Rabbit Blood Agar Research Articles

Hemolysins profile of clinical isolates of S. aureus from tertiary care hospitals in India

Background: Staphylococcus aureus is an important pathogenic bacterium causing both hospital and community acquired infections. Hemolysins are important virulence factors of S. aureus which help in the pathogenesis of diseases. Screening of hemolysins is important in infection control and prevention. Methods: Aim of the study was to characterize hemolysins of Staphylococcus aureus (n=152) isolated from tertiary hospitals in India, by phenotypic and genotypic methods. Phenotypic characterization of hemolysins of Staphylococcus aureus was done on either sheep or rabbit blood agar plates. Genes encoding hemolysin were amplified with specific primers in a multiplex Polymerase Chain Reaction (PCR) and analyzed by agarose gel electrophoresis. Results: Genes of all four hemolysins namely hla, hlb, hlg and hld were screened by multiplex PCR. Genotyping revealed that the isolates contained patterns of four, three or two hemolysin genes in multiple combinations. The most common profile comprised of all four hemolysin genes, namely hla, hlb, hlg, hld in a majority of isolates (47.3%, 72/152). Sixty one isolates (40.1%, 61/152) carried three hemolysin genes in three different patterns: 56 (37%) isolates contained hla, hlb, hld genes, second pattern combined 3 genes namely (1.9%) hla, hlg, hld and the third pattern was found in 2 (1.3%) isolates with hlb, hlg, hld. Five (3.28%) isolates in this study had only two hemolysin genes in three different combinations: 2 (1.3%) isolates with hlb:hld, 2 (1.3%) isolates with hla:hld pattern and 1 (0.7%) isolate with hlg:hld pattern. 14 (9.2%) isolates carried only hld gene. Phenotyping showed that all the isolates had α, β and δ activities and high activity of α, β and δ hemolysins were seen in isolates which were obtained from pus, blood and other body fluids. All these sites suffer enormous tissue damage in severe S. aureus infection. The genotype data correlated well with the phenotype analysis. Conclusions: Four hemolysin genes were detected in all clinical isolates of S. aureus. We could identify the activity of α, β and δ hemolysins on blood agar media. We found out that the isolates displayed varying combination of the four hemolysin genes. These differences in genotype of four genes and the hemolysin activity may indicate a preference mandated by the host tissue or site during the pathogenesis of the disease since each hemolysin has different activity profile. Body fluids in general, pus, blood, sputum in particular carried S. aureus with higher hemolysin activities compared to isolates of specimen from other sites. The results of this study reinforce the importance of hemolysins in S. aureus diseases and suggest that the tissue damages and symptoms are related to the hemolysins activity during pathogenesis.

Identification and safety assessment of Enterococcus thailandicus TC1 isolated from healthy pigs.

Enterococci have the dual characteristics of being opportunistic pathogens and promising probiotics. The isolation from patients of CDC PNS-E2, a newly described Enterococcus species Enterococcus sanguinicola, may pose potential hazards. Enterococcus thailandicus from fermented sausage is a senior subjective synonym of E. sanguinicola. In this study, Enterococcus thailandicus TC1 was first isolated in healthy pigs in Tongcheng, China and identified by phenotypic analysis and 16S rRNA-based techniques. To evaluate the strain safety, an approach including virulence factors, antibiotic resistance, and animal experiments was adopted. The results show that cylA, gelE, esp, agg, ace, efaAfm, efaAfs, ptsD genes were undetected, and that the strain was sensitive or poorly resistant to some clinically relevant antibiotics. However, the isolated strain demonstrated β-hemolytic activity in rabbit blood agar plates. Analysis of animal experiments revealed that the isolated strain had no adverse effect on translocation and the internal organ indices, though significant differences in histology (villi height, crypts height) of ileum were observed. The data acquired suggest that E. thailandicus TC1 may be associated with a potential health risk.

227 Effect of triamcinolone & manuka honey on Staphylococcus aureus growth & hemolytic activity

Atopic dermatitis (AD) is an inflammatory skin disease commonly treated with topical corticosteroids (TCS). Manuka honey (MH) is an alternative therapy made from nectar of the Leptospermum scoparium, native to New Zealand. The high abundance of Staphyloccocus aureus (S. aureus) on AD skin enhances disease severity, in part due to the effects of hemolysins. To address the effect TCS & MH have on S. aureus, we quantified growth & hemolytic activity of USA300 (MRSA strain) with & without exposure to triamcinolone acetonide (TAC) or MH. USA300 was cultured in Tryptic soy broth (TSB) containing 0.1% w/v TAC or MH (ManukoraTM; 1.25%, 2.5%, 5% & 10% w/v). Growth after MH exposure was evaluated by measuring optical density at 600nm (OD600). Due to TAC’s low solubility which interfered with OD readings, we evaluated growth by colony forming units (CFUs) after 5 hrs. After 24 hrs, supernatants were filtered (0.22-μm) and spotted onto rabbit blood agar (RBA) to quantify hemolytic activity (diameter of zones of clearance). TAC exposure resulted in a slightly lower CFUs (avg 0.82 x 109) than TSB alone (avg 1.26 x 109 CFU; n=2/condition), suggesting that TAC did not significantly inhibit USA300 growth. In contrast, MH markedly reduced OD600 values by 5, 23, 82 & 92% in 1.25, 2.5, 5, & 10% MH, respectively, compared to TSB alone (mean; n=3, p<0.03) after 5 hrs. This inhibitory effect of MH on USA300 growth was notable with the most significant reduction (mean 35%; n=3, p=0.001) observed at the highest concentration (10%) that was still observed at 24 hrs. Supernatants from TAC-incubated USA300 reduced clearance zones on RBA (avg 1.8±0.4 mm) compared to TSB alone (avg 5.8±0.4 mm; n=2/condition). Whereas, MH, inhibited all hemolytic activity, even at the lowest concentration 1.25% (n=3). Collectively, these findings demonstrate that at even at low MH concentrations, commonly found in cosmetic products, there is a greater effect on S. aureus growth as well as nearly complete inhibition of hemolytic activity compared to TAC, the most commonly used topical TCS.

Survival, Virulent Characteristics, and Transcriptomic Analyses of the Pathogenic Vibrio anguillarum Under Starvation Stress.

Many bacteria have developed strategies for metamorphosis into sophisticated survival forms to survive extended periods of environmental stress. As a global causative agent of vibriosis in marine fish farming, Vibrio anguillarum (V. anguillarum) can efficiently grow and proliferate under environmental stress, but the specific mechanism is not clear. In the present study, survival, virulent characteristics, and transcriptomic analysis of the V. anguillarum BH1 were performed under starvation stress. The results demonstrated that V. anguillarum was still culturable and showed rippled surface after 6 months of starvation. Starved cells maintained their infectivity in half-smooth tongue sole (Cynoglossus semilaevi). Detection of virulence factors and virulence-associated genes in starved cells showed that the starved strain still produced β-hemolysis on rabbit blood agar, caseinase, dnase, and gelatinase, and possessed empA, vah1, vah2, vah3, vah4, vah5, rtxA, flaA, flaD, flaE, virC, tonB, mreB, toxR, rpoS, and ftsZ virulence-related genes. In addition, we first reported the RNA-seq study for V. anguillarum with and without starvation treatment for a period of 6 months and emphasized the regulation of gene expression at the whole transcriptional level. It indicated that V. anguillarum expressed 3,089 and 3,072 genes in the control group and starvation stress group, respectively. The differently expressed genes (DEGs) of the starved strain were thereby identified, including 251 up-regulated genes and 272 down-regulated genes in comparison with the non-starved strain. Gene Ontology (GO) analysis and Kyto Encyclopedia Genes and Genomes (KEGG) enrichment analysis of DEGs were also analyzed. GO functional classification revealed that among the significantly regulated genes with known function categories, more genes affiliated with signal transducer activity, molecular transducer activity, and cell communication were significantly up-regulated, and more genes affiliated with cellular macromolecule, cellular component, and structural molecule activity were significantly down-regulated. In addition, the DEGs involved in the pathway of two-component system was significantly up-regulated, and the pathways of ribosome and flagellar assembly were significantly down-regulated. This study provides valuable insight into the survival strategies of V. anguillarum and suggests that a portion of the bacterial populations may remain pathogenic while persisting under starvation stress by up-regulating or down-regulating a series of genes.

Isolation of Marine Bacillus sp. with Antagonistic and Organic-Substances-Degrading Activities and Its Potential Application as a Fish Probiotic.

We report on the isolation and characterization of an acid- and bile-tolerant bacterial strain, Bacillus sp. YB1701 with antibacterial and quorum-quenching activity. Strain YB1701 was isolated from coastal sediment samples and characterized by biochemical tests and 16S rRNA sequencing. In vitro study indicated that strain YB1701 can survive at pH 2.0 for up to 3 h and tolerate bile up to 2.0% concentration even after 12 h of exposure. Strain YB1701 showed antimicrobial activity against fish pathogens Aeromonas hydrophila and Vibrio parahemolyticus using an agar well diffusion assay. The trial test showed dietary supplementation of YB1701 significantly improved the resistance of Carassius auratus gibelio against A. hydrophila challenge. The safety assessment revealed that the isolate Bacillus sp. YB1701 was not cytotoxic to Carassius auratus gibelio or mice and did not exhibit hemolytic activity on rabbit blood agar plate. Disc-diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that YB1701 was susceptible to selected antibiotics. Under laboratory conditions, the degradation rate of organic waste (predominately fish excrement) for 14 days by YB1701 was 79.69%. Results from the present study suggest that strain YB1701 is a potential probiotic strain and can be used in aquaculture for degrading organic waste and improving disease resistance of fish against bacterial infection. Further study is needed to assess the utility of strain YB1701 on a commercial scale.

Performance evaluation of oxacillin-resistant Staphylococcus aureus genotypes and taxa on human and animal blood agar culture media

The performance characteristics of growth, morphological aspects and hemolytic activities of oxacillin-resistant S. aureus (ORSA) strains were studied on twelve types of blood agar (BA) culture media (sheep, bovine, horse, rabbit and human). ORSA isolates were also previously characterized by isoenzymes genotyping and genetic and grouping analysis. Variations in the diameter of the colonies were detected among seven sets of BA media. In terms of morphology, 99, 53 and 98% presented shiny, yellow, and glossy colonies, respectively, regardless of the type of BAs. The rabbit BA favored the expression of hemolysins for most isolates (74%), followed by the human BA and other animal BAs. Certain BA media promoted the expression of hemolysins; however, the expression was correlated with a deficit in the colonial growth potential and vice-versa. The data point to the existence of two or more isolates genetically identical or highly related: (i) that either share or do not share the same wild species-specific phenotypes related to appearance without any influence from the external environment, and that are (ii) potentially virulent depending on the external environment. This study also suggestes the use of rabbit BA for the phenotypical characterization of S. aureus. Key words: Colonial morphology, hemolysis, human and animal blood agar, oxacillin-resistant Staphylococcus aureus, clinical microbiology.

Characterization of hemolysins of Staphylococcus strains isolated from human and bovine, southern Iran.

The staphylococci are important pathogenic bacteria causing various infections in animals and human. Hemolysin is one of the virulence factors of coagulase-positive (CPS) and coagulase-negative staphylococci (CNS). The aims of the study were to characterize hemolysins of Staphylococcus spp. isolated from human and bovine origin, phenotypic- and genotypically. Characterization of hemolysin phenotypically based on hemolysis pattern of Staphylococcus spp. was done on the sheep, horse and rabbit blood agar plates. Genes encoding hemolysin were amplified with specific primers by using polymerase chain reaction (PCR) technique. Hemolytic activities phenotypically were determined in 60 and 90% of the total bovine and human isolates, respectively. All non hemolytic isolates were CNS (P≤0.05). In all isolates, hla and hld genes were determined by PCR amplification. None of the bovine and human isolates showed phenotypically and genotypically gamma hemolysin. The results from this study suggest that, in accordance with what is generally believed, some differences are apparent in hemolysin types among Staphylococcus strains of bovine and human origin. Furthermore, this study showed that CNS can be important as new pathogens.

Effect of Blood Agar from Different Animal Blood on Growth Rates and Morphology of Common Pathogenic Bacteria

Sheep and horse blood are the most commonly used blood for the isolation of microorganisms from human tissue and fluids. However, in many developing countries such as Nigeria, expired human blood from blood banks is still used despite the risk of exposure to HIV and other blood-borne infections, because it is easy to obtain. Blood agar made from blood of rams (similar to sheep), cows, chickens and goats, which are very common in Nigeria, were therefore evaluated. The isolation rates, colony size and morphology as well as haemolytic characteristics of common hospital pathogens such as Pseudomonas aeruginosa, Klebsiella pneumonae, Staphylococcus aureus, and Streptococcus spp, were tested on blood agar prepared from the different animal blood types. All reactions were observed at 24 hrs and 48 hrs respectively. Good growth was achieved by all isolates on rabbit, sheep and chicken blood agar though the best growth was achieved on ram blood agar but there was no significant variation in their morphology. There were differences in their abilities to distinguish haemolytic patterns. Beta Haemolytic Streptococci remained the same on all the blood agar, but the haemolysis of Staphylococci aureus and Pseudomonas aeruginosa varied on different media while haemolysis was least consistent on chicken and cow blood agar. Ram blood agar gave the best reactions in terms of good growth rates of organisms, good morphological characterization as well as good haemolytic reactions. Besides, it is easily available and large quantities of blood can be obtained. Despite the good qualities of ram blood agar observed in this study, however, there is a need for it to be tested further for its ability to support more fastidious organisms.

Hemolytic and mice acute oral toxicity evaluation of a new antimicrobial peptide APNT-6

In this paper, the hemolytic and mice acute oral toxicity of a new antibacterial peptide APNT-6 with antibacterial lipopeptide surfactin, iturin and fengycin as the main components was evaluated. At first, hemolytic activity of the antibacterial peptide was studied in vitro; a 100-fold dilution of the antibacterial peptide was added to an Oxford cup placed on rabbit blood agar plate, after 37 ℃ overnight, the hemo-lytic activity was determined. The results showed that, 100-fold dilution of the antibacterial peptide in the Oxford cup formed 32 mm hemolytic circle on the plate medium, indicating that it has a strong hemolytic toxicity in vitro that is possible barriers to actual use. Meanwhile, a study for acute oral toxicity of the an-tibacterial peptide in mice was conducted, 60 male and female Kunming mice were randomly divided into six groups, with normal saline as a control, each group of mice was respectively fed 8, 40, 200, 1 000 and 5 000 mg/kg of the antibacterial peptide dose at one-time, within 7 d, observing toxicity body signs and death situation of mice and determining feed intake, water intake, weight gain and organ index, and exam-ining pathological changes for the abnormal organs. The results showed that in each dose group of mice death and any signs of poisoning were not seen. The oral median lethal dose (LD50) is greater than 5 000 mg/kg body weight; feed intake and water intake of each group mice have no significant change; except that the highest dose group had slightly lower weight gain, there were no significant differences in weight gain between the other groups and the control group; the spleen, kidney, lung and cardiac index of each group had no statistically significant differences and liver index of more than 1 000 mg/kg dose group was higher compared with the control group; mice organs and tissues of each dose group have no obvious pa-thological changes. These results suggest that high doses of the antibacterial peptide by oral administration can not achieve an effective hemolytic concentration in mice; in accordance with the acute toxic standard of disinfectant, it really has no toxicity, and that oral administration of the antibacterial peptide in mice does not constitute significant acute toxic effects. Based on the above results, it can be concluded that the new antibacterial peptide APNT-6 has strong hemolytic toxicity in vitro, but no acute oral toxicity in mice.

Re-assessment of phenotypic identifications of Bacteroides putredinis to Alistipes species using molecular methods

Alistipes (previously Bacteroides) are strictly anaerobic gram-negative rods that resemble the Bacteroides fragilis group in that most species are bile-resistant and indole-positive; however, they are only weakly saccharolytic and most species produce light brown pigment only on laked rabbit blood agar. In this retrospective study, we re-identified 18 organisms previously identified phenotypically as “ Bacteroides putredinis-like”, but that did not produce pigment on routine media. The strains were identified with 16S rDNA sequencing and pigment production was evaluated on several different culture media. Only 12/18 strains had molecular identifications of Alistipes species, while the remaining strains phylogenetically resembled Butyricimonas and Odoribacter spp. Pigment production was not a reliable test for those Alistipes strains that are described as pigment producers.

Phenotypic and molecular characterization of Photobacterium damselae, a pathogen of the cultured tongue sole Cynoglossus semilaevis in China

Significant numbers of half-smooth tongue sole (Cynoglossus semilaevis) raised on a marine fish farm in the P.R. China presented erratic swimming, loss of appetite and increased ventilation rate. Internally, diseased fish exhibited redness of liver, stomach and intestines, and white materials may be observed in the intestine. Mortality rates reached levels of up to 10% of the total fish on the farm per day. The dominant bacterial colonies were isolated from the liver, kidney and spleen of affected fish, and high pathogenicity of the isolates to Cynoglossus semilaevis, Paralichthys olivaceus and Synechogobius hasta was confirmed. The phenotypic characteristics of the isolates were studied extensively, including morphological, physiological and biochemical characteristics. The 16S rRNA and gyrB genes of a representative strain (ST1) were sequenced, and phylogenetic trees representing genetic relatedness between the isolate and publicly available 16S rRNA and gyrB gene sequence from the GenBank database was constructed as an aid to identification. The sequenced 16S rRNA gene (GenBank accession No. GU228793) and the gyrB gene of strain ST1 (GenBank accession No. GU228794) exhibited high similarity (98–99%) to those of Photobacterium damselae from the GenBank database. In addition, the activities of extracellular enzymes and haemolysin were also studied, the results showed that the isolates produced beta-haemolysis on rabbit blood agar, lecithinase, proteinase and DNase but diastase, lipase and gelatinase were not produced. Antibiotic susceptibility tests showed that the isolates exhibited around 80% of susceptibility to 36 various antibiotics tested.

Inhibitory effect of cranberry juice on the colonization of Streptococci species: An in vitro study

Background:Dental biofilm harboring oral bacteria is highly correlated with the progression of dental diseases. The existence of micro organisms as polyspecies in an oral biofilm and dental plaque has profound implications for the etiology of periodontal disease. The adhesion of streptococci to the tooth surface is the first step in the formation of dental plaque. Antiadhesive agents which can disrupt the biofilm formation can be an effective alternative to antibacterial therapy.Materials and Methods:A total of 10 patients both male and female between the age group of 20–40 years were included in the study. Plaque samples were taken and subjected to culturing on rabbit's blood agar plate for the growth of streptococci colonies for 24–48 h. The streptococci colonies were identified and was further subjected to subculturing for 24–48 h with disc containing ethyl alcohol+cranberry extract (Group A) and water+cranberry extract (Group B). Both these groups A and B were further divided into subgroups I, II, III, IV, and V according to concentration 1:2, 1:4, 1:40, 1:100 and 1:600 respectively.Results:Zone of inhibition of Group A with subgroup I disc was 2 mm. while with subgroups II, III, IV, V disc was 4mm. Whereas the zone of inhibition seen of Group B was same (2mm) in subgroups I, II, III, IV discs however a significant zone of inhibition (10mm) was observed in subgroup V disc.Conclusion:Cranberry juice (active ingredient: Non dialyzable material) inhibits the adhesion and reverses the coaggregation of various oral micro organisms. The present study revealed that cranberry gel in highly concentrated (1:600) form has an inhibitory effect on the colonization of the streptococci species, and thus can be beneficial in the inhibition of dental plaque formation.

Molecular and phenotypic characterization of Vibrio aestuarianus, a pathogen of the cultured tongue sole, Cynoglossus semilaevis Günther

Studies were conducted to determine the cause of high mortalities of cultured half-smooth tongue sole Cynoglossus semilaevis. Gross signs of disease included loss of appetite, erratic swimming, and haemorrhages on the head, opercula and base of fins, dorsal fin rot, swollen abdomen filled with ascitic fluid and herniation of the intestine. Histological examination of the liver showed focal areas of necrosis and extensive haemorrhages. Virtually pure, dense bacterial cultures were obtained from liver, kidney and spleen tissues, and high pathogenicity of the isolates to tongue sole was confirmed. The phenotypic characteristics of the isolates including morphological, physiological and biochemical characteristics were determined. The 16S rRNA and gyrB genes of the isolates were sequenced, and the phylogenetic trees representing genetic relatedness between the isolate and publicly available 16S rRNA and gyrB gene sequences from GenBank were constructed. The results confirmed that the diseased tongue soles were infected with Vibrio aestuarianus. The sequenced 16S rRNA gene of strain TS1 (GenBank Accession No. GQ372983) was 1446bp, the gyrB gene of strain TS1 (GenBank Accession No. GQ372984) was 1200bp and the two genes exhibited high similarity (98~99%) to those of V. aestuarianus from GenBank. In addition, the activities of extracellular enzymes and haemolysin were also studied; the results showed that the isolates produced beta haemolysis on rabbit blood agar, lecithinase, proteinase, DNase and lipase, but gelatinase was not produced.

Virulence factors of Vibrio metschnikovii strains isolated from fish in Brazil

Vibrio metschnikovii is a natural inhabitant of the aquatic environment and has been isolated from seafood, and associated with disease in humans. This study presents results for virulence factors produced by V. metschnikovii strains isolated from fish. Results for virulence factors demonstrated that 38.5%, 92.3% and 100% of the strains were hemolytic on sheep, rabbit, and human blood agar, respectively. Verotoxin was produced by 100% of the strains tested and 76.9% were skin test positive. We concluded that potentially pathogenic Vibrio species, including V. metschnikovii, must be considered when investigating food borne diseases related to consumption of raw or undercooked seafood, mainly fish.

Detection of partial virulence determinants and phenotypes in Enterococci

目的 调查临床分离的肠球菌6种致病基因和2种表型的分布. 方法应用聚合酶链反应和斑点杂交技术检测临床分离的145株肠球菌的6种致病基因,用7%兔血平板和3%明胶平板检测β溶血和明胶溶解表型.结果粪肠球菌和屎肠球菌6种致病基因的检出率分别为gelE 72.9%、30.6%;efaA 79.2%、36.7%;cylA 54.2%、34.7%;esp 34.4%、36.7%;agg 18.8%、0;ace28.1%、0.粪肠球菌和屎肠球菌β溶血分别为45.8%、20.4%;明胶溶解分别为35.4%、16.3%.结论粪肠球菌6种致病基因和2种表型的检出率高于屎肠球菌;尿标本比痰标本肠球菌致病基因的检出率高;gelE、efaA和cylA在粪肠球菌中的检出率高于其他致病基因。

Evaluation of isolation media for the detection of Bartonella henselae--isolation of Bartonella henselae from domestic cats

Bartonella henselae is a causative agent of cat scratch disease. We preliminarily tested four media for the bacterial growth, including agar plates with sheep, horse or rabbit blood, and chocolate agar. Of these media, rabbit blood and chocolate agar plate were found to be more excellent for the growth than the medium with sheep or horse blood. Blood samples from 60 domestic cats in Yamaguchi Prefecture were then cultured using 7% rabbit blood agar plates and BACTEC9050 (BD), automated blood culture microbial detection system. B. henselae was isolated from six of the 60 (10%) blood samples. Tiny colonies of B. henselae were visible on the agar medium after one week of culture at 35 degrees C in the 5% CO2 atmosphere. BACTEC 9050 detected B. henselae in one of the 10 blood samples and it took two weeks to detect the bacteria automatically, though gram stain failed to show organisms in the blood culture bottle. In conclusion, rabbit blood or chocolate agar and incubation of agar media more than one week and of BACTEC more than two weeks are recommended for the detection of B. henselae.

Infection with Bartonella weissii and detection of Nanobacterium antigens in a North Carolina beef herd.

Very recently, Bartonella organisms have been isolated from large ruminants (deer, elk, and dairy and beef cattle) located in the United States and in France. In this study, we report the serologic, microbiologic, and molecular findings related to the isolation of a Bartonella species in North Carolina beef cattle and the detection of nanobacterial antigen using a commercially available enzyme-linked immunosorbent assay. Between August 1998 and September 1999, blood was collected from 38 cattle ranging in age from 1 month to 6.5 years. After a 1-month incubation period, a Bartonella sp. was isolated on a 5% rabbit blood agar plate from three of six EDTA blood samples. PCR amplification of the 16S rRNA gene from all three isolates resulted in a DNA sequence that was 100% identical to that of B. weissii 16S rRNA (GenBank no. AF199502). By IFA testing, 36 of 38 cattle had antibodies (> or =1:64) to Bartonella weissii (bovine origin) antigens. Nanobacterial antigen was detected in 22 of 22 serum samples. We conclude that infection with an organism similar or closely related to B. weissii can occur in North Carolina cattle and that although their actual existence is still controversial Nanobacterium antigens were detected with a commercially available test kit. The epidemiology, vector biology, and potential pathogenicity of these organisms in cattle deserve future consideration.

Acute clinical disease in cats following infection with a pathogenic strain of Bartonella henselae (LSU16).

Bartonella henselae is the causative agent of human cat scratch disease as well as several serious sequelae of infections, including bacillary angiomatosis and bacillary peliosis. Conflicting reports describe the pathogenesis of B. henselae in the cat. In this study, we characterized a strain of B. henselae termed LSU16. This strain was isolated on rabbit blood agar from a naturally infected 10-month-old female cat during a recurrent episode of bacteremia. The bacterial species was confirmed by PCR-restriction fragment length polymorphism analysis. Nine cats were infected intradermally with 5 x 10(7) CFU of LSU16, and clinical signs, antibody responses, and bacteremia were monitored. All nine cats developed raised, erythematous areas at the site of inoculation within 72 h postinoculation; the swelling peaked at 14 days postinfection and was not palpable by 28 days postinfection. Fever developed in all nine cats between 6 and 16 days postinfection and lasted for 1 to 8 days. Between 6 and 16 days postinfection, all nine cats experienced lethargy which persisted 5 to 18 days. Seven of nine cats were bacteremic by day 7, and all nine cats had become bacteremic by 14 days postinfection. Bacteremia peaked at 14 to 28 days postinfection in all cats. In six of the nine infected cats, bacterial numbers reached nondetectable levels during the 7th week postinfection; however, a single animal maintained bacteremia to 18 weeks postinfection. All nine cats developed strong antibody responses to B. henselae, as determined by Western blot analysis and enzyme-linked immunosorbent assay. Subsequently, three naive cats were injected intradermally with blood from cats infected with LSU16 from a pure culture, and five naive cats were injected with feces from fleas which had been feeding on cats infected with a pure culture of LSU16. These cats developed signs similar to those described in the previous experiment and were euthanized at 5 weeks postinfection. We conclude that B. henselae LSU16 is a virulent strain of B. henselae in cats and propose that the virulence of B. henselae in cats is strain dependent.

Prevalence of Bartonella henselae and Bartonella clarridgeiae in an urban Indonesian cat population.

We studied evidence of Bartonella henselae and Bartonella clarridgeiae infection in 54 cats living in Jakarta, Indonesia. By using an indirect immunofluorescence assay, we found immunoglobulin G antibody to B. henselae in 40 of 74 cats (54%). The blood of 14 feral cats was cultured on rabbit blood agar plates for 28 days. Bartonella-like colonies were identified as B. henselae or B. clarridgeiae by using restriction fragment length polymorphism analysis and direct sequencing of the PCR amplicons. Of the cats sampled in the study, 6 of 14 (43%; all feral) were culture positive for B. henselae; 3 of 14 (21%; 2 feral and 1 pet) culture positive for B. clarridgeiae. This is the first report that documents B. henselae and B. clarridgeiae infections in Indonesian cats.

Behaviour of Aeromonas spp. after animal passage.

The Aeromonas genus have been consideredas important infectious agents in humans and ani-mals (JM Janda 1991 Clin Microbiol Reviews 4:397-410). The biological characteristics such as,toxins production (e. g. hemolysins, cytotoxins andenterotoxins) and also cell-associated features, thatappear to play important role in infectious pro-cesses in humans and animals, have been studiedin the attempt of elucidate patogenicity of the dif-ferent Aeromonas species (Janda loc. cit.). Evi-dences indicate that the animal passage may influ-ence in the expression of biological characteris-tics in many organisms such as, Plesiomonasshigelloides (SC Sanyal et al. 1980 J Med Microbiol13: 401-409) and Vibrio cholera O1 biotype ElTor (A Tikoo et al. 1994 J Med Microbiol 40: 246-251). DV Singh and SC Sanyal (1992 J MedMicrobiol 37: 262-267) reported that passagethrough rabbit instestines may control the expres-sion of the genes responsible for toxins produc-tion in Aeromonas spp.In this communication we report on the alter-ation of the hemolytic and enterotoxigenic charac-ter and also surface characteristics in one strain ofAeromonas isolated from environment, after ani-mal passage by endovenous route.Four samples of Aeromonas were used in thisstudy, three from polluted estuary water ( A. caviae- 030, Aeromonas sp. - 057, A. trota - 058) and onefrom drain treatment station supplyed by FundacaoOswaldo Cruz, Rio de Janeiro (A. hydrophila -T336). Those strains were maintend in nutrient agar(NA) with 1% of NaCl (FW Hickman-Brenner etal. 1987 J Clin Microbiol 25: 900- 906) at roomtemperature.All samples were tested with regar to hemol-ysin production, through β hemolysis zones aroundthe colonies in rabbit blood agar with 5% (v/v) oferitrocytes and the haemolitic activity was analyzedwith the metodology described by M Cumberbatchet al. (1979 Infect Immun 23: 829-837). The suck-ling mouse test (WA Dean et al. 1972 Infect Dis125: 407), was used for enterotoxin detection, theautoagglutination capacity for self pelleting (SP)and for preciptation after boiling (PAB) as de-scribed by JM Janda et al. (1987 Infect Immun 55:3070-77). The hidrofobic profile by phase parti-tioning with hydrocarbon solvents described by MRosenburg et al. (1980 Fems Microbiol Lett 9: 29-33).Strains for animal experiments were cultivatedin 5ml of BHI (OXOID) at 28