strains were more similar to Nocardia kampachi than to the other representative species. However, 12 distinct differences were found between the isolates and N. kampachi, including growth on nutrient agar, sensitivity to 250 and 500 μg hydroxylamine/mL and 0.01% phenol, tolerance of 0.05% salicylate and 0.1% picric acid in brain-heart infusion medium, hydrolysis of starch, nicotinamidase reaction, and acid from fructose, sucrose, and dextrin. Accordingly, the isolates from the diseased giant gourami were classified as a new species of Nocardia.     

Effect of Dietary Lipid Sources on Production of Leukotriene B by Head Kidney of Channel Catfish Held at Different Water Temperatures

Abstract

Separate and combined effects of dietary fatty acids and water temperature on the production of leukotriene B (LTB) by the head kidney of channel catfish Ictalurus punctatus were investigated. Fish were fed semipurified diets containing 7% lipid as beef tallow, corn oil, linseed oil, menhaden oil, or a mixture (1:1:1) of menhaden oil, beef tallow, and corn oil. At 28°C, fish fed corn oil produced the greatest amount of LTB, and fish fed beef tallow produced the least. At 17°C, production of LTB by fish fed the beef tallow diet was again lowest; production of LTB by fish fed the corn oil diet was highest but was not significantly different from that obtained with the mixed-oil or menhaden oil diet. Production of LTB for the other diets was intermediate. This is the first report that the head kidney of channel catfish produces LTB; this production was affected by dietary lipid sources but not by the two water temperatures tested. Production of LTB was not related to weight gain. Moreover, the pattern of LTB production does not explain survival rates of channel catfish observed in a previous experiment in response to pathogen challenge. However, LTB concentration does appear to be roughly proportional to the amount of eicosanoid precursors (arachidonic acid and eicosapentaenoic acid) previously reported for liver phospholipids of channel catfish fed diets containing different amounts of essential fatty acids.     

Nested Polymerase Chain Reaction Assay for Detection of Mycobacterium shottsii and M. pseudoshottsii in Striped Bass

Abstract

Wild striped bass Morone saxatilis in Chesapeake Bay are experiencing a high prevalence of mycobacteriosis, which produces granulomatous lesions of the skin and visceral organs. Culture-based studies have indicated that the newly described species Mycobacterium shottsii and M. pseudoshottsii are the dominant isolates from diseased fish. The classical fish pathogen M. marinum is also found, albeit at much lower frequencies. Both M. shottsii and M. pseudoshottsii are extremely slow-growing on standard selective media, and up to 12 months may be required for isolation and characterization. Epidemiological studies of mycobacteriosis in Chesapeake Bay would therefore benefit from rapid molecular assays with which to detect these species in fish. In this paper, we describe the development of polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assays capable of detecting M. shottsii, M. pseudoshottsii, and, in most instances, coinfections thereof in striped bass tissues. In addition, PCR–RFLP assays were designed to detect M. marinum and other as-yet-undescribed Mycobacterium spp. present in Chesapeake Bay striped bass. Comparison of these molecular assays with culture-based techniques using splenic tissue from wild striped bass yielded generally concordant results and demonstrated the applicability of these techniques to the study of wild fish.     

Preliminary Morphometrics of Spleen and Kidney Macrophage Aggregates in Clinically Normal Blue Gourami Trichogaster trichopterus and Freshwater Angelfish Pterophyllum scalare

Abstract

Macrophage aggregate (MA) morphometry and pigment composition are believed to be dependent on the species, age, and health status of the fish. The aim of this study was to characterize a “normal” morphometry baseline of spleen and kidney MAs in blue gourami Trichogaster trichopterus and freshwater angelfish Pterophyllum scalare. Three size-classes of clinically normal fish were analyzed. Blue gourami and freshwater angelfish were obtained from three local ornamental fish farms; for each size-class, 10 fish from each farm were analyzed. Hematoxylin- and eosin-stained tissue sections were analyzed by light microscopy at 100× magnification and an image analysis program. The percentage of tissue occupied by MAs, MA size, and MA number were calculated on three arbitrarily selected fields of view from each spleen and kidney. In clinically normal blue gourami, increases in the percentage of tissue occupied by MAs and in MA size were associated with an increase in fish size, but in clinically normal angelfish no correlation was observed. Furthermore, in angelfish, a high variability in MA morphometry was observed, even among fish from the same sample group. In both species, a significant difference in the value of the morphometric parameters was observed among farms. Because iridoviruses inhibit macrophage activity and (possibly) proliferation, MAs in 25 clinical cases of iridovirus-infected blue gourami were analyzed. Preliminary data indicate that in iridovirus-infected blue gourami, there is a decrease in MA size and MA number compared with those of healthy fish.     

Predominant Bacteria Associated with Red Snapper from the Northern Gulf of Mexico

Abstract

Since the Deepwater Horizon oil spill in 2010, anecdotal observations of Red Snapper Lutjanus campechanus from the northern Gulf of Mexico exhibiting unusual external lesions have been reported. Two opportunistic bacterial fish pathogens, Vibrio vulnificus and Photobacterium damselae, were recovered from the fish and were deemed responsible for the abnormalities. However, the culturable microbiota of healthy Red Snapper has not yet been characterized. We analyzed the heterotrophic bacteria associated with healthy Red Snapper caught off the Louisiana coast. In total, 179 isolates from 60 fish were recovered from skin and mucus, and 43 isolates were obtained from anterior kidney. All isolates were identified by 16S ribosomal RNA gene sequencing. The Proteobacteria was the predominant phylum in both external and internal samples, followed by the Firmicutes and the Actinobacteria. Within the Proteobacteria, most isolates were members of the genera Vibrio and Photobacterium, and V. natriegens and P. damselae were the predominant species. The results of this study suggest that both Vibrio spp. and Photobacterium spp. are associated with the normal microbiota of healthy Red Snapper. Thus, the opportunistic fish pathogens recovered in previous studies cannot be deemed lesion-forming until Koch's postulates are fulfilled.

Received March 1, 2013; accepted September 4, 2013     

Phenotypic and Genotypic Heterogeneity among Streptococcus iniae Isolates Recovered from Cultured and Wild Fish in North America,Central America and the Caribbean Islands

Abstract

Streptococcus iniae, the etiological agent of streptococcosis in fish, is an important pathogen of cultured and wild fish worldwide. During the last decade outbreaks of streptococcosis have occurred in a wide range of cultured and wild fish in the Americas and Caribbean islands. To gain a better understanding of the epizootiology of S. iniae in the western hemisphere, over 30 S. iniae isolates recovered from different fish species and geographic locations were characterized phenotypically and genetically. Species identities were determined biochemically and confirmed by amplification and sequencing of the 16S rRNA gene. Repetitive-element palindromic PCR fingerprinting as well as biochemical and antimicrobial susceptibility profiles suggest that a single strain of S. iniae was responsible for two different disease outbreaks among reef fishes in the Caribbean, one in 1999 and another in 2008. Interestingly, a majority of the isolates recovered from cultured fish in the Americas were genetically distinct from the Caribbean isolates and exhibited a trend toward higher minimal inhibitory concentration with respect to several antibiotics as well as greater genetic variability. The biological significance of this genetic variability is unclear, but it could have implications for future vaccine development and treatment.

Received April 20, 2014; accepted July 7, 2014     

Predicted Redistribution of Ceratomyxa shasta Genotypes with Salmonid Passage in the Deschutes River,Oregon

Abstract

A series of dams on the Deschutes River, Oregon, act as migration barriers that segregate the river system into upper and lower basins. Proposed fish passage between basins would reunite populations of native potamodromous fish and allow anadromous fish of Deschutes River origin access to the upper basin. We assessed the potential redistribution of host-species-specific genotypes (O, I, II, III) of the myxozoan parasite Ceratomyxa shasta that could occur with fish passage and examined the influence of nonnative fish on genotype composition. To determine the present distribution of the parasite genotypes, we exposed eight salmonid species—three native and five stocked for sport fishing—in present and predicted anadromous salmonid habitats. We monitored fish for infection by C. shasta and sequenced a section of the parasite ribosomal DNA gene from fish and water samples to determine parasite genotype. Genotype O was present in both upper and lower basins and detected only in steelhead Oncorhynchus mykiss. Genotype I was spatially limited to the lower basin, isolated predominately from Chinook salmon O. tshawytscha, and lethal for this species only. Genotype II was detected in both basins and in multiple species, but only as a minor component of the infection. Genotype III was also present in both basins, had a wide host range, and caused mortality in native steelhead and multiple nonnative species. Atlantic salmon Salmo salar and kokanee O. nerka were the least susceptible to infection by any genotype of C. shasta. Our findings confirmed the host-specific patterns of C. shasta infections and indicated that passage of Chinook salmon would probably spread genotype I into the upper Deschutes River basin, but with little risk to native salmonid populations.

Received April 20, 2012; accepted July 19, 2012     

Pathogenicity of Mycobacterium fortuitum and Mycobacterium smegmatis to goldfish, Carassius auratus

Despite the ubiquitous presence of atypical mycobacteria in the environment and the potential risk of infection in humans and animals, the pathogenesis of diseases caused by infection with atypical mycobacteria has been poorly characterized. In this study, goldfish, Carassius auratus were infected either with the rapidly growing fish pathogen, Mycobacterium fortuitum or with another rapidly growing mycobacteria, Mycobacterium smegmatis. Bacterial persistence and pathological host response to mycobacterial infection in the goldfish are described. Mycobacteria were recovered from a high percentage of inoculated fish that developed a characteristic chronic granulomatous response similar to that associated with natural mycobacterial infection. Both M. fortuitum and M. smegmatis were pathogenic to fish. Fish infected with M. smegmatis ATCC 19420 showed the highest level of giant cell recruitment compared to fish inoculated with M. smegmatis mc(2)155 and M. fortuitum. Of the three strains of mycobacteria examined, M. smegmatis ATCC 19420 was the most virulent strain to goldfish followed by M. fortuitum and M. smegmatis mc(2)155, respectively.     

A SURVEY OF MYCOBACTERIOSIS OF FERAL PIGS IN THE NORTHERN TERRITORY

Seven hundred and fifty-one feral pigs from the subcoastal plains of the Northern Territory were examined. The sample population consisted of 52.4% females and 47.6% males. They ranged in age from newborn piglets to mature animals of over 72 months. Of the pigs examined 47.7% had macroscopic abscesses and of these 80.2% were probably caused by mycobacteria. Tissues from 193 pigs were examined bacteriologically and 93 strains of mycobacteria were isolated. These were typed as M. bovis (37 strains); M. avium serotype 2 (1); M. intracellulare serotypes 6 (2), 7 (3), 9 (1) and 18 (1); M. intracellulare double serotypes 6 + 12 (1), 8 + 12 (1) and 11 + 25 (1); M. intracellulare unclassified serotype (4); M. scrofulaceum serotype 41 (1); M. scrofulaceum unclassified serotype (7); M. gordonae (2); M. kansasii (1); M. simiae (2); M. szulgai (2); M. vaccae (1); and M. xenopi (2). Additionally, 3 strains were unidentifiable members of the M. avium-M. intracellulare-M. scrofulaceum (MAIS) complex, one strain was a Runyon's group IV and 4 strains were typed as members of the genus Rhodococcus. Five strains were non-viable on subculture and 10 did not conform to any currently recognised species of mycobacteria. Of the 93 strains, 3 were isolated from tissue that did not contain macroscopic lesions, viz. M. simiae, Runyon's group IV and an unidentifiable member of the MAIS complex. It was concluded that the feral pig is probably an end host for both M. bovis and atypical mycobacteria and not a significant source of infection for cattle. M. bovis is not a significant cause of mortality in feral pigs but mycobacterioses are a significant cause of morbidity. With increasing age, the proportion of pigs having lesions increased whereas the proportion of lesions from which mycobacteria could be isolated decreased.     

Occurrence and Distribution of Aeromonas salmonicida Infections on Swedish Fish Farms, 1951–1987

Abstract

The occurrence of typical and atypical Aeromonas salmonicida infections on Swedish fish farms was surveyed, and the methods for controlling the disease are described here. This review is based on data for a period of 36 years from fish health control studies conducted by the National Veterinary Institute and the Salmon Research Institute. The epizootiology and routes of transmission of the pathogens are described, and policy and methods for disease control are discussed. The distribution of atypical A. salmonicida infections was entirely different from that of typical infections. There was also circumstantial evidence of different routes of transmission for typical and atypical A. salmonicida infections. Although subclinical carriers of atypical A. salmonicida occurred among Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss on fish farms, acute disease outbreaks were most frequent among Arctic char Salvelinus alpinus and brown trout Salmo trutta. On the other hand, acute outbreaks of typical A. salmonicida infections were frequent among Atlantic salmon and rainbow trout.     

Influence of Dietary Lipid and Temperature on Bactericidal Activity of Channel Catfish Macrophages

Abstract

We fed juvenile channel catfish Ictalurus punctatus purified diets containing different lipid sources for 90–110 d to examine the influence of dietary lipids on intracellular killing of Edwardsiella ictaluri by pronephros macrophages. The diets contained either menhaden oil, soybean oil, beef tallow, or a combination ofall three lipids. We performed the study with nonimmune and immunized fish acclimated to optimal (28°C) and suboptimal (19°C) temperatures. In both temperature trials, bactericidal activity was positively correlated with the level of n-3 fatty acids in the diet (particularly long-chain highly unsaturated fatty acids). Vaccination resulted in substantially enhanced bactericidal activity, except at suboptimal temperature in fish fed the diet containing beef tallow (primarily saturated and monoenoic fatty acids). Overall results were similar at optimal and suboptimal temperatures. This study demonstrates the potential for nutritional manipulation of disease resistance in fish and illustrates the need for standardized diets in fish health research.     

Antibiotic Resistance of Aeromonas spp. Isolated from Tropical Fish Imported from Singapore

Abstract

To determine the scope of antibacterial resistance among Aeromonas spp., bacterial cultures were taken from a variety of tropical fish species imported from Singapore. The samples were plated on Rimler-Shotts medium for bacterial isolation and identified with both the API20E and Nonfermenter Test Strip systems. Aeromonas hydrophila, A. sobria, and A. caviae were identified in monomicrobic and concomitant infections. Following identification of bacterial isolates, 11 antimicrobials routinely used in the tropical fish industry and 1 new fluoroquinolone were tested for their effectiveness against Aeromonas spp. with the Kirby-Bauer disk-diffusion technique. Aeromonas sobria proved to be the most resistant, often showing susceptibility to only 3 of 12 test drugs. Aeromonas hydrophila was consistently the least resistant. Based on these results, antimicrobial resistance appears to be a rapidly emerging problem in the pet fish industry.     

Aeromonas hydrophila Isolated from Wild Freshwater Fish in Croatia

Aeromonas hydrophila was recovered from fish living in lake Vrana on the Croatian island of Cres. The occurrence of the bacterium in the fish was assessed and related to gross signs of disease and findings at necropsy as a potential health hazard for fish. Isolated bacteria were subjected to morphological, physiological, biochemical and antibiotic susceptibility tests. A total of 26 A. hydrophila isolates were obtained. There was a clear seasonality, as no isolates were recovered in the summer months. Most of the isolates were sensitive to all the antimicrobials used in the study except novobiocin and penicillin G. Affected fish manifested haemorrhages over the skin, in the liver, kidney and swim bladder, spleen infarcts, fatty liver, ascitic fluid and swollen haemopoietic tissues. A. hydrophila does not appear to pose a major threat for the fish in the lake at present but under unfavourable and stressful conditions it could seriously compromise fish health.