Immunodominance of LipL3293-272 peptides revealed by leptospirosis sera and therapeutic monoclonal antibodies.

BACKGROUND/PURPOSE: Leptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL3293-147 and LipL32148-184 middle domain of LipL3293-184, and LipL32171-214, and LipL32215-272 of c-terminal LipL32171-272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera. RESULTS: IgM-dominant was directed to highly surface accessible LipL32148-184 and Lipl32171-214. IgG dominance of LipL32148-184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32148-184 truncation whereas two LipL32148-184 and LipL32215-272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32148-214 and IgG-dominant LipL32148-184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32243-253 and LipL32122-130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera. CONCLUSION: The IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.

The preventable efficacy of ß-glucan against leptospirosis.

Leptospirosis, caused by pathogenic Leptospira species, has emerged as an important neglected zoonotic disease. Few studies have reported the preventable effects of immunoregulators, except for antibiotics, against leptospirosis. Generally, immunostimulatory agents are considered effective for enhancing innate immune responses. Many studies have found that beta-glucan (ß-glucan) could be a potent and valuable immunostimulant for improving immune responses and controlling diseases. In this study, we investigated the preventable role of ß-glucan against Leptospira infection in hamsters. First, ß-glucan was administered 24 h prior to, during and after infection. The results showed that ß-glucan increased the survival rate to 100%, alleviated tissue injury, and decreased leptospire loads in target organs. Additionally, we found using quantitative real-time PCR that application of ß-glucan significantly enhanced the expression of Toll-like receptor (TLR) 2, interleukin (IL)-1ß and iNOS at 2 dpi (days post infection) and reduced the increase of TLR2, IL-1ß and iNOS induced by Leptospira at 5 dpi. Furthermore, to induce memory immunity, ß-glucan was administered 5 days prior to infection. ß-Glucan also significantly increased the survival rates and ameliorated pathological damage to organs. Moreover, we demonstrated that ß-glucan-trained macrophages exhibited elevated expression of proinflammatory cytokines (IL-1ß and IL-6) in vitro, indicating that ß-glucan induces an enhanced inflammatory response against Leptospira infection. These results indicate that administration of ß-glucan and other immunostimulants could be potential valuable options for the control of Leptospira infection.

Advances & challenges in leptospiral vaccine development.

Considerable progress has been made in the field of leptospiral vaccines development since its first use as a killed vaccine in guinea pigs. Despite the fact that the immunity conferred is restricted to serovars with closely related lipopolysaccharide antigen, certain vaccines have remained useful, especially in endemic regions, for the protection of high-risk individuals. Other conventional vaccines such as the live-attenuated vaccine and lipopolysaccharide (LPS) vaccine have not gained popularity due to the reactive response that follows their administration and the lack of understanding of the pathogenesis of leptospirosis. With the recent breakthrough and availability of complete genome sequences of Leptospira, development of novel vaccine including recombinant protein vaccine using reverse vaccinology approaches has yielded encouraging results. However, factors hindering the development of effective leptospiral vaccines include variation in serovar distribution from region to region, establishment of renal carrier status following vaccination and determination of the dose and endpoint titres acceptable as definitive indicators of protective immunity. In this review, advancements and progress made in LPS-based vaccines, killed- and live-attenuated vaccines, recombinant peptide vaccines and DNA vaccines against leptospirosis are highlighted.

Pre-treatment with Lactobacillus plantarum prevents severe pathogenesis in mice infected with Leptospira interrogans and may be associated with recruitment of myeloid cells.

Recent estimates on global morbidity and mortality caused by Leptospirosis point to one million cases and almost 60,000 deaths a year worldwide, especially in resource poor countries. We analyzed how a commensal probiotic immunomodulator, Lactobacillus plantarum, affects Leptospira interrogans pathogenesis in a murine model of sub-lethal leptospirosis. We found that repeated oral pre-treatment of mice with live L. plantarum restored body weight to normal levels in mice infected with L. interrogans. Pre-treatment did not prevent L. interrogans access to the kidney but it affected the inflammatory response and it reduced histopathological signs of disease. Analysis of the immune cell profiles in lymphoid tissues of mice pre-treated with L. plantarum showed increased numbers of B cells as well as naïve and memory CD4+ helper T cell populations in uninfected mice that shifted towards increased numbers of effector CD4+ helper T in infected mice. CD8+ cytotoxic T cell profiles in pre-treated uninfected and infected mice mirrored the switch observed for CD4+ except that CD8+ memory T cells were not affected. In addition, pre-treatment led to increased populations of monocytes in lymphoid tissues of uninfected mice and to increased populations of macrophages in the same tissues of infected mice. Immunohistochemistry of kidney sections of pre-treated infected mice showed an enrichment of neutrophils and macrophages and a reduction of total leucocytes and T cells. Our results suggest that complex myeloid and T cell responses orchestrate the deployment of monocytes and other cells from lymphoid tissue and the recruitment of neutrophils and macrophages to the kidney, and that, the presence of these cells in the target organ may be associated with reductions in pathogenesis observed in infected mice treated with L. plantarum.

[A Case of Leptospirosis in which the Causative Pathogen was Detected Using Cerebrospinal Fluid PCR Eight Days after Onset].

We report a patient with leptospirosis caused by infection with Leptospira interrogans serovar Rachmati. A 30-year-old Japanese man took part in a survival camp on Iriomote Island, Okinawa, from July 9 to July 15, 2014. During the camp, he swam in the river and kayaked. He developed a high fever and fatigue 7 days after completing his trip and was admitted to our hospital on July 22. On admission, he complained of a posterior cervical pain and a loss of appetite. Laboratory findings revealed granulocytosis, mildly elevated AST and ALT levels, elevated BUN and Cr levels, and a significantly elevated CRP level. No pathogenic bacteria were isolated from blood, urine, or cerebrospinal fluid cultures. We included leptospirosis in the differential diagnosis because of the patient's history of participating in a survival camp on Iriomote Island. Minocycline 200 mg, p.o. showed an excellent efficacy. The Leptospira flagellar gene FlaB was detected using a cerebrospinal fluid PCR. A microscopic agglutination test (MAT) during the convalescent stage demonstrated significant increases in antibodies against L. interrogans serovar Rachmati, confirming the diagnosis of leptospirosis. A medical history including occupation and recent travel history, and an adequate specimen sampling are crucial for the accurate and early diagnosis of leptospirosis.

Work Environment-Related Risk Factors for Leptospirosis among Plantation Workers in Tropical Countries: Evidence from Malaysia.

BACKGROUND: Leptospirosis is a zoonotic disease that is recognized as a re-emerging global public health issue, especially in tropical and subtropical countries. Malaysia, for example, has increasingly registered leptospirosis cases, outbreaks, and fatalities over the past decade. One of the major industries in the country is the palm oil sector, which employs numerous agricultural workers. These laborers are at a particularly high risk of contracting the disease. OBJECTIVE: To identify the work environment-related risk factors for leptospirosis infection among oil palm plantation workers in Malaysia. METHODS: A cross-sectional study involving 350 workers was conducted. The participants were interviewed and administered a microscopic agglutination test. Seropositivity was determined using a cut-off titer of ≥1:100. RESULTS: 100 of 350 workers tested positive for leptospiral antibodies, hence, a seroprevalence of 28.6% (95% CI 23.8% to 33.3%). The workplace environment-related risk factors significantly associated with seropositive leptospirosis were the presence of cows in plantations (adjusted OR 4.78, 95% CI 2.76 to 8.26) and the presence of a landfill in plantations (adjusted OR 2.04, 95% CI 1.22 to 3.40). CONCLUSION: Preventing leptospirosis incidence among oil palm plantation workers necessitates changes in policy on work environments. Identifying modifiable factors may also contribute to the reduction of the infection.

Immunomodulatory treatment with thalidomide in experimental leptospirosis in Golden Syrian hamsters (Mesocricetus auratus).

BACKGROUND: The benefit of antibiotics in leptospirosis is limited when treatment is started four days after symptoms appear, and new adjuvant therapeutic options are urgently needed. METHODS: Hamsters (Mesocricetus auratus) were infected by Leptospira interrogans strain L1-130, and groups were assigned based on no treatment (NONE), thalidomide only (TAL), ampicillin only (AMP) or both (AMP-TAL). Treatment was started two days after the onset of symptoms (experiment 1) and immediately after detection of the first death (experiment 2). RESULTS: Experiment 1: all hamsters from the groups AMP and AMP-TAL survived (n=8), while all hamsters from groups NONE (n=6) and TAL (n=8) died. The AMP and the AMP-TAL groups showed no renal or liver pathology and absent or very low leptospiral burden in target organs. Experiment 2: lethal outcome was observed in 6/6 hamsters in the NONE group, 8/8 in the TAL group, and 6/8 in both the AMP and AMP-TAL groups. Thalidomide showed no survival benefit when compared to hamsters treated with ampicillin alone. The TAL, AMP and AMP-TAL groups had very low tissue leptospiral counts. CONCLUSION: Thalidomide had minimal impact on survival in the late treatment of leptospirosis hamster model.

Duration of immunity of a multivalent (DHPPi/L4R) canine vaccine against four Leptospira serovars.

Despite effective vaccines against common Leptospira serovars, the development of new products with long duration of immunity is still important to protect dogs against leptospirosis. The results from four challenge studies performed one year after vaccination of dogs with a multivalent vaccine containing four Leptospira antigens are reported. Six week old dogs received two vaccinations, three weeks apart, and were challenged 367 days later. Clinical observations were recorded, while blood (culture, biochemistry and haematology), urine (culture) and liver and kidney (culture) samples were collected throughout the study or at necropsy. All control dogs remained seronegative until challenge, when they seroconverted. Antibody titres to Leptospira antigens were seen in vaccinated dogs 21 days after first vaccination and peaked three to six weeks after the second vaccination. Titres decreased in all studies over the following 12 months, until challenge when anamnestic responses were observed. In all studies control dogs demonstrated various abnormal clinical signs, while no vaccinated dogs were affected; differences between groups were only significant following L. bratislava challenge. Analysis of blood cultures showed all control and five of the 24 vaccinated dogs were Leptospira positive after challenge; all studies showed significant differences between treatment groups in mean number of days with positive cultures. Significant differences between vaccinated and control groups in mean number of days with positive urine cultures were also observed, with all non-vaccinated and one vaccinated dog Leptospira positive. The urine culture positive vaccinated dog also gave positive culture from kidney and liver samples. All except one control dog also showed positive Leptospira isolation from kidney or liver, with significant differences between vaccinated and control groups observed. The results demonstrate that administration of a new vaccine to six week old puppies induces immunity which is still effective up to one year later as demonstrated by challenge.

Increased Immunogenicity to LipL32 of Leptospira interrogans when Expressed as a Fusion Protein with the Cholera Toxin B Subunit

Leptospirosis is one of the most widespread zoonosis in the world. The development of a recombinant leptospira vaccine remains a challenge. In this study, we cloned the Leptospira interrogans open reading frame (ORF) coding the external membrane protein LipL32, an immunodominant antigen found in all pathogenic leptospira, downstream of the highly immunogenic cholera toxin B subunit (CTB) ORF. Expression and assembly of the CTB-LipL32 fusion protein into oligomeric structures of pentameric size were observed in soluble fractions by Western blot analysis. The CTB-LipL32 protein demonstrated strong affinity for monosialotetrahexosylgaglioside (GM1-ganglioside) in an enzyme-linked immunosorbent assay (ELISA), suggesting that the antigenic sites for binding and proper folding of the pentameric CTB structure were conserved. Furthermore, antisera against LipL32 also recognized the CTB-LipL32 fusion protein, suggesting that LipL32 also conserved its antigenic sites, a fact confirmed by an ELISA assay showing soluble CTB-LipL32 recognition by sera from convalescent patients. In addition, soluble CTB-LipL32 generated higher specific titers in mice immunized without external adjuvant than co-administration of CTB with LipL32. The data presented here provide support for CTB-LipL32 as a promising antigen for use in the control and study of leptospirosis.

Evaluation of colostral immunity in swine with commercial anti-leptospira polyvalent whole-bacteria vaccine.

The intensity and duration of passive immunity against swine leptospirosis were investigated by the microscopic agglutination test and in vitro leptospira growth inhibition. Twenty-one females at first parturition were divided into three groups: Group A (n=08): received two doses with 30 days interval of the commercial anti-leptospira bacterin A. Group B (n=06) received two doses with 30 days interval of the commercial anti-leptospira bacterin B and Group C (n=07) was the control. In all groups the colostrums were collected. Blood collection of piglets was performed in four different ages. Agglutinin antibodies were equally detected in sera and colostrums for serovars Canicola, Grippotyphosa, Copenhageni, Icterohaemorrhagiae and Pomona (Group A) and Canicola, Copenhageni, Icterohaemorrhagiae, Pomona and Hardjo (Group B). Mean neutralizing antibodies titers were low. Passive immunity was low duration.

[Evaluation of an homologous bacterin against bovine leptospirosis]. / Evaluación de una bacterina homóloga contra la leptospirosis bovina.

48 adult bovine females dividided into 6 groups were used aimed at characterizing the immune response induced in breastfeeeding cows by an homologous bacterin formulated with different adjuvants. They were intramuscularly administered 2 milliliters of a bacterin formulated with Leptospira interrogans serovars uam, wolffi, hardjo, bratislava, grippotyphosa and panama added with different adjuvants, such as aluminum hydroxide, Freud's complete adjuvant, Freud's incomplete adjuvant, liposoluble vitamins, bacterin plus disparasitization with levamisol. The control group was administred only with bacterin. Immunization took place in 2 occasions at a time interval of 28 days. Blood samples were taken every 7 days during the first month after vaccination, and every 28 days for the next 8 months. All the sera were analyzed by the microscopic agglutination test. The results were transformed into Log10 and they were analyzed by NLIN and GLM of SAS. The period of greater response was estimated by the prediction model (Wood). The bacterin did not produce alteration either in the physiological constants, or in milk production. The serovars of Leptospira interrogans that induced higher titers were uam, hardjo and wolffi. The statistical difference between treatments and between serovars was determined.

Respuesta serológica por ELISA y MAT en voluntarios cubanos vacunados con vax SPIRAL® / Serological response by ELISA and MAT in Cuban volunteers vaccinated with vax SPIRAL@

Se estudió la seroconversión en 408 individuos vacunados y 135 placebo, incluidos en 2 ensayos clínicos de la vacuna cubana contra la leptospirosis humana. Se estudiaron 2 dosis vacunales y 5 esquemas. Seroconvirtieron de 38 vacunados (Fase I), 11 (29 por ciento) por MAT y 12 (32 por ciento) por ELISA, y de 33 placebo, 2 (6 por ciento) y 3( 9 por ciento) respectivamente. En la Fase II de 68 vacunados (dosis de 0,25 mL) y de 65 (dosis de 0,50 mL), seroconvirtieron 21 (31 por ciento) y 16 (25 por ciento) por ELISA respectivamente, por MAT 9 (13 por ciento) y 7 (11 por ciento) individuos fueron positivos. No hubo diferencias significativas entre las dosis utilizadas. La seroconversión por MAT, en 237 individuos vacunados con diferentes esquemas, fue de 22,4 por ciento, no existiendo diferencias significativas entre estos. En la mayoría de los individuos reactivos, se encontraron niveles de anticuerpos al menos a una de las cepas vacunales. Se recomendó, buscar y evaluar otros métodos para demostrar in vivo, el nivel de protección de esta vacuna(AU)

Neumonía por leptospira, presentación de dos casos / Pneumonia for leptospira, presentation of two cases

Se describen dos casos de neumonía donde los estudios serológicos y epidemiológicos confirmaron una leptospirosis. Se analizan los síntomas y signos de los pacientes, los exámenes complementarios y los resultados de los sueros pareados de leptospira. Se recomienda que se debe estudiar más frecuentemente los cuadros respiratorios agudos con medios diagnósticos de leptospirosis

Use of doxycycline for leptospirosis after high-risk exposure in São Paulo, Brazil.

A clinical trial pilot study, double-blinded, randomized, and controlled with a placebo to assess the effectiveness of oral doxycycline (200 mg, single dose) in preventing leptospirosis after high exposure to potentially contaminated water was performed in São Paulo, SP, Brazil. Confirmed cases were defined as those with leptospira IgM antibody and symptoms; asymptomatic cases were those presenting with IgM antibodies but no symptoms; and suspected cases were individuals with symptoms but no IgM antibody. Forty subjects were given doxycycline and 42 were given placebo. In the drug-treated group there were 2 confirmed cases, 11 asymptomatic cases, and 6 suspected cases. In the placebo group there were 5 confirmed, 6 symptomatic, and 5 suspected cases. Even though we found a protective association of doxycycline for confirmed leptospirosis cases (RR = 2.3) and seroconversion only (RR = 2.0), the association was not statistically significant because of the small number of individuals enrolled in this pilot study. We observed that the 22% of the volunteers already had IgM antibodies to leptospirosis at the first sampling. Finally, the attack rate to confirmed, asymptomatic, and suspected cases of Leptospirosis was 8.5%, 22%, and 13%, respectively, in this population.

Effect of streptomycin treatment on the shedding of and the serologic responses to Leptospira interrogans serovar hardjo subtype hardjobovis in experimentally infected cows.

Shedding patterns of and serologic responses to Leptospira interrogans serovar hardjo subtype hardjobovis (L. hardjobovis) have been studied in experimentally infected cows treated with streptomycin in comparison to experimentally infected cows receiving no such treatment. Fourteen cows were experimentally infected with L. hardjobovis, and blood and urine samples were collected weekly for 24 weeks. The microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA) were used to determine serologic responses. The polymerase chain reaction (PCR) was used to determine bacterial shedding in urine. Six weeks after infection six cows were treated with the antibiotic streptomycin (25 mg/kg body weight/day); three cows were treated only once, and the remaining three were treated for five consecutive days. After treatment all six cows had lower serologic responses compared to the untreated cows. The treated cows became also PCR-negative two days after the first treatment, whereas the eight untreated cows remained PCR-positive for at least 70 days. Cows that stopped shedding did not resume shedding within the observation period. Since streptomycin treatment reduces the period of shedding, transmission of leptospira via contaminated urine might be prevented by a single treatment of an infected herd.

Limulus lysate positivity and Herxheimer-like reactions in leptospirosis: a placebo-controlled study.

Jarisch-Herxheimer reactions are characteristic of some spirochetal diseases and have been reported in leptospirosis, but their pathogenesis and relationship to endotoxin remain unclear. Serial limulus amebocyte lysate assays (LAL) for endotoxin were performed on 40 patients with proven leptospirosis who were monitored for reactions after receiving either intravenous penicillin (24) or saline placebo (16). No Herxheimer-like reactions were observed, although 78% of patients had at least one positive LAL. Serum creatinine, serum bilirubin, and white blood cell counts were significantly higher (P less than .01) in simultaneously drawn LAL-positive specimens than in negative ones. Delayed hepatic clearance of endotoxin due to liver dysfunction may explain the high LAL positivity rate, since assay results correlated with severity of disease but not with the presence or absence of spirochetes. Fear of a Herxheimer-like reaction should not dissuade clinicians from administering antibiotics to patients with leptospirosis.

Effect of maternal vaccination on the susceptibility of growing pigs to leptospiral infection.

Serum samples were collected from 30 piglets, derived from 17 litters, whose dams had been vaccinated against leptospirosis. Microscopic agglutination test (MAT) titres against Leptospira interrogans serovar pomona varied greatly from pig to pig; there was less variation among littermates. Titres declined between 4 and 10 weeks of age, with an uncorrected half-life of 15.5 days, consistent with IgG being the main antibody class involved. Twelve pigs, 4 derived from unvaccinated sows and 8 from sows vaccinated against leptospirosis, were challenged intravenously at 8 weeks of age with leptospires of serovar pomona. Colostrum-derived antibody protected 4 out of 8 pigs, and in 1 of the remaining 4 the serological response was reduced. Three of the protected pigs showed reduced serological responses and in the fourth the response was strong, but delayed. All of the pigs derived from unvaccinated sows developed leptospiraemia and leptospiruria and showed strong serological responses. Protection by colostrum-derived antibody bore an inexact relationship to MAT titre, but a titre of 16 appeared to be sufficient for protection.

Effect of cyclophosphamide treatment on clinical and serologic response of rats to infection with Leptospira interrogans serovar icterohaemorrhagiae.

Young albino rats were immunosuppressed with cyclophosphamide and exposed to virulent and low-virulence Leptospira interrogans serovar icterohaemorrhagiae 820K. In rats exposed to virulent and low virulence leptospires, microscopic agglutinating antibody responses occurred later, and longer leptospiremic phase and more massive tissue invasion by the organisms were observed in immunosuppressed rats than in immunocompetent controls. Clinical and pathologic signs of illness were more severe in the immunosuppressed animals than in immunocompetent controls. When exposed to low-virulence leptospires, immunosuppressed rats became infected and developed signs of illness and 2 of 16 died. Immunocompetent rats rapidly developed a humoral response and did not develop any signs of illness, and with 1 exception, organisms were not recovered from any tissues.