Anti-erythrocyte IgG in hamsters with acute experimental infection by Leptospira interrogans serovar Canicola

Accepted Manuscript Anti-erythrocyte IgG in hamsters with acute experimental infection by Leptospira interrogans serovar Canicola Ânderson O. Sobroza, Guilherme L. Dorneles, Márcia S.N. Machado, Cássia B. Silva, Letícia Petry, Andressa Schafer, Raqueli T. França, Cinthia M. de Andrade, Alfredo Q. Antoniazzi, Alexandre A. Tonin, Sonia T.A. Lopes, Micheli M. Pillat PII:

S0882-4010(17)30280-2

DOI:

10.1016/j.micpath.2017.06.032

Reference:

YMPAT 2324

To appear in:

Microbial Pathogenesis

Received Date: 17 March 2017 Revised Date:

18 June 2017

Accepted Date: 21 June 2017

Please cite this article as: Sobroza ÂO, Dorneles GL, Machado MáSN, Silva CáB, Petry Letí, Schafer A, França RT, de Andrade CM, Antoniazzi AQ, Tonin AA, Lopes STA, Pillat MM, Anti-erythrocyte IgG in hamsters with acute experimental infection by Leptospira interrogans serovar Canicola, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.06.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Anti-erythrocyte IgG in hamsters with acute experimental infection by Leptospira interrogans serovar Canicola

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Departament of small animals clinic, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil. b

Department of large animal clinic, Federal de Santa Maria, Rio Grande do Sul, Brazil.

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Ânderson O. Sobrozaa*, Guilherme L. Dornelesa, Márcia S. N. Machadoa,Cássia B. Silvaa, Letícia Petrya, Andressa Schafera, Raqueli T. Françaa, Cinthia M. de Andradea, Alfredo Q. Antoniazzib, Alexandre A. Toninc Sonia T. A. Lopesa Micheli M. Pillatd

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Master in Animal Health and Production Applied to Small Properties, University of Western Santa Catarina (UNOESC), Santa Catarina, Brazil; dDepartment of Veterinary Medicine, Uninorte, Amazonas, Brazil.

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Department of Biochemistry, Institute of Chemistry, Universidade de São Paulo São Paulo, Brazil

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*Corresponding author: phone/fax: +55 55 3220 8819. E-mail address:

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[email protected] (Sobroza, Â.O.)

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ACCEPTED MANUSCRIPT Abstract

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The aim of this study was to evaluate the behavior of erythrocyte and platelet,

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as immunological markers, as well as evaluate the involvement of these factors

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in hemolytic and hemorrhagic reactions in hamsters experimentally infected by

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Leptospira interrogans Serovar Canicola. Our experimental design was

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composed by two randomized groups: Infected Group (IG) (n=12) and control

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group (CG) (n=6). Ninety-six hours after the inoculation, the presence of

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immunoglobulins (IgG and IgM) and complement C3 levels, related to

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erythrocytes and platelets, was assessed. Platelet’s microparticles marked by

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CD61, reticulocytes and reticulated platelets were also quantified. Additionally,

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fibrinogen, prothrombin time, partially activated thromboplastin time and sera

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levels of IgG and IgM were assessed. Our results showed that levels of platelet

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decreased in IG (P < 0.001); as well as, there was presence of IgG and C3

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associated with erythrocyte surface in the infected animals (P<0.01, P<0.05,

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respectively). Levels of prothrombin time and Activated Partial Thromboplastin

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Time were increased, while fibrinogen level was decreased (P < 0.01) in IG.

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CD61 microparticles were higher (P<0.05) in IG due to platelet activation. Thus,

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it was established a positive correlation (P<0.01) between platelets count and

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fibrinogen (Figure 3, R=0.84, P<0.001). Therefore, the platelet consumption

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component was preponderant in relation to autoimmune causes. Finally,

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regarding the erythrocytes, the autoimmune component played an important

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role, did not causing hemolytic reaction in this acute experimental time.

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Key words: Microparticle; platelets; immunoglobulins; leptospirosis; hemolysis.

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1. Introduction

Leptospirosis is a worldwide public health problem with great relevance

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in several countries, especially in Latin America and Southeast Asia.

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Epidemiologic studies indicate the occurrence of more than one million cases of

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severe human leptospirosis per year throughout the world, with a fatality rate of

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approximately 10% [1]. Pathophysiologically it is characterized by vasculitis,

54

and the damage to the capillary endothelial cells is the main cause of clinical

55

manifestations, such as renal tubular dysfunction, liver damage and pulmonary

ACCEPTED MANUSCRIPT hemorrhage [2]. The injury is most likely due to deposits of immune complexes

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in the small vessels of the organs affected. Activation of immuno-inflammatory

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response determines the release of various humoral factors, which cause the

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inflammatory process [2]. Several different studies [3.4] have shown an

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important correlation of Serovar canicola and infection in dogs, since it is more

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adapted to dogs as hosts [5], with special significance in urban areas and,

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consequently, leading to human infections.

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The increase of microparticles (MPs) has been the subject of studies in

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sepsis and in diseases with activation of the coagulation cascade, such as

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acute leptospirosis. Microparticles are defined as a population of vesicles

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originated from different cell types after activation or apoptosis. They measure

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between 50 nm and 1000 nm and contain cellular material, such as proteins,

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mRNA and lipoproteins, which are keys to their identification by flow cytometry

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[6]. All blood cells produce MPs, however platelets release the highest number

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of circulating MPs, corresponding to 70%-90% of total MP in the plasma of

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healthy individuals [7,8]. When appropriate technology and techniques are used

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to mark MPs, by CD 61, along with anexin as an apoptosis marker, they are an

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important tool and can be used as explanatory factor in studies of pathogenesis,

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since the attention is focused on the activation and platelet consumption

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phenomena [9].

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Hemostatic disorders and clinical presentations with loss of erythroid

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mass, by either hemorrhages or hemolytic events, have been described in

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severe studies of infection by L. interrogans [10-12]. Such studies, however,

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have not been focused on the phenomena of platelet and/or erythrocyte

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opsonization. Therefore, this study aimed to: i) evaluate IgG, IgM and

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complement system activation by C3 in the erythrocyte and platelet membrane

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of hamsters experimentally infected by Leptospira interrogans sorovar Canicola;

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ii) analyze the medullary response against infection leading hematic and

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platelet loss (through the percentage of reticulocytes and reticulated platelets,

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respectively); iii) evaluate the micro expression, by CD61 binding, in platelet-

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poor plasma (PPP); and iv) perform a hemogram, quantification of serum IgG

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and IgM, assess prothrombin time (PT) and activated partial thromboplastin

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time (APTT), as well as plasma fibrinogen levels.

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2. Material and Methods 2.1. Experimental model

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This study was approved by the Animal Welfare Committee of Federal

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University de Santa Maria (UFSM), under number 4923201015 and it is in

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accordance to Brazilian laws and ethical principles published by the Colégio

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Brasileiro de Experimentacão Animal (COBEA). As experimental models, we

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used hamsters (Mesocricetus auratus), since it is the ideal model for virulence

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and pathogenicity investigations in leptospirosis infection [13,14], providing a

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similar pattern when compared with to severe human leptospirosis. Hamsters

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underwent to a 15-day adaptation period in which they received commercial

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feed and water ad libitum. After adaptation, 2 animals were used for bacterial

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activation, and 18 for the establishment of the experimental groups (total of 20

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hamsters). All of them were adults, males, with an average age of 60 days and

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weighing 80 to 96 grams. Hamsters were randomly divided into 2 groups [CG =

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control (n=6) and IG = infected (n=12)], housed in cages (3/each cage) in an

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experimental room (under controlled temperature and humidity, 25°C and 70%,

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respectively).

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2.2. Virulence recovery, inoculum, inoculation process and success of experimental infection.

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Leptospira interrogans sorovar Canicola used in our study was a virulent

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strain (LO4). In order to obtain a fully virulent pattern, 0.5 mL of culture,

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containing approximately 2 x 108 leptospires (quantified through Petroff-

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Hausser counting chamber in

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intraperitoneally in one adult hamster (H1). When H1 showed severe clinical

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signs of leptospirosis (5 days post-infection), we performed its euthanasia,

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recovering samples of liver and kidney for further re-inoculation. A pool of these

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organs were obtained (composing a macerate) and, then, it was reinoculated in

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a second animal (H2) following a protocol of 1/10 proportion (g tissue/mL of

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distilled water at 28ºc). The onset of clinical signs of disease in H2 was 4 days

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post-infection, period that was carried out the euthanasia.

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darkfield microscope),

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Thus, after the second passage (virulence recovery), it was performed

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the liver and kidney collection for Leptospira recovery, carrying out the

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inoculation

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subcutaneously according to a consolidated methodology [15]. The animals in

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IG received, as inoculum, 0.5 mL of supernatant (pool macerated of

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kidney/liver) at 28° C and containing 20 to 30 lept ospiras per microscopic

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darkfield (quantified through Petroff-Hausser counting chamber) at 400x

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magnification. Animals from CG received, also subcutaneously, 0.5 mL of sterile

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saline solution at 28 °C. Prostration, food and wat er ingestion and presence of

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blood in the cages were evaluated every 12 hours after inoculation.

our

infected

group

(IG).

Inoculation

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For confirmation of infection success, it was performed two protocols: 1st:

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leptospire culture of urine aspirated from bladder. One drop of urine was placed

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between slide and coverslip and examined under darkfield microscopy at a

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magnification of 400x. 2nd: inoculation of renal and hepatic macerated tissue in

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EMJH media. Briefly, samples of kidney and liver (01 gram of pool) were mixed

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in modified EMJH medium with 5-fluorouracil (300 mg/L – Sigma USA) and

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nalidixic acid (20 mg/L), using a serial dilution technique, to evaluate the

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percentage of recovery of the added microorganisms. Other characteristics

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findings of severe leptospirosis were observed during necropsies and were also

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taken into account [free liquid (ascites) into the abdominal cavity, jaundice,

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blood in the intestinal lumen, hepatosplenomegaly and macroscopic renal

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changes].

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2.3. Sampling and auxiliary analysis

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Blood samples collection were carried out (by cardiac puncture with

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hamsters under anesthesia) on the fourth day post-infection (PI) in both groups.

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In average 5 ml of blood was obtained and divided into tubes containing

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ethylenediaminetetraacetic acid (EDTA), sodium citrate and tubes without

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anticoagulant. Euthanasia was conducted by cervical dislocation after a deep

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anesthesia. Complete blood count and platelet count was carried out through a

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blood cell counter (BC 2800 Vet). Erythrocyte and platelet morphological

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evaluation was performed on blood smear stained with Fast Paranotico®, using

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optical microscopy.

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Quantification

of

IgG

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IgM

in

serum

was

performed

by

immunonephelometric assay, using a Behring BN II Nephelometer (Dade

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Behring - USA) with reagents from Dade Behring. To measure Prothrombin time

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(PT) and Activated Partial Thromboplastin Time (APTT), a commercial reagent

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containing rabbit brain extract was used, with preservative and plasma activator

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for PT and ellagic acid and calcium chloride for APTT. For determination of

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Fibrinogen, via commercial kit (Labtest, Minas Gerais, BR), a standardized

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amount of Thrombin was added to a sample of diluted citrated plasma and the

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clotting time was measured. The coagulation time of diluted citrated plasma was

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found to be inversely proportional to the concentration of Fibrinogen. In order to

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obtain the concentration of Fibrinogen, coagulation time of plasma being tested

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was compared with the coagulation times for a series of dilutions of plasma

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containing a known concentration of Fibrinogen. For PT, APTT and Fibrinogen

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the formation of fibrin was monitored in a semi-automated device (Quick

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timer®).

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2.4. Antibodies and C3 detection on the surface of erythrocytes

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The detection of IgG, IgM and C3 in surface of erythrocytes was

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performed in samples containing, at least, 1 ml of whole blood. These samples

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were centrifuged at 1,500 g for 5 min, in order to obtain the fraction of plasma-

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free erythrocytes. Upon removing the proteins from the plasma, 100 µL of

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erythrocytes were added to 4 mL of phosphate buffered saline solution

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(Phosphate Buffered Saline; PBS; Ca2 +, Mg2 +-free PBS, pH 7.4). The

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erythrocytes were washed three times with PBS to remove any remaining

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plasma proteins. After the last centrifugation, the pellet was resuspended in 5

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mL of PBS. Aliquots containing 1,000,000 cells were incubated with the specific

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antibodies (anti-IGM, anti-IgG and anti-C3 conjugated with FITC fluorophore;

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Bethyl Laboratories, Inc.) in the dilution of 1:50 at 4°C for 45 min. After the

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incubation period, the samples were washed three times with PBS and then

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resuspended in 300 µ L of PBS for data acquisition in the flow cytometer BD

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FACSVerse (BD Bioscience), with 15,000 events being acquired per sample.

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A gate in the dot-plot of the forward scatter channel (FS- forward

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scatter) versus the side scatter channel (SS-side scatter) was used to select the

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erythrocytes. To confirm the location of the gate, 7.3 µm Beads were used.

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From the negative samples, it was possible to map out a region in fluorescence

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detector 1 (FL1-FITC), where events observed on the right were considered

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positive. The analyses were carried out on the software Flowjo V10.

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2.5. Determination of the percentage of reticulocytes

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In order to determine the percentage of reticulocytes, a thiazole orange

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solution at a concentration of 100 ng/mL was used. An aliquot of 5 µ L of whole

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blood with EDTA was added to 1 ml of thiazole orange solution. For all the

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samples, tubes that served as negative control were also prepared using 5 µ L

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of whole blood with EDTA added to 1 ml of PBS. The samples were incubated

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and protected at room temperature for 1 h [16]. After the incubation period, the

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acquisition of the data was performed in BD FACSVerse Flow cytometry in

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which 15,000 events were acquired per sample. The selection/gate of the

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population of erythrocytes was performed in the same way described in the

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subsection 2.4. From the negative samples, it was possible to trace a region in

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the FL1, where events observed on the right were considered positive for

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thiazole orange. The analyses were carried out on the software Flowjo V10.

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2.6. Antibodies and C3 detection on the surface of platelets

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The detection of antibodies on the surface of the platelets was

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determined in one aliquot of whole blood with EDTA containing 106 cells. The

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samples were washed three times with PBS containing 3 mM of EDTA and

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centrifuged at 1,500 g for 5 minutes. After washings, the cells were incubated

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for 30 min with specific antibody (anti-IGM, IgG and anti-C3 in conjunction with

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FITC, both in the dilution of 1:50) at room temperature and protected from light.

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After the incubation period, the samples were washed three times with PBS and

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then resuspended in 400 µ L of the same solution for data acquisition in BD

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FACSVerse Flow cytometry. Fifteen thousand events per sample were acquired

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at the gate of the platelets, with one gate in the dot-plot of FSC versus SSC

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used to select the platelets. Beads of 2 µm diameter and samples incubated

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with the antibody anti-FITC CD61 (1:100; GeneTex) were used to assist in

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ACCEPTED MANUSCRIPT 218

selecting the population regarding the platelets (Figure 1). Negative sample,

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consisted of cells not exposed to the antibodies [17]. The analyses were carried

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out using software Flowjo V10.

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2.7. Reticulated platelet count

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For reticulated platelet count, the thiazole orange solution, at a

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concentration of 20 ng/mL was used. An aliquot of whole blood with EDTA

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containing 106 cells was added to 560 µL of thiazole orange solution. The

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samples were protected from light and incubated for 30 min at room

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temperature [18]. After the incubation period, data acquisition was performed in

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BD FACSVerse Flow cytometry, with events per sample. Gate selection of the

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platelet population was conducted in the same manner as described in the

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subsection 2.6. From the negative samples, it was possible to trace a region in

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the FL1, where events observed on the right were considered positive for

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thiazole orange. The analyses were carried out using Flowjo V10 software.

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2.8. Determination of microparticles marked by CD61

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For determination of microparticles marked by CD61, it was necessary to

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prepare platelet-poor plasma (PPP). In order to achieve this, 1 mL of

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anticoagulated blood (by using sodium citrate) was centrifuged at 1500 g for 10

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min at 20°C, in order to obtain platelets rich in p lasma (PRP). Thereafter, a new

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centrifugation at 13,000 g, for 10 min, at 20° C wa s conducted resulting in the

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oblation of PPP [19]. The MPs were isolated from the PPP by using two

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ultracentrifugation at 100,000 g for 60 min. The MPs pellet was resuspended

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and gently homogenized in vortex using wash buffer. Two aliquots of MPs were

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incubated, respectively, with annexin-V antibody and anti CD61 conjugated with

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FITC for 15 min in the dark at room temperature. The MPs were determined by

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adding a known number of beads (Figure 1) to the sample and adjusted to the

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final dilution of the original blood sample [19].

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2.9. Statistical analysis

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The data were submitted to the Student test (test t) for comparison of

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mean values. The significance level was 5%, and P values below 5% were

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considered statistically different. The effect of platelets count on fibrinogen

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concentration was examined by linear correlation. P values lower than 5% were

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considered to be statistically different.

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3. Results

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3.1. Clinical evaluation, characteristics of autopsy and cultures

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Hamsters of IG presented clinical signs such as bristling fur, prostration,

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apathy, hypersensitivity to light and eye hyperemia, in average 48 h PI. Fecal

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blood loss was observed after 72 h. The cultures of the material used for

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bacterial recovery were positive, reaffirming the successful in our experimental

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model of infection. On necropsies, the infected animals confirmed the changes

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previously described (jaundice, hepato-splenomegaly, ascites).

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3.2.

Hematology

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Platelet count was considered lower (P < 0.001) in the animals of IG

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(Table 1). Hematocrit parameter showed no statistical difference between both

ACCEPTED MANUSCRIPT groups (P>0.05, Table 2). The presence of some schistocytes and rare

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microspherocytes (less than 1%) were observed in IG.

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3.3. Platelet immunoglobulins, coagulation tests, CD61+ microparticles and reticulated platelets

The markers of immunoglobulins and platelet complement (IgG, IgM and

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C3), did not show significant difference between both groups (P>0.05), (Figure

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2). In IG, PT and APTT results were increased and plasma fibrinogen was

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decreased (Table 1, P<0.001). MPs, marked by CD61, were increased in

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Platelet-poor plasma on hamster of IG (Table 1, P<0.05). However, the

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percentage of reticulate platelets did not differ between groups (Table 2,

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P>0.05). It was found a positive correlation (P<0.01) between levels of platelets

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and fibrinogen (r = 0.84, P<0.001),(Figure 3).

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3.4. Erythrocyte immunoglobulins, C3 erythrocyte and reticulocyte

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Hamsters of IG showed higher percentage of IgG binding (Figure 4,

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P<0.001). No significant difference on IgM was noticed between both groups

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(Figure 4, P>0.05). A higher percentage of binding was also observed for C3 in

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IG (Figure 4, P<0.05). There was no difference in the percentage of

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reticulocytes between both groups (Table 2, P>0.05).

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3.5. Serum immunoglobulins

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The dosage of IgM and IgG in serum was not significantly changed by

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the experimental infection (Table 2, P>0.05).

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4. DISCUSSION

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The lack of significant decrease in the hematimetric values between

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infected animals might is because the animals were not hydrated enough in the

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short period of the study. Losses were clinically confirmed by necropsy and

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blood found in intestines. Fluid loss followed the decrease in erytroid mass, thus

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obscuring laboratory results. Even though, clinical signs of acute disease were

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observed during our experimental study.

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In this sense, studies involving acute infection by pathogenic serovars of

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L. interrogans also presented thrombocytopenia, having as the main

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justifications splenic sequestration and peripheral consumption [10, 20, 21].

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Efforts to clarify thrombocytopenia, as a possible consequence of autoimmune

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involvement, in experimental models of leptospirosis has not been frequent;

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although other infectious diseases, that cause thrombocytopenia, involved

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immune disruption as part of the cause for this change [22]. By using the

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appropriate technique, our results demonstrate the absence of increase in

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platelet immunoglobulin’s on IG. This observation brings important contribution

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on

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thrombocytopenia as explanatory basis for this condition.

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the

pathophysiological

point

of

view,

eliminating

autoimmune

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By eliminating the autoimmune activity as a cause of the decrease in

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platelet count, and considering the results presented in the remaining

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assessments of coagulation cascade, it is possible to recognize that this

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collection of information is compatible with consumptive coagulopathy. Other

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studies of pathogenesis with leptospirosis feature the prolongation of

ACCEPTED MANUSCRIPT prothrombin time and activated partial thromboplastin, as well as decrease by

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consumption of coagulating proteins such as fibrinogen [20, 21,23]. One must

336

consider that fibrinogen, since it is an acute phase protein [24], could be

337

increased by an intense inflammatory situation, as proposed by the present

338

experiment. However, its exaggerated consumption is linked to bacterial

339

mechanisms of pathogenicity, and such use, in a disseminated manner, of

340

fibrinogen and other coagulation factors, provoke the extension perceived in in

341

vitro tests such as the ones carried out (PT and APTT).

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On the one hand, non-compliance of the autoimmune component, in our

343

results, may be evidenced by the absence of platelet opsonization markers. On

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the other hand, it may be ratified by the behavior of conventional coagulation

345

tests. There seems to have been insufficient time for a proper spinal cord

346

regenerative response, since the percentage of reticulate platelets did not differ

347

statistically between the groups. Microparticles were found in large amount in

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situations of vascular damage and hypercoagulability, such as in disseminated

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intravascular coagulation [25-29]. Hematopoietic cells MP producers contribute

350

significantly to the formation of fibrin [30]. Our results (MPsCD61

351

IG) demonstrate important platelet activation, which is consistent with

352

performance in microthrombus consumers. Platelet disorders are often reported

353

in sepsis and the role and behavior of the platelet MPs has been the target of

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revision studies. In contrast to our results, there may be an autoimmune

355

component associated with the overconsumption of platelets, and in this case,

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also with presence of activation monitored by CD61 [31].

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When attention is focused on erythrocyte conditions, resulting from

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experimental infection, in which there was increased positivity for IgG and C3

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among the infected, the results lead to a condition of opsonization for IgG with

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complement activation, however without causing clinical haemolysis. The

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following facts reinforce this perspective i) the microscopic findings in the blood

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smear analysis showed the presence of some schistocytes, ii) rare

363

microspherocytes, which may not accumulate in the blood if the formation

364

speed is below its removal speed [32]. Both alterations tend to be present in

365

situations of intra and extra-vascular hemolysis.

ACCEPTED MANUSCRIPT Anemia, of acute or chronic onset, has been described as a relevant

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signal in the onset and aggravation of leptospirosis in pets, production animals

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as well as in humans. Some studies demonstrate severe hemolytic activity [10]

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without deepening the hemolytic mechanisms. In this study, anti-erythrocyte IgG

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seems to act nonspecifically, since the acute onset of the experimental infection

371

would have insufficient time for the seroconversion of IgG anti-leptospira [33].

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What seems to occur is a clutter in the immune system and important pre-

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hemolytic opsonization. Leptospiras in circulation are avid by heme groups [34],

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leading to preliminary injury on erythrocyte membrane, which exposes self

375

antigens, forming membrane immunocomplexes; thus, triggering opsonization,

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and targeting extravascular hemolysis, usually splenic.

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Another possible mechanism demonstrated, by the presence of

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erythrocyte C3, which shows the participation of the complement system, is an

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intravascular hemolysis, producing schistocytes, which apparently began to

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accumulate in the peripheral blood. Io our study, there was no erythrocyte

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regenerative medullary response, since no difference was found between

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groups for percentage of reticulocytes, due to there being a short amount of

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time between the first clinical signs and material collection.

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The non-occurrence of difference in the quantification of serum

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immunoglobulins, between the two groups, possibly is because there was

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insufficient time for seroconversion, which has been estimated between 5 and 7

387

days. [33]. Furthermore, the degree of immunoglobulin consumption, by

388

opsonization (evidenced by cytometry techniques), was not enough to decrease

389

IgG serum levels.

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conclusions: i) we excludes the direct autoimmune factors as a cause of platelet

392

decrease in acute infection by L. interrogans Serovar Canicola and

393

demonstrated the participation of the IgG and complement system in pre-

394

hemolytic opsonization (at this point we believe that a model of chronic

395

experimental infection may be of great explanatory value); ii) we observed that

396

there was no erythrocyte and platelet regenerative medullary response as a

397

consequence of clinical hemorrhage; and iii) It is also possible to conclude that

398

inflammatory endothelial causes have been motivating the activation of the

399

cascade, the consumption of coagulation factors and clinical hemorrhage for

ACCEPTED MANUSCRIPT 400

this experimental infection. Microparticles, marked by CD61, proved to be an

401

important tool for monitoring Platelet Activation, which strengthens the

402

possibility of CD61 uses as an auxiliary marker of diagnostic and/or as a

403

possible therapeutic target in future studies of infectious diseases with clinical

404

bleeding.

RI PT

405

5.Acknowledgments:

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This study was supported by the Conselho Nacional de Desenvolvimento

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Científico (CNPq). We also acknowledge fellowships from CNPq and CAPES.

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M.M.P. is grateful for a postdoctoral fellowship from FAPESP (2015/19478-3).

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ACCEPTED MANUSCRIPT Highlights

We excludes the direct autoimmune factors as a cause of platelet decrease in acute infection by L. interrogans Serovar Canicola.

RI PT

Was demonstrated the participation of the IgG and complement system in pre-hemolytic opsonization.

Inflammatory endothelial causes have been motivating the activation of

AC C

EP

TE D

M AN U

SC

the coagulation cascade in acute infection by L. interrogans Serovar Canicola.