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PubMed1995

Detection of aerolysin gene in Aeromonas strains isolated from drinking water, fish and foods by the polymerase chain reaction.

A polymerase chain reaction (PCR) technique was used to assay the presence of the aerolysin gene in a total of 89 Aeromonas hydrophila and A. sobria strains isolated from drinking water, fish and foods. These strains were also characterized for the production of virulence factors such as haemolysin, protease and cytotoxin. The primers used in the PCR targeted a 209-bp fragment of the aer gene coding for the beta-haemolysin and detected template DNA only in haemolytic A. hydrophila strains. The cell-free culture supernatants of these aerolysin-positive A. hydrophila strains were also cytotoxic to the HeLa and McCoy cells. The haemolytic A. sobria and non-haemolytic A. hydrophila were consistently negative in the PCR assay. Primer specificity was determined in the PCR by using a control haemolytic Escherichia coli, Streptococcus pyogenes and a restriction endonuclease assay. The PCR clearly identified the aerolysin-producing strains of A. hydrophila and may have application as a rapid species-specific virulence test.

Aeromonas↗
PubMed1996

Ecological relationship between Aeromonas and Vibrio spp. and planktonic copepods in the coastal marine environment in southern Italy.

The colonisation of planktonic copepod integument by bacteria belonging to the family of Vibrionaceae is a well described phenomenon. In this study, besides reporting on the occurrence of Vibrionaceae and other enteropathogens, we further report on the bacterial attachment to the Estuarine copepod Acartia margalefi in a faecal polluted coastal lagoon near Naples, Southern Italy. In addition, we also performed a laboratory experiment to study the ability of 7 bacterial strains (Vibrio cholerae non-Ol, V. mimicus, V. parahaemolyticus, V. alginolyticus, Aeromonas hydrophila, Escherichia coli and Pseudomonas sp.) to colonise the copepod integument. For this laboratory study, 4 different species of copepods, namely Temora stylifera, A. clausi, Centropages typicus and Paracalanus parvus sampled from the Gulf of Naples (Southern Italy) were used. Scanning electron microscope (SEM) studies on the copepods sampled from the lagoon indicated that the bacterial attachment on the integument of Acartia margalefi was mainly on the ventro-lateral body region of the host and in the joints of the thoracic segments, as well as on the swimming and feeding appendages. This infestation, made by rod-like bacteria, was absent in winter but reached peak values of 70% frequency in June. The laboratory studies showed that while V. cholerae non-O1 and A. hydrophila attached on live and dead copepods, respectively, the V. parahaemolyticus, V. alginolyticus, V. mimicus, E. coli and Pseudomonas sp. failed to colonise either live or dead copepods. Thus, this study provides further valuable information about the ecological relationship between different microorganisms (pathogens) and copepods in the coastal marine environment in Southern Italy.

Aeromonas↗
PubMed1986

Interaction between Aeromonas salmonicida and peritoneal macrophages of brook trout (Salvelinus fontinalis).

The phagocytic and bactericidal properties of peritoneal macrophages obtained from brook trout (Salvelinus fontinalis), injected with either glycogen or a modified Freund's complete adjuvant (MFCA), were evaluated against an avirulent and a virulent strain of Aeromonas salmonicida. Avirulent bacteria were effectively phagocytized and killed by macrophages obtained from fish injected with both irritants. With glycogen-elicited macrophages, no enhancement of killing was observed following opsonization of avirulent bacteria with specific antibodies. A killing index (K.I.) of 38 was obtained, compared to a K.I. of 39 for unopsonized bacteria. When avirulent bacteria were opsonized with complement, the K.I. was increased to 67. Virulent bacteria were less susceptible to the phagocytic and the bactericidal activities of glycogen-elicited macrophages, even after opsonization with antibodies and/or complement, K.I. of 9 to 15. In contrast, MFCA-elicited macrophages showed increased phagocytic and bactericidal activities against both strains. The K.I. of unopsonized virulent bacteria was increased to 47 and 46 compared to K.I. of 4 and 7 obtained with glycogen-elicited macrophages.

Aeromonas↗
PubMed1986

Effect of an immunopotentiator on Aeromonas salmonicida infection in rainbow trout (Salmo gairdneri).

The immunoactive peptide FK-565 (heptanoyl-y-D-glutamyl-(L)-mesodiaminopimelyl-(D)-alanine) was found to induce protection against intraperitoneal Aeromonas salmonicida infection in rainbow trout (Salmo gairdneri Richardson). The survival rate was as high as 60% when FK-565 was given intraperitoneally as a single dose (1mg/kg) one day before bacterial challenge. A non-specific stimulation of phagocytic cells by FK-565 at an early stage of the bacterial infection may contribute to the resistance observed. The phagocytic activity of peritoneal phagocytic cells as well as phagocytic cells of the pronephros were stimulated by FK-565 in vivo and in vitro, respectively, as compared to untreated control fish. Furthermore, decreased activity of phagocytic cells previously immunosuppressed with cyclophosphamide was rapidly restored by application of FK-565.

Aeromonas↗
PubMed1986

Blood changes in carp (Cyprinus carpio) induced by ulcerative Aeromonas salmonicida infections.

Only recently Aeromonas salmonicida has been recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. Our attempts to formulate a vaccine based on bacterial surface antigens were unsuccessful in conferring reliable protection against lethal challenge. This lead us to study pathological changes in the humoral defense system during ulcerative A. salmonicida infection in carp. High numbers of opportunist pathogens such as A. hydrophila and Pseudomonas sp. were frequently recovered from the internal organs of moribund fish, in addition to A. salmonicida. These findings together with leucopenia in moribund fish suggest that pathogenesis is characterized by a state of immune suppression. In addition, fish which had sustained a sublethal infection were not protected against a subsequent lethal challenge. However, fish previously injected with a concentrated and inactivated culture supernatant showed protection. Differential blood cell counts did not differ between experimental and control groups during sublethal infection in contrast to serum proteins. Furthermore infected non-immune carp showed a progressive decrease of immunoglobulin and total serum protein levels before the day of peak mortality whereas protected carp maintained the immunoglobulin concentration despite a decrease in protein. Our observations suggest the involvement of multiple pathogenic events, affecting different parts of the humoral defense system during ulcerative A. salmonicida infection. The immunosuppressive effects can be minimized by prior vaccination with culture supernatant.

Aeromonas↗
PubMed1986

Does Aeromonas salmonicida affect the immune system of carp, Cyprinus carpio L.?

Aeromonas salmonicida is a significant bacterial pathogen of cyprinid and salmonid fishes causing the systemic disease furunculosis. Several observations led us to believe that A. salmonicida was able to evade or suppress the immune system of the fish: injection of whole bacteria or surface antigens was unsuccessful at protecting fish against lethal challenges; memory did not develop in survivors of sublethal infections; diseased fish often carried other opportunistic bacterial pathogens in addition to A. salmonicida, and serum protein and particularly immunoglobulin significantly decreased during A. salmonicida infections. We tested the ability of fish sublethally infected with virulent and avirulent A. salmonicida to mount a humoral immune response to sheep erythrocytes and found fewer plaque forming cells in the pronephros and lower serum anti-SRBC antibodies in infected fish as compared to controls. We also monitored the cellular immune response of diseased fish by skin allograft rejection and found an enhancement of the response that increased as the disease progressed. However, the extend of inflammation was reduced in infected fish as compared to non-infected animals. At this moment these preliminary observations are difficult to explain. Our future research will focus more specifically on cell populations that may be affected by A. salmonicida.

Aeromonas↗
PubMed1986

Protective efficacy of Aeromonas hydrophila vaccines in nile tilapia.

Protection and serum antibody production against Aeromonas hydrophila was examined in nile tilapia, Tilapia nilotica (L.). Intraperitoneally injected formal in-killed and Freunds complete adjuvant vaccines were compared using different doses (2.9 X 10(7) and 2.9 X 10(9) cfu/ml). Upon challenge, the protective ability and antibody titers resulting were significantly different between vaccinated and unvaccinated groups. A relative level of protection of 100% was obtained within two-weeks, and a maximum level of 53-61% protection was found one-week post-vaccination.

Aeromonas↗
PubMed1992

Immunostimulants added to injected Aeromonas salmonicida bacterin enhance the defense mechanisms and protection in rainbow trout (Oncorhynchus mykiss).

Immunostimulants were given to rainbow trout for assaying effects on modulating non-specific defense mechanisms, specific immune response, and protection levels against pathogen challenge with Aeromonas salmonicida. Three drugs, levamisole (an approved veterinary drug in the USA), a quaternary ammonium compound (QAC), and a short-chain polypeptide (ISK) were found to affect the non-specific defense mechanism activities, which were measured by changes in circulatory neutrophil and phagocytic activity levels, and the specific immune response factors, which were measured by numbers of plaque-forming cells, and circulatory antibody levels. When given alone, the immunostimulants elevated the non-specific factors. When injected in combination with an A. salmonicida O-antigen bacterin, the non-specific factors were further elevated, and the specific response was raised over samples taken from fish given the bacterin without the immunostimulants. Challenge tests with the virulent pathogen, A. salmonicida, showed a 5-6 day delay in the onset of mortalities in the fish given the immunostimulants alone, and a 12-14 day delay when immunostimulants given were combined with the bacterin. In the groups given the QAC or ISK with the bacterin, there was a 20% and 40% survival rate, respectively.

Adjuvants, Immunologic↗
PubMed1994

Genetic variation in the humoral immune response in Atlantic salmon (Salmo salar) against Aeromonas salmonicida A-layer.

Antibody responses to Aeromonas salmonicida A-layer were analysed in family material of Atlantic salmon (Salmo salar), consisting of 791 fish belonging to 34 full-sib groups within 12 paternal half-sib groups. The fish were immunized twice and blood samples were collected three times. Significant increase in antibody titre from first to second, and from second to third sampling, was observed. Genetic variation in antibody titres was observed at the three samplings with estimated heritabilities ranging from 0.16 to 0.20, and a significant heritability estimate was recorded in the antibody response after the second immunization (h2 = 0.16). Moderate to high genetic (r = 0.5-0.72) and phenotypic (r = 0.29-0.57) correlations were found between the titre values at different samplings, and between selected titres and titre increases. Production parameters, such as mean slaughter weight and mean survival rate were obtained for fish which were reared commercially in the sea, and which belonged to the same full-sib groups as those analysed for A. salmonicida A-layer antibodies. No significant correlation between the mean antibody titre to A. salmonicida A-layer in this study and mean slaughter weight and survival rate in full-sib family material in the sea was observed.

Aeromonas↗
PubMed1995

Study of the humoral response of Atlantic salmon (Salmo salar L.), naturally infected with Aeromonas salmonicida ssp. achromogenes.

The humoral antibody response of healthy Atlantic salmon and of two groups of salmon, naturally infected with Aeromonas salmonicida ssp. achromogenes, was examined in some detail. One diseased group was chronically infected and the other recently infected. It was found that the humoral response of these two infected groups was quite different. The chronically infected fish showed poor specific response to the causative agent whereas the recently infected salmon produced strong specific antibody response. The chronically infected fish showed evidence of increased unspecific response including an elevated level of natural antibodies. The specific humoral response of the recently infected fish was primarily directed against two cell-associated antigens of the A. salmonicida ssp. achromogenes bacterium, the A-layer protein and the o-polysaccharide component of LPS. In the chronically infected fish the humoral response was primarily directed against the A-layer protein.

Aeromonas↗
PubMed1996

The effect of temperature and water quality on antibody response to Aeromonas salmonicida in sunshine bass (Morone chrysops x Morone saxatilis).

The kinetics of the antibody response to Aeromonas salmonicida were determined in sunshine bass (hybrid striped bass: female Morone chrysops X male Morone saxatilis) acclimated to 10, 18, 24, 29 degrees C, and to 0.15 mg 1(-1) un-ionized ammonia and 200 mg 1(-1) nitrate levels. Temperature and water quality factors affected the immune response of sunshine bass. Temperatures of 10 degrees C and 18 degrees C decreased the magnitude and delayed the time of the antibody response to A. salmonicida. The antibody response was not affected at 29 degrees C, which is above the optimal temperature for sunshine bass. Elevated un-ionized ammonia concentrations of 0.15 mg 1(-1) also did not affect the antibody response. Elevated nitrate levels of 200 mg 1(-1), however, decreased the antibody response to the same extent as 18 degrees C water.

Aeromonas↗
PubMed1996

Multiple regulation of carp (Cyprinus carpio L.) macrophages and neutrophilic granulocytes by serum factors: influence of infection with atypical Aeromonas salmonicida.

Normal carp serum contains inhibitory and stimulatory factors for macrophage and neutrophilic granulocyte respiratory burst activity. As stimulatory factors were only effective in combination with phorbol myristate actetate (PMA) activation, it is concluded that they are probably linked to protein kinase C activation. Both the stimulatory and inhibitory factors are heat stable. Macrophage- and neutrophilic granulocyte-enriched cell fractions from the pronephros of carp had high respiratory burst- and high bactericidal in vitro responses to virulent atypical Aeromonas salmonicida bacteria. Serum factors were inhibitory for the A. salmonicida induced respiratory burst activity. No change in inhibitory or stimulatory serum factors could be observed during a 12-day challenge experiment with A. salmonicida, or during a rechallenge of survivors from a previous sub-lethal infection. The sensitivity of macrophages and neutrophilic granulocytes to stimulation of respiratory burst activity by PMA was not significantly altered. Culture supernatants from PHA pre-treated lymphocytes stimulated the respiratory burst activity of macrophages and neutrophilic granulocytes suggesting that serum factors may partially be lymphocyte derived.

Aeromonas↗
PubMed1988

The growth of Aeromonas hydrophila K144 in ground pork at 5 degrees C.

The influence of NaCl, pH, atmosphere, and background microflora on the growth and/or survival of Aeromonas hydrophila K144 was studied in ground pork held at 5 degrees C. In ground pork, A hydrophila was sensitive to pH values below 6.0 in the form of either a low starting pH in the pork itself or induced by lactic acid bacteria action on added glucose. Growth of the organism is inhibited by NaCl levels of 3% (w/w) (approx. 4% brine content). A hydrophila grew in vacuum-packaged ground pork; its growth was diminished by the presence of the naturally occurring meat microflora. Except for pH values below 6.0, conditions which inhibited growth permitted survival of the organisms for extended periods. Data indicate that the growth of A. hydrophila in ground pork can be controlled by factors such as NaCl, pH, and background microflora. In general, measures designed to control other foodborne pathogens appear adequate to limit A. hydrophila.

Aeromonas↗
PubMed1989

Enterotoxigenicity and drug sensitivity of Aeromonas hydrophila isolated from well water in Sweden: a case study.

A large number of Aeromonas spp. have been found in drinking water from a drilled well in Sweden. Isolates identified as A. hydrophila were tested for production of enterotoxin, hemolysin, enzymes and for resistant patterns to different antibiotics. The enterotoxin-producing A. hydrophila could be responsible for the long-term diarrhoeal case of a 1 1/2 year old child who consumed the contaminated water.

Aeromonas↗
PubMed1989

Toxin production by strains of Aeromonas hydrophila grown in laboratory media and prawn purée.

Toxin production by four strains of Aeromonas hydrophila grown at 30 and 37 degrees C in two laboratory media and prawn purée was studied. Three different cell lines were used to test for cytotoxic activity, haemolytic activity was tested against rabbit and guinea pig erythrocytes, proteolytic activity was assayed with azo-casein and enterotoxic activity using the suckling mouse assay. Results showed reduced cytotoxic and haemolytic activities in prawn purée compared with the two media, but in most cases increased proteolytic activity. No enterotoxic activity was observed in prawn purée although it was occasionally detectable in both laboratory media.

Aeromonas↗
PubMed1989

Effect of temperature on hemolysin production in Aeromonas spp. isolated from warm and cold environments.

The effect of growth temperature on the hemolysin production in 97 Aeromonas isolates was studied. Only 9% of the isolates obtained from low-temperature sources such as refrigerated foods, drinking water and trout aquaculture were able to produce high hemolysin titers (titer greater than 128) at 37 degrees C compared with 65% of the isolates from warm sources such as human clinical cases and warm water aquaculture (p less than 0.001). In contrast, 61% of the low-temperature isolates were producing high hemolysin titers at 10 degrees C while only 24% of the isolates from high temperature sources produced high titers at this temperature (p less than 0.05). Hemolytic activity could decrease markedly during longer (48 h) incubation at 37 degrees C. Some of the filtrates from growth at 37 degrees C and 10 degrees C were tested for enterotoxin-like activity in the suckling mice test. Of the culture filtrates produced at 37 degrees C, 40% of A. hydrophila were positive for enterotoxin-like activity, irrespective of hemolysin titer. Of A. sobria 88% of the filtrates with high hemolytin titers and none of the low-titer filtrates were enterotoxigenic. Due to a high mortality rate of mice injected with filtrates produced at 10 degrees C the enterotoxic effect of these filtrates could not be established. Autoagglutination after boiling was observed in 31% of the clinical isolates and 2% of the strains of environmental origin.

Aeromonas↗
PubMed1990

Growth characteristics of motile Aeromonas spp. isolated from different environments.

The growth of 80 strains of motile Aeromonas spp. derived from environments with temperatures above 25 degrees C and below 15 degrees C, respectively, were examined at five temperatures (5 degrees C, 10 degrees C, 25 degrees C, 37 degrees C and 44 degrees C) and four salt levels (0.05%, 2%, 4% and 6% NaCl). Sixty-one strains were further examined at two pH levels (pH 7.3 and pH 5.3). All strains grew at 25 degrees C and 10 degrees C with the majority of the isolates proliferating from approx. 10(2) to approx. 10(7) cfu/ml within 1 and 3 days, respectively. In contrast, there were significant differences in the proportion of isolates able to grow at 5 degrees C and 37 degrees C depending on the temperature of their source of isolation. The ecological background of the organisms thus influences their thermal growth range and their ability to proliferate at body temperature, a highly significant factor in infective disease. At 25 degrees C and pH 7.3, all strains grew in 0.05% NaCl, 96% grew in 2% NaCl, 96% grew in 2% NaCl while few grew in broth containing 4% or 6% NaCl. Lowering the pH to 5.3 with lactic acid caused a marked increase in the lag phase at 25 degrees C and prevented growth of a large number of isolates at suboptimal conditions. Thus, none of the isolates from warm environments and only 8% of the isolates from cold environments grew at this pH at 5 degrees C. The observed differences in growth optima between strains from different environments are discussed in relation to food- or waterborne infection.

Aeromonas↗
PubMed1993

Growth of Listeria monocytogenes, Aeromonas hydrophila and Yersinia enterocolitica in pâté and a comparison with predictive models.

A reference or type strain and a food derived-strain of the cold-tolerant pathogens Listeria monocytogenes, Aeromonas hydrophila and Yersinia enterocolitica were individually inoculated into samples of commercial pâté and incubated at 4 and 10 degrees C. The organisms were periodically estimated by presumptive counts, then values for the lag and generation times were calculated. Both strains of L. monocytogenes grew at both temperatures. The food strain of A. hydrophila grew only at 10 degrees C, and the type strain did not grow at either temperature. Similarly, the type strain of Y. enterocolitica did not grow at either temperature, whereas the food strain grew at both 4 and 10 degrees C. In some cases growth of non-test organisms may have inhibited the growth of these latter two species. The measured values of lag and generation times did not, in general, correlate well with those predicted by response surface models, taken from the literature and produced in this laboratory. It may be that the pâté contained an inhibitor that affected the growth of the organisms. The two strains of A. hydrophila and Y. enterocolitica showed significantly different growth characteristics, reinforcing the value of using a 'cocktail' of strains in growth experiments. Differences in predicted kinetic values from the models indicate that a model for any particular strain may not reflect the growth of naturally occurring contaminants of the same species.

Aeromonas hydrophila↗