Animal leptospirosis is a zoonotic disease that affects multiple domestic and wild species. It causes generalized vasculitis, which in pregnant females results in placentitis and triggers abortion. Furthermore, it causes hemoglobinuria, icterus (jaundice), and congestion in different mucosa. The objective of this study was to establish whether the occurrence of hemoglobinuria and changes in the pigmentation of the vaginal mucosa was associated with the presence of Leptospira spp. DNA in the urine. Therefore, 38 urine samples from lactating female buffaloes showing hemoglobinuria, alterations in the pigmentation of the vaginal mucosa, history of abortion, and low-weight calves were evaluated. Additionally, hematocrit was determined by blood centrifugation in a capillary tube, and the presence of hemoparasites was determined by staining of blood smears. The DNA of Leptospira spp. was detected by PCR using two pairs of primers for the markers G1-G2, derived from a sequence from the genomic library of L. interrogans serovar icterohaemorragiae, strain RGA, which was described by Gravekamp et al. (1993). Additionally, Internal 1 and Internal 2 were derived from the sequence of the gene encoding the LipL32 protein and specific to identify pathogenic leptospira, described by Haake et al. (2000). Data analysis was performed with R (Fisher’s exact test). The hematocrit mean was 21%, and all females were negative for hemoparasites (Anaplasma mar ginale, Babesia spp., and Trypanosoma spp.). The percentage of Leptospira spp. DNA-positive samples with at least one of the markers used was 63%. The marker G1-G2 detected more positive samples in comparison with the marker Internal 1-Internal 2 (60.5% vs 7.9%, p<0.0001 respectively). A significant association was observed between the mucosa’s appearance and the presence of Leptospira spp. DNA in urine when the G1-G2 was used (p <0.0001). In the case of females with icteric vaginal mucosa, the percentage of positive samples (85%, 17/20, p = 0.001227) was higher than that in buffaloes with normal mucosa (26.66%, 4/15). In addition, in females with icteric vaginal mucosa, the odds of a positive sample for Leptospira spp. DNA (using the G1-G2 marker) were 14 (95% CI: 2.3427-119.1974) times more than in females with normal vaginal mucosa. There was no significant difference in the percentage of positive samples between buffaloes grouped as icteric mucosa, and congestive mucosa (p= 0.4529), nor between the females grouped as congestive and normal mucosa (p= 0.2451). When the Internal 1-2 marker was used, there was no association between the appearance of the mucosa and the percentage of buffaloes with Leptospira spp. DNA in the urine (p= 0.1714). The presence of hemoglobinuria was not associated with Lep tospira spp. DNA in the urine, regardless of the marker used for its detection (p>0.05). In conclusion, the data from this study indicates that the G1-G2 marker was more efficient in determining the presence of Leptospira spp. DNA in urine, and using this marker, the icteric vaginal mucosa is associated with Lep tospira spp. DNA in urine, thus suggesting that this clinical sign should be considered suggestive of the disease.

The test most used for leptospirosis diagnosis is the Microagglutination Test (MAT). However, the low antibody titers in chronic infections induced by strains adapted to bovines can limit the interpretation of the correct results. The bacteriological culture of Leptospira spp. is the gold standard test because it can demonstrate the agent’s presence. Therefore, this research aimed to reveal the presence of Leptospira spp. in aborted fetuses, genital-urinary secretions of female and male Bubalus bubalis, food raw materials, and rodent feces using bacteriological culture. The analyses were performed on a farm located in the state of Cojedes, Venezuela. This farm has an abortion rate of 17,05% and 2,98% mortality at birth. For this, 18 blood samples (16 females and two males) were extracted by puncture of the jugular vein, and these were allowed to coagulate to obtain the serum and urine samples from 2 female buffaloes, one male buffalo, and semen from 2 other animals, were collected. Additionally, two fetuses of 120 and 75 days of gestation were collected. Also, after the necropsy, fluid samples were obtained from the abdomen, umbilical artery, ocular and thoracic cavities, and amniotic liquid, and two samples of liver, spleen, kidney, lung, heart, and brain were collected. Furthermore, samples of food ration and of the four by-products (corn silage, hay, urea, mineral) that compose it were collected, besides the rodent feces found in the storage where the food was preserved. The standard serological MAT was performed with a panel of strains of 7 Leptospira serovars. The fluids and tissue samples were inoculated in Ellinghausen–McCullough–Johnson–Harris medium (EMJH), a liquid culture medium selective for Leptospira. A portion of the tissue sample was fixed in 10% buffered formalin for histological slices and routine H-E staining. The MAT results indicated titers of 1:50 for the Icterohaem morragiae serovar at 5.5%, Pomona at 5.5%, and Bratislava at 5.5%. The serovars Canicola, Grippotyphosa, Harjo bovis, and Harjo pratjino were negative. There was a growth of Leptospira spp. in fetal fluids from the abdomen, umbilicus, thorax, liver, spleen, lung, kidney, and heart tissues; in the same way, Lepto spira spp. growth was obtained after the culture of urine and semen samples, food, corn silage, hay, urea-mineral and rodent feces. Histopathology analysis showed lesions in the spleen as extra-medullary hematopoiesis and hemorrhage; in the kidney (multifocal hemorrhages, tubular degeneration, and edema) while in the lungs, perivascular hemorrhages and degeneration of the vascular wall were observed. In conclusion, these preliminary results suggest that the MAT cannot diagnose all Leptospirosis cases correctly, especially chronic infections characterized by low antibodies. At the same time, the culture in EMJH seems to solve this weakness, as Leptospira spp. growth was obtained from different biological samples. Additionally, these preliminary findings confirm Leptospira is an agent involved in abortions, as we obtained Leptospira spp. growth from aborted fetus samples. Finally, it is likely that this bacterium growing in the bacteriological cultures belongs to a serovar different from those in the panel of strains used in MAT, so the cultures were preserved for subsequent studies that contemplate molecular identification.

The Vaccine Administration Law of the People's Republic of China and other relevant laws require that vaccine recipients or their guardians be educated about vaccines and how they work, and described in general the methods and contents of such vaccination education. With the new law and "Standard Operational Procedures for Immunization" as foundation documents, and in consultation with experts at home and abroad, the Chinese Preventive Medicine Association developed a consensus statement about informed consent for vaccination. This consensus statement is written for disease control and prevention health care personnel in vaccination services and describes the educational content of informed consent, a theoretical framework for immunization and immunization knowledge, the informed consent processes, principles of planning for vaccination, and an informed consent form. Part Two of the consensus includes influenza vaccine, pneumococcal vaccine, haemophilus influenzae type b containing vaccine, enterovirus type 71 inactivated vaccine, rotavirus vaccine, varicella attenuated live vaccine, herpes-zoster vaccine, human papillomavirus vaccine, rabies vaccine, hemorrhagic fever with renal syndrome vaccine, leptospira vaccine, anthrax vaccine, hepatitis E vaccine, cholera vaccine, typhoid vaccine, and tick-borne encephalitis vaccine.

"Leptospira calling“ from TMAID artist in residence Raffaela Pitzurra

We present a case of a 25-year-old previously healthy man who came into our emergency department with decreased verbal output and fever. Patient came in febrile, tachycardic and tachypneic. Physical examination was essentially normal; however, patient was noted to be aphasic. Initial diagnostics were requested. Complete blood count (CBC) showed leukocytosis with white blood cell count of 15 × 10 9 /L and normal platelet count of 157 × 10 9 /L. Chest X-ray was normal, and so was the urinalysis. Leptospira IgG and IgM showed negative results. Patient was suspected to have bacterial meningitis. He was then given empiric therapy and was started with ceftriaxone and vancomycin. The patient was then referred to the neurology service where a magnetic resonance angiography of the intracranial vessels was made. Results were highly suggestive of viral encephalitis. Lumbar tap for cerebrospinal fluid analysis was performed showing negative test results for cryptococcal antigen, gram stain, acid fast stain, culture and susceptibility, tuberculosis (TB) culture, negative staining, herpes simplex virus 1 and 2, and TB polymerase chain reaction (PCR). Patient was referred to the infectious disease service and a repeat CBC was requested showing leukopenia and thrombocytopenia. Dengue duo was positive together with the CSF dengue RNA-PCR. Platelet went down to as low as 14 × 10 9 /L and patient was transfused with six units of platelet concentrate. Supportive therapy was given and daily monitoring of blood counts was made. The patient started to recover on the fifth day and completely recovered and discharged on the ninth hospital day with normal blood counts and no neurologic deficit. Clin Infect Immun. 2020;5(1):10-13 doi: https://doi.org/10.14740/cii93

Leptospirosis is a major threat in tropical and subtropical countries as well as temperate countries. The disease is caused by pathogenic Leptospira species and considered to be an emerging or re-emerging disease in many countries of the world. Infection in domestic animals and wildlife can lead to economic loss and pose a potential spread to the communities. In the present study recombinant LigB protein is employed in latex agglutination test, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiral serovars. It was employed for serodiagnosis of leptospirosis. The 46KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in P QE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against Leptospirosis suspected wildlife field sera. A total of 80 wildlife sera samples were collected, including 27 wild feline sera samples (18 tigers, 8 lions, and 1 jaguar) obtained from Chhatbir zoo, Chandigarh, 42 serasamples ( 8 tigers, 4 lions and 6 leopards, 2 cheethals, 1 black buck, 12 buffaloes and 9 zoo staff) sera and 3 live rodents ) were received from Jodhpur zoo Rajasthan, 8 sera samples (4 tigers, 3 leopards, 1 lion) sera from Van Vihar National park, Bohpal, Madhya Pradesh and 3 sera samples (2 lions,and 1 tiger) received from Biwani Mini zoo, Haryana, India. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, pen site, reliable diagnostic tool of high sensitivity and specificity, under laboratory and field conditions, for the detection of Leptospirosis .

Objective To investigate the dynamic changes of antibodies induced by leptospiral vaccines. Methods Antigens for antibody detection were screened out. ELISA was used to analyze antibody responses induced at different time points after immunizing guinea pigs with different batches of leptospiral vaccines from different manufacturers. To investigate the relationship between antibody responses induced by leptospiral vaccines and their protective effects in animal model, guinea pigs were challenged with Leptospira after immunization. Results There was no significant antigen-antibody reaction between the LigA protein or Patoc Ⅰ antigen and the serum samples of guinea pigs immunized with leptospiral vaccines. Notable IgG and IgM antibody reactions were observed in all vaccination groups when using bacterial proteins from seven Leptospira reference strains which were used for the preparation of leptospiral vaccines as envelope antigens. Antigen-specific IgG antibodies peaked at 35 d after the last immunization, and the highest peak of antigen-specific IgM antibodies was reached 11 d after the last immunization. Results of the challenge test showed that non-diluted leptospiral vaccines induced significant IgG and IgM antibody reactions in guinea pigs as compared with those diluted three or nine times, showing good protective effects. Conclusions Analysis of the dynamic changes of antibodies induced by leptospiral vaccines revealed that there was correlation between the induced serum antibody responses and the protective effects. This study provided reference for further study on alternative methods for evaluating leptospiral vaccine potency. Key words: Leptospira; Vaccine; Antibody; Protection

Objective To analyze the enzymatic activity of Leptospira interrogans (L.interrogans) LA_2144 gene product to hydrolyze platelet activating factor acetylhydrolase (PAF-AH) and phosphatidase A2 (PLA2). Methods Bioinformatic softwares were used to predict transmembrane regions, signal peptides and domains of the LA_2144 gene of L. interrogans strain Lai. A prokaryotic expression system for signal peptide-free LA_2144 gene was established. The expressed target recombinant protein rLep2144 was extracted by Ni-NTA affinity chromatography and then renatured. Spectrometry was used to detect the activity of rLep2144 to hydrolyze PAF-AH substrate 2-thio PAF and the Km and Kcat values as well as the activity to hydrolyze PLA2 substrate arachidonoyl 2-thio PC. Real-time fluorescence quantitative RT-PCR and Western blot were performed to detect the transcription, protein expression and secretion of LA_2144 gene during infection of human and mouse vascular endothelial cells (HUVEC and EOMA) with L. interrogans. Results L. interrogans LA_2144 gene contained a signal peptide and a domain belonging to SGNH hydrolase superfamily, but no transmembrane regions. The established prokaryotic expression system for signal peptide-free LA_2144 gene could efficiently express rLep2144. The extracted rLep2144 was shown as a single protein fragment in separation gel and then successfully renatured. rLep2144 had a stronger PAF-AH activity with the Km and Kcat values of 688.235 μmol/L and 0.976/s, but its PLA2 activity was relatively weak. Expression of the LA_2144 gene at mRNA and protein levels in HUVEC and EOMA was rapidly increased after the cells were infected with L. interrogans (P<0.05) and the secretion of LA_2144 gene product could be detected. Conclusions L. interrogans LA_2144 gene product had a stronger PAF-AH and a certain PLA2 activity, which might involve in the hemorrhage and inflammatory response in leptospirosis. Key words: Leptospira interrogans; LA_2144 gene; PAF-AH; PLA2; Enzymatic activity

Leptospirosis is a common zoonosis, an infectious disease that infects both humans and animals, which is caused by spirochete bacteria from genus Leptospira . Approximately 30% of children in urban Detroit and 16% of adults in Baltimore demonstrated serologic evidence of previous leptospirosis infections. The Detroit study showed correlation between degree of rat infestation and seropositivity rates. This finding suggests rats are major vectors for human leptospirosis in mainland United States. The leptospires from infected animals survive best in fresh water, damp alkaline soil, vegetation, and mud with temperatures higher than 22 °C. Although leptospirosis is a well documented clinical condition, the sequelae of polyneuritis has not yet been reported to our knowledge. The following case is an example of such an occurrence. A 49-year-old Caucasian female presented to the emergency department with flu-like symptoms after volunteering in an urban Detroit neighborhood clean-up project. She admitted to picking up wood from stagnant water without wearing personal protective equipment. The primary differential diagnosis being Weil’s disease from leptospirosis and was proven positive with serologic testing via the Center for Disease Control. A computed tomography (CT) abdomen and pelvis demonstrated lymphadenopathy throughout the abdomen and near the spinal column. She was treated with ceftriaxone and doxycycline for 12 days but developed chronic neuralgia and polyneuritis. Leptospires are known to cause vasculitis. This patient developed polyneuritis either from direct invasion of the nerves from leptospires or compromised blood supply to the nerves. In addition to antibiotics, patients with severe cases of leptospirosis also require supportive therapy and careful management of renal, hepatic, hematologic, and central nervous system complications. If renal failure ensues, early initiation of hemodialysis or peritoneal dialysis may reduce mortality. Rapid identification of symptoms and a high clinical suspicion are paramount because early intervention can save their life. J Med Cases. 2019;10(6):183-187 doi: https://doi.org/10.14740/jmc3320

Objective To understand the differences in engulfing ability and phagolysosome formation between mononuclear-macrophages and neutrophils during Leptospira interrogans infection. Methods Human THP-1 monocytes and HL-60 cells were pretreated with PMA (phorbol-12-myristate-13-acetate) and ATRA (all-trans retinoic acid) to differentiate them into mononuclear-macrophages and neutrophils, respectively. The phagocytosis of Leptospira interrogans in THP-1-PMA mononuclear-macrophages and HL-60-ATRA neutrophils was detected by confocal microscopy. The morphology of intracellular Leptospira was determined by transmission electron microscopy. The viability of phagocytized Leptospira and the percentages of dead Leptospira were analyzed by confocal microscopy and spectrofluorimetry, respectively. Confocal microscopy was used to measure the formation of phagolysosomes in different phagocytes. Results Both THP-1-PMA mononuclear-macrophages and HL-60-ATRA neutrophils could phagocytize Leptospira interrogans, but the phagocytic ability of the former was notably stronger than that of the latter (P<0.05). Intracellular Leptospira were surrounded by phagocytic vesicles in both types of phagocytes. THP-1-PMA mononuclear-macrophages were better than HL-60-ATRA neutrophils in killing intracellular Leptospira (P<0.05). More phagolysosomes were formed in THP-1-PMA mononuclear-macrophages than in HL-60-ATRA neutrophils (P<0.05). Conclusions Human mononuclear-macrophages but not neutrophils act as major phagocytes that play an important role in phagocytizing and killing Leptospira during infection. Less fusion of the phagosomes with lysosomes may be responsible for the lower Leptospira-killing ability of neutrophils. Key words: Leptospira interrogans; Mononuclear-macrophages; Neutrophils; Phagolysosome

Objective To analyze the activity of diadenylate cyclase (DAC) encoded by LA3304 gene of Leptospira interrogans (L.interrogans) and to investigate the influence of CdaR encoded by LA3303 gene on DAC activity. Methods The LA3304 gene in L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR and inserted into a prokaryotic expression system for expressing DAC. The expressed recombinant protein, rDAC, was purified by Ni-NTA affinity chromatography. High Performance Liquid Chromatography (HPLC) was used to measure the synthesis of c-di-AMP from ATP by rDAC in vitro. Bacterial two-hybrid analysis was used to detect the interaction between CdaR and DAC. Prokaryotic co-expression system was constructed and used in combination with HPLC to analyze the role of CdaR in activating DAC. Results The constructed prokaryotic expression system for LA3304 gene of L. interrogans strain Lai could highly express the rDAC upon the induction of IPTG. The purified rDAC showed high purity with a single protein band in gel as indicated by SDS-PAGE. rDAC could synthesize c-di-AMP from ATP in vitro. CdaR interacted with DAC and enhanced the activity of DAC (P<0.05). Conclusion DAC encoded by LA3304 gene was a diadenylate cyclase that could convert ATP into c-di-AMP. CdaR promoted the activation of DAC and formed a CdaR-DAC system with DAC. The system was involved in the synthesis of c-di-AMP in L. interrogans. Key words: Leptospira; c-di-AMP; DAC; CdaR

Background. Leptospirosis is a zoonotic disease of great impact and worldwide distribution due to which it appears in an isolated way or in outbreaks, it is common in tropical areas and presents consequences for the health of people, coming to cause including death. This is a febrile illness, caused by bacteria of the genre Leptospira spp infects wild and domestic animals but nevertheless the man it can become infected in contact with contaminated water, urine of these animals and variety of factors as they are exposed throughout the article. Methods. the objective is done a bibliographic review in different databases and languages of the multiple associated epidemiological risk factors to present Leptospira infection, both internationally as a national, including they mean the most outstanding and with a level of significance, some factors of housing and contact with rodents also of different occupations like agriculture. Conclusions. It was found that there are multiple sociodemographic and epidemiological factors associated with Leptospira infection, oriented towards men, perhaps due to the occupational risk, in addition to the rural area origin in Colombia.

Leptospira - Jaundice - soft and coiled.

Objective To investigate the influences of Leptospira interrogans (L.interrogans) infection on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Methods Expression of ICAM-1 and VCAM-1 at mRNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR) after infecting human umbilical vein endothelial cells (HUVEC) with L. interrogans strain Lai. Silver staining was used to detect leptospires in lung, liver and kidney tissues of L. interrogans-infected C3H/HeJ mice. Expression of ICAM-1 and VCAM-1 in lung, liver and kidney tissues of L. interrogans-infected mice was measured with immunohistochemistry. Results L. interrogans infection increased the expression of ICAM-1 and VCAM-1 on HUVEC (P<0.05). Moreover, the expression of VCAM-1 at mRNA level was significantly higher than that of ICAM-1 (P<0.05). Silver-stained leptospires could be found in lung, liver and kidney tissues of L. interrogans-infected C3H/HeJ mice. Results of the immunohistochemical examination showed that increased expression of both ICAM-1 and VCAM-1 could be detected in ling, liver and kidney tissues of L. interrogans-infected mice, and the VCAM-1 level was significantly higher than that of ICAM-1 in every tissue sample (P<0.05). Conclusion L. interrogans infection could induce the expression of ICAM-1 and VCAM-1 on endothelial cells and increase the expression of VCAM-1 to a level significantly higher than that of ICAM-1, which mediated the infiltration of specific inflammatory cells to the site of infection. Key words: Leptospira interrogans; Adhesion molecule; ICAM-1; VCAM-1

Epidemiology of leptospirosis in Yogyakarta and Bantul Purpose The purpose of this study was to determine the relationship knowledge, attitude, and environmental factors with the incidence of leptospirosis. Methods A case-control study was conducted in November 2016 in Yogyakarta city and Bantul district on 242 respondents. The case was leptospirosis patients that recorded in the Yogyakarta and Bantul District health offices within the period of June 2014 to June 2016. Controls were households in the same neighborhood as the case and had no history of leptospirosis, with case and control comparisons 1:1. Rat-catching was done inside the house and in the rice fields. Results Knowledge (OR: 1, 95% CI: 0.58-1.71, p-value: 1) and attitude (OR: 0.8, 95% CI: 0.47-1.40, p-value: 0 .44) had no significant association with leptospirosis incidence. The presence of rats in the home has no significant relationship with leptospirosis incidence (OR: 0.7, 95% CI: 0.15-3.78, p-value: 0.73). The multivariate analysis found that the distance of house to open sewer (OR: 2.96, 95% CI: 1.22-7.14) and the presence of in-house waste (OR: 2.03, 95% CI: 1.14-3.62) had a significant association with leptospirosis incidence. Conclusion There was no statistically significant relationship between knowledge and attitude with leptospirosis incidence. Environmental factors such as the distance of house to open sewer and the presence of in-house waste have a statistically significant relationship with the incidence of leptospirosis. Two of the 4 serum mice examined with a positive MAT method contained Leptospira sp . with Benjamin serovar Benjamini strain.

Objective To analyze the enzymatic properties of alkyl hydroperoxide reductase subunit C (AhpC) from Leptospira interrogans (L.interrogans) and to elucidate its physiological roles in host-pathogen interactions in macrophages during Leptospira infection. Methods A prokaryotic expression system for ahpC gene of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was established to express the recombinant AhpC (rAhpC). After purified by Ni-NTA affinity chromatography, the enzymatic activity of the rAhpC and its role in protecting DNA from oxidation were analyzed. The importance of each cysteine in its molecule was evaluated through site-directed mutation. L. interrogans strains were pretreated with or without Conoidin A, a covalent inhibitor of peroxiredoxin, and then were used to infect macrophages. Changes in oxidative status in leptospires and survival rates of L. interrogans strains were analyzed by fluorescence-activated cell sorting and colony counting method. Results The rAhpC was successfully expressed in the established prokaryotic expression system. It had peroxiredoxin activity that was able to catalyze the reduction of hydrogen peroxide. Its ability of reducing hydrogen peroxide depended on the thioredoxin/thioredoxin reductase system. Cys47 (a peroxidatic cysteine) and Cys167 (a resolving cysteine) were critical to maintaining the enzymatic activity of AhpC. AhpC could protect DNA from hydrogen peroxide induced-oxidative damage. When L. interrogans strains were pretreated with Conoidin A, the oxidative status in leptospires was elevated and the survival of L. interrogans in macrophages was significantly reduced in a dose-dependent manner. Conclusion The AhpC of L. interrogans is a thioredoxin-dependent peroxiredoxin that plays an important role in protecting L. interrogans against oxidative stress in macrophages. Key words: Leptospira interrogans; ahpC gene; Oxidative stress; Survival

Objective To investigate the syphilitic infection condition of outpatients in sexually transmitted diseases (STD) clinics in recent years. Methods A total of 4 162 cases of serum specimen from outpatients of STD clinics were detected by two kinds of serologic syphilitic leptospira test[rapid plasma regain circle card test (RPR) and microhemagglutination assay for antibody to Treponema pallidum (TPPA)]simultaneously. Results Among the 4 126 serum specimens, there were 1 591 cases of syphilis positive sample, and positive rate was 38.23% (1 591/4 162); and there were 2 661 cases had high-risk sexually behavior, which accounted for 63.94% (2 661/4 162) of all selected patients and had syphilis positive rate of 49.79% (1 325/2 661); syphilis infection had correlation with high-risk sexually behavior (χ2=5.047, P 0.05). Conclusions High-risk sexually behavior is the important factor of syphilis infection. All kinds of people should be strengthened the knowledge of syphilis prevention and control to reduce the incidence of syphilis. Key words: Outpatient; Syphilis; Positive rate; Serological test

Data set containing test results of screening blood and serum samples from five populations of Brown Bears in Alaska. Samples were tested to detect current or previous exposure to bacteria (Brucella spp., Francisella tularensis, and Leptospira spp.), viruses (canine adenovirus, canine distemper virus, canine parvovirus, and influenza A virus), and parasites (Toxoplasma gondii, and Trichinella spp.).

Objective To understand and determine the biological activity and pathogenicity of metalloendopeptidases encoded by LA2582 and LA2901 genes of Leptospira interrogans(L.interrogans) serogroup Icterohaemorrhagiaeserovar Lai strain Lai. Methods Structures and functions of LA2582 and LA2901 genes were analyzed by using bioinformatic software. Prokaryotic expression systems for expressing the extracellular regions of LA2582 and LA2901 genes were generated. The target recombinant expression products, rLA2582 and rLA2901, were extracted by Ni-NTA affinity chromatography. The Azo-casein-hydrolyzingactivity of rLA2582 and rLA2901 was detected by spectrophotometry. Activities of rLA2582 and rLA2901 in the hydrolysis of Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans, a fluorescence-labeling pentapeptide substrate, were detected by fluorospectrophotometry, and then the Km and Kcat values were determined. SDS-PAGE and spectrophotometry were performed to detect the activities of rLA2582 and rLA2901 in hydrolyzing extracellular matrix molecules such as collagen type-Ⅰ (COL1), fibronectin (FN) and Congo red-labeling elastin (ELN). Real-time fluorescent quantitative RT-PCR (qRT-PCR) and Western blot were respectively used to measure the expression of LA2582 and LA2901 genes at mRNA and protein levels after infecting human umbilical vein endothelial cells(HUVEC) with L.interrogans strain Lai. Results The gene products of LA2582 and LA2901 genes were identified as the signal peptide and matrix metalloproteinase motif HXH-containing Zn2+ -dependent Gly-Gly metalloendopeptidases belonging to the M23 superfamily. rLA2582 and rLA2901 did not hydrolyze Azo-casein (Km=126.54 μmol/L, Kcat=4.67/s), but could hydrolyze the pentapeptide substrate (Km=190.25 μmol/L, Kcat 4.86/s). rLA2582 and rLA2901 could hydrolyze COL1, FN and ELN. Expression of LA2582 and LA2901 genes at both mRNA and protein levels was significantly increased after infection of HUVEC with L.interrogans strain Lai (P<0.05). Conclusion The products of LA2582 and LA2901 genes of L.interrogans strain Lai are Zn2+ -dependent M23 metalloendopeptidases, which can hydrolyze multiple ECM molecules and are closely associated with the leptospiral invasiveness. Key words: Leptospira interrogans; M23 superfamily; Metalloendopeptidase; Biological activity; Pathogenecity

Objective To investigate the regulatory effects of cyclic diguanylate (c-di-GMP) signaling on CheB and CheR, which were chemotaxis regulatory proteins relating to the motility of Leptospira interrogans. Methods Real-time PCR was used to determine the expression of cheB1, cheB2, cheB3, cheR1 and cheR2 genes at mRNA level during Leptospira interrogans infection. Fragments of these genes were amplified and cloned into the expression vector pET-28a, respectively, to construct the prokaryotic expression system for them. Colony morphologies of Escherichia coli (E.coli) strains that overexpressed the target genes were observed to determine the regulatory effects of c-di-GMP on CheB and CheR. Results The expression of cheB1 gene at mRNA level increased 60 min after infection and reached the peak at 90 min. Compared with the control group, the expression of cheB3 gene at mRNA level were up-regulated, while no significant difference in the expression of cheB2 and cheR genes was observed 60 min after infection. The prokaryotic expression system for the five genes was successfully constructed and the purified proteins were obtained. CheB1, CheB3 and CheR2 improved the motility of E. coli, but that was inhibited by the inhibitor of diguanylate cyclase (DGC) or phosphodiesterase (PDE). Conclusion CheB and CheR regulate the swarming motility of E. coli and are affected by intracellular c-di-GMP. Key words: Leptospira interrogans; Chemotaxis; CheB; CheR; c-di-GMP