Alleviation of renal and pulmonary injury by immunomodulation in leptospirosis: hamster model.

OBJECTIVE: Severe leptospirosis manifestations include acute renal failure, caused by acute interstitial nephritis and pulmonary hemorrohage. Spirochete invasion and toxicity of outer membrane cause robust inflammatory host responses. These responses lead to the generation of cytokines, chemokines, and inflammatory cell infiltrations which result in severe organ dysfunctions. The immunomodulation by the modulation of host immune response may alleviate the renal and pulmonary injury. The authors determined whether the current immunosuppressive agents could alleviate the inflammation and minimize the organ injury in hamster model. MATERIAL AND METHOD: The animal experiments were conducted with the approval of The Ethical Research Committee of Chulalongkorn University Hospital. The leptospira interrogan serovar pyrogenese was isolated from a wild rat. The spirochete was grown in Fletcher's semisolid media and after subcultures were transferred to the Fletcher's liquid media. An amount of 0.5 ml of the spirochete culture media containing 1 x 10(8) leptospires/ml was intraperitoneally injected to golden Syrian hamsters (Mesocrietus auratus), age 4-6 weeks, weighing 60-80 grams. The hamsters were randomed into 5 groups (n = 4 in each group) namely, 1) Normal group (Control group), 2) Leptospira group, 3) CsA group (leptospira with cyclosporine feeding, 100 mg/kg/ day), 4) Rapa group (leptospira with rapamicin feeding, 0.6 mg/kg/day), and 5) Irra group (leptospira with irradiation). Cyclosporine and rapamicin were started at day 0 after the spirochete injection. Gamma ray dose 200 cGy was irradiated to the hamster 3 days before the spirochete inoculation. The animals were autopsied or euthanized if expired or at day 5 post inoculation. The blood samples for BUN, and creatinine were drawn before the inoculation and at autopsy or euthanasia. RESULTS: The inoculation of L Interrogan 0.5 ml (1 x 10(8) leptospires/ml) without immunomodulation cause mortality of all animals at day 4 or day 5 post inoculation. The blood chemistry showed acute severe azotemia. The autopsy findings revealed severe interstitial nephritis and severe pulmonary hemorrhage. The hamsters in the Rapa group had only minimal pulmonary hemorrhage and minimal focal interstitial inflammation of kidney. There were cytoadherance of inflammatory cells to the endothelial cells in lungs and kidneys without the intrusion into the interstitium. The blood chemistry in Rapa group showed mild elevation of BUN and Cr. The immunomodulation by cyclosporine and irradiation did not alleviate the disease. On the contrary, cyclosporine and irradiation caused more severe histopathology. CONCLUSION: The immunomodulation by rapamicin in leptospirosis in hamsters could alleviate the kidney and pulmonary injuries. The up-regulation of IL-2 in peripheral blood lymphocytes did not result in the kidney and pulmonary injuries.

Duplex PCR-hybridization based detection of pathogenic Leptospira in environmental water samples obtained from endemic areas in northeast region of Thailand.

Leptospirosis, a major health problem worldwide, is known to be endemic in the northeastern part of Thailand with the risk of infection by exposure to pathogenic Leptospira in contaminated aquatic environment. A method based on PCR-hybridization detection of pathogenic Leptospira in water was established. The method included filtration of water sample through membrane filters of two pore sizes, DNA extraction from filters using a guanidine thiocyanate extraction method, a duplex-PCR assay with two primer pairs, and hybridization with a synthetic LipL32 DNA probe. The duplex-PCR allowed detection of two products of 279 bp for LipL32 gene and 430 bp for 16S rRNA gene. In water samples artificially seeded with serovar bratislava, at least 10(3) cells could be analyzed by PCR-agarose gel electrophoresis and 1-10 cells by PCR-Southern blot hybridization. The protocol was applied to the detection of pathogenic Leptospira in environmental waters collected from endemic areas in the northeast region of Thailand. Of 100 water samples analyzed, 23 samples were positive for pathogenic Leptospira with PCR performed with Southern blot hybridization only, but none was detected by PCR-agarose gel-electrophoresis. However, PCR performed with the chemiluminescent LipL32 probe using the Fluorescein ULS labeling facilitated the detection of low numbers of pathogenic Leptospira in water. This method should prove useful for monitoring of pathogenic Leptospira pollution in environmental waters, and has the potential to become a valuable tool to the surveillance of leptospirosis in endemic areas, thus leading to enhanced public health protection.

A histopathological study of hearts and spleens of hamsters (Mesocricetus auratus) infected with Leptospira interrogans, serovar pyrogenes.

The effects of Leptospira interrogans on the heart and spleen of hamsters were studied histopathologically. Infected hamsters were sacrificed at 1 hour, 6 hours and on days 1, 2, 3, 4, 5 and 6 after inoculation with Leptospira interrogans serovar pyrogenes. The heart and spleen of each of the sacrificed animals were removed and processed for routine conventional light microscopy. Infected hearts showed degenerative change of the cardiac muscle cells composed of cellular swelling, condensation of chromatin granules, pyknotic nuclei and acidophilic cytoplasm. Congestion of the cardiac blood vessels and hemorrhagic areas were found. Necrosis of the cardiac muscle cells was surrounded by numerous inflammatory cells. In the spleen, cellular necrosis was found scattered throughout the splenic cord. The splenic sinusoids were dilated and congested with many hemorrhagic areas. Inflammatory cell infiltration was also noted in the splenic parenchyma and the splenic sinusoids.

Development of a duplex-polymerase chain reaction for rapid detection of pathogenic Leptospira.

A duplex-polymerase chain reaction (PCR) for the rapid detection of pathogenic leptospires was developed by using two sets of newly designed primers which amplified in the same reaction two different DNA fragments simultaneously: 279-bp of LipL32 and 430-bp of 16S rRNA. For DNA extraction from bacterial cultures, the silica-based spin column method was found to be more suitable and was selected for the extraction of DNAs from all 92 bacterial strains including 56 strains of pathogenic Leptospira, 15 strains of non-pathogenic Leptospira and 21 other strains of bacteria. The PCR products were analyzed by agarose gel-electrophoresis with confirmation by Southern and dot hybridization using synthetic DNA probe prepared from LipL32 gene of a pathogenic reference strain, L. interrogans serovar pyrogenes. The duplex-PCR allowed detection of two products of 279 bp and 430 bp in all pathogenic Leptospira. Non-pathogenic Leptospira generated a single product of 430 bp. Other bacterial strains failed to reveal any amplification products. As little as 1 pg of pure DNA corresponding to 100 cells could be detected by agarose gel-electrophoresis, and 1-10 fg of pure DNA by hybridization.

A test strip IgM dot-ELISA assay using leptospiral antigen of endemic strains for serodiagnosis of acute leptospirosis.

A test strip IgM dot-ELISA assay for the detection of leptospire-specific IgM antibodies in human sera was developed. Antigen dotted on a nitrocellulose paper strip was the pool sonicated antigen prepared from three predominant reactive Leptospira serovars currently in endemic area, i.e., Bratislava, Sejroe and Pyrogenes. The ability of the test to diagnose acute leptospiral infection was assessed by testing 343 single serum samples from 96 laboratory-confirmed leptospirosis case patients with positive result in the standard microscopic agglutination test (MAT), 55 serum samples from patients with various diseases other than leptospirosis, and 192 serum samples from healthy individuals. Using the results of the MAT as a gold standard, the sensitivity and specificity of the test strip IgM dot-ELISA assay were 98.96 and 93.93 per cent, respectively. The assay offered relatively high negative predictive values (99.57%) thus making the assay ideally suited for rapid screening. The stability of the test strip was assessed with a panel of five positive and five negative control sera after storage at 4 degrees C and -20 degrees C at different times. The results showed a good performance of the test strip at both storage temperatures for up to one year. In conclusion, the test strip IgM dot-ELISA assay was sufficiently sensitive for use as a screening test for serodiagnosis of acute leptospirosis. The assay was simple, inexpensive, and easy to perform for both a single test format and a large number of specimens. However, further studies are still needed to improve the stability of the test strip and assay reagents at ambient temperature, and to make the assay more rapidly and more user friendly.

Two simple immunoassays using endemic leptospiral antigens for serodiagnosis of human leptospirosis.

Two simple enzyme immunoassays, a conventional microplate and dot-ELISA, were developed to detect specific IgM antibodies using pool sonicated antigen prepared from three of the most reactive serovars of Leptospira associated with disease in Thailand. Both assays were evaluated and compared with the standard microscopic agglutination test (MAT) performed with 343 serum samples. A battery of 16 pathogenic serovars of L. interrogans were used as antigens in the MAT assay. The result of MAT at serum titers > or = 1:400 showed three pathogenic serovars of leptospira, Bratislava (71.88%), Sejroe (63.54%) and Pyrogenes (36.46%), were among the most commonly reacted serovars and they were selected for preparation of pool sonicated antigen for both IgM ELISA tests. The microplate IgM-ELISA, performed with sera at 1:80 dilution using the cutoff OD of 0.60, demonstrated sensitivity, specificity and efficiency of 87.50, 97.57, and 94.75%, respectively. The same values for IgM dot-ELISA performed with sera at 1:160 dilution were 98.96, 93.93, and 95.33%, respectively. Both ELISA methods showed results with statistically significant differences from MAT (p < 0.05). The agreement rate of IgM dot-ELISA compared with microplate IgM-ELISA was 0.85 by Kappa analysis. Both assays offered relatively high negative predictive values (95.26-99.57%), thus making the assays ideally suited for rapid screening. Future applications of the IgM dot-ELISA as a test kit would be suitable for use at the peripheral level as a rapid screening test for human leptospirosis.

The development of rabbit’s urinary system serial sections study of 12-14 mm rabbit embryos and a 10 mm pig embryo.

The urogenital system develops from the intermediate mesoderm, the coelomic epithelium and the endoderm of the urogenital sinus. The urinary system of mammal is characterized by three sets of kindneys, i.e. the nonfunctional pronephros, the mesonephros and the functional metanephros. The metanephros or the permanent kidney develops from the ureteric bud and metanephrogenic tissue. At the beginning the kidney is located in the pelvic region but later gradually ascends to the abdomen. The urinary bladder development of human urinary system in the Department of Anatomy, Siriraj Hospital, we have employed serial sections of 10-15 mm pig embryos as laboratory models. This method of laboratory study is helpful in understanding and recognizing how the kidney forms. It is quite regrettable that nowadays the pig embryos are no longer available. Thus, it is necessary to find another suitable laboratory model and study the details of its normal kidney development. In this study, we demonstrated that 12-14 mm rabbit embryos can be used instead of pig embryos as the development of the metanephros is very similar. Although the rabbit’s mesonephros is smaller than that of the pig, it resembles the human mesonephros more closely. For this reason, the rabbit embryo is suitable for use as a laboratory model for embryology education of urinary system.

Antibodies to leptospirosis in rodents from Thailand using a modified human diagnostic assay.

The number of reported cases of Leptospirosis in Thailand has grown since 1996. Identification of major reservoirs and endemic areas is essential in surveillance of Leptospira species in Thailand. To assist in the effort of surveillance, a dipstick assay for detecting Leptospira antibodies in mammals was adapted from a human diagnostic assay and tested in a field trial in Thailand. Antibodies to Leptospira were detected in 18 of 60 wild rodents. Four of 9 culture positive rodents were positive by the dipstick assay. The proportion of sera positive for antibodies by dipstick was correlated with positive culture outcome using McNemar test for correlated proportions (0.83, P> 0.05). The dipstick assay was effective in detecting antibodies to Leptospira in mammals and may be useful in resource poor areas or under circumstances where the microagglutination test (MAT) is not practical.

A Microanatomical study of the organs of hamsters (Mesocricetus auratus) infected with Leptospira interrogans, serovar pyrogenes which caused an outbreak in Thailand 1999.

The effects of Leptospira interrogans on various organs of hamsters were studied microanatomically. Three infected hamsters were sacrificed at 1 hour, 6 hours and on days 1, 2, 3, 4, 5 and 6 after infection with Leptospira interrogans serovar pyrogenes. The kidneys, lungs, liver, gastrocnemius and hamstring muscles of all the sacrificed animals were removed and processed for routine conventional light microscopy. The microscopic change of the infected kidney showed degenerative changes of the renal tubular cells, including vacuolar degeneration, cellular swelling of proximal tubules, dilatation of the distal tubular lumen and necrosis. The glomeruli had many pathological appearances including congestion and swelling of the glomerular tuft, imflammatory cell infiltration, hemorrhage in the glomerular tuft and the urinary space. This phenomenon may have been related to glomerular damage. Congestion of the renal blood vessels was demonstrated in both the cortex and the medulla. There were many other hemorrhagic areas including the interstitium and the renal tubule. Interstitial nephritis and pyelonepritis were also found. In the lung, the alveolar and interalveolar capillaries were distended and engorged with red blood cells. A small number of alveoli were filled with inflammatory cells which represented bronchopneumonitis. Most areas of the lungs showed intersitital and intra-alveolar hemorrhage as well as thickening of the alveolar septum. The interalveolar septum was also thickened by accumulation of inflammatory cells which is a sign of interstitial pneumonitis. The infected liver showed enlarged and vacuolated hepatocytes being related to cloudy swelling the hepatocytes. Vascular and sinusoidal congestion, prominent Kupffer cells, and inflammatory cell infiltration in the hepatic parenchyma and hepatic sinusoids were also demonstrated. The portal area showed a number of inflammatory cells. Hepatocellular necrosis was found scattered throughout the hepatic lobules which is a sign of hepatocellular damage and disorganization of the liver structure and function. In the gastrocnemius and hamstring muscles, dilation and congestion of blood vessels was shown in some hamsters in the infected groups. The congestion of blood vessels is a sign of hyperemia. One hamster of the infected group showed inflammatory cell infiltration in the perimysium of the gastrocnemius muscle. Another one showed necrosis of some muscle fibers together with inflammatory cell infiltration which are signs of muscular inflammation. The results of this research correspond with previous similar studies, however, the pathogenesis of this study was quicker and the infection was more severe than in other studies. This may be due to the difference in serovar studied.

Rapid detection of haemophilus influenzae type B by PCR.

Rapid and reliable diagnosis of meningitis caused by Haemophilus influenzae type b (Hib) is essential for early treatment to reduce the mortality rate and neurological damage among survivors. In this study, a PCR assay for Hib was developed as a reliable method in the clinical laboratory. Two methods for DNA extraction from H. influenzae isolates were compared. The extraction by a commercial “DNAzol reagent” was more rapid and convenient than conventional phenol-chloroform extraction. DNA yield from both methods was not significantly different. DNA from standard strains was used for optimizing the PCR reaction with our new designed primers based on the genes coding for capsule type-specific Hib. The sensitivity, specificity and agreement rate of the primers were tested by comparison with one pair of the published primers. There was a perfect agreement between the newly designed and the published primers with K = 1; however, the new primers had higher sensitivity and could detect as low as 1 pg of DNA. When the blind colonies of 187 bacterial meningitis isolates were used in the PCR assay, the sensitivities, specificities and efficiencies of the PCR with both primer sets, comparing the results of the culture and slide agglutination with Hib specific antiserum as the “gold” standard, were 100, 99.32 and 99.47%, respectively. The one capsule-deficient type b mutant strain could be detected by PCR while the serological result with Hib antiserum was negative. The PCR developed in this study shows rapidity, high sensitivity and specificity and may be a useful adjunct to conventional methods for early diagnosis of Hib meningitis in the clinical microbiology laboratory.