Construction and evaluation of type III secretion system mutants of the catfish pathogen Edwardsiella piscicida.

Catfish is the largest aquaculture industry in the United States. Edwardsiellosis is considered one of the most significant problems affecting this industry. Edwardsiella piscicida is a newly described species within the genus Edwardsiella, and it was previously classified as Edwardsiella tarda. It causes gastrointestinal septicaemia, primarily in summer months, in farmed channel catfish in the south-eastern United States. In the current study, we adapted gene deletion methods used for Edwardsiella to E. piscicida strain C07-087, which was isolated from a disease outbreak in a catfish production pond. Four genes encoding structural proteins in the type III secretion system (T3SS) apparatus of E. piscicida were deleted by homologous recombination and allelic exchange to produce in-frame deletion mutants (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT). The mutants were phenotypically characterized, and virulence and vaccine efficacy were evaluated. Three of the mutants, EpΔssaV, EpΔyscR and EpΔesaM, were significantly attenuated compared to the parent strain (p < .05), but EpΔescT strain was not. Vaccination of catfish with the four mutant strains (EpΔssaV, EpΔesaM, EpΔyscR and EpΔescT) provided significant protection when subsequently challenged with wild-type strain. In conclusion, we report methods for gene deletion in E. piscicida and development of vaccine candidates derived from a virulent catfish isolate.

Intraspecific diversity of Edwardsiella ictaluri isolates from diseased freshwater catfish, Pangasianodon hypophthalmus (Sauvage), cultured in the Mekong Delta, Vietnam.

A molecular epidemiology study was conducted on 90 Edwardsiella ictaluri isolates recovered from diseased farmed freshwater catfish, Pangasianodon hypophthalmus, cultured in the Mekong Delta, Vietnam. Thirteen isolates of E. ictaluri derived from diseased channel catfish, Ictalurus punctatus, cultured in the USA were included for comparison. All the E.ictaluri isolates tested were found to be biochemically indistinguishable. A repetitive (rep)-PCR using the single (GTG)(5) primer was shown to possess limited discriminatory power, yielding two similar DNA profiles categorized as (GTG)(5) -PCR group 1 or 2 among the Vietnam isolates and (GTG)(5) -PCR group 1 within the USA isolates. Macrorestriction analysis identified 14 and 22 unique pulsotypes by XbaI and SpeI, respectively, among a subset of 59 E. ictaluri isolates. Numerical analysis of the combined macrorestriction profiles revealed three main groups: a distinct cluster formed exclusively of the USA isolates, and a major and minor cluster with outliers contained the Vietnam isolates. Antibiotic susceptibility and plasmid profiling supported the existence of the three groups. The results indicate that macrorestriction analysis may be regarded as a suitable typing method among the E. ictaluri species of limited intraspecific diversity. Furthermore, the findings suggest that E. ictaluri originating from Vietnam may constitute a distinct genetic group.

Photodynamic therapy against common bacteria causing wound and skin infections.

Photodynamic therapy (PDT) uses the combination of a photosensitising (PS) agent and visible light. Historically, various injectable PS agents have been used with medical grade lasers to treat neoplasia. The objective of this in vitro study was to determine whether PDT using topical aminolevulinic acid (ALA) with a non-coherent light source would kill common wound infecting bacteria, namely, Staphylococcus intermedius, Streptococcus canis, Pseudomonas aeruginosa, and Escherichia coli. Bacterial strains were sensitised to light with ALA before exposing to the non-coherent light source. Colony counts were performed in triplicate and compared to controls. When compared with controls there was a significant decrease in bacterial survival following PDT for all organisms except E. coli. A single treatment required 2-3h of light exposure. These data suggest that PDT may be a possible treatment option for wound infections but repeated treatments or alterations in the PS or its carrier will be needed to decrease treatment times.

Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops x M. saxatilis and zebrafish Danio rerio.

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified. Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops x M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish.

Use of a putative transcriptional regulator gene as target for specific identification of Staphylococcus epidermidis.

AIMS: To investigate the use of a Staphylococcus epidermidis transcriptional regulator gene as target for species-specific determination. METHODS AND RESULTS: Staph. epidermidis genes encoding putative transcriptional regulators were retrieved from GenBank and those showing no homology with other bacterial sequences were selected. Of the four PCR primer sets analysed, the primers Serp0107F/R from serp0107 amplified a specific product of 581 bp from Staph. epidermidis DNA only, and they did not cross-react in PCR with nonepidermidis staphylococci and other common bacteria. CONCLUSION: Being uniquely present in Staph. epidermidis, putative transcriptional regulator gene serp0107 offers a valuable target for specific identification of Staph. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: As a member of a specialized gene group, putative transcriptional regulator gene serp0107 may be important to Staph. epidermidis adaptation to its niche environment. Further analysis of serp0107 and its related protein may help reveal new insights on the molecular regulation of Staph. epidermidis survival and virulence.

Evaluation of PCR primers from putative transcriptional regulator genes for identification of Staphylococcus aureus.

AIMS: To examine if PCR primers derived from putative transcriptional regulator genes can be useful for Staphylococcus aureus identification. METHODS AND RESULTS: Staphylococcus aureus gene sequences that encode transcriptional regulators were retrieved from GenBank and compared with other DNA sequences via BLAST searches. Two uniquely present, putative transcriptional regulator genes (i.e. Sa0836 and Sa0856) were selected as a consequence and PCR primers (Sa0836F/R and Sa0856F/R) were then designed from these genes for evaluation. A total of 84 bacterial strains/isolates including 23 Staph. aureus, 18 nonaureus Staphylococcus and 43 other common bacterial isolates were examined. The results indicated that PCR primers from Sa0836 and Sa0856 recognized genomic DNA from Staph. aureus only, but not from other non-aureus Staphylococcus or common bacteria. CONCLUSIONS: PCR detection of the putative transcriptional regulator genes Sa0836 and Sa0856 represents a useful means of identifying Staph. aureus from other bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The existence of species-species transcriptional regulator genes may be a common phenomenon in bacteria. Besides their value as novel diagnostic markers, further investigation on the putative transcriptional regulator genes Sa0836 and Sa0856 and their related products may shed light on the molecular mechanisms of Staph. aureus adaptation and virulence.

Identification of a gene encoding a putative phosphotransferase system enzyme IIBC in Listeria welshimeri and its application for diagnostic PCR.

AIMS: To identify a Listeria welshimeri-specific gene that can be used for identification of this species by PCR. METHODS AND RESULTS: Through comparative analysis of genomic DNA from Listeria species using dot blot hybridization, an L. welshimeri-specific clone was isolated that contained a gene segment whose translated protein sequence is similar to enzyme IIBC from phosphotransferase systems in other bacteria. Using oligonucleotide primers derived from this L. welshimeri-specific clone, a 608-bp fragment was amplified from L. welshimeri genomic DNA and not from other Listeria species or other Gram-negative and Gram-positive species. CONCLUSION AND SIGNIFICANCE: The PCR employing L. welshimeri-specific primers shows promise as a useful method for differentiating L. welshimeri from other Listeria species and related bacteria.

Pathogenicity and production of virulence factors by Listeria monocytogenes isolates from channel catfish.

Pathogenicity of four channel catfish Listeria monocytogenes isolates (CCF1, CCF4, HCC7, and HCC23) was examined in a comparative manner with virulent type strains L. monocytogenes ATCC 19115 and EGD and avirulent type strain ATCC 15313 in BDF and A/J mice. Isolates HCC7 and CCF1 (both serovar 1) caused similar percent mortalities and 50% lethal concentration values when compared with virulent type strains and were therefore considered pathogenic. The presence of the virulence factors listeriolysin (LLO), phosphotidylcholine-phospholipase (PC-PLC), and phosphotidylinositol-phospholipase (PI-PLC) was determined using specific activity tests. The virulent catfish isolates were positive for production of LLO, PC-PLC, and PI-PLC. However, catfish isolate HCC23 was not virulent in mice despite being hemolytic, suggesting that not every hemolytic L. monocytogenes strain is virulent. With the exception of HCC7, all virulent strains displayed enhanced LLO production in a special stress medium, whereas almost undetectable LLO activity was present when catfish isolates and virulent type strain L. monocytogenes were grown in a rich medium such as brain heart infusion. Avirulent strains were found to lack or have decreased expression of LLO, PC-PLC, or PI-PLC.

Comparison of the immunofluorescent assay and reverse transcription-polymerase chain reaction to detect and type infectious bronchitis virus.

Indirect fluorescent antibody (IFA) assay and reverse transcription-polymerase chain reaction (RT-PCR) are two current methods commonly used for the detection of infectious bronchitis virus (IBV) and its serotypes. The objectives of this study were to compare the two methods relative to detecting IBV in chicken embryos that were artificially inoculated with Mass41, Ark99, or Mass41/Ark99 in serial embryo passages and in tracheas and cecal tonsils collected from vaccinated commercial flocks with and without respiratory problems. The tissues collected from these birds were used to inoculate chicken embryos. The chorioallantoic membranes collected from the inoculated embryos were examined by the IFA assay, and the allantoic fluids collected from the same embryos were subjected to the RT-PCR. The IFA assay and RT-PCR both were able to detect and type IBV when only one IBV strain was inoculated into embryos, regardless of the number of passages. When embryos were inoculated with Mass41 and Ark99 strains simultaneously, both strains were 100% detected by the RT-PCR. By the IFA assay, 20% of samples were positive for two strains and 80% were positive for the Ark only. Two of eight samples collected from healthy flocks that were vaccinated with Mass/Conn/Ark were positive by the IFA assay, and four of eight were positive by the RT-PCR. Sixteen percent (12/75) of the samples from birds experiencing respiratory problems were positive by the IFA assay and 35% (26/75) were positive by the RT-PCR. Results suggest that the RT-PCR is more sensitive than the IFA assay, especially when more than one strain of IBV is involved, but the IFA assay is rapid and cheaper than the RT-PCR.

Production of monoclonal antibodies to Listeria monocytogenes and their application to determine the virulence of isolates from channel catfish.

We produced monoclonal antibodies (MAbs) to the extracellular proteins of Listeria monocytogenes EGD grown in Chelex-treated improved minimal medium. Ten of the positive hybridomas generated were chosen for further characterization. Seven of the MAbs reacted with a protein having a molecular mass of 60 kDa. These MAbs inhibited listeriolysin (LLO)-mediated hemolysis, and two of them were specific for LLO and none of the other thiol-activated toxins tested. In an enzyme-linked immunosorbent assay and Western blot analysis, five of the anti-LLO MAbs reacted with ivanolysin from Listeria ivanovii. Three of the 10 MAbs reacted with a 29-kDa protein on Western blots and neutralized the phosphatidylcholine-specific phospholipase C (PC-PLC) activity of L. monocytogenes. These three anti-PC-PLC MAbs did not react with phospholipases from five different gram-positive bacteria. However, the anti-PC-PLC MAbs recognized a 27-kDa extracellular protein from L. ivanovii and neutralized sphingomyelinase activity in a hemolysis test that demonstrates the antigenic relatedness of listerial phospholipases. These data indicate that listerial thiol-activated toxins possess species-specific epitopes and share group-specific epitopes. This is the first description of MAbs that neutralize listerial PC-PLC, and the data suggest that there is antigenic similarity between L. monocytogenes PC-PLC and L. ivanovii sphingomyelinase. The reactions of the MAbs with catfish isolates of L. monocytogenes suggested that some of the isolates examined lack the LLO and/or PC-PLC required for pathogenicity. The MAbs described here differentiated some catfish isolates from previously described type strain-pathogenic isolates and could be useful for detecting and determining the virulence of L. monocytogenes in food and clinical samples and for detecting L. ivanovii in veterinary clinical samples.

Derivation of extracellular polysaccharide-deficient variants from a serotype A strain of Pasteurella multocida.

The production of serotype A extracellular polysaccharide is thought to be associated with expression of an approximately 40-kDa lipoprotein (P1p-40) present on the outer surface of Pasteurella multocida strains of avian origin. The tendency of certain strains to undergo colonial dissociation concomitantly with serial passaging on laboratory growth media was exploited to derive two variant strains exhibiting the capsule-deficient phenotype from a heavily capsulated parental strain. Assessments of colonial consistency, iridescence, gentian violet binding, and hyaluronidase sensitivity were consistent with cellular observations indicating little or no capsulation of derivative strains. Fluorographic analysis of electrophoretically resolved cellular lipoproteins labeled with [3H]-palmitate revealed capsular loss occurred with a concomitant diminution of P1p-40 production in the variant strains. In contrast, a phenotypically stable strain that did not undergo colonial dissociation under identical conditions exhibited no decrease in P1p-40 content. This work provides a model system for investigating the role of extracellular polysaccharide in the cell surface physiology and pathogenicity of P. multocida. The present results strongly support the notion that P1p-40 is associated with serotype A capsular material and suggest coordinate regulation of their biosynthesis.

Characterization of Listeria monocytogenes isolated from channel catfish (Ictalurus punctatus).

OBJECTIVE: To characterize Listeria monocytogenes from tissues of channel catfish for their ability to cause hemolysis and grow intracellularly in mouse macrophages. SAMPLES: 15 isolates from processed fillets and 15 isolates from the brain, spleen, and kidneys. PROCEDURE: Serotype and hemolytic activity of L monocytogenes isolates were evaluated, using plate agglutination and CAMP tests, respectively. Invasiveness of L monocytogenes was determined by inoculating each strain or isolate on J774A.1 macrophage cells. Infected cells were incubated for 0 or 3 hours and lysed; then 100 gli of the lysate was plated onto a brain heart infusion agar plate. Colony counts for each strain or isolate were analyzed statistically. RESULTS: Of 30 isolates, 19 were serotype 1 and 11 were serotype 4. Mouse J774A.1 macrophages were inoculated with catfish isolates, a wild-type (EGD) or a nonhemolytic strain of L monocytogenes. Seventy-three percent (11/15) of isolates originating from catfish organs and 100% (15/15) of isolates originating from fillets were not significantly different from the wild-type EGD strain. The nonhemolytic L monocytogenes strain used as a negative control failed to replicate. Intracellular growth of all L monocytogenes isolates decreased after an additional 3-hour incubation period with medium containing 50 [microg/ml of gentamicin. CONCLUSIONS: Similar to the wild-type EGD strain, most channel catfish L monocytogenes isolates were hemolytic, serotype 1 or 4, and were invasive for mouse J774A.1 macrophages. CLINICAL RELEVANCE: monocytogenes growth in mouse macrophages may serve as an in vitro model for determining virulence of isolates from food products or environments.

The effect of added complex carbohydrates or added dietary fiber on necrotic enteritis lesions in broiler chickens.

Two trials utilizing two corn diets and four wheat diets were conducted. In Trial 2, all chicks were crop-infused at 9 d of age with Eimeria acervulina. In both trials, a broth culture of Clostridium perfringens was mixed with the diets for 3 consecutive d. Necrotic enteritis lesion scores were lowest in chickens consuming the corn diet with no C. perfringens and highest in chickens fed the wheat diets with C. perfringens. Chickens consuming a wheat diet with no added complex carbohydrates or added fiber exhibited the highest lesion score. Chickens on wheat diets with 4% new, ground, pine shavings had intestinal lesion scores intermediate to those of chickens that consumed the wheat or corn diets. Chickens consuming corn diets yielded the lowest lesion scores. Chickens provided diets containing either guar gum or pectin were not fully consumed and thus probably reduced the number of challenge organisms ingested.

Effect of drinking water containing ammonium chloride or sodium bicarbonate on Mycoplasma gallisepticum isolation in experimentally infected broiler chickens.

In each of three trials, 150 day-old broiler chicks were eyedrop inoculated with 0.04 ml of high-passage F strain Mycoplasma gallisepticum (MG) and housed in biological isolation units at 10 chicks per unit. At 4 wk of age, 50 chickens were designated as controls and remained on tap water (pH 7.30), 50 chickens were provided tap water containing 0.63% ammonium chloride (NH4Cl, pH 6.91), and 50 chickens were provided tap water containing 1.26% sodium bicarbonate (NaHCO3, pH 8.17). Fluids were supplied for ad libitum consumption. At 5 wk of age, all chickens were swabbed from the choanal cleft for MG isolation and bled from the left cutanea ulnea vein for pH determination. As a percent of total swabs obtained, significantly fewer chickens that consumed water containing NH4Cl (38.3%) were positive for MG by culture compared with either the NaHCO3 group (61.3%) or the control group (67.6%). Nonmycoplasmal swab contamination was significantly higher for chickens that consumed water containing NH4Cl (59.7%) compared with either controls (31.8%) or NaHCO3-treated chickens (38.7%). When contaminated cultures were discarded, MG isolations from the tap water group were not significantly different from MG isolations from either the NH4Cl or NaHCO3 group. However, MG isolations from the NH4Cl group (95%) were significantly less compared with the NaHCO3 group (100%). Mortality was significantly higher in chickens that consumed water containing NaHCO3 (8.7%) compared with either controls (1.3%) or the NH4Cl-treated chickens (0.7%). Blood pH values were lower for the NH4Cl group (7.927), higher for the NaHCO3 group (8.093), and intermediate for controls (8.035). Results of this study suggest that water containing NH4Cl hinders the bacteriological recovery of MG from the choanal cleft.

Isolation of Yersinia pseudotuberculosis and Listeria monocytogenes serotype 4 from a gray fox (Urocyon cinereoargenteus) with canine distemper.

In February 1993, clinical, pathological, and microbiological investigations were performed on an adult female gray fox (Urocyon cinereoargenteus) from northern Mississippi (USA). The fox had clinical signs consistent with canine distemper virus encephalitis. Eosinophilic inclusions characteristic of canine distemper virus were in the nuclei and cytoplasm of cerebral neurons and glial cells and in the cytoplasm of urinary, gastric, pancreatic and biliary epithelial cells. The liver contained multifocal microscopic nodular foci of granulomatous to pyogranulomatous inflammation and necrosis with large colonies of small Gram-negative coccobacilli. A low number of small Gram-positive bacilli were within viable-appearing Kupffer cells and hepatocytes. Yersinia pseudotuberculosis and Listeria monocytogenes serotype 4 were isolated from the liver and a mesenteric lymph node.

The effects of nonautoclaved and autoclaved water-soluble wheat extracts on the growth of Clostridium perfringens.

Clostridium perfringens is the causative agent of necrotic enteritis, a commonly diagnosed disease in chickens that is also observed in turkeys and geese. Two trials were conducted to determine the in vitro effect of filter-sterilized, water-soluble wheat extracts on the growth of C. perfringens. The extracts were either nonautoclaved or autoclaved at 121 C for 40 min and were used to reconstitute thioglycolate broth media. Results of this study suggest that growth of C. perfringens is suppressed in vitro by inclusion of either extract. Glycosyl composition analysis revealed no significant differences in arabinose, xylose, or mannose content between the nonautoclaved and autoclaved extracts. Galactose, glucose, and total glycosyl content were significantly higher in the nonautoclaved extract.

Survey for selected diseases in nutria (Myocastor coypus) from Louisiana.

Thirty-two trapper-caught nutria (Myocastor coypus) from East Baton Rouge, Iberville, Tangipahoa, and St. Helena Parishes in Louisiana (USA) were sampled for several disease agents. Antibodies against Toxoplasma gondii, Chlamydia psittaci, Francisella tularensis, Leptospira spp., and encephalomyocarditis virus were detected in 7%, 14%, 0%, 7%, and 0% of nutria, respectively. Both animals seropositive for leptospirae were positive for L. interrogans serovar canicola. No Salmonella spp. were isolated from feces, and no Giardia spp. were seen in trichrome-stained fecal preparations.

Purification of a Pasteurella haemolytica serotype 1-specific polysaccharide epitope by use of monoclonal antibody immunoaffinity.

A murine IgM monoclonal antibody causing bacterial agglutination was used in an immunoaffinity procedure to purify a serotype 1-specific polysaccharide epitope from Pasteurella haemolytica. The P haemolytica serotype 1-specific antibody was precipitated from peritoneal ascitic fluid, dialyzed, and covalently attached to cyanogen bromide-activated Sepharose 4B beads. Retention of purified antibody activity and coupling efficiency were > 99% when evaluated by ELISA, agglutination testing, and protein determination. Potassium thiocyanate was selected as an eluant on the basis of reversible dissociation of bacterial agglutination and was titrated for the lowest effective concentration. Immunobead activity was observed microscopically by immobilization of encapsulated P haemolytica serotype 1 and its reversible dissociation after elution with 0.4M potassium thiocyanate. Specificity of immobilization was visualized, using P haemolytica serotypes 2 and 5, which were not bound, and by blocking serotype-1 binding with homologous capsular material. Saline-extractable capsular material from P haemolytica serotype 1 was used as an antigen source. After elution of the serotype 1-specific polysaccharide epitope, the product was dialyzed and analyzed, using chemical and immunologic methods. The immunoaffinity product contained no detectable protein and greater than half the original hexosamine content. Using defined monoclonal antibodies in ELISA, titration of the original capsular material and the immunoaffinity product revealed specific retention of lipopolysaccharide, a 10- to 30-kd polysaccharide antigen common to all P haemolytica and P multocida serotypes, and serotype 1-specific capsular polysaccharide, indicating possible epitope sharing among polysaccharide antigens of P haemolytica serotype 1.

Detection of humoral antigen and antibody by enzyme-linked immunosorbent assay in horses with experimentally induced Ehrlichia equi infection.

An enzyme-linked immunosorbent assay (ELISA) was used to detect antigen in plasma and antibody in serum of 3 horses inoculated with Ehrlichia equi. Clinical signs, including rectal temperature, were correlated with the antigen and antibody detection. ELISA was very efficient in detection of serum antibody. Antigen detection using monoclonal antibodies to E. equi and ELISA should be considered as a diagnostic method.

A characterization of monoclonal antibodies prepared against Pasteurella haemolytica serotype 1 surface antigens.

The production and characterization of monoclonal antibodies against Pasteurella haemolytica serotype 1 is described. Ten monoclonal antibodies were produced and divided, on the basis of their properties, into six different groups. One produced bacteria agglutination only of P. haemolytica serotype 1. Three antibodies bound with P. haemolytica serotypes 1, 5-8 and 12 and the antigen was identified in immunoblots as lipopolysaccharide. Two antibodies bound P. haemolytica serotypes 1, 2, 5-8 and 12 and P. multocida serotypes 1-7, 9, 12, 15 and 16, recognizing an epitope present on a 29 kDa outer membrane protein. One antibody bound all P. haemolytica and P. multocida serotypes. The antigen was a hexosamine less than 30 kDa which contained a formalin sensitive epitope. One antibody bound only to P. haemolytica serotype 1 and the antigen was identified as a 66 kDa outer membrane protein. Two antibodies bound P. haemolytica serotypes 1, 2, 5-9 and 12 and the antigen, while not identified, was localized on the outer membrane. This study identified antigens which contribute to the cross-reactions among P. haemolytica and P. multocida serotypes and the antibodies may be useful in investigating the pathogenesis of pneumonic pasteurellosis.