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Abstract This study evaluated the seropositivity of Brucella abortus and Leptospira interrogans in ewes with reproductive disturbances in southern Brazil and verified the creatine kinase (CK) activity and oxidation status via assessment of superoxide dismutase, glutathione peroxidase and glutathione transferase in serum of seropositive animals for L. interrogans serovar Icterohaemorrhagiae. For Leptospira infection 381 animals with clinical history of reproductive disturbance from Planalto Serrano de Santa Catarina (Brazil) were analyzed, showing an occurrence for L. interrogans of 20.2% from which 81.8% were seropositive for L. interrogans Icterohaemorrhagiae. Serovars Wolfii, Grippothyphosa, Bratislava, Canicola and Butembo were also identified. In the case of B. abortus, positive cases were identified by buffered acidified antigen, finding 14 positive samples, but none of them were positive after a second test (2-mercaptoethanol), showing the absence of relationship between infection with B. abortus and abortion in the tested individuals. Serum reactive oxygen species (ROS) levels and CK activity were found higher in animals positive for Leptospira infection, presenting higher titrations (1:320) than non-infected individuals. Serum glutathione peroxidase activity was higher in positive animals with titrations 1:160 and 1:320, while serum glutathione S-transferase was higher in positive individuals only for titrations 1:320. Serum superoxide dismutase showed lower activity in infected animals with titrations of 1:320. Our results show the region of Planalto Serrano de Santa Catarina with a high occurrence levels of sheep infected by L. interrogans serovar Icterohaemorrhagiae, from which animals with high titrations (1:320) present oxidative stress elicited by excessive ROS production, triggering the stimulation of antioxidant systems to counter this excess. In summary, ovine with higher titrations (1:320) present oxidative damage that can contribute to disease pathophysiology. Highlights • No sheep were found to be positive for Brucella abortus. • High incidence of sheep infected by L. interrogans serovar Icterohaemorrhagiae. • Case of animals with highest titrations (1:320) presented an increased oxidative stress. • Seropositive ewes has excessive ROS production, and stimulation of the antioxidant system in order to counteract this excess. [ABSTRACT FROM AUTHOR]

Abstract Leptospirosis is an important zoonotic disease and, in urban areas, rodents are considered the main reservoir of Leptospira to human hosts. It has been described that capybaras, the world largest rodent, also harbor and shed leptospires by urine. Although not virulent to their hosts, strains of rodent origin are virulent for the hamster. In this context we aim to investigate the virulence of Leptospira kirschneri strains of serogroup Icterohaemorrhagiae recovered from capybaras in Brazil in the hamster model. Five isolates of Leptospira recovered from asymptomatic capybaras were submitted to virulence tests following the suggested protocols and the 3Rs policy for experimental science. Briefly, 1 ml of 1 × 108 leptospires was inoculated intraperitoneally four times in one hamster for each strain. Four days after inoculation, a blood sample was collected via the gingival route for confirmation of blood culture infection. The infected animals were kept isolated in microisolators to observe clinical signs and monitored daily till day 21 post-inoculation. None strain caused acute disease in hamsters but were able to colonize their kidneys. The present study demonstrated that although Icterohaemorrhagiae strains are often reported as virulent, not all strains of that serogroup are indeed aggressive. Concluding, we report that strains of L. kirschneri serogroup Icterohaemorrhagiae recovered from healthy capybaras presented an atypical virulence to the hamster model, what reinforces that virulence is an intrinsic strains characteristic. Highlights • Not all strains of serogroup Icterohaemorrhagiae are indeed aggressive. • L. kirschneri serogroup Icterohaemorrhagiae infects hamster but do not lead to death. • Virulence of strains is not directly related to of the same serogroup. [ABSTRACT FROM AUTHOR]

Leptospira enters humans and animals through injured skin or mucous membranes by direct or indirect contact with urine excreted from infected reservoirs. Individuals with cut or scratched skin are at high risk of infection and are recommended to be protected from contact with Leptospira , but the risk of infection via skin without apparent wounds is unknown. We hypothesized that the stratum corneum of the epidermis might prevent percutaneous invasion of leptospires. We established a stratum corneum deficient model of hamsters using the tape stripping method. The mortality rate of hamsters lacking stratum corneum that were exposed to Leptospira was higher than that of controls with shaved skin, and was not significantly different from an epidermal wound group. These results indicated that the stratum corneum plays a critical role in protecting the host against leptospiral entry. We also examined the migration of leptospires through the monolayer of HaCaT cells (human keratinocyte cell line) using Transwell. The number of pathogenic leptospires penetrating the HaCaT cell monolayers was higher than that of non-pathogenic leptospires. Furthermore, scanning and transmission electron microscopic observations revealed that the bacteria penetrated the cell monolayers through both intracellular and intercellular routes. This suggested that pathogenic Leptospira can migrate easily through keratinocyte layers and is associated with virulence. Our study further highlights the importance of the stratum corneum as a critical barrier against the invasion of Leptospira found in contaminated soil and water. Hence, preventative measures against contact infection should be taken, even without visible skin wounds. • Hamsters without stratum corneum exposed to Leptospira showed a high mortality rate. • Pathogenic Leptospira penetrated the HaCaT cell, human keratinocyte, monolayer. • Leptospira would be able to pass through the epidermis without the stratum corneum. • Skin should be covered to prevent leptospirosis, even if there are no visible wounds. [ABSTRACT FROM AUTHOR]

Leptospirosis is a disease that negatively affects the productive and reproductive indices of ruminants. Sheep are considered highly resistant to infection, although susceptibility may vary among breeds. Thus, the aim of the present study was to analyze the susceptibility between sheep breeds to the experimental infection by leptospires of the Pomona serogroup. Pomona serogroup, Kennewicki serovar strain (1 × 107 bacteria) was inoculated via the conjunctival route in 12 sheep divided into two groups, one comprising Santa Inês ewes and the other comprising crossbred sheep. In each group, five ewes were challenged with the bacterial strain and one was used as control. All sheep were monitored for 60 days, during which blood samples were collected for serological diagnosis and urine and vaginal fluid samples for molecular and microbiological analyses. Finally, as ewes were submitted to euthanasia and necropsy, some tissues of interest were collected for microbiological, molecular, and histopathological diagnoses. The groups were compared regarding the number of positive reactions according to diagnostic tests. All sheep in each group presented antibodies to Leptospira in all serological analyses, except animals of the control group. However the Santa Inês sheep presented higher concentration and duration of the titers, and their positive reactions were detected earlier than those in crossbred sheep. The antibody titers in group A (median 200, geometric mean 317.48) were significantly different from the group B (median 800, geometric mean 918.96) at D60 post-infection ( P  = 0,032). The Santa Inês sheep presented a higher number of positive reactions than did the crossbred sheep in the molecular diagnostic tests. According to the molecular diagnosis, the Santa Inês sheep presented more reactions (urine and vaginal fluid) compared to crossbred ewes, but there was no predominance in the detection of leptospiral DNA when comparing urine and vaginal fluid results, nor even between the number of positive kidneys and uterus. The Santa Inês sheep presented a higher number of positive bacteriological cultures. No sheep in either group presented alterations in anatomopathological and histopathological findings. Pure-bred sheep may be more susceptible than crossbred sheep to infection by Leptospira sp. Our findings emphasize the importance of the genital tract as a site of extraurinary infection and indicate the possibility of venereal transmission in the species. [ABSTRACT FROM AUTHOR]

Good vaccines should confer protection against specific pathogens in experimental and field conditions. However, some commercial vaccines are not capable to confer protection to animals, being inefficient in a bovine vaccine program. In this sense, the aim of this study was to evaluate the antibody levels involved in the immune response in vaccinated cows against leptospirosis, as well as acute phase protein and the immunological markers in a vaccine program in beef cattle. Twenty non-lactating cows, negative for leptospirosis and without vaccination against this disease were evaluated during five months. The herd was divided into two groups named as A (the control group) and B (the vaccinated group). Ten cows from the group B received an initial dose (5 mL) of vaccine on day 0 and one booster dose (5 mL) on day 29. In order to evaluate humoral response (MAT – titration 1:25), cytokine levels (tumor necrosis factor (TNF) and interleukin-10 (IL-10)), and C-reactive protein levels (an acute phase protein), blood samples were evaluated on days 0, 29, 40, 83 and 144 after vaccination. In none of the evaluated periods it was observed specific antibodies to any of the six serovars presents in the vaccine, as well as no difference between groups regarding cytokine and C-reactive protein levels. Therefore, the vaccine used did not stimulate the immune response of cattle, inferring the absence of protection against infections by L. interrogans. [ABSTRACT FROM AUTHOR]

Leptospirosis is a severe worldwide zoonotic disease caused by pathogenic Leptospira spp. It has been demonstrated that pathogenic leptospires are resistant to the bactericidal activity of normal human serum while saprophytic strains are susceptible. Pathogenic strains have the ability to bind soluble complement regulators and these activities are thought to contribute to bacterial immune evasion. One strategy used by some pathogens to evade the complement cascade, which is not well explored, is to block the terminal pathway. We have, thus, examined whether leptospires are able to interact with components of the terminal complement pathway. ELISA screening using anti-leptospires serum has shown that the pathogenic, virulent strain L. interrogans L1-130 can bind to immobilized human C8 (1 μg). However, virulent and saprophyte L. biflexa strains showed the ability to interact with C8 and C9, when these components were employed at physiological concentration (50 μg/mL), but the virulent strain seemed more competent. Lsa23, a putative leptospiral adhesin only present in pathogenic strains, interacts with C8 and C9 in a dose-dependent mode, suggesting that this protein could mediate the binding of virulent Leptospira with these components. To our knowledge, this is the first work reporting the binding of Leptospira to C8 and C9 terminal complement components, suggesting that the inhibition of this pathway is part of the strategy used by leptospires to evade the innate immunity. [ABSTRACT FROM AUTHOR]

Leptospirosis causes severe clinical signs more frequently in men than in women, but the mechanism underlying the gender differences in leptospirosis remains unclear. In this study, petechial hemorrhage was observed in male but not in female hamster lung tissues infected with Leptospira interrogans serovar Hebdomadis at 120 h pi, demonstrating that male hamsters were more susceptible to the development of a severe disease upon Leptospira infection. No leptospiral DNA was detected in the lung tissues at 120 h pi when pulmonary hemorrhage was observed, indicating that pulmonary hemorrhage is attributable to the immune reactions of the host rather than from the direct effect of leptospires. The upregulation of nitric oxide synthase genes in the hamsters without pulmonary hemorrhage, inos and enos in female hamsters at 96 h pi and enos in male animals without hemorrhage at 120 h pi, may suggest that nitric oxide has a suppressive effect on leptospirosis-associated pulmonary hemorrhage. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the behavior of erythrocyte and platelet, as immunological markers, as well as evaluate the involvement of these factors in hemolytic and hemorrhagic reactions in hamsters experimentally infected by Leptospira interrogans Serovar Canicola. Our experimental design was composed by two randomized groups: Infected Group (IG) (n = 12) and control group (CG) (n = 6). Ninety-six hours after the inoculation, the presence of immunoglobulins (IgG and IgM) and complement C3 levels, related to erythrocytes and platelets, was assessed. Platelet's microparticles marked by CD61, reticulocytes and reticulated platelets were also quantified. Additionally, fibrinogen, prothrombin time, partially activated thromboplastin time and sera levels of IgG and IgM were assessed. Our results showed that levels of platelet decreased in IG (P < 0.001); as well as, there was presence of IgG and C3 associated with erythrocyte surface in the infected animals (P < 0.01, P < 0.05, respectively). Levels of prothrombin time and Activated Partial Thromboplastin Time were increased, while fibrinogen level was decreased (P < 0.01) in IG. CD61 microparticles were higher (P < 0.05) in IG due to platelet activation. Thus, it was established a positive correlation (P < 0.01) between platelets count and fibrinogen (Figure 3, R = 0.84, P < 0.001). Therefore, the platelet consumption component was preponderant in relation to autoimmune causes. Finally, regarding the erythrocytes, the autoimmune component played an important role, did not causing hemolytic reaction in this acute experimental time. [ABSTRACT FROM AUTHOR]

Bovine Genital Leptospirosis (BGL) is an important syndrome that leads to reproductive failures. The present study aimed to perform a molecular analysis of Leptospira spp. identified from genital and urine samples from in vivo naturally infected cows with poor reproductive performance. A total of 48 cows destined for culling due to low reproductive efficiency were selected and submitted to sampling. Uterine fragments, cervicovaginal mucus (CVM), and urine were collected from all of the cows and processed for culturing and PCR. One isolate was recovered from the uterus of one cow. Other 25 animals were PCR-positive, totaling 26 positive cows. Of them, 18 animals were positive in lipL 32-PCR to genital samples, while only seven animals were positive in urine. From those, sequencing of sec Y gene was performed. Of the 21 good sequences obtained, 16 were L. interrogans, two were L. noguchii, two were L. santarosai and one was L. borgpetersenii. In order to evaluate the genetic similarity of sequences found herein and other sequences from bovines worldwide, a phylogenetic analysis and haplotype networks were performed. Cows with reproductive failures had a significant association (p < 0.05) with positive PCR of genital samples when compared to PCR of urine. None of the animals were positive for genital samples and urine simultaneously. A high diversity of leptospiral strains were found, even in animals of the same epidemiological region. Haplotype networks of L. interrogans showed clusters of sequences from the uterus and CVM with high similarity to other genital sequences originating from previous studies. L. borgpetersenii haplotype networks presented two major clusters with high similarity, even from worldwide sequences, while L. santarosai showed clusters with high genetic distances, even with all the sequences being from Brazil. This study reinforces the theory that BGL and renal infection are distinct diseases, as well as, genital samples are crucial for the diagnosis of cows with reproductive failures caused by leptospires. In addition, haplotype networks confirmed a high genetic similarity between sequences from the present study and Sejroe strains, reinforcing Sejroe strains as the main BGL agents. • Subfertility is significantly associated with genital PCR positivity. • Sequencing demonstrated a high diversity of leptospiral strains, mostly Sejroe. • Genital samples are a lot more reliable than urine for diagnosing subfertility. • This study reinforces that BGL and renal infection are distinct diseases. [ABSTRACT FROM AUTHOR]

Salmonella is a momentously zoonotic and food-borne pathogen that seriously threats human and animal health around the world. Salmonella pathogenicity is closely related to its virulence genes profile. However, conventional virulence gene analysis methods cannot truly reveal whole virulence genes carried by Salmonella. In this study, whole genome sequencing in combination with Virulence Factor Database were applied to investigate whole virulence gene profiles of 243 Salmonella isolates from animals and humans in China from 2004 to 2019. The results showed that a total of 670 virulence genes were identified in Salmonella , among them, 319 virulence genes were found in all the Salmonella tested isolates, and 9 virulence genes were unique to Salmonella. The 670 virulence genes were classified into 14 categories according to their functions, and the genes related to adherence, effector delivery system, immune modulation, motility and nutritional/metabolic factors accounted for 84.63%. Relationships between virulence genes and serovars, sequence types indicated that strains belonged to the same serovar or sequence type had similar virulence genes profiles, however, isolates from different sources, years and locations of isolation had variable virulence gene profiles. In addition, copy number of virulence genes and homologous virulence genes shared with other pathogens were also analyzed in this study. In summary, we investigated pan-genomic virulence gene profiles and molecular epidemiology of Salmonella isolates from humans and animals in China from 2004 to 2019. These findings are beneficial for pathogenic monitoring, investigation of virulence evolution as well as prevention and control of Salmonella. • 670 virulence genes were identified in Salmonella , and 319 virulence genes accounted for 100%, 9 virulence genes were unique. • The adhesion related and type III secretory system virulence genes accounted for the largest proportion (84.63%). • Isolates of the same serovar had similar virulence gene profiles, and sources of isolation affectedvirulence gene profiles. • Numerous virulence genes shared with other pathogens and gene copy number heterogeneity was also manifested. [ABSTRACT FROM AUTHOR]

Reproductive tract is an important site of infection for chronic leptospirosis and cooperate in pathogenesis of reproductive failure, leading to economic losses. Since serology techniques cannot detect chronic carriers, the molecular analysis of clinical samples is an alternative to detect these animals on livestock. The aim of the present study was to perform a retrospective study in order to detect leptospiral uterine carriers in slaughtered cows. Tissue samples were collected from 50 post-pubertal, nonpregnant cows. These samples were fixed in 10% buffered formalin, paraffin-embedded and stored. PCR targeting lip L32 gene and molecular characterization by sec Y sequence was performed. Leptospiral DNA was identified in 18% (9/50) examined blocks. Two sequences were characterized as L. interrogans . These findings suggest that the presence of infectious leptospires in uterus is associated with the physiopathogenesis of the reproductive failure. [ABSTRACT FROM AUTHOR]

Studies related to the prevalence of leptospirosis in the semiarid region showed that even during long periods of drought, the disease has a remarkable frequency in herds in the region. It is a neglected disease and the extent of its effects in the Brazilian semiarid region is not known. The dynamics of this agent is well studied in the urinary tract, however, there are not many studies regarding the genital tract in female goats. Observing this scenario, the present work aimed to diagnose Leptospira spp. in female goats kept in the Brazilian semiarid region by means of serological, molecular and isolation techniques. Blood samples, vaginal fluid, urine and fragments of organs from the genitourinary tract were collected from 40 goats destined for slaughter. Microscopic agglutination test (MAT) was used as a serological technique, with a battery of 24 serovars. The Polymerase Chain Reaction (PCR) of the vaginal fluid, urine and organ fragments was performed, as well as the bacterial growth of these same products in a selective medium. Isolation positive samples were subjected to PCR. It was observed that two (5%) animals were serologically positive for the Pyrogenes serogroup. A total of 29 (72.5%) animals were PCR positive, with DNA present in 51/160 (31.8%) samples from the genital tract and 34/120 (28.3%) from the urinary tract, with no statistical difference. For bacterial growth, 22/40 (55%) animals were positive for growth, with morphology being observed in 19/160 (11.8%) for the genital tract and 16/120 (13.3%) for the urinary tract, with no statistical difference. Two uterus samples showed 99% similarity with L. interrogans after sequencing. Thus, female goats kept under semiarid conditions were positive for Leptospira spp, with positive samples from both the urinary and genital tracts, which possible is an alternative way of adapting and maintaining the agent for severe and adverse conditions. • Leptospira spp. is present in the genital tract of female goats. • Genital tract is an extrarenal site for female goats. • Two uterus samples had 100% similarity with L. interrogans. [ABSTRACT FROM AUTHOR]

Leptospira interrogans ( L. interrogans ), a worldwide zoonosis, infect humans and animals. In dogs, four syndromes caused by leptospirosis have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups), and also it caused inflammation. Extracellular matrix (ECM) is a complex mixture of matrix molecules that is crucial to the reproduction. Both inflammatory response and ECM are closed relative to reproductive. The aim of this study was to clarify how L. interrogans affected the uterus of dogs, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans . In the present study, 27 dogs were divided into 3 groups, intrauterine infusion with L. interrogans , to make uterine infection, sterile EMJH, and normal saline as a control, respectively. The uteruses were removed by surgical operation in 10, 20, and 30 days, respectively. The methods of histopathological analysis, ELISA, Western blot and qPCR were used. The results showed that L. interrogans induced significantly inflammatory responses, which were characterized by inflammatory cellular infiltration and high expression levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in uterine tissue of these dogs. Furthermore, L. interrogans strongly down-regulated the expression of ECM (collagens (CL) IV, fibronectins (FN) and laminins (LN)) in mRNA and protein levels. These data indicated that strongly inflammatory responses, and abnormal regulation of ECM might contribute to the proliferation of dogs infected by L. interrogans . [ABSTRACT FROM AUTHOR]

Sporotrichosis is a cosmopolitan mycosis caused by pathogenic species of Sporothrix genus, that in Brazil is often acquired by zoonotic transmission involved infected cats with S. brasiliensis. Previous studies showed that the Sporothrix spp. recombinant enolase (rSsEno), a multifunctional protein with immunogenic properties, could be a promising target for vaccination against sporotrichosis in cats. Nevertheless, the considerable sequence identity (62%) of SsEno with its feline counterpart is a great concern. Here, we report the identification in silico, chemical synthesis and biological validation of six peptides of SsEno with low sequence identity to its cat orthologue. All synthesized peptides exhibit B-cell epitopes on the molecular surface of SsEno and proved to be highly reactive with the serum of infected mice with S. brasiliensis and sera of cats with sporotrichosis. Interestingly, our study revealed that anti-peptide sera did not react with the recombinant enolase from Felis catus (cats, rFcEno), thus, may not trigger autoimmune response in these felines if used as a vaccine antigen. The immunization with peptide mixture (PeptMix) formulated with Freund adjuvant (FA), induced high levels of antigen-specific IgG, IgG1 and IgG2b antibodies that conferred protection upon passive transference in infected BALB/c mice with S. brasiliensis. We also observed, that the FA+PeptMix formulation induced a Th1/Th2/Th17 cytokine profile ex vivo, associated with protecting effect against the experimental sporotrichosis. Our results suggest that the six SsEno-derived peptides here evaluated, could be used as safe antigens for the development of vaccine strategies against feline sporotrichosis, whether prophylactic or therapeutic. [Display omitted] • PeptMix is a mixture of six peptides derived from Sporotrix spp enolase. • PeptMix formulated with Freund's adjuvant proved to be immunogenic. • FA+PeptMix induced high titers of specific IgG, IgG1 and IgG2b in mice. • FA+PeptMix increased the ex vivo release of IL-12, IL-6, IFN-γ and IL-17 cytokines. • FA+PeptMix induced protective antibodies against S. brasiliensis infection in mice. [ABSTRACT FROM AUTHOR]

Canine leptospirosis is a worldwide zoonosis, varying from asymptomatic and chronic infections to clinical acute disease. In many parts of the world Leptospira interrogans serogroup Icterohaemorrhagiae strains have great epidemiological importance, being the most prevalent on dogs. The present study aims to characterize and compare strains/sequences belonging to the serogroup Icterohaemorrhagiae recovered from clinically ill and asymptomatic dogs. Based on sec Y gene sequences of L. interrogans serovar Icterohaemorrhagiae, we have studied genetic diversity of strains obtained from 13 dogs, including dogs with clinical signs of acute leptospirosis, asymptomatic dogs and animals with chronic kidney disease, all of them from the same geographical area, the state of Rio de Janeiro, Brazil. No genetic variations on sec Y gene were observed between strains/groups. No significant associations were observed between clinical status and age, sex or vaccinal status. The same strain leads to different clinical outcomes on canine leptospirosis. The answer for this will rise from deep studies regarding whole genomic sequencing of the strains, as well as proteomics. Those studies may provide key information for understanding of the clinical manifestation of the disease. • The same strain leads to different clinical outcomes on canine leptospirosis. • There is no significant associations between clinical status and age, sex or vaccinal status. • The severity of leptospirosis is defined by a complex interaction among leptospires, their hosts and the environment. • Key for the development of the acute disease is still unknown, and varies from individual to individual. [ABSTRACT FROM AUTHOR]

Leptospirosis, caused by pathogenic Leptospira , is one of the most common zoonotic diseases in the world. It is transmitted to humans through the skin and mucous membranes by contact with water or soil contaminated with urine excreted from infected animals. In human infections, gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea have been frequently observed, but there have been no reports analyzing gastrointestinal lesions in leptospirosis, and the pathological mechanism of gastrointestinal symptoms in leptospirosis remains unclear. In this study, we investigated the pathological changes and the distribution of leptospires in the intestinal wall, and the presence of leptospires in the intestinal contents and feces, of hamsters subcutaneously infected with Leptospira interrogans. Results showed that infected hamsters had macroscopic redness in the jejunum and ileum. Submucosal hemorrhage was observed histologically, and there was no infiltration of inflammatory cells such as neutrophils. There were no obvious changes in the colon, either macroscopically or histologically, and the feces were normal (solid stools). Leptospira was isolated from all the intestinal walls from the small intestine to the colon, the intestinal contents, and the feces. These findings suggest that the invasion of leptospires into the intestinal wall and the associated submucosal hemorrhage may be the cause of the gastrointestinal symptoms observed in leptospirosis. Furthermore, not only the urine of infected animals but also the feces could be a source of infection. • Macroscopic redness was observed in the jejunum and ileum of hamsters subcutaneously infected with Leptospira interrogans. • Histologically, submucosal hemorrhage was observed in the reddened intestine. • In the culture, Leptospira was isolated from the intestinal wall of the jejunum to the colon and in the feces. • Not only the urine but also feces of infected animals could be a source of infection and could contaminate the environment. [ABSTRACT FROM AUTHOR]

Abstract: Leptospirosis is a re-emergent zoonosis, caused by pathogenic spirochetes from the genus Lepstospira. To date, there is no protein described to be involved in leptospiral hemorrhagic manifestations, although several proteases have been reported for other bacterial infections. In this study we identified 12 putative metalloproteases from the genome of Leptospira interrogans, and characterized for the first time a putative metalloprotease, here named Leptallo I, as a potential Zn2+ dependent glycylglycine protease belonging to the M23 metalloendopeptidase family. The native protein was detected in extracts from several pathogenic Leptospira species and further shown to be secreted to the culture medium. We expressed the recombinant protein and its C-terminal fragment containing the metalloprotease domain, and both presented regular secondary structures. The sera of humans with leptospirosis were able to recognize rLeptallo I, indicating that the native protein is expressed and presented to the immune system during infection. The recombinant proteins displayed a significant, though relatively low, elastinolytic activity, and the challenge of hamsters immunized with rLeptallo I conferred 33% protection, suggesting a significant importance of this protein in the pathogenesis. The elastinolytic activity may be important for leptospires–host interaction, because elastin constitutes a significant proportion of total lung and blood vessel proteins. [Copyright &y& Elsevier]

Abstract: We report the study of a predicted outer-membrane leptospiral protein encoded by the gene lic11207. This protein is conserved in several pathogenic leptospiral strains but is absent in the saprophyte Leptospira biflexa. This putative outer-membrane protein has a domain of unknown function (DUF) 1565 found in several phylogenetically diverse bacteria and in the archaeon Methanosarcina acetivorans. The gene was cloned and expressed in Escherichia coli BL21 (SI) strain using the expression vector pDEST17. The 34 kDa recombinant protein was tagged with N-terminal hexahistidine and purified by metal-charged chromatography. The purified protein was used to assess: reactivity with human convalescent sera; in vivo expression; ability to activate endothelial cells (EC); and ability to modulate the apoptosis of polymorphonuclear cells (PMNs). The LIC11207 coding sequence was identified in vivo in the hamster renal tubules during experimental infection with Leptospira interrogans. The rLIC11207 showed significant antigenicity against human convalescent sera when compared with sera from healthy donors. The recombinant protein did not alter the surface expression of E-selectin or intercellular adhesion molecule 1 (ICAM-1) in EC and failed to induce the release of von Willebrand factor (vWF). Interestingly, rLIC11207 delayed apoptosis of PMNs suggesting a possible role of this protein during the infection. [Copyright &y& Elsevier]

Abstract: Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K D ) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. [Copyright &y& Elsevier]

Abstract: Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)–LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx–LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions. [Copyright &y& Elsevier]

Leptospira species are the etiological agent of an emerging zoonotic disease known as "Leptospirosis" that substantially affects both human health and economy across the globe. Despite the global importance of the disease, pathogenetic features, host-adaptation and proper diagnosis of this bacteria remains lacking. To accomplish these gaps, pan-genome of Leptospira genus was explored in the present study. The pan-genome of Leptospira genus was comprised of core (692) and accessory parts (softcore:1804, shell:6432, cloud:16,600). The functional analysis revealed the abundancy of "Translation, ribosomal structure and biogenesis" COG class in core-genes; whereas in accessory parts, genes involved in signal transduction was the most abundant. Furthermore, pathogen-host interaction (PHI) analysis of core and accessory proteins with human proteins showed the presence of a total of 599 and 510 interactions, respectively. There were eight hubs in core PHI network and five hubs in PHI network of accessory proteins. The human's proteins involved in these interactions were found functionally enriched in metabolic processes, responses to stimulus and immune system processes. Further, pan-genome based phylogeny separated the Leptospira genus in three major clades (belonging to P1, P2 and S) which relates with their pathogenicity level. Additionally, pathogenic and saprophytic clade specific genes of Leptospira have also been identified and functionally annotated for COG, KEGG and virulence factors. The results revealed the presence of 102 pathogenic and 215 saprophytic group specific gene clusters. The COG functional annotation of pathogen specific genes showed that defence mechanism followed by signal transduction mechanisms category were most significantly enriched COG categories; whereas in saprophytic group, signal transduction mechanisms was the most abundant COG, suggesting their role in adaptation and hence important for microbe's evolution and survival. In conclusion, this study provides a new insight of genomic features of Leptospira genus which may further be implemented for development of better control actions of the disease. • A total of 24,836 pan-genes and 692 core-genes were identified among Leptospira genus. • Pan-genome of pathogenic Leptospira was found to be more open in nature. • A total of 599 and 510 interactions were found for leptospiral core and accessory proteins with human proteins, respectively. • 102 pathogenic and 215 saprophytic specific gene clusters were identified, which can further be used as biomarker for species. • These findings could upgrade our current knowledge of genomic diversity and host interaction of Leptospira. [ABSTRACT FROM AUTHOR]

Leptospirosis, caused by pathogenic Leptospira , is a global critical zoonotic disease in terms of mortality and morbidity. Vaccines are often used to prevent leptospirosis. However, few studies have reported the therapeutic effect of a vaccine against Leptospira infection. This study demonstrates the efficacy of the emergency vaccine immunization against acute leptospirosis in hamsters. Treatment with a whole-cell vaccine (Leptospira interrogans serovar Lai) at 24 h post-infection improved the survival rate of hamsters with lower leptospiral burden and minor pathological damage to organs. The vaccine also protected against multiple Leptospira serotypes acute infection. However, the protective effect of the vaccines was lost when beginning treatment at 36 h or 48 h post-infection. These results indicated that vaccines could treat acute leptospirosis in hamsters, but only if immunization is within 24 h after infection. • Treatment with vaccines against acute homologous Leptospiral (56601) acute infection. • Treatment with vaccines elevates gene expression of TLR2, TLR4 and inflammatory factors during leptospira infection. • Treatment with vaccines reduces the leptospiral burden of organs and relieves pathological damage. • Treatment with vaccines against heterologous leptospiral acute infection. Author Summary: Leptospirosis is one of the most important neglected emerging zoonotic diseases; it usually spreads in bursts due to natural disasters or large-scale water pollution. Vaccines are often used to protect against leptospirosis; it has not been reported whether leptospiral vaccines can play a therapeutic role in post-infection. This study demonstrated that treatment with a whole dead vaccine within 24 h post-infection improved the survival rate of hamsters with lower leptospiral burden and minor pathological damage in their organs. These results indicated that vaccines may be useful for prevention but also therapeutic action against leptospirosis. [ABSTRACT FROM AUTHOR]

Abstract: Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell–cell or cell–extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. [Copyright &y& Elsevier]

Shiga toxin-producing Escherichia coli (STEC) is a water- and food-borne pathogenic agent that causes diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS), and end-stage renal disease. As the annual incidence of STEC increases, disease control is also becoming important in Korea. In this study, we aimed to analyze the incidence trends and characteristics of STEC isolated from diarrheal patients over 10 years. From 2009 to 2018, STECs were collected by the Enteric Pathogens Active Surveillance Network (Enter-Net) and analyzed according to clinical epidemiological information (month of isolation, age, and sex of patient), O serogroup, and shiga toxin type. Shiga toxin genes (stx 1 and stx 2) and O serogroups of isolates were determined using multiplex PCR and an agglutination method with the available O antisera, respectively. A total of 418 strains were isolated over 10 years. The isolation rate according to age group and season was highest in children ≤4 years old (38.1%) and in the summer season (June to August). Among the 418 isolates, the major serogroups were divided O157 (20.3%), O103 (13.6%), O26 (7.7%), O111 (5.5%), O91 (4.3%), O108 (2.4%), and O8 (2.2%). The most frequently isolated O157 showed a lower isolation rate compared to that isolated from other developed countries. The profiles of stx genes were distinct among serogroups. In O157 and O91, stx 1+ stx 2 was detected more frequently than either stx 1 or stx 2 alone. Particularly, most of the O157 (98%) isolates harbored the stx 2 gene, which is an important factor in severe diseases, including HUS. In O103, O26, O111, and O108, stx 1-only was more frequently present than stx 2-only or stx 1+ stx 2. As a result of analyzing domestic STECs collected through Enter-Net, it was confirmed that patients ≤4 years of age and in the summer months require attention, and that STEC with a serogroup of O157 is highly likely to cause diseases such as HUS. Therefore, the pathogen active surveillance network for characterization and provision of STEC isolates must be operated continuously. [Display omitted] • Shiga toxin-producing E.coli (STEC) infections were analyzed from Enter-Net (Korea). • STEC prevalence was highest in summer months and patients aged ≤ 4 years. • O157 serogroup, common worldwide, was not prevalent in Korea. • STEC serogroup is correlated with pathogenicity and stx gene type. [ABSTRACT FROM AUTHOR]

Leptospirosis, caused by pathogenic Leptospira species, is an essential but neglected zoonosis. There are more than 300 serovars of pathogenic Leptospira , while inactivated bacteria offers only short-term serovar-specific protection. Leptospirosis treatment is mainly dependent on the use of antibiotics. However, the side effects of antibiotics and the risk of antibiotic resistance remain major problems. Thus, alternative agents which are fewer side effects on humans and efficient in leptospirosis would be welcome. Many studies have reported that polysaccharides could be used as immunostimulants in treating infection and cancer. In this study, we examined the protective effect of polysaccharides isolated from Iris against leptospirosis. To our knowledge, it is the first time to report Iris polysaccharides (IP) as an immunostimulant in treating infection. The results showed that IP treatment significantly increased the survival rate of hamsters challenged by a lethal dose of leptospires. Besides, the tissue injury and leptospiral load were reduced in IP-treated infection group compared with the untreated infection group at 4 days post-infection (p.i.). Intriguingly, IP treatment sustained intense immune response at 4 days p.i. analyzed by qPCR. The results exhibited that the gene expression of TLR2 and TLR4 was significantly increased in the group coinjected with IP and leptospires than in the infected controls. And the expression of IL-1β and TNF-α were also up-regulated after IP treatment, except the expression of IL-1β in the kidney. Our results not only broaden the medicinal value of Iris, but also provide a competent candidate for the control of Leptospira infection. • Iris polysaccharide-treated hamsters showed improved survival rate during Leptospira infection. • Iris polysaccharide treatment reduced bacterial load and ameliorated Leptospira-induced organ injury in hamsters. • Iris polysaccharide treatment could prolong intense immune response during infection. [ABSTRACT FROM AUTHOR]

Chronic leptospirosis usually occurs during sublethal doses infection of susceptible animal and reservoir host, which typical symptom is interstitial nephritis, and leptospira urine, contaminating the environment and threatening other susceptible animals and humans. Dipotassium glycyrrhizinate (DG) is a replacement for glycyrrhizic acid, which exhibits anti-inflammation, immunomodulation effects. This study is to investigate whether DG relieves leptospira-induced nephritis. In vitro , DG inhibited the leptospira-induced transcription levels of IL-1β, IL-6, TNF-α, RANTES, MCP-1 and iNOS, and protein levels of IL-1β and TNF-α, and downregulated NF-κB and MAPK pathway in TCMK-1 cells. In vivo , DG attenuated the kidney histopathological change and downregulated the expression of IL-1β and TNF-α, as well as reduced kidney leptospiral burden. In summary, DG alleviated leptospira-induced inflammation through inhibitory NF-κB and MAPK pathway, and DG decreased the renal colonization of leptospires in mice. • DG downregulates the expression of cytokines and chemokines in leptospira-infected TCMK-1 cells. • DG inhibits the NF-κB and MAPK signaling pathways activation caused by leptospira-infection in vitro. • DG alleviates leptospira-induced nephritis in chronic leptospirosis model. • DG reduces the colonization of leptospires in mice. [ABSTRACT FROM AUTHOR]

Linguatulosis, as a zoonotic disease, can infect most ruminants and cause accidental infections in humans. The objective of this study was to explore the epidemiological, histopathological and phylogenetic profiles of Linguatula serrata infection in sheep and goats and its public health importance during 2015–2018. Mesenteric lymph nodes (MLNs) and liver tissue of goats and sheep were selected randomly in Kerman slaughterhouse. Nymphal samples were used for DNA extraction, amplification and subsequently phylogenetic analysis using 18s rRNA and cytochrome C oxidase subunit 1 (cox1). Overall, of 828 examined livestock, 179 (42.4%) goats and 71 (17.5%) sheep were found to be infected with the nymphal stage of L. serrata. A significant difference was observed between linguatulosis and age. In the histopathological assessment, longitudinal and transverse sections of L. serrata nymphs were observed within the cyst-like spaces surrounded by a wall of fine fibrosis and compact lymphocytes. Moreover, comparing with the L. serrata reference sequences, we found only a single nucleotide change in our goat haplotype in 18s genetic region; while much nucleotide variations were observed in cox1 gene sequences. The results of the present study showed a high infection rate among goats and sheep in southeastern Iran. A better understanding of the disease could be achieved when the parasite species, their molecular characterization and the extent of infection in the area are determined. It is fundamental to select a comprehensive control program in order to take proper preventive and therapeutic measures against the infection. • High infection rate of linguatulosis was identified in mesenteric lymph nodes (MLNs) of goats and sheep. • A significant difference was observed between linguatulosis and age in both groups. • Longitudinal and transverse sections of Linguatula serrata nymphs were observed within the cyst-like spaces in MLNs. • Phylogenetic analysis represented more nucleotide variations in the sequences of cox1 gene region than 18s gene region. [ABSTRACT FROM AUTHOR]

Leptospirosis is a global disease that affects humans and animals, impacting public health and the economy. The symptoms caused by Leptospira infection can vary from mild to severe, affecting liver, lungs, and kidneys. The host-pathogen interaction in leptospirosis is still poorly understood, but there is evidence for the role of the host immune response in the pathogenesis. Chemokines are a family of structurally-related low-molecular-mass proteins (8–14 kDa) that signal the recruitment of leukocytes. In this study the profile of 22 chemokines were evaluated in liver and kidney of three mice strains with different phenotypes of susceptibility to leptospirosis. We extended our previously reported observations showing that expression of chemokines with homeostatic function, activation and chemotaxis of leukocytes are essential to modulate and to induce resistance to leptospirosis. Our findings support that an early induction of CXC chemokines in resistant BALB/c mice can be associated with the control of the infection. The correlation of chemokine expression between liver and kidney observed in BALB/c suggests that a balance of chemokine induction in the organs may contribute to resistance to leptospirosis. [ABSTRACT FROM AUTHOR]

Leptospirosis is considered as a neglected tropical disease which is caused by pathogenic Leptospira spp. The precise mechanisms of leptospirosis pathogenesis are unclear and hence, the progress in development of treatment modalities has been dismal. The present study aimed to identify novel virulent factors of leptospires to understand the disease pathogenesis and to develop treatment modalities. Leptospira interrogans contains two chromosomes and encodes for ~3703 genes, but the functions of several open reading frames have not yet been explored. Among them, novel virulent associated leptospiral proteins (LIC11334, LIC11542, LIC11436, LIC11120 and LIC12539) were identified using VirulentPredict and the antigenicity of these targets was explored by VaxiJen server. Domain architecture of the pathogen specific proteins revealed that LIC11334 had potential to evoke significant immune response against leptospiral infection and LIC11436 contains four folds of immunoglobulin-like domain and plays a vital role in pathogenesis. Therefore, B-cell epitopes were predicted and the epitope of high virulence (and VaxiJen score from LIC11334) was chemically synthesized as peptide (KNSMP01) and labeled with Biotin (Biotin-SGSGEVENPDPKVAQEC). Binding affinity of KNSMP01 with MHC molecules was predicted and the molecule was discovered to have potential to elicit both humoral and cell mediated immune responses and found to interact with host components via hydrophobic interaction, hydrogen bonding and salt bridges. Rabbit antisera was raised against KNSMP01 and found to elicit antigenicity using Western, ELISA and dot blot assays. In silico and in vitro experiments show KNSMP01 to be a promising immunogen and may be a better vaccine candidate for leptospirosis. • The functions of several open reading frames of Leptospira genome are undetermined. • VirulentPredict and VaxiJen were used to explore the antigenic LIC11334 among them. • Antigenic epitope of LIC11334 was chemically synthesized as peptide KNSMP01. • Antisera of KNSMP01 found to elicit immunogenicity by Western, ELISA and dot blot assays. • In silico and in vitro experiments show KNSMP01 to be a promising immunogen. [ABSTRACT FROM AUTHOR]

To adapt into the host system from moist environment Leptospira alter their gene expression by inducing differential expression of the genes encoding virulence factors. Knowledge about the molecular pathogenesis and virulent evolution remains limited to Leptospira. The pathogenic organism sense the environmental changes mainly through their outer membrane proteins that in-turn activates the signal transduction pathways to overcome the stress to adaptation into host system and to evade immunity. In this present study, we analyzed the expression profile of virulence associated OMPs regulated under various stress conditions like temperatures, iron deprivation, osmotic stress and low to high passages in single scale and characterized the selected proteins by MALDI-TOF MS/MS and their role in pathogenesis were predicted by implying in-silico analysis. To identify differential expression profile, the extracted OMPs were resolved through 2DE and compared the OMPs profile from various in-vivo like conditions in single scale and found 61 upregulated OMPs and three potentially virulent proteins were earmarked for their significance in pathogenesis. Further, the in-silico analysis revealed that differentially expressed protein has MHC-I T-cell, MHC-II T-cell and B-cell epitopes which showed an interaction between human TLR2 proteins confirmed by CABS docking and interaction network unveiled to understand the leptospiral virulent mechanism and host adaptation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Leptospira infection is influenced by the host species and the bacterial strain involved. Important differences on their tissue distribution are referred, also depending on the host and the studied strain. Considering tissue distribution of leptospires the majority of the studies focus on a single strain, or strains from the same serogroup with different inoculation doses. Nevertheless, none had associated different serogroups with their tissue distribution. Thus, the present study aims to investigate the hypothesis that tissue distribution of Leptospira spp. on experimentally infected hamsters may vary according to the strain serogroup. Females of Golden Syrian hamsters were used for the experimental infection with Leptospira isolates (n = 13) belonging to serogroups Icterohaemorrhagiae (n = 8) and Sejroe (n = 5). PCRs were conducted in renal, hepatic and uterine tissue. Seven out of the thirteen studied hamsters presented acute clinical signs and were euthanized before the 21° day p.i. (strains VF237, VF52, U81, UFF-SG001, UFF-G19, UFF-B15, L1-130), while the others presented a subclinical infection. Regarding to the PCR results it was possible to observe that, all but one strain (UFF-SG001), which was detected on all the studied tissues, presented some differences on their tissue distribution. All strains could be detected on at least one tissue and a significant association was observed for the occurrence of the strains of serogroup Sejroe in uterus (p = 0.044). The results of the present study indicate that strains of serogroup Sejroe are strongly associated to uterine infection on experimentally infected hamsters. • All strains of Leptospira could be detected on at least one tissue of the experimentally infected hamsters. • A significant association was observed for the occurrence of the strains of serogroup Sejroe in uterus (p = 0.044). • Strains of serogroup Sejroe are strongly associated to uterine infection on experimentally infected hamsters. [ABSTRACT FROM AUTHOR]

Leptospirosis is a zoonotic disease caused by a bacterium of the genus Leptospira that affects domestic animals, including cattle. Despite of this importance in livestock, little is known about the epidemiology of leptospirosis in cattle in Brazil and other tropical countries. In this context, the objective of this study was to perform the culture of urine and kidney (n = 372 animals) of slaughtered bovines of Minas Gerais state (Brazil), in order to obtain pure cultures of Leptospira strains and carry out its characterization by serological (microscopic agglutination test) and molecular techniques (rrs, sec Y sequencing and variable-number tandem-repeat genotyping). One strain, named UFU 02, was obtained from a male bovine without apparent alterations in ante-mortem examination. The strain was characterized belonging to species Leptospira kirschneri and serogroup Grippotyphosa. The serogroup Grippotyphosa is most often described in wild species, suggesting a link between domestic and wild cycles/environments. Considering the importance of the country in livestock breeding, the knowledge about leptospiral strains could help to control the disease in the herds. [ABSTRACT FROM AUTHOR]