Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway.

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif ((161)DDDDDGDD(168)). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp(132), Thr(133), Asp(164), Asp(165), and Tyr(178). The goals of this study were to determine possible roles of the Ca(2+)-binding cluster for the interaction of LipL32 and Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca(2+)-binding residues of LipL32 to Ala, and their effects subsequently were observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells.

Dynamic Compression of the Left Coronary Artery by a Left Ventricular Pseudoaneurysm after Myocarditis.

UNLABELLED: A 69-year-old man with a recent diagnosis of suspected leptospirosis infection-related myocarditis presented with antecedent arthralgia, myalgia, fever, intermittent anterior chest pain, yellowish sclera, yellowish skin and shortness of breath. His symptoms improved after antibiotic treatment with penicillin for 14 days. However, recurrent chest pain and progressive dyspnea upon exertion developed 2 months later. A newly developed left ventricular outflow tract pseudoaneurysm was identified by cardiac sonography and multi-detector computed tomography of the heart. A subsequent coronary arteriogram demonstrated an left ventricular (LV) pseudoaneurysm causing compression to both the left circumflex coronary artery and the left anterior descending coronary artery with significant stenosis. To the best of our knowledge, this is the first reported case of a LV pseudoaneurysm developing after a clinical course of suspected leptospirosis-related myocardit is causing dynamic compression of the left coronary artery. KEY WORDS: Dynamic compression of coronary artery; Left ventricular pseudoaneurysm; Leptospirosis; Myocarditis.

Development and validation of a new loop-mediated isothermal amplification for detection of pathogenic Leptospira species in clinical materials.

Leptospirosis, a global zoonotic disease, is often neglected but has a significant impact on human health. Here we reported a new loop mediated isothermal amplification (LAMP) assay targeting lipL32 gene of pathogenic Leptospira spp. Polymerase chain reaction (PCR), nested-PCR and real-time PCR assays were included in this study for the comparison of analytic and diagnostic sensitivity and specificity. LipL32 LAMP we designed enables detection of 10 copies of L. interrogans and has a higher diagnostic sensitivity (91.67%) and specificity (100%) than other PCR-based methods. The high sensitivity, specificity and flexible reaction conditions of the lipL32 LAMP assay makes it feasible for resource-limited countries and on-site application.

Immunohistochemical study of lymph nodes in patients with cat scratch disease.

BACKGROUND/PURPOSE: Bartonella henselaeis the causative agent of cat scratch disease (CSD), manifesting as fever and acute regional lymphadenopathy. Although serologic testing is the reference method for diagnosis, successful use of immunohistochemical (IHC) stain of regional lymph nodes for the diagnosis of CSD has been reported. To determine the characterization and diagnostic potential of IHC in lymphadenopathy of CSD, lymph nodes were excised from patients with suspected CSD for further evaluation. METHODS: Polyclonal antibody-based IHC studies were performed for the detection of B. henselae. Between January 2001 and December 2004, the reference laboratory of the Center for Disease Control, Taiwan, received a total of 377 sera from 352 reported suspected CSD cases. Twenty-three formalin-fixed paraffin-embedded lymph nodes from 16 patients and two skin biopsies from two patients suspected of having CSD were included in this study. Nine of them were serologically confirmed to have CSD and the others were seronegative but suspected to have CSD by the attending physicians. Seven lymph node specimens were obtained from tuberculosis patients for comparison. RESULTS: We demonstrated that the microorganisms existed in the cytoplasm of histiocytes within the granulomatous lesions in nine lymph nodes and one skin biopsy. Among the nine lymph nodes with IHC (+) stains, three were seronegative. On the other hand, three cases were IHC (+) and six cases were IHC (-) among nine seronegative patients. In addition, two seronegative patients with skin biopsy showed one IHC (+) and one IHC (-). CONCLUSION: IHC can contribute to the etiologic diagnosis of B. henselaelymphadenopathy when serology and molecular techniques are not available.

Omp52 is a growth-phase-regulated outer membrane protein of Leptospira santarosai serovar Shermani.

We report the expression and characterization of the omp52 gene of Leptospira santarosai serovar Shermani strain CCF that is isolated in Taiwan. omp52 was identified among pathogenic leptospires but not among non-pathogenic leptospires by using suppression subtractive hybridization in our previous study. With an open reading frame of 1371 bp that encodes 456 amino acids and a predicted molecular mass of 52.6 kDa, Omp52 was shown to be an outer membrane protein containing a C-terminal OmpA consensus domain and exposed on the cell surface. Furthermore, Omp52 increases dramatically during the stationary phase, indicating that the expression of Omp52 is environmentally regulated. By using immunoblotting analysis, we proved that Omp52 was expressed in human patients infected with leptospires. These observations suggest that Omp52 may play roles in the interaction of host cells and pathogens during infection.

Clinical distinction and evaluation of leptospirosis in Taiwan--a case-control study.

BACKGROUND: Leptospirosis is the most widespread zoonosis. Leptospirosis remains underreported in Taiwan because of ignorance and the broad spectrum of clinical manifestations. Acute renal failure (ARF) is a prominent feature of leptospirosis. This investigation conducted a case-control study to obtain information to distinguish leptospirosis from other conditions with similar presentations. METHODS: Leptospirosis surveillance was performed at Chang Gung Memorial Hospital, Taiwan, between September 2000 and December 2001. Suspected clinical cases were included in the sample and investigated. Diagnosis was confirmed with four-fold or greater increase of microscopic agglutination test (MAT) titer in paired sera; positive immunoglobulin M (IgM) dipstick with single MAT > or =400; or isolation of leptospira. Cases were classified as excluded based on confirmed etiology other than leptospirosis or negative paired serologic test. RESULTS: Twenty-two confirmed cases and 21 excluded cases of leptospirosis were identified from among 169 suspected cases. An outbreak was observed during the flooding from Typhoon Nali. L. shermani, the most common serovar in Taiwan, was identified in 78.5% of confirmed cases. In the confirmed group, mean age was 44 +/- 14.5 (21-66) yrs similar to that of the excluded group, with male predominance (86.4 vs. 57.1%, p<0.05). The most common presentations in the confirmed group were fever (95.5%), ARF (86.4%), myalgia (72.7%) and jaundice (63.6%). Ten patients were infected through occupational or recreational exposure, and in six patients, the infection was associated with flooding. The most distinct presentations of leptospirosis cases compared with excluded cases were increased incidence of hemorrhagic diathesis (odds ratio (OR): 10, p=0.04), myalgia (OR: 8.0, p=0.02), bilateral enlarged kidneys (OR: 7.5, p=0.0004), risk factor exposure (OR: 6.9, p=0.005), sterile pyuria (OR: 6.3, p=0.017), hypokalemia (OR: 5.0, p=0.035) and thrombocytopenia (OR: 4.8, p=0.04). Hospitalization days correlated well with levels of peak creatinine (Cr) (p=0.0362) and platelet nadir (p=0.0039) reached. Penicillin treatment was followed by rapid symptoms and renal function improvement. CONCLUSIONS: Prompt recognition of the characteristic presentations of leptospirosis, followed by timely antibiotic treatment, can dramatically save the patients even with severe multiple organ damage.

Disproportional exaggerated aspartate transaminase is a useful prognostic parameter in late leptospirosis.

AIM: To evaluate the hepatic dysfunction in leptospirosis is usually mild and resolved eventually. However, sequential follow-up of liver biochemical data remained lacking.. METHODS: The biochemistry data and clinical symptoms of 11 sporadic patients were collected and analyzed, focusing on the impacts of leptospirosis upon liver biochemistry tests. RESULTS: The results disclosed that of the 11 cases, 5 or 45% died. The liver biochemistry data in the beginning of the disease course were only mildly elevated. Nevertheless, late exaggerated aspartate transaminase (AST) elevations were noted in three cases who finally died when compared with the typical course. Besides, significant higher AST/alanine transaminase (ALT) ratios (AARs) of the peak levels for transaminase were also noted in the cases who eventually succumbed. The mean+/-SD of AARs for the survival group and dead group were 5.65+/-2.27 (n = 5) and 1.86+/-0.64 (n = 6) respectively (P = 0.006). The ratios of the cases who finally died were all more than 3.0. Conversely, the survival group's ratios were less than 3.0. CONCLUSION: Serial follow-up of transaminase might provide evidence to predict some rare evolutions in leptospirosis. If AST elevated progressively without a concomitant change of ALT, it might indicate an acute disease course with ensuing death. Additionally, AAR is another prognostic parameter for leptospirosis. Once the value was higher than 3.0, a grave prognosis is inevitable.

Identification Leptospira santarosai serovar shermani specific sequences by suppression subtractive hybridization.

In Taiwan, leptospirosis is caused mainly by Leptospira santarosai serovar shermani. Suppression subtractive hybridization was employed to isolate DNA fragments present in pathogenic L. santarosai serovar shermani but absent in non-pathogenic L. biflexa serovar patoc. Analysis of 23 subtracted DNA clones revealed 25 gene fragments by BLASTX program. Eight clones showed similarity to transposase genes and three clones displayed homology with either translation or metabolism related genes. Four clones were similar to outer membrane protein, penicillin-binding protein, CreD-like protein and the protein of two-component signal transduction system, respectively. One clone had TPR repeat domain and five clones had significant similarity with hypothetical proteins of unknown functions. The remaining four clones exhibited no homology with any known genes. These results indicate that subtractive hybridization can successfully identify genes that are absent from the non-pathogenic Leptospira and provide a starting point for clarifying the differential genes expression between pathogenic and non-pathogenic Leptospira species.

Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus.

A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS-PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40 degrees C to 50 degrees C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10-phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N-terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat-labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.

Detection of Anaplasma phagocytophilum and Anaplasma bovis in small wild mammals from Taichung and Kinmen Island, Taiwan.

A total of 138 animals from 7 species (Apodemus agrarius, Bandicota indica, Crocidura suaveolens, Mus caroli, Mus formosanus, Rattus losea, and Suncus murinus) captured in Taichung, located in central Taiwan, and Kinmen Island, an island off the shore of China, were examined for the presence of Anaplasma phagocytophilum. The presence of the bacteria, which causes human granulocytic anaplasmosis, was examined by nested PCR targeting the16S rDNA. Twelve animals (8.7%) from M. caroli and R. losea, and 25 (18.1%) from A. agrarius, B. indica, M. caroli, and R. losea were infected with A. phagocytophilum and Anaplasma bovis, respectively. Phylogenetic analysis revealed that partial 16S rDNA sequences in the 12 aforementioned animals showed higher similarity to the sequences related to A. phagocytophilum detected in wild rodents (Rattus and Niviventer) from southeast China. The sequences of the other 25 animals belonged to the A. bovis clade. We demonstrated that small wild mammals were infected with A. phagocytophilum and A. bovis in Taichung and Kinmen Island, Taiwan.

Serological and PCR detection of feline leptospira in southern Taiwan.

Taiwan is in the subtropical zone and has typhoons every year. Leptospirosis is an endemic disease in Taiwan, and feline leptospirosis in Taiwan remains unknown so far. From January, 2010, to September, 2011, 233 cats in south Taiwan (159 stray cats and 74 household cats) were sampled in this research. Leptospira antibody titer was detected by the serology gold standard, the microscopic agglutination test (MAT). Both serum and urine were examined for Leptospira DNA by polymerase chain reaction (PCR) with two sets of primers. In this study, the serological survey showed 21 (9.3%) examined sera contained antibodies specific for pathogenic Leptospira serogroups. The results of PCR revealed that 25 (19.1%) serum and 80 (67.8%) urine samples were found positive for leptospiral DNA sequences. All products amplified from PCR reactions were sequenced by an automated method for further confirmation. This is the first study concerning the epidemiology of pathogenic Leptospira in stray and household cats' urine, and the results demonstrate that some of the cats are susceptible to pathogenic Leptospira and have the potential to shed pathogenic Leptospira into the environment. This could be an issue of public health.

Potential impact on kidney infection: a whole-genome analysis of Leptospira santarosai serovar Shermani.

Leptospira santarosai serovar Shermani is the most frequently encountered serovar, and it causes leptospirosis and tubulointerstitial nephritis in Taiwan. This study aims to complete the genome sequence of L. santarosai serovar Shermani and analyze the transcriptional responses of L. santarosai serovar Shermani to renal tubular cells. To assemble this highly repetitive genome, we combined reads that were generated from four next-generation sequencing platforms by using hybrid assembly approaches to finish two-chromosome contiguous sequences without gaps by validating the data with optical restriction maps and Sanger sequencing. Whole-genome comparison studies revealed a 28-kb region containing genes that encode transposases and hypothetical proteins in L. santarosai serovar Shermani, but this region is absent in other pathogenic Leptospira spp. We found that lipoprotein gene expression in both L. santarosai serovar Shermani and L. interrogans serovar Copenhageni were upregulated upon interaction with renal tubular cells, and LSS19962, a L. santarosai serovar Shermani-specific gene within a 28-kb region that encodes hypothetical proteins, was upregulated in L. santarosai serovar Shermani-infected renal tubular cells. Lipoprotein expression during leptospiral infection might facilitate the interactions of leptospires within kidneys. The availability of the whole-genome sequence of L. santarosai serovar Shermani would make it the first completed sequence of this species, and its comparison with that of other Leptospira spp. may provide invaluable information for further studies in leptospiral pathogenesis.

Sequence of Leptospira santarosai serovar Shermani genome and prediction of virulence-associated genes.

Leptospirosis, a widespread zoonosis, is a re-emerging infectious disease caused by pathogenic Leptospira species. In Taiwan, Leptospira santarosai serovar Shermani is the most frequently isolated serovar, causing both renal and systemic infections. This study aimed to generate a L. santarosai serovar Shermani genome sequence and categorize its hypothetical genes, particularly those associated with virulence. The genome sequence consists of 3,936,333 nucleotides and 4033 predicted genes. Additionally, 2244 coding sequences could be placed into clusters of orthologous groups and the number of genes involving cell wall/membrane/envelope biogenesis and defense mechanisms was higher than that of other Leptospira spp. Comparative genetic analysis based on BLASTX data revealed that about 73% and 68.8% of all coding sequences have matches to pathogenic L. interrogans and L. borgpetersenii, respectively, and about 57.6% to saprophyte L. biflexa. Among the hypothetical proteins, 421 have a transmembrane region, 172 have a signal peptide and 17 possess a lipoprotein signature. According to PFAM prediction, 32 hypothetical proteins have properties of toxins and surface proteins mediated bacterial attachment, suggesting they may have roles associated with virulence. The availability of the genome sequence of L. santarosai serovar Shermani and the bioinformatics re-annotation of leptospiral hypothetical proteins will facilitate further functional genomic studies to elucidate the pathogenesis of leptospirosis and develop leptospiral vaccines.

Identification and characterization of OmpA-like proteins as novel vaccine candidates for Leptospirosis.

Leptospira is an important infectious Gram-negative bacterium causing Leptospirosis in mammals. Outer membrane proteins (OMPs) are key molecules in the interface between the cell and its environment. A group of putative leptospiral outer membrane proteins with an OmpA-like domain, comprising Lp0056, Lp0222, Lp3615, Lp3685, Lp4337 and Lbp328, were identified by bioinformatic methods and expressed as GST-tag fusion proteins. All these recombinant proteins were screened for immune-protective potential in hamsters challenged with Leptospira interrogans serovar Pomona. Out of these six proteins, three fusion proteins including Lp4337, Lp3685 and Lp0222 were found to be protective on the basis of survival. The immune response generated against these three recombinant proteins was evaluated on the basis of antibody production, lymphocyte proliferation and cytokine profiles in response to recall antigens whereas protective efficacy was assessed on the basis of survival and histopathological lesions in the vital organs (kidney, liver, and lung). Our results show that all three recombinant proteins generated strong immune responses, enhanced survival and reduced the severity of histopathological lesions. Of the proteins studied, Lp4337 generated the strongest immune response and was able to impart maximum protection (75%), followed by Lp3685 (58%), whereas Lp0222 generated lowest immune response correlating to protection (42%) against lethal infection of leptospira in the immunized animals. In contrast, control animals injected with PBS demonstrated low survival and had significant lesions. All these results clearly indicate that these three OmpA-like proteins may serve as novel vaccine candidates for leptospirosis.

Leptosome-entrapped leptospiral antigens conferred significant higher levels of protection than those entrapped with PC-liposomes in a hamster model.

We prepared novel liposomes from total polar lipids of non-pathogenic Leptospira biflexa serovar Potac (designated leptosomes) and evaluated their vaccine delivery/adjuvant potential with novel protective antigens (Lp0607, Lp1118 and Lp1454) of L. interrogans serovar Pomona in a hamster model. The immune response induced by three individual antigens and protective efficacy were evaluated and compared to those induced by same antigens entrapped with PC-liposomes and E. coli lipid liposomes (escheriosomes). Four-week-old hamsters were immunized subcutaneously twice at a 3-week interval, bled at various time points to evaluate antibody response and sacrificed to isolate splenocytes for lymphocyte proliferation and cytokine profiles in response to recall antigen. For the challenge test, 10x MLD(50) (modified lethal dose 50%) of virulent L. interrogans serovar Pomona were administered intraperitoneally. Our results demonstrate that leptosome are better adjuvant than PC-liposomes as revealed by enhanced long term antibody response, lymphocyte proliferation and significant enhancement of both Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines. Additionally, leptosomes and escheriosomes induced significantly higher level of memory responses than PC-liposome did. Moreover, the novel leptosomal vaccine induced significantly higher levels of protection than those prepared with PC-liposomes as revealed by enhanced survival, reduced histopathological lesions in vital organs and reduced leptospiral load in kidneys. Taken together, the results of the present study clearly reveal that both leptosomes and escheriosomes have emerged as promising delivery vehicles/adjuvants that can be widely exploited with newly discovered antigens in future leptospira vaccines.

Immunogenicity and protective efficacy of recombinant Leptospira immunoglobulin-like protein B (rLigB) in a hamster challenge model.

Leptospiral immunoglobulin-like protein (LigB) was truncated into conserved (LigBcon) and variable (varB1, varB2) fragments and expressed as GST/His-tag fusion proteins. Four-week-old hamsters were immunized with equal amounts of each fragment individually or combined in alum adjuvant at days 0 and 21 and subsequently challenged three weeks after the booster with 2.5 LD(50) live virulent Leptospira interrogans serovar Pomona. Our results demonstrate that immunization with LigB produced strong humoral immune responses as revealed by high titers against each fragment and significant enhancement in Th2 cytokines (IL-4, IL-10). A significant activation of CMI is revealed by enhanced proliferation of lymphocytes and up regulation of Th1 cytokines (IL-12p40, IFN-gamma) was also noted. Of the peptides studied, rLigBcon was able to impart maximum protection (71%), followed by rVarB1 (54%), whereas rVarB2 was not able to impart a significant level of protection (33%) against lethal infection as revealed by enhanced survival and reduced severity of histopathological lesions in vital organs (viz. kidney, liver, spleen) of the immunized animals. Moreover, concurrent administration of all three fragments significantly enhanced the protective efficacy of the vaccine (83%). Overall, our results clearly demonstrate that LigB has emerged as novel protective antigen that can be used in future subunit vaccines against leptospirosis.

Immunogenicity of the recombinant leptospiral putative outer membrane proteins as vaccine candidates.

Leptospiral putative outer membrane proteins (OMPs) are likely to be essential components of more effective vaccines. Recently completed genomic sequences of Leptospira allowed us to target putative OMPs for the development of recombinant vaccines. We focused on 12 putative OMPs that had no homology with other organisms listed in the NCBI database except MceI and MceII of Leptospira, which are approximately 25% homologous to MceI of Mycobacterium tuberculosis. All putative OMPs were cloned, expressed and purified as glutathione-S-transferase (GST) fusion proteins. Primary screening for immunoprotective potential was performed in hamsters challenged with an LD50 inoculum of low passage serovar Pomona. Out of these 12 OMPs three fusion proteins viz. rLp1454, rLp1118 and rMceII were found to be protective in a hamster model of leptospirosis. The protective efficacy was evaluated on the basis of survival, histopathological lesions in vital organs and antibody responses against each antigen. All the recombinant proteins were able to enhance the survival and reduce the histopathological lesions. In contrast, control animals immunized with rGST demonstrated low survival and had significant lesions. Further, these three proteins were evaluated for synergistic protective efficacy as compared to LigA, which has already been established as a protective antigen. Our results indicate that rLp1454, rLp1118, and rMceII showed protection individually and synergistically against serovar Pomona infection, which may help us to develop a multicomponent vaccine for leptospirosis.

The efficacy of mouse-brain inactivated Nakayama strain Japanese encephalitis vaccine--results from 30 years experience in Taiwan.

An intensive mandatory vaccination program has been underway, combating Japanese encephalitis (JE) since 1968 in Taiwan. Long-term collection of immunization records has been developed from 1967 to 2000 in this study to retrospectively assess the efficacy of the mouse-brain inactivated Nakayama JE vaccine. The vaccine efficacy (VE) of completing at least two doses of the JE vaccine was 96.98%. Among 1 to 14-year-old children, the efficacy of completing 1, 2, and 3 doses of immunization was 85.59%, 91.07% and 98.51%, respectively. Furthermore, the long-term efficacy for a single dose vaccinated at least 25 years was 86.79%, and for 2 and 3 doses it was 88.10% and 95.54%, respectively. In contrast to previous studies that recommended at least two doses of JE vaccination to acquire necessary protection, the empirical results in this study indicated that even immunization with one single dose provides sufficient protection to the population. However, a single dose of JE vaccine might still be beneficial for some JE epidemic or endemic developing countries with limited resources for infectious disease control.

Cat-scratch disease in veterinary-associated populations and in its cat reservoir in Taiwan.

In Taiwan, the first human case of cat-scratch disease (CSD) was diagnosed by a serologic test in 1998. Since then, no studies have been conducted to understand the epidemiology of the infection in Taiwan. Therefore, this study is the first epidemiologic survey of CSD in cats and humans in this country. Using veterinary-associated individuals as the study population, it was identified that 1.7% of them were seropositive for B. henselae, and residence was the only factor associated with seropositivity. Bartonella species were successfully isolated from 25 (19.1%) of the 131 cats tested. Only B. henselae and B. clarridgeiae were obtained from bacteremic cats. Furthermore, 9.2% of 131 cats were dually-infected with genotypes I and II of B. henselae. It is the highest prevalence of co-infection that has ever been reported worldwide. In cats, the seroprevalence was 23.7% by indirect immunofluorescence antibody assay with B. henselae Houston-1 (type I) as the antigen. When 12 bacteremic but seronegative cats were re-tested by IFA slides coated with B. henselae U-4 antigen (type II), 9 cats were identified to be seropositive. Our study further suggested that using only direct PCR of 16S-23S rRNA intergenic region or the combination of the PCR method and indirect immuno-fluorescence test will be useful to diagnose Bartonella-free cats.