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Alveolar Septal Deposition of Immunoglobulin and Complement Parallels Pulmonary Hemorrhage in a Guinea Pig Model of Severe Pulmonary Leptospirosis
2004; Elsevier BV; Volume: 164; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63198-7
ISSN1525-2191
AutoresJarlath E. Nally, Chavit Chantranuwat, Xiaoyang Wu, Michael C. Fishbein, Martha María Pereira, João José Pereira da Silva, D R Blanco, Michael Lovett,
Tópico(s)Leptospirosis research and findings
ResumoHuman patients suffering from leptospirosis present with a diverse array of clinical manifestations, including the more severe and often fatal pulmonary form of the disease. The etiology of pulmonary hemorrhage is unclear. Isolates of Leptospira acquired from patients suffering from pulmonary hemorrhage were used to develop a guinea pig model of pulmonary hemorrhage. Gross findings post-infection confirmed extensive hemorrhage in the lungs and on peritoneal surfaces as the likely cause of death. Immunohistochemistry confirmed the presence of large numbers of leptospires in kidney, liver, intestinal tissues, and spleen, but few inflammatory cells were seen. In marked contrast, few leptospires were detected in infected hemorrhagic lung tissue. Blood chemistries and hematology did not reveal the etiology of the hemorrhage observed. There was no chemical or microscopic evidence for disseminated intravascular coagulation. To ascertain an immunopathologic role during disease, immunofluorescence was performed on infected lung tissues and confirmed the presence of IgM, IgG, IgA, and C3 along the alveolar basement membrane. This suggests that an autoimmune process may be the etiology of fatal pulmonary hemorrhage in leptospirosis. Human patients suffering from leptospirosis present with a diverse array of clinical manifestations, including the more severe and often fatal pulmonary form of the disease. The etiology of pulmonary hemorrhage is unclear. Isolates of Leptospira acquired from patients suffering from pulmonary hemorrhage were used to develop a guinea pig model of pulmonary hemorrhage. Gross findings post-infection confirmed extensive hemorrhage in the lungs and on peritoneal surfaces as the likely cause of death. Immunohistochemistry confirmed the presence of large numbers of leptospires in kidney, liver, intestinal tissues, and spleen, but few inflammatory cells were seen. In marked contrast, few leptospires were detected in infected hemorrhagic lung tissue. Blood chemistries and hematology did not reveal the etiology of the hemorrhage observed. There was no chemical or microscopic evidence for disseminated intravascular coagulation. To ascertain an immunopathologic role during disease, immunofluorescence was performed on infected lung tissues and confirmed the presence of IgM, IgG, IgA, and C3 along the alveolar basement membrane. This suggests that an autoimmune process may be the etiology of fatal pulmonary hemorrhage in leptospirosis. Leptospirosis continues to be a leading zoonotic infection throughout the world.1Vinetz JM Leptospirosis.Curr Opin Infect Dis. 2001; 14: 527-538Crossref PubMed Scopus (210) Google Scholar, 2Levett PN Leptospirosis.Clin Microbiol Rev. 2001; 14: 296-326Crossref PubMed Scopus (2321) Google Scholar The purpose of this study was to address the etiology and pathogenesis of the hemorrhagic diathesis seen in leptospirosis, and in particular, in the severe pulmonary form of the disease.3O'Neil KM Rickman LS Lazarus AA Pulmonary manifestations of leptospirosis.Rev Infect Dis. 1991; 13: 705-709Crossref PubMed Scopus (91) Google Scholar, 4Vijayachari P Sehgal SC Goris MG Terpstra WJ Hartskeerl RA Leptospira interrogans serovar Valbuzzi: a cause of severe pulmonary haemorrhages in the Andaman Islands.J Med Microbiol. 2003; 52: 913-918Crossref PubMed Scopus (37) Google Scholar, 5Trevejo RT Rigau-Perez JG Ashford DA McClure EM Jarquin-Gonzalez C Amador JJ de los Reyes JO Gonzalez A Zaki SR Shieh WJ McLean RG Nasci RS Weyant RS Bolin CA Bragg SL Perkins BA Spiegel RA Epidemic leptospirosis associated with pulmonary hemorrhage: Nicaragua, 1995.J Infect Dis. 1998; 178: 1457-1463Crossref PubMed Scopus (331) Google Scholar, 6Silva JJ Dalston MO Carvalho JE Setubal S Oliveira JM Pereira MM Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis.Rev Soc Bras Med Trop. 2002; 35: 395-399Crossref PubMed Google Scholar, 7Seijo A Coto H San Juan J Videla J Deodato B Cernigoi B Messina OG Collia O de Bassadoni D Schtirbu R Olenchuk A de Mazzonelli GD Parma A Lethal leptospiral pulmonary hemorrhage: an emerging disease in Buenos Aires, Argentina.Emerg Infect Dis. 2002; 8: 1004-1005Crossref PubMed Scopus (47) Google Scholar The hemorrhagic potential of leptospirosis has been noted since the first description of the features of severe human infection by Weil in 1886.8Weil A Ueber eine eigenthumliche, mit Milztumor, Icterus und Nephritis einhergehende, acute Infectionskrankheit.Dtsch Arch Klin Med. 1886; 39: 209Google Scholar When the causative spirochete was first cultivated in 1916 by Inada, he accordingly named it "spirocheta icterohaemorrhagiae."9Feigin RD Anderson DC Human leptospirosis.CRC Crit Rev Clin Lab Sci. 1975; 5: 413-467Crossref PubMed Scopus (121) Google Scholar, 10Inada R Ido Y Hoki R Kaneko R Ito H The etiology, mode of infection, and specific therapy of Weil's disease. (Spirochaetosis icterohaemorrhagica).J Exp Med. 1916; 23: 377-402Crossref PubMed Scopus (107) Google Scholar Nonetheless, appreciation that pulmonary hemorrhage can be the most prominent and often fatal manifestation of severe leptospirosis in humans has emerged relatively recently.5Trevejo RT Rigau-Perez JG Ashford DA McClure EM Jarquin-Gonzalez C Amador JJ de los Reyes JO Gonzalez A Zaki SR Shieh WJ McLean RG Nasci RS Weyant RS Bolin CA Bragg SL Perkins BA Spiegel RA Epidemic leptospirosis associated with pulmonary hemorrhage: Nicaragua, 1995.J Infect Dis. 1998; 178: 1457-1463Crossref PubMed Scopus (331) Google Scholar A case of pulmonary hemorrhage in humans was first reported in 1943 in Switzerland,11Moeschlin S Lungeninfiltrate beim Ikterus infeciosus Weil.Schweitz Med Wochenschr. 1943; 73: 1227-1230Google Scholar by 1953 it was regarded as an infrequent but potentially severe feature of leptospirosis in the United States.12Silverstein CM Pulmonary manifestations of leptospirosis.Radiology. 1953; 61: 327-334PubMed Google Scholar Small numbers of leptospirosis cases marked by severe pulmonary hemorrhage were recognized in China and Singapore by 1970.13Poh SC Soh CS Lung manifestations in leptospirosis.Thorax. 1970; 25: 751-755Crossref PubMed Scopus (20) Google Scholar However, in 1975 a comprehensive clinical review of leptospirosis did not mention pulmonary hemorrhage per se.9Feigin RD Anderson DC Human leptospirosis.CRC Crit Rev Clin Lab Sci. 1975; 5: 413-467Crossref PubMed Scopus (121) Google Scholar Since that time there has been growing recognition of severe cases of leptospirosis in which pulmonary hemorrhage is the most prominent and often fatal feature.6Silva JJ Dalston MO Carvalho JE Setubal S Oliveira JM Pereira MM Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis.Rev Soc Bras Med Trop. 2002; 35: 395-399Crossref PubMed Google Scholar, 7Seijo A Coto H San Juan J Videla J Deodato B Cernigoi B Messina OG Collia O de Bassadoni D Schtirbu R Olenchuk A de Mazzonelli GD Parma A Lethal leptospiral pulmonary hemorrhage: an emerging disease in Buenos Aires, Argentina.Emerg Infect Dis. 2002; 8: 1004-1005Crossref PubMed Scopus (47) Google Scholar, 14Sehgal SC Murhekar MV Sugunan AP Outbreak of leptospirosis with pulmonary involvement in north Andaman.Indian J Med Res. 1995; 102: 9-12PubMed Google Scholar, 15Yersin C Bovet P Merien F Clement J Laille M Van Ranst M Perolat P Pulmonary haemorrhage as a predominant cause of death in leptospirosis in Seychelles.Trans R Soc Trop Med Hyg. 2000; 94: 71-76Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 16Im JG Yeon KM Han MC Kim CW Webb WR Lee JS Han YC Chang WH Chi JG Leptospirosis of the lung: radiographic findings in 58 patients.AJR Am J Roentgenol. 1989; 152: 955-959Crossref PubMed Scopus (81) Google Scholar, 17Zaki SR Shieh WJ Leptospirosis associated with outbreak of acute febrile illness and pulmonary haemorrhage, Nicaragua, 1995: The Epidemic Working Group at Ministry of Health in Nicaragua.Lancet. 1996; 347: 535-536Abstract PubMed Scopus (135) Google Scholar In particular, a 1995 Nicaraguan outbreak was noteworthy because the few fatal cases presented as a hemorrhagic fever, and unlike classic Weil's disease, renal failure was not observed.5Trevejo RT Rigau-Perez JG Ashford DA McClure EM Jarquin-Gonzalez C Amador JJ de los Reyes JO Gonzalez A Zaki SR Shieh WJ McLean RG Nasci RS Weyant RS Bolin CA Bragg SL Perkins BA Spiegel RA Epidemic leptospirosis associated with pulmonary hemorrhage: Nicaragua, 1995.J Infect Dis. 1998; 178: 1457-1463Crossref PubMed Scopus (331) Google Scholar Pulmonary hemorrhage was the most frequent cause of death in this outbreak. Recent reviews of human leptospirosis have highlighted the significance of pulmonary involvement.1Vinetz JM Leptospirosis.Curr Opin Infect Dis. 2001; 14: 527-538Crossref PubMed Scopus (210) Google Scholar, 2Levett PN Leptospirosis.Clin Microbiol Rev. 2001; 14: 296-326Crossref PubMed Scopus (2321) Google Scholar It is clear that the severe pulmonary form of leptospirosis (SPFL)6Silva JJ Dalston MO Carvalho JE Setubal S Oliveira JM Pereira MM Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis.Rev Soc Bras Med Trop. 2002; 35: 395-399Crossref PubMed Google Scholar may occur as distinct outbreaks,5Trevejo RT Rigau-Perez JG Ashford DA McClure EM Jarquin-Gonzalez C Amador JJ de los Reyes JO Gonzalez A Zaki SR Shieh WJ McLean RG Nasci RS Weyant RS Bolin CA Bragg SL Perkins BA Spiegel RA Epidemic leptospirosis associated with pulmonary hemorrhage: Nicaragua, 1995.J Infect Dis. 1998; 178: 1457-1463Crossref PubMed Scopus (331) Google Scholar, 14Sehgal SC Murhekar MV Sugunan AP Outbreak of leptospirosis with pulmonary involvement in north Andaman.Indian J Med Res. 1995; 102: 9-12PubMed Google Scholar in which a high percentage of cases are marked by pulmonary hemorrhage or as sporadic cases,2Levett PN Leptospirosis.Clin Microbiol Rev. 2001; 14: 296-326Crossref PubMed Scopus (2321) Google Scholar, 6Silva JJ Dalston MO Carvalho JE Setubal S Oliveira JM Pereira MM Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis.Rev Soc Bras Med Trop. 2002; 35: 395-399Crossref PubMed Google Scholar, 7Seijo A Coto H San Juan J Videla J Deodato B Cernigoi B Messina OG Collia O de Bassadoni D Schtirbu R Olenchuk A de Mazzonelli GD Parma A Lethal leptospiral pulmonary hemorrhage: an emerging disease in Buenos Aires, Argentina.Emerg Infect Dis. 2002; 8: 1004-1005Crossref PubMed Scopus (47) Google Scholar while in other instances, outbreaks of leptospirosis occur in which pulmonary involvement is not striking.18Ko AI Galvao Reis M Ribeiro Dourado CM Johnson Jr, WD Riley LW Urban epidemic of severe leptospirosis in Brazil: Salvador Leptospirosis Study Group.Lancet. 1999; 354: 820-825Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar It is not known what factors influence the relative virulence and spectrum of clinical manifestations seen in human leptospirosis. Although serovar Icterohaemorrhagiae is most often associated with severe disease, it is well established that infections with diverse Leptospira can present with similar clinical manifestations.1Vinetz JM Leptospirosis.Curr Opin Infect Dis. 2001; 14: 527-538Crossref PubMed Scopus (210) Google Scholar, 2Levett PN Leptospirosis.Clin Microbiol Rev. 2001; 14: 296-326Crossref PubMed Scopus (2321) Google Scholar, 19Sasaki DM Pang L Minette HP Wakida CK Fujimoto WJ Manea SJ Kunioka R Middleton CR Active surveillance and risk factors for leptospirosis in Hawaii.Am J Trop Med Hyg. 1993; 48: 35-43PubMed Google Scholar It is unknown whether the seeming emergence of cases of severe pulmonary hemorrhagic leptospirosis reflects unique virulence properties of specific strains. A variety of Leptospira strains can cause disease in rodents with features similar to the most severe human cases. In 1957, Faine20Faine S Virulence in Leptospira. I. Reactions of guinea pigs to experimental infection with Leptospira icterohaemorrhagiae.Br J Exp Pathol. 1957; 38: 1-7PubMed Google Scholar reported that guinea pigs infected with serovar Icterohaemorrhagiae exhibited hemorrhages most prominent in lung, abdominal wall, and retroperitoneal fat. Since Faine's report,20Faine S Virulence in Leptospira. I. Reactions of guinea pigs to experimental infection with Leptospira icterohaemorrhagiae.Br J Exp Pathol. 1957; 38: 1-7PubMed Google Scholar many investigators have reported similar findings with serovar Icterohaemorrhagiae infection of guinea pigs.21Higgins R Cousineau G The pathogenesis of leptospirosis I: hemorrhages in experimental leptospirosis in guinea pigs.Can J Comp Med. 1977; 41: 174-181PubMed Google Scholar, 22De Brito T Bohm GM Yasuda PH Vascular damage in acute experimental leptospirosis of the guinea pig.J Pathol. 1979; 128: 177-182Crossref PubMed Scopus (76) Google Scholar, 23da Silva JJ Netto BA Lilembaum W Alvim ME de Oliveira AV The hemorrhagic syndrome of leptospirosis: an experimental study in guinea pigs.Rev Soc Bras Med Trop. 1995; 28: 169-177Crossref PubMed Google Scholar, 24Merien F Truccolo J Rougier Y Baranton G Perolat P In vivo apoptosis of hepatocytes in guinea pigs infected with Leptospira interrogans serovar Icterohaemorrhagiae.FEMS Microbiol Lett. 1998; 169: 95-102Crossref PubMed Google Scholar Pulmonary hemorrhage has been reported in hamsters including infections with serovar Icterohaemorrhagiae,25Miller NG Allen JE Wilson RB The pathogenesis of hemorrhage in the lung of the hamster during acute leptospirosis.Med Microbiol Immunol (Berl). 1974; 160: 269-278Crossref PubMed Scopus (36) Google Scholar, 26Sanger VL Hamdy AH Fizetle WB Bohl EH Ferguson LC Leptospira pomona infection in hamsters.Cornell Vet. 1961; 51: 489-498PubMed Google Scholar serovar Pomona26Sanger VL Hamdy AH Fizetle WB Bohl EH Ferguson LC Leptospira pomona infection in hamsters.Cornell Vet. 1961; 51: 489-498PubMed Google Scholar serogroup Canicola,27Oliva R Infante JF Gonzalez M Perez V Sifontes S Marrero O Valdes Y Farinas M Estevez L Gonzalez I Pathologic-clinical characterization of leptospirosis in a golden Syrian hamster model.Arch Med Res. 1994; 25: 165-170PubMed Google Scholar and serogroups Hardjo and Szwaijizak.28Badiola J Thiermann AB Cheville NF Pathologic features of leptospirosis in hamsters caused by Leptospira interrogans serovars Hardjo and Szwajizak.Am J Vet Res. 1983; 44: 91-99PubMed Google Scholar In contrast, experimental infection of adult outbred mice or rats results in asymptomatic infection and the development of chronically infected carrier hosts.29Adler B Faine S Susceptibility of mice treated with cyclophosphamide to lethal infection with Leptospira interrogans Serovar pomona.Infect Immun. 1976; 14: 703-708PubMed Google Scholar, 30Faine S The growth of Leptospira australis B in the kidneys of mice in the incipient experimental carrier state.J Hyg (Cambridge). 1962; 60: 435-442Crossref PubMed Scopus (22) Google Scholar, 31Faine S Factors affecting the development of the carrier state in leptospirosis.J Hyg (Cambridge). 1962; 60: 427-434Crossref PubMed Scopus (17) Google Scholar It is generally believed that neither thrombocytopenia nor diminution of hepatically synthesized clotting factors seen in human leptospirosis were sufficient per se to account for the bleeding diathesis observed.1Vinetz JM Leptospirosis.Curr Opin Infect Dis. 2001; 14: 527-538Crossref PubMed Scopus (210) Google Scholar, 9Feigin RD Anderson DC Human leptospirosis.CRC Crit Rev Clin Lab Sci. 1975; 5: 413-467Crossref PubMed Scopus (121) Google Scholar There have been two reports that disseminated intravascular coagulation (DIC) was associated with the hemorrhage caused by serogroup Icterohemorrhagiae in guinea pigs.21Higgins R Cousineau G The pathogenesis of leptospirosis I: hemorrhages in experimental leptospirosis in guinea pigs.Can J Comp Med. 1977; 41: 174-181PubMed Google Scholar, 23da Silva JJ Netto BA Lilembaum W Alvim ME de Oliveira AV The hemorrhagic syndrome of leptospirosis: an experimental study in guinea pigs.Rev Soc Bras Med Trop. 1995; 28: 169-177Crossref PubMed Google Scholar These reports, however, did not demonstrate significant small vessel fibrin deposition, fibrinogen consumption, or convincing release of fibrin degradation products. DIC is not mentioned as a feature of human leptospirosis.1Vinetz JM Leptospirosis.Curr Opin Infect Dis. 2001; 14: 527-538Crossref PubMed Scopus (210) Google Scholar, 2Levett PN Leptospirosis.Clin Microbiol Rev. 2001; 14: 296-326Crossref PubMed Scopus (2321) Google Scholar, 9Feigin RD Anderson DC Human leptospirosis.CRC Crit Rev Clin Lab Sci. 1975; 5: 413-467Crossref PubMed Scopus (121) Google Scholar Recently SPFL characterized by alveolar hemorrhage and acute respiratory failure was described in four patients, three of whom died within 48 hours of presentation with respiratory symptoms; blood cultures from each of the four patients were positive.6Silva JJ Dalston MO Carvalho JE Setubal S Oliveira JM Pereira MM Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis.Rev Soc Bras Med Trop. 2002; 35: 395-399Crossref PubMed Google Scholar The recovery of such isolates from these patients has provided an opportunity to study the causative agent of SPFL and the pathogenic mechanisms underlying pulmonary hemorrhage. Isolates of Leptospira interrogans serovar Copenhageni were obtained from blood cultures of patients suffering from SPFL who were admitted to Antonio Pedro University Hospital, Rio de Janeiro, Brazil.6Silva JJ Dalston MO Carvalho JE Setubal S Oliveira JM Pereira MM Clinicopathological and immunohistochemical features of the severe pulmonary form of leptospirosis.Rev Soc Bras Med Trop. 2002; 35: 395-399Crossref PubMed Google Scholar Cultures were maintained in EMJH liquid (Becton Dickinson, Cockeysville, MD) or EMJH semi-solid media (EMJH liquid media containing 0.2% noble agar). Isolates were passaged through guinea pigs to maintain virulence as described below. Hartley male guinea pigs (Charles River Laboratories, Kingston, NY), weighing 150 to 200 grams, 10 to 15 days of age, were housed in individual cages and fed standard guinea pig chow and water ad libitum. Guinea pigs were injected intraperitoneally with 103, 105, or 107 of low-passage RJ15958 or RJ16441 in a final volume of 500 μl. Infected guinea pig kidney tissue, cultured in EMJH semi-solid media in the presence of 200 μg/ml 5-fluorouracil, and sub-cultured to liquid EMJH media not more than two times provided low-passage isolates. Negative control animals were injected with EMJH media alone. Animals were monitored daily for signs of illness including weight loss and loss of mobility, and were euthanized when they appeared moribund. All animal studies were approved by the Animal Research Committee of the University of California Los Angeles. Normal and infected guinea pig viscera were fixed in neutral-buffered 4% formaldehyde (Medical Chemical Corporation, Torrance, CA), processed routinely, embedded in paraffin, cut into 4-μm serial sections and stained with hematoxylin and eosin (H&E). Paraffin-embedded tissue sections were stained for the presence of spirochetes using the modified Steiner silver stain as previously described.32Garvey W Fathi A Bigelow F Modified Steiner for the demonstration of Spirochetes.J Histotechnol. 1985; 8: 15-17Crossref Google Scholar Paraffin-embedded tissue sections were incubated at 60°C for 30 minutes. Two xylene washes were applied for 5 minutes, followed by 2 × 100% ethanol for 1 minute, 2 × 95% ethanol for 1 minute, 1 × 70% ethanol for 1 minute and incubation in 3% H2O2 in methanol for 15 minutes. After 2 × 5-minute washes in phosphate-buffered saline (PBS), sections were incubated in 0.2 mg/ml trypsin at 37°C for 15 minutes. Sections were again washed × 2 in PBS for 5 minutes before blocking with 10% normal goat serum (NGS) (Vector Laboratories, Burlingame, CA) for 30 minutes. Primary rabbit antibody specific for outer membrane vesicles (OMV) of RJ15958 was then added at a dilution of 1:500 in 3% NGS for 90 minutes. OMV was prepared using a modified procedure as previously described.32Garvey W Fathi A Bigelow F Modified Steiner for the demonstration of Spirochetes.J Histotechnol. 1985; 8: 15-17Crossref Google Scholar In brief, Leptospira were re-suspended in 0.1 mol/L citrate buffer (pH 3.0) and shaken vigorously for 3 hours. Samples were then passed through a French press (Thermo Spectronic, Rochester, NY) × 4 at 12,000 psi and the supernatant was centrifuged over a continuous sucrose gradient (20 to 60%). The upper OMV band was used to immunize 3.5 to 4 kg New Zealand White rabbits. After again washing tissues, biotinylated goat anti-rabbit IgG, diluted 1:200 in 3% NGS, was added for 40 minutes. Sections were again washed × 2 in PBS and incubated in avidin D-horseradish peroxidase (Vector Laboratories), diluted 1:1000 in PBS for 30 minutes. After washing, staining was visualized using 3, 3′-diaminobenzidine (Vector Laboratories) for 10 minutes and the reaction was stopped using H2O. Before mounting, slides were washed in H2O, dehydrated by dipping in 95% ethanol 40 times followed by 100% ethanol for 40 times and 3 × 5-minute washes in xylene. Sections were counterstained in weak hematoxylin. Fresh lung tissue from infected guinea pigs obtained at autopsy was embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA) and snap-frozen on dry ice. Four-μm thick frozen sections were serially cut and fixed in cold acetone for 10 minutes. Sections were air dried, washed in PBS for 5 minutes and 0.3% hydrogen peroxide in dH2O for 10 minutes. After 3 × 5-minute washes in PBS, tissue sections were blocked with 10% normal serum of the same species as the following secondary antibodies for 30 minutes. Sections were incubated in rabbit anti-guinea pig IgG, 1:50, (Sigma, St. Louis, MO), IgM μ-chain and IgA α-chain (ICN Biomedicals, Inc., Aurora, Ohio), or goat anti-guinea pig C3 (Accurate Chemical & Scientific Corporation, Westbury, NY) at 1:50 for 90 minutes and then washed in PBS for 5 minutes × 3. Biotinylated goat anti-rabbit IgG or horse anti-goat IgG was then applied for 40 minutes at 1:200. After washing in PBS for 5 minutes × 3, tissue sections were incubated with 1:100 FITC-Avidin D (Vector Laboratories) for 30 minutes in the dark. After final washing with PBS for 5 minutes × 3, sections were mounted with Vectashield mounting medium (Vector Laboratories) and examined by fluorescence microscopy. Negative controls included negative tissue samples processed as described, and positive tissues processed without primary antibody. Tissues were minced into 1- to 2-mm pieces and immersed for more than 2 hours in 2% glutaraldehyde in PBS (pH 7.0). Tissue was then washed in PBS (20 minutes × 3) and post-fixed in 1% osmium tetroxide. Tissues were washed again and dehydrated in graded ethanols, and embedded in Epon. One-μm sections were cut and mounted and stained with toluidine blue. From these sections, regions of interest were selected for ultrastructural study. Seventy-nm sections were cut and stained with uranyl acetate and lead citrate and examined with a JEOL JEM-100CX electron microscope. Blood was collected from moribund guinea pigs under anesthesia by cardiac puncture. Hematological parameters were determined by IDEXX Laboratories (Sacramento, CA). Data from seven separate experiments were pooled for analysis using the non-parametric Wilcoxon test. L. interrogans serovar Copenhageni SPFL isolates RJ15958 and RJ16441 proved highly virulent when injected intraperitoneally into guinea pigs at doses as low as 103 leptospires (Table 1). Isolate RJ16441 was consistently more virulent than RJ15958 at similar doses resulting in all RJ16441 infected guinea pigs being euthanized sooner than RJ15958 infected guinea pigs, P < 0.0001 (seven separate experiments, n = 50).Table 1Time of Euthanasia of Guinea Pigs after Infection with Different Amounts of SPFL Isolates RJ15958 and RJ16441Number of days post-infection until euthanasiaIsolateRJ15958RJ16441Dose103106–710575–610764–5Isolate RJ16441 was consistently more virulent that RJ15958 at similar doses resulting in all RJ16441 infected guinea pigs being euthanized sooner than RJ15958 infected guinea pigs, p < 0.0001. Open table in a new tab Isolate RJ16441 was consistently more virulent that RJ15958 at similar doses resulting in all RJ16441 infected guinea pigs being euthanized sooner than RJ15958 infected guinea pigs, p < 0.0001. Gross autopsy revealed distinct, well-circumscribed, multi-focal areas of hemorrhage visible on surfaces of the lungs, and extensive hemorrhage on peritoneal surfaces after infection with doses as low as 103 leptospires (Figure 1). The hemorrhages involved the intestinal mesenteric surfaces, but not the kidneys, liver, or spleen. H&E staining of infected liver tissue demonstrated hepatocyte necrosis observed as groups or as scattered individual hypereosinophilic cells with pyknotic nuclei (Figure 2). Cellular discohesion of hepatocytes ranged from focal to diffuse. Increased mitotic activity and increased binucleated hepatocytes, representing hepatocyte regeneration, were observed in all animals, but were more evident in RJ15958-infected guinea pigs. Activated Kuppfer cells characterized by an expanded foamy cytoplasm were commonly seen in the sinusoids. Mild to moderate increases in numbers of monocytes and neutrophils were observed in portal tracts. Silver and immunohistochemical staining confirmed large numbers of Leptospira in infected livers (Figure 2). More spirochetes and less background staining were observed with immunohistochemical staining compared to silver stains. Thus, immunohistochemical staining with rabbit antiserum specific for outer membrane vesicles (OMV) of RJ15958 provided a higher degree of sensitivity and specificity for the detection of Leptospira compared to silver staining. Immunohistochemistry provided two patterns of reactivity; pattern 1 in which the staining pattern was similar in shape to whole intact Leptospira, and pattern 2 in which the staining appeared as granular debris, presumably remnants of OMV or degenerated Leptospira. Leptospira were observed primarily along cell membranes of hepatocytes. Livers of guinea pigs infected with RJ16441 had larger numbers of intact leptospires identified by immunohistochemistry compared to those infected with RJ15958. However, large amounts of reactive debris were seen within the cytoplasm of activated Kuppfer cells in the livers of guinea pigs infected with RJ15958. In the liver, and in all other organs, there was no evidence of vasculitis or intravascular fibrin thrombi that would occur in the presence of DIC. Infected kidneys were characterized by tubular cell necrosis with occasional apoptotic cells (Figure 3). Increased mitoses indicative of tubular regeneration were noted. Occasionally, acute inflammatory cells were present in and around the renal tubules. There was also mild to marked infiltration by histiocytes. However, glomeruli appeared normal. Silver and immunohistochemical staining revealed large numbers of intact Leptospira (Figure 3). Intact Leptospira were observed along the tubular basement membrane, between tubular cells, within the tubular lumens, within the interstitium, and in some cases, a few were present within glomeruli. Fragments of spirochetes were found within histiocytes, in the interstitium, and in tubules. Degenerated spirochetes were rarely found in the glomeruli. Focal mucosal hemorrhage and edema were noted (Figure 4). The large intestine showed an increased number of foamy macrophages in the lamina propria and apoptotic bodies in the overlying epithelium. Immunohistochemical staining confirmed the presence of large numbers of intact Leptospira within the mucosa of the large and small intestines of guinea pigs infected with RJ15958 and RJ16441. Infected spleens showed reticuloendothelial system activation with increased hemophagocytosis ranging from mild to marked (Figure 4). Activated histiocytes with expanded pale to foamy cytoplasm were observed predominantly within red pulp. Immunohistochemical staining confirmed the presence of large numbers of intact Leptospira in the spleens of guinea pigs infected with RJ16441 (not shown). Few intact Leptospira were identified in guinea pigs infected with RJ15958, although reactive leptospiral debris was observed in the spleens of animals with either RJ15958 (Figure 4k), or RJ16441 and associated with the contents of enlarged foamy histiocytes. All infected guinea pigs had significant microscopic hemorrhage in lungs involving an estimated 5 to 70% of total lung parenchyma. Early hemorrhage appeared randomly as small foci. As severity increased, coalesced areas of hemorrhage developed. Hemophagocytic histiocytes were seen in hemorrhagic alveolar spaces. Generally, there was a mild to moderate increase of interstitial cellularity of the alveolar septae due to increased numbers of mononuclear cells and occasional neutrophils (Figure 5, a to h). Increased numbers of neutrophils in capillaries were also observed. Intact leptospires were only rarely detected by immunohistochemistry. Reactive debris was occasionally detected associated with monocytes or polymorphonuclear cells. Guinea pigs injected with 103 or 107 of isolate RJ15958 or RJ16441 (n = 10 including 2 negative controls), were sacrificed when they appeared moribund. Blood was taken by cardiac puncture for analysis of coagulation parameters and blood chemistry (Figure 6). Fibrinogen levels in infected guinea pigs, though elevated compared to normal controls, were within the normal range of 178 to 422 mg/dl.34Kaspareit J Messow C Edel J Blood coagulation studies in guinea pigs (Cavia porcellus).Lab Anim
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