Investigations on the role of the salmon louse, Lepeophtheirus salmonis (Caligidae), as a vector in the transmission of Aeromonas salmonicida subsp. salmonicida.

A bacteria-parasite challenge model was used to study the role of sea lice, Lepeophtheirus salmonis (Copepoda), as a vector of Aeromonas salmonicida subsp. salmonicida. Three hypotheses were tested: (i) L. salmonis can acquire A. salmonicida subsp. salmonicida via water bath exposure; (ii) L. salmonis can acquire the bacteria via parasitizing infected Atlantic salmon, Salmo salar; and (iii) L. salmonis can transmit the bacteria to naïve Atlantic salmon via parasitism. Adult L. salmonis exposed to varying A. salmonicida subsp. salmonicida suspensions (10(1) -10(7)  cells mL(-1) ) for 1.0, 3.0 or 6.0 h acquired the bacteria externally (12.5-100%) and internally (10.0-100%), with higher prevalences associated with the highest concentrations and exposures. After exposure to 10(7)  cells mL(-1) , viable A. salmonicida subsp. salmonicida could be isolated from the external carapace of L. salmonis for 120 h. Lepeophtheirus salmonis also acquired the bacteria externally and internally from parasitizing infected fish. Bacterial transmission was observed only when L. salmonis had acquired the pathogen internally via feeding on 'donor fish' and then by parasitizing smaller (<50 g) 'naive' fish. Under specific experimental conditions, L. salmonis can transfer A. salmonicida subsp. salmonicida via parasitism; however, its role as a mechanical or biological vector was not defined.

Aeromonas salmonicida infection levels in pre- and post-stocked cleaner fish assessed by culture and an amended qPCR assay.

Due to increasing resistance to chemical therapeutants, the use of 'cleaner fish' (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de-licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A-layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real-time PCR (qPCR), targeting the A. salmonicida A-layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A-layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild-caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre- and post-stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded.

Detection of variants of the pRAS3, pAB5S9, and pSN254 plasmids in Aeromonas salmonicida subsp. salmonicida: multidrug resistance, interspecies exchanges, and plasmid reshaping.

The ubiquitous water-borne Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a worldwide disease in fish farms. Plasmids carrying antibiotic resistance genes have already been described for this bacterium. The aim of the present study was to identify and characterize additional multidrug resistance plasmids in A. salmonicida subsp. salmonicida. We sequenced the plasmids present in two multiple antibiotic-resistant isolates using high-throughput technologies. We also investigated 19 other isolates with various multidrug resistance profiles by genotyping PCR and assessed their resistance to tetracycline. We identified variants of the pAB5S9 and pSN254 plasmids that carry several antibiotic resistance genes and that have been previously reported in bacteria other than A. salmonicida subsp. salmonicida, which suggests a high level of interspecies exchange. Genotyping analyses and the antibiotic resistance profiles of the 19 other isolates support the idea that multiple versions of pAB5S9 and pSN254 exist in A. salmonicida subsp. salmonicida. We also identified variants of the pRAS3 plasmid. The present study revealed that A. salmonicida subsp. salmonicida harbors a wide variety of plasmids, which suggests that this ubiquitous bacterium may contribute to the spread of antibiotic resistance genes in the environment.

Pedicure-associated rapidly growing mycobacterial infection: an endemic disease.

BACKGROUND: Pedicure-associated nontuberculous mycobacterial furunculosis has been reported in the setting of either outbreaks or sporadic case reports. The epidemiology of these infections is not well understood. METHODS: Systematic surveillance for pedicure-associated nontuberculous mycobacterial furunculosis was conducted in 2 North Carolina counties from 1 January 2005 through 31 December 2008. A subset of implicated nail salons and control salons was inspected and sampled for nontuberculous mycobacteria. RESULTS: Forty cases of suspected or confirmed pedicure-associated nontuberculous mycobacterial furunculosis were reported during the 4-year study period. Furunculosis incidence in the surveillance region was 1.00, 0.96, 0.83, and 0.89 cases per 100,000 population in 2005, 2006, 2007, and 2008, respectively. The responsible organisms primarily belonged to the Mycobacterium chelonae/abscessus group (30 [91%] of 33 isolates). Thirteen implicated salons and 11 control salons were visited and environmentally sampled. An assortment of nontuberculous mycobacteria was cultured from footbaths, but there was no association between the species distribution of the environmental isolates and implication of the salon in human infection. Evidence of suboptimal cleaning (visible debris or surface biofilms) was observed in at least 1 footbath for 11 of 13 implicated salons and 4 of 11 control salons (P = .032). CONCLUSIONS: Pedicure-associated mycobacterial furunculosis was endemic in these 2 North Carolina counties during 2005-2008. Suboptimal footbath cleaning may have contributed to these infections, which suggests straightforward means of potential prevention. The relative rarity of this type of infection in the setting of nearly ubiquitous exposure to these pathogens suggests that as yet undefined host-specific or procedure-related factors may be involved in susceptibility to these infections.

Familial furunculosis associated with community-acquired leukocidin-positive methicillin-susceptible Staphylococcus aureus ST152.

Panton-Valentine leukocidin (PVL) is associated with staphylococcal skin and pulmonary infections. Community dissemination of PVL-producing Staphylococcus aureus strains constitutes a public health concern. Family transmission and spread of community-acquired leukocidin-positive methicillin-susceptible S. aureus ST152 isolates associated with severe clinical symptoms are herein described. Remarkably, ST152 isolates usually are methicillin resistant.

Tri-trophic linkages in disease: pathogen transmission to rainbow trout through stonefly prey.

Relationships between macroinvertebrates and microorganisms in aquatic environments are only poorly understood despite the fact that many aquatic macroinvertebrates feed on microbial biofilms during some life stage. Better understanding of trophic interactions between microbial biofilms, macroinvertebrates, and fish may also help control fish diseases and loss of natural resources. It has also been suggested that pollution, habitat fragmentation, and poor water quality may contribute to increased pathogenesis and mortality in fish. Increased disease incidence is difficult to assess, however, in part because of the complexity of pathogen transmission dynamics. Several environmental pathogens exist whose reservoir(s) and means of transmission remain poorly understood, highlighting the need to study pathogen ecology and interactions with organisms other than susceptible hosts. Aeromonas salmonicida is rarely isolated from freshwater sediments. However, stonefly nymphs were found to frequently harbor A. salmonicida and were shown to preferentially feed on the bacterium. Rainbow trout juveniles were presented with different feeding regimes to determine the transmission capacity of nymphs, and all fish fed stoneflies harboring A. salmonicida expressed symptoms of disease. Although current rates of furunculosis in freshwater ecosystems are unknown, trout primarily feed on stoneflies when water oxygen levels are high and temperatures are low (winter months), which is presumed to correspond to high resistance to the pathogen. Given that furunculosis is associated with physiological stress and higher water temperatures, its natural incidence may change in response to global or regional climatological effects.

A stochastic modelling approach to describing the dynamics of an experimental furunculosis epidemic in Chinook salmon, Oncorhynchus tshawytscha (Walbaum).

A susceptible-infected-removed (SIR) stochastic model was compared to a susceptible-latent-infectious-removed (SLIR) stochastic model in terms of describing and capturing the variation observed in replicated experimental furunculosis epidemics, caused by Aeromonas salmonicida. The epidemics had been created by releasing a single infectious fish into a group of susceptible fish (n = 43) and progress of the epidemic was observed for 10 days. This process was replicated in 70 independent groups. The two stochastic models were run 5000 times and after every run and every 100 runs, daily mean values of each compartment were compared to the observed data. Both models, the SIR model (R(2) = 0.91), and the SLIR model (R(2) = 0.90) were successful in predicting the number of fish in each category at each time point in the experimental data. Moreover, between-replicate variability in the stochastic model output was similar to between-replicate variability in the experimental data. Generally, there was little change in the goodness of fit (R(2)) after 200 runs in the SIR model whereas 500 runs were necessary to have stable predictions with the SLIR model. In the SIR model, on an individual replicate basis, approximately 80% of 5000 simulated replicates had R(2) = 0.7 and above, whereas this ratio was slightly higher (82%) with the SLIR model. In brief, both models were equally effective in predicting the observed data and its variance but the SLIR model was advantageous because it differentiated the latent, i.e. infected but not having the ability to discharge pathogen, from the infectious fish.

Translocation of viable Aeromonas salmonicida across the intestine of rainbow trout, Oncorhynchus mykiss (Walbaum).

The pathogenic bacterium Aeromonas salmonicida is the causative agent of the destructive disease furunculosis in salmonids. Horizontal transmission in salmonids has been suggested to occur via the skin, gills and/or intestine. Previous reports are contradictory regarding the role of the intestine as a route of infection. The present study therefore investigates the possibility of bacterial translocation across intestinal epithelia using Ussing chamber technology, in vitro. Intestinal segments were exposed for 90 min to fluorescein isothiocyanate-labelled pathogenic A. salmonicida. Sampling from the serosal side of the Ussing chambers showed that bacteria were able to translocate across the intestinal epithelium in both the proximal and distal regions. Plating and subsequent colony counting showed that the bacteria were viable after translocation. During the 90 min exposure to A. salmonicida, the intestinal segments maintained high viability as measured by electrical parameters. The distal region responded to bacterial exposure by increasing the electrical resistance, indicating an increased mucus secretion. This study thus demonstrates translocation of live A. salmonicida through the intestinal epithelium of rainbow trout, suggesting that the intestine is a possible route of infection in salmonids.

Effects of host density on furunculosis epidemics determined by the simple SIR model.

To determine the transmission dynamics of furunculosis, Chinook salmon, Oncorhynchus tshawytscha held at various densities were challenged with a single infectious fish. Data from the in vivo experiments were compared to predicted values from a susceptible--infected--removed (SIR) model. Seven different densities were tested including 9.13, 4.56, 0.72, 0.36, 0.19, 0.06 and 0.03 fish/L and each produced different transmission coefficients (beta) of 0.01, 0.019, 0.0051, 0.0076, 0.0001, 0.0005 and 0.00, respectively. Furunculosis related mortality rates decreased as density of the host decreased. Mortality rates at the highest fish densities where disease specific mortality (DSM) was observed were 0.42, 0.44, 0.18 and 0.02 infected animals per day. The natural mortality rate also followed a similar pattern with 0.005, 0.005, 0.001, 0.005, 0.0001, 0.000 and 0.000 surviving animals per day. The results indicated that the transmission coefficient and the DSM of furunculosis was dependent on host density. It is possible that fish behavioral changes at the lower densities caused the reproductive rate of disease to be persistent even at very low densities. In summary, the simple SIR model suggested that low mortality does not always mean low prevalence of furunculosis in a given population. This is the first study evaluating host density as a risk factor for fish diseases using experimental furunculosis data.

A deterministic model for the dynamics of furunculosis in chinook salmon Oncorhynchus tshawytscha.

Studies were undertaken to determine the parameters of transmission of Aeromonas salmonicida in chinook salmon Oncorhynchus tshawytscha, and to develop a deterministic model of the dynamics of experimental furunculosis. For determination of disease transmission coefficient (beta), disease-related mortality rate (alpha) and natural mortality rate (gamma), fish in 70 tanks (approximately 42 fish tank(-1)) were each exposed to a single infectious donor fish, 7 tanks were randomly selected daily and all individuals were examined for the presence of A. salmonicida in the kidney. The proportion of susceptible (S), infected (I) and removed (R, dead) individuals were determined daily. The parameters beta, alpha, gamma, reproductive ratio (R0) and threshold density were estimated to be 0.0214 infected ind. d(-1), 0.29 infected ind. d(-1), 0.00015 ind. d(-1), 3.23 and 13.56 ind., respectively. Using these parameters, a deterministic disease model of A. salmonicida infection as a cause of furunculosis was constructed. The net rate at which new individuals became infected (the incidence rate) per unit time was proportional to S x I x beta. The model-produced data for S were significantly associated with experimental data (r2 = 0.92). In brief, a simple SIR (susceptible-infected-removed) model was successfully utilized to simulate observed data

Phenotypic and molecular characteristics of Aeromonas salmonicida subsp. salmonicida isolated in southern and northern Finland.

Aeromonas salmonicida subsp. salmonicida causes outbreaks of furunculosis in salmonid fish. Furunculosis was first detected in Finland in 1986 on fish farms located on the Finnish coast of the Bothnian Bay. Molecular methods, SDS-PAGE, ribotyping, randomly amplified polymorphic DNA (RAPD) and plasmid profile analysis as well as phenotypic characteristics (biochemical characteristics, maximum growth temperature, pigment and elastase production) were used both for typing the strains and to study the possible routes of transmission of the organism to Finland and the spread of infection within Finland from 1986 to 1993. Ribopattern analysis of chromosomal DNA digested with SmaI, BglI, PstI and ClaI of 28 Finnish strains and eight foreign strains (Denmark, Sweden, Norway or Canada) showed that all Finnish strains and the Swedish strain originating from the Swedish coast of the Bothnian Bay had identical ribopatterns. All other foreign strains had distinct, unique ribotypes except for the second Swedish strain studied, the ribotype of which was identical with that of one Danish strain. RAPD typing, based on the results of two arbitrary primers, yielded 15 types for the Finnish strains. Except for both Danish strains, which had the RAPD type which was identical with that of one Finnish strain, the foreign strains had RAPD patterns differing from those of the Finnish strains. Plasmid profile typing and RAPD profile typing did not correlate. Ribotyping with four different enzymes proved to be the most sensitive method for studying genetically homogeneous Aer. salmonicida subsp. salmonicida. Ribopattern analysis showed that the infection which first started in 1984/1985 on the Swedish coast of the Bothnian Bay may have been transmitted to Finland where the first outbreaks occurred in 1986. The strains infecting Finnish fish farms were very homogeneous, with most differences seen, for example, in maximum growth temperature, plasmid profiles and the RAPD profiles of the strains.

An outbreak of furunculosis among high school athletes.

Furuncles (boils) are common among teenagers; however, few outbreaks have been documented. We investigated an outbreak of furuncles that occurred among male athletes of a Kentucky high school during the 1986 to 1987 school year. The overall attack rate was 25% (31/124). The risk of developing a furuncle increased two to three times in those who had skin injury. Athletes who sustained abrasions more than twice per week (P less than 0.01), who had a cut that required bandaging (P = 0.01), or had an unspecified injury causing a missed practice or game (P = 0.04) were at increased risk. The risk of developing furunculosis did not appear to be related to contact with formites, but rather, to contact with furuncles. Although athletes shared common areas (showers, locker rooms, practice areas, the attack rates for varsity football (36%) and varsity basketball (33%) were four times greater than for nonvarsity teams (P less than 0.01). Players who had a friend with a furuncle were more than twice as likely to also have had a furuncle (P less than 0.01). Exposure to furuncles appeared to increase the risk of furunculosis independently of reported skin injury. Control and prevention should, therefore, focus on both reducing skin injury and reducing exposure to furuncles, rather than attempting to sterilize inanimate objects.

Recurrent staphylococcal furunculosis in families.

Recurrent familial staphylococcal furunculosis causes severe physical and especially socio-psychological problems to the families involved. In the present study 6 families with 28 persons were not treated with antibiotics but with a combination of chlorhexidine bathing, nasal 1% chlorhexidine gel and improved cleaning and hygiene in the surroundings. The infecting Staphylococcus aureus strain was eradicated from the surroundings and the skin in all 6 families, but several of the family members still retained the strain in the nose. All family members were, however, cured and remained without infections during an observation period of 2 years.

Prevalence of staphylococcal zoonosis in pyogenic skin infections.

S. aureus aetiology was found in 76.47% of the 102 pyoderma cases under study, with a high frequency of incidence in children below 5 years of age. The majority of the S. aureus isolates was nontypable (50.0%) and among the typable strains, phage group-III (31.0%), Group-I (20.7%) and a mixed phage group (34.5%) were predominant. 28.2% of the isolates were S. aureus var. bovis and 2.6% were S. aureus var. canis subgroups. Zoonoses in staphylococcal pyogenic skin infections were proved in 9 of the cases, which showed the history of direct animal contact supported by the isolation of S. aureus of animal host origin. Dogs, cattle and poultry were found to be the most important reservoirs transmitting S. aureus to humans, with folliculitis, furunculosis, impetigo as the frequent clinical manifestations.

Furunculosis in a high school football team.

An outbreak of Staphylococcus aureus furuncles (boils) occurred among 26 members of a central Illinois high school football team. A total of 55 boils were reported with a diameter of induration ranging from 0.25 to 6 inches (mean = 1.25 inches). Eighty-nine percent of the boils were located on the extremities, and 70% were known to have developed following a bruise or break in the skin. The development of boils was associated with prior bruises and open wounds. Use of skin lubricant, elbow and forearm pads, whirlpool bath, and athletic tape were also associated with the development of boils, but were not judged to be causal. Rather, these factors most likely reflected attempts to protect areas of compromised skin which were at high risk of developing boils. Lack of hot water and soap for showering may have facilitated the occurrence of the outbreak by discouraging players from showering. Other variables examined, but excluded as important factors, were the sharing of football pads, the sharing of towels, the laundering of athletic clothes, and the position played on the football team.