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1.
BMC Microbiol ; 21(1): 99, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33789603

RESUMEN

BACKGROUND: Leptospirosis is a zoonotic disease caused by infection with spirochetes from Leptospira genus. It has been classified into at least 17 pathogenic species, with more than 250 serologic variants. This wide distribution may be a result of leptospiral ability to colonize the renal tubules of mammalian hosts, including humans, wildlife, and many domesticated animals. Previous studies showed that the expression of proteins belonging to the microbial heat shock protein (HSP) family is upregulated during infection and also during various stress stimuli. Several proteins of this family are known to have important roles in the infectious processes in other bacteria, but the role of HSPs in Leptospira spp. is poorly understood. In this study, we have evaluated the capacity of the protein GroEL, a member of HSP family, of interacting with host proteins and of stimulating the production of cytokines by macrophages. RESULTS: The binding experiments demonstrated that the recombinant GroEL protein showed interaction with several host components in a dose-dependent manner. It was also observed that GroEL is a surface protein, and it is secreted extracellularly. Moreover, two cytokines (tumor necrosis factor-α and interleukin-6) were produced when macrophages cells were stimulated with this protein. CONCLUSIONS: Our findings showed that GroEL protein may contribute to the adhesion of leptospires to host tissues and stimulate the production of proinflammatory cytokines during infection. These features might indicate an important role of GroEL in the pathogen-host interaction in the leptospirosis.


Asunto(s)
Chaperonina 60/inmunología , Citocinas/inmunología , Interacciones Huésped-Patógeno/inmunología , Leptospira/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología
2.
Infect Immun ; 83(3): 888-97, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25534939

RESUMEN

Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion.


Asunto(s)
Proteínas Bacterianas/química , Leptospira interrogans/química , Leptospira/química , Fragmentos de Péptidos/química , Vitronectina/química , Anticuerpos Monoclonales/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/inmunología , Sitios de Unión , Unión Competitiva , Activación de Complemento , Proteína de Unión al Complemento C4b/química , Proteína de Unión al Complemento C4b/inmunología , Complemento C9/química , Complemento C9/inmunología , Factor H de Complemento/química , Factor H de Complemento/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/química , Heparina/química , Humanos , Evasión Inmune , Leptospira/inmunología , Leptospira/patogenicidad , Leptospira interrogans/inmunología , Leptospira interrogans/patogenicidad , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/inmunología , Unión Proteica , Vitronectina/inmunología , Zinc/química
3.
J Infect Dis ; 205(6): 995-1004, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22291192

RESUMEN

Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular matrix components, coagulation cascade proteins, and human complement regulators. Here we demonstrate that acquisition of factor H (FH) on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes, besides interacting with FH, FH related-1, and C4b binding protein (C4BP), also acquire FH like-1 from human serum. We also demonstrate that binding to these complement regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins, previously shown to interact with fibronectin, laminin, collagen, elastin, tropoelastin, and fibrinogen. Factor H binds to Lig proteins via short consensus repeat domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins. Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by factor I. In conclusion, Lig proteins are multifunctional molecules, contributing to leptospiral adhesion and immune evasion.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas Inactivadoras del Complemento C3b/metabolismo , Antígenos de Histocompatibilidad/metabolismo , Leptospira/patogenicidad , Leptospirosis/inmunología , Adhesión Bacteriana , Proteínas Bacterianas/genética , Sitios de Unión , Clonación Molecular , Complemento C3b/metabolismo , Proteína de Unión al Complemento C4b/metabolismo , Factor H de Complemento/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Evasión Inmune , Inmunoglobulinas/química , Leptospira/genética , Leptospira/metabolismo , Leptospirosis/microbiología , Plásmidos
4.
Front Microbiol ; 13: 1051698, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36519163

RESUMEN

Pathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still remain unknown. Toxins and adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines with cross-protection and long-lasting effect against leptospirosis. For this aim, we used the shotgun phage display technique to unravel new proteins with adhesive properties. A shotgun library was constructed using fragmented genomic DNA from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and pG8SAET phagemid vector. Selection of phages bearing new possible cell-binding antigens was performed against VERO cells, using BRASIL biopanning methodology. Analysis of selected clones revealed the hypothetical protein LIC10778, a potentially exposed virulence factor that belongs to the virulence-modifying (VM) protein family (PF07598), composed of 13 members in the leptospiral strain Fiocruz L1-130. Prediction of LIC10778 tertiary structure indicates that the protein contains a cellular-binding domain (N-terminal portion) and an unknown domain of no assigned activity (C-terminal portion). The predicted N-terminal domain shared structural similarities with the cell-binding and internalization domain of toxins like Ricin and Abrin, as well as to the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin in Mycoplasma pneumoniae. Interestingly, recombinant portions of the N-terminal region of LIC10778 protein showed binding to laminin, collagens I and IV, vitronectin, and plasma and cell fibronectins using overlay blotting technique, especially regarding the binding site identified by phage display. These data validate our preliminary phage display biopanning and support the predicted three-dimensional models of LIC10778 protein and other members of PF07598 protein family, confirming the identification of the N-terminal cell-binding domains that are similar to ricin-like toxins. Moreover, fluorescent fused proteins also confirmed that N-terminal region of LIC10778 is capable of binding to VERO and A549 cell lines, further highlighting its virulence role during host-pathogen interaction in leptospirosis probably mediated by its C-terminal domain. Indeed, recent results in the literature confirmed this assumption by demonstrating the cytotoxicity of a closely related PF07598 member.

5.
Artículo en Inglés | MEDLINE | ID: mdl-33174979

RESUMEN

Leptospirosis is an acute infection caused by pathogenic species of the genus Leptospira, which affects humans and animals in all world. In severe forms of the disease, kidneys, liver and lungs are the main affected organs, resulting in acute kidney injury, jaundice and pulmonary hemorrhage. Previous post-mortem studies have shown that lesions are not limited to these organs. Cardiac and striated muscle injuries have already been reported, but the pathophysiology of cardiac and skeletal lesions in leptospirosis is not fully understood. It has been suggested that the tissue damage observed in leptospirosis could be directly mediated by leptospires or by their toxic cellular components. LipL32 and Lp25 are leptospira membrane proteins with unknown functions, that are present only in pathogenic strains of Leptospira spp. Both proteins induce skeletal muscle lesions similar to those observed when normal guinea pigs are inoculated with leptospires. Through immunohistochemistry, this study showed the presence of LipL32 and Lp25 proteins on muscle cell membranes and in the underlying cytoplasm of skeletal muscles, as well as focal lesions in cardiac tissues of fatal cases of leptospirosis. Altogether, these results reinforce that both proteins can be important factors in the pathogenesis of leptospirosis.


Asunto(s)
Lesión Renal Aguda/patología , Proteínas de la Membrana Bacteriana Externa/genética , Riñón/patología , Leptospira/genética , Leptospirosis/complicaciones , Lipoproteínas/genética , Miocardio/patología , Lesión Renal Aguda/microbiología , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Femenino , Genes Bacterianos , Cobayas , Humanos , Leptospira/metabolismo , Leptospirosis/metabolismo , Lipoproteínas/metabolismo , Masculino , Persona de Mediana Edad , Músculos/patología
6.
Front Immunol ; 11: 572562, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33240263

RESUMEN

Properdin (P) is a positive regulatory protein that stabilizes the C3 convertase and C5 convertase of the complement alternative pathway (AP). Several studies have suggested that properdin can bind directly to the surface of certain pathogens regardless of the presence of C3bBb. Saprophytic Leptospira are susceptible to complement-mediated killing, but the interaction of properdin with Leptospira spp. has not been evaluated so far. In this work, we demonstrate that properdin present in normal human serum, purified properdin, as well as properdin oligomers P2, P3, and P4, interact with Leptospira. Properdin can bind directly to the bacterial surface even in the absence of C3b. In line with our previous findings, AP activation was shown to be important for killing non-pathogenic L. biflexa, and properdin plays a key role in this process since this microorganism survives in P-depleted human serum and the addition of purified properdin to P-depleted human serum decreases the number of viable leptospires. A panel of pathogenic L.interrogans recombinant proteins was used to identify putative properdin targets. Lsa30, an outer membrane protein from L. interrogans, binds to unfractionated properdin and to a lesser extent to P2-P4 properdin oligomers. In conclusion, properdin plays an important role in limiting bacterial proliferation of non-pathogenic Leptospira species. Once bound to the leptospiral surface, this positive complement regulatory protein of the AP contributes to the formation of the C3 convertase on the leptospire surface even in the absence of prior addition of C3b.


Asunto(s)
Complemento C3b/metabolismo , Factor B del Complemento/metabolismo , Leptospira interrogans/fisiología , Leptospira/fisiología , Leptospirosis/metabolismo , Properdina/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Procesos de Crecimiento Celular , Vía Alternativa del Complemento , Citotoxicidad Inmunológica , Humanos , Leptospira/patogenicidad , Leptospira interrogans/patogenicidad , Leptospirosis/inmunología , Properdina/inmunología , Unión Proteica , Virulencia
7.
Biotechnol Lett ; 30(5): 807-12, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18071638

RESUMEN

Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of approximately 7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 microg porcine aprotinin inhibited 6 microg trypsin whereas 1 microg commercial soybean inhibitor inhibited only 1 microg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.


Asunto(s)
Aprotinina/genética , Aprotinina/aislamiento & purificación , Pulmón/metabolismo , Secuencia de Aminoácidos , Animales , Aprotinina/metabolismo , Secuencia de Bases , Bovinos , Cromatografía de Afinidad , ADN/genética , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Porcinos , Tripsina/metabolismo
8.
Artículo en Inglés | MEDLINE | ID: mdl-29846473

RESUMEN

Leptospirosis is an acute bacterial septicemic febrile disease caused by pathogenic leptospires, which affect humans and animals in all parts of the world. Transmission can occur by direct contact with infected animals or, more commonly, through indirect contact with water or soil contaminated with urine from infected animals. Leptospires enter the body by penetrating mucous membranes or skin abrasions and disseminate through the hematogenic route. In humans, leptospirosis may cause a wide spectrum of symptoms. Most cases have a biphasic clinical presentation, which begins with the septicemic phase followed by immune manifestations. The severe forms of the disease may be life threatening with multisystem damage including renal failure, hepatic dysfunction, vascular damage, pulmonary hemorrhage and muscle lesions. In this review, we present and discuss the pathogenesis of the human disease and the mechanisms of cell membrane injuries, which occur mainly due to the presence of leptospires and/or their antigen/s in the host tissues.


Asunto(s)
Cadherinas/metabolismo , Membrana Celular/parasitología , Riñón/parasitología , Leptospirosis/etiología , Leptospirosis/patología , Hígado/parasitología , Enfermedades Musculares/parasitología , Animales , Membrana Celular/patología , Humanos , Riñón/patología , Leptospirosis/metabolismo , Hígado/patología , Enfermedades Musculares/patología
9.
J Proteomics ; 186: 56-70, 2018 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-30026101

RESUMEN

Intraspecific venom variability has been extensively reported in a number of species and is documented to be the result of several factors. However, current evidence for snake venom variability related to captivity maintenance is controversial. Here we report a compositional and functional investigation of individual and pooled venoms from long-term captive (LTC) and recently wild-caught (RWC) B. jararaca snakes. The composition of individual venoms showed a remarkable variability in terms of relative abundance of toxins (evidenced by 1-DE and RP-HPLC), enzymatic activities (proteolytic, PLA2, and LAAO) and coagulant activity, even among captive specimens. Thus, no compositional and functional pattern could be established to assign each individual venom to a specific group. Conversely, pooled venom from LTC and RWC snakes showed no significant differences regarding protein composition (characterized by 1-DE and shotgun proteomics), enzymatic activities (proteolytic, PLA2 and LAAO) and biological function (coagulant, hemorrhagic and lethal activities), except for edematogenic activity, which was more prominent in RWC venom pool. Additionally, both pooled venoms displayed similar immunoreactivity with the bothropic antivenom produced by Instituto Butantan. Taken together, our results highlight the complexity and the high intraspecific variation of B. jararaca venom, that is not influenced at a discernible extent by captivity maintenance. BIOLOGICAL SIGNIFICANCE: Bothrops jararaca snakes are one of the main causes of snakebites in Southeastern Brazil. Due to its medical interest, the venom of this species is the most studied and characterized among Brazilian snakes and captive B. jararaca specimens are maintained for long periods of time in our venom production facility. However, knowledge on the influence of captivity maintenance on B. jararaca venom variability is scarce. In this report, we described a high compositional and functional variability of individual venoms from LTC and RWC B. jararaca snakes, which are not observed between LTC and RWC pooled venoms. This intraspecific variability is more likely to be due to genetic/populational differences rather than "captivity vs wild" conditions. In this regard, data generated by the present work support the use of venom from captive and wild snakes for antivenom production and scientific research. Moreover, the data generated by this study highlight the importance of analyzing individual venom samples in studies involving intraspecific venom variability.


Asunto(s)
Bothrops/inmunología , Venenos de Crotálidos/química , Proteínas/análisis , Proteómica/métodos , Animales , Animales Salvajes/inmunología , Animales de Zoológico/inmunología , Antivenenos/inmunología , Biodiversidad , Venenos de Crotálidos/enzimología , Venenos de Crotálidos/inmunología , Enzimas/análisis , Enzimas/fisiología , Proteínas/fisiología , Especificidad de la Especie
10.
Immunobiology ; 221(5): 679-89, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26822552

RESUMEN

Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host.


Asunto(s)
Proteínas Bacterianas/inmunología , Complemento C3b/inmunología , Complemento C5/inmunología , Fibrinógeno/metabolismo , Fibrinolisina/metabolismo , Evasión Inmune , Antígenos Bacterianos/inmunología , Sitios de Unión , Complemento C3b/metabolismo , Proteína de Unión al Complemento C4b/metabolismo , Complemento C5/metabolismo , Activación Enzimática , Interacciones Huésped-Patógeno/inmunología , Humanos , Leptospira interrogans/inmunología , Leptospirosis/inmunología , Leptospirosis/metabolismo , Concentración Osmolar , Unión Proteica , Proteolisis
11.
Braz J Microbiol ; 45(3): 1083-8, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25477946

RESUMEN

We investigated the existence of cross-protection between two anti-leptospirosis monovalent experimental bacterins produced with two strains of Leptospira serogroup Pomona: Fromm strain of serovar Kennewicky, isolated from pigs in the United States, and strain GR6 of serovar Pomona isolated from pigs in Brazil. Both were added of aluminum hydroxide as an adjuvant. Experimental bacterins were tested with the hamster potency test in order to assess protection provided against the disease and against the establishment of kidney infection. Controls were polyvalent commercial vaccine produced with Leptospira strains isolated outside Brazil, which included a representative of Pomona serovar, or Sorensen solution added of aluminum hydroxide adjuvant. The challenge was performed with cross-strains of serogroup Pomona tested in accordance with international standards established for the potency test. After 21 days of the challenge, survivors were killed to evaluate the condition of Leptospira renal carrier. Experimental bacterins protected hamsters against homologous and heterologous strains, demonstrating the existence of cross-protection. The commercial vaccine protected the hamsters challenged with both strains, but there was a high proportion of animals diagnosed as renal carriers when the challenge was performed with strain GR6, isolated from pigs in Brazil.


Asunto(s)
Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Protección Cruzada , Leptospirosis/inmunología , Leptospirosis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Animales , Portador Sano/microbiología , Portador Sano/prevención & control , Cricetinae , Riñón/microbiología , Leptospira/aislamiento & purificación , Resultado del Tratamiento
12.
PLoS One ; 7(6): e37625, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22701573

RESUMEN

BACKGROUND: Leptospirotic renal lesions frequently produce a polyuric form of acute kidney injury with a urinary concentration defect. Our study investigated a possible effect of the glycolipoprotein, (GLPc) extracted from L. interrogans, on vasopressin (Vp) action in the guinea pig inner medullary collecting duct (IMCD). METHODS: The osmotic water permeability (Pf µm/s) was measured by the microperfusion in vitro technique. AQP2 protein abundance was determined by Western Blot. Three groups were established for study as follows: Group I, IMCD from normal (ngp, n = 5) and from leptospirotic guinea-pigs (lgp-infected with L. interrogans serovar Copenhageni, GLPc, n = 5); Group II, IMCD from normal guinea-pigs in the presence of GLPc (GLPc group, n = 54); Group III, IMCD from injected animals with GLPc ip (n = 8). RESULTS: In Group I, PFS were: ngp--61.8±22.1 and lgp--8.8±12.4, p<0.01 and the urinary osmolalities were: lgp--735±64 mOsm/Kg and ngp--1,632±120 mOsm/Kg. The lgp BUN was higher (176±36 mg%) than the ngp (56±9 mg%). In Group II, the Pf was measured under GLPc (250 µg/ml) applied directly to the bath solution of the microperfused normal guinea-pig IMCDs. GLPc blocked Vp (200 pg/ml, n = 5) action, did not block cAMP (10(-4) M), and Forskolin (Fors--10(-9) M) action, but partially blocked Cholera Toxin (ChT--10(-9) M) action. GLP from L.biflexa serovar patoc (GLPp, non pathogenic, 250 µg) did not alter Vp action. In Group III, GLPc (250 µg) injected intraperitoneally produced a decrease of about 20% in IMCD Aquaporin 2 expression. CONCLUSION: The IMCD Pf decrease caused by GLP is evidence, at least in part, towards explaining the urinary concentrating incapacity observed in infected guinea-pigs.


Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/farmacología , Túbulos Renales Colectores/efectos de los fármacos , Leptospira interrogans/metabolismo , Leptospirosis/patología , Equilibrio Hidroelectrolítico/efectos de los fármacos , Animales , Acuaporina 2/metabolismo , Western Blotting , Regulación de la Expresión Génica/fisiología , Glicoproteínas/aislamiento & purificación , Cobayas , Túbulos Renales Colectores/fisiología , Leptospirosis/microbiología , Permeabilidad , Equilibrio Hidroelectrolítico/fisiología
13.
Braz. j. microbiol ; 45(3): 1083-1088, July-Sept. 2014. tab
Artículo en Inglés | LILACS | ID: lil-727041

RESUMEN

We investigated the existence of cross-protection between two anti-leptospirosis monovalent experimental bacterins produced with two strains of Leptospira serogroup Pomona: Fromm strain of serovar Kennewicky, isolated from pigs in the United States, and strain GR6 of serovar Pomona isolated from pigs in Brazil. Both were added of aluminum hydroxide as an adjuvant. Experimental bacterins were tested with the hamster potency test in order to assess protection provided against the disease and against the establishment of kidney infection. Controls were polyvalent commercial vaccine produced with Leptospira strains isolated outside Brazil, which included a representative of Pomona serovar, or Sorensen solution added of aluminum hydroxide adjuvant. The challenge was performed with cross-strains of serogroup Pomona tested in accordance with international standards established for the potency test. After 21 days of the challenge, survivors were killed to evaluate the condition of Leptospira renal carrier. Experimental bacterins protected hamsters against homologous and heterologous strains, demonstrating the existence of cross-protection. The commercial vaccine protected the hamsters challenged with both strains, but there was a high proportion of animals diagnosed as renal carriers when the challenge was performed with strain GR6, isolated from pigs in Brazil.


Asunto(s)
Animales , Cricetinae , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Protección Cruzada , Leptospirosis/inmunología , Leptospirosis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Hidróxido de Aluminio/administración & dosificación , Portador Sano/microbiología , Portador Sano/prevención & control , Riñón/microbiología , Leptospira/aislamiento & purificación , Resultado del Tratamiento
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