Tissue apoptosis in mice infected with Leptospira interrogans serovar Icterohaemorrhagiae

Background: This investigation aimed to evaluate the occurrence of some apoptotic features induced by Leptospira interrogans serovar Icterohaemorrhagiae infection in young BALB/c mice during 2, 4, 7, 10, 14 and 21 days post-infection (dpi). Methods: The animals were euthanized and lung, liver and kidneys were harvested to histopathology analysis and immunohistochemistry to caspase-3 antigen detection was performed. Results: Chromatin condensation in kidney and liver tissues, but not in lung tissue, was observed. Caspase-3 reactive cells, mainly characterized as renal epithelial cells, were detected in the days 14 and 21 at high levels when compared to days 2,4 and 7 (p = 0.025; p <0.05). Lung sections revealed caspase-3 labeled alveolar cells in 10 and 14 days post-infection was higher than observed at 7 days (p = 0.0497; p < 0.05). Liver sections demonstrated reactive cells at a highest level at 14 and 21 days post-infection when comparison to 2,4, 7 and 10 days (p = 0.0069; p<0.05). Conclusions: Our results suggest that infection of L interrogans induce in kidney, liver and lung an activation of apoptosis mediated by caspase-3 dependent pathway in later phases of infectious process.

Isolation of Salmonella enterica and serologic reactivity to Leptospira interrogans in opossums (Didelphis virginiana) from Yucatán, México.

The presence of Salmonella enterica and serologic evidence of infection by Leptospira interrogans, were detected in the opossum Didelphis virginiana in a semi-urban locality of the Yucatán State, México. Ninety-one opossums were captured during the period April 1996 and May 1998. From a total of 17 feces samples, four Salmonella enterica subsp. enterica serotypes (Sandiego, Newport, Anatum, and Minnesota), and one Salmonella enterica subsp. arizonae serovar O44:Z4,Z23:- were isolated. Some opossums presented mixed infections. From 81 sera samples, four (4.9%) were positive to antibodies to Leptospira serovars pomona and wolfii. Both animals infected with Salmonella enterica and those serologically positive to Leptospira interrogans were captured in peridomestic habitat. Opossums infected with Salmonella enterica, were captured in dry season, and those seropositive to Leptospira interrogans during the rainy season. The implications of infection and reactivity of these zoonotic pathogens in D. virginiana in the Yucatan state are briefly discussed.

[Leptospira interrogans serovar Pomona: detection of antigenic differences among 3 regional isolates from cattle and a reference strain]. / Leptospira interrogans serovar Pomona: detección de diferencias antigenicas entre tres aislamientos regionales de bovinos y una cepa de referencia.

Leptospirosis is a zoonosis caused by Leptospira interrogans. This disease is diagnosed by quantification of specific immunoglobulins in serum by the microagglutination test (MAT). The aims of this research were: a) to compare the protein profiles of 3 clinical isolates of bovine leptospirosis with the reference strain used for the MAT, and b) to identify the immunodomain antigens of the regional isolates through PAGE and immunoblotting techniques of bovine sera from infected, vaccinated and MAT-negative animals. Coomassie-blue stained gels revealed extensive protein similarities between pathogenic and reference strain. Most infected (8/10) and vaccinated animal sera (4/7) showed by immunoblotting a similar reactivity against the proteins from pathogenic leptospires, with a strong band of 25-30 kDa which was not detected in the reference strain. The lack of correlation between MAT and immunoblotting techniques for infected animals could be due either to the infection stage at which the diagnosis was made or to the immunoglobulin isotype involved in the response. Results obtained would confirm the antigenic differences between the 3 isolates and the reference strain.

Leptospira interrogans serovar Pomona: detección de diferencias antigenicas entre tres aislamientos regionales de bovinos y una cepa de referencia / Leptospira interrogans serovar Pomona: detection of antigenic differences among 3 regional isolates from cattle and a reference strain

Leptospirosis is a zoonosis caused by Leptospira interrogans. This disease is diagnosed by quantification of specific immunoglobulins in serum by the microagglutination test (MAT). The aims of this research were: a) to compare the protein profiles of 3 clinical isolates of bovine leptospirosis with the reference strain used for the MAT, and b) to identify the immunodomain antigens of the regional isolates through PAGE and immunoblotting techniques of bovine sera from infected, vaccinated and MAT-negative animals. Coomassie-blue stained gels revealed extensive protein similarities between pathogenic and reference strain. Most infected (8/10) and vaccinated animal sera (4/7) showed by immunoblotting a similar reactivity against the proteins from pathogenic leptospires, with a strong band of 25-30 kDa which was not detected in the reference strain. The lack of correlation between MAT and immunoblotting techniques for infected animals could be due either to the infection stage at which the diagnosis was made or to the immunoglobulin isotype involved in the response. Results obtained would confirm the antigenic differences between the 3 isolates and the reference strain.

Serological study of the frequency of leptospiral infections among dairy cows in farms with suboptimal reproductive efficiency in Galicia, Spain.

Between March 1996 and September 1997, 15 farms located in Galicia (NW Spain) and experiencing suboptimal reproductive efficiency were visited and blood samples were obtained from all the lactating cows (n=442). Additionally, 1060 samples were obtained monthly from a cohort of 219 lactating cows belonging to nine of the farms between March 1996 and April 1997. All the samples were tested by microscopic agglutination (MAT) using live antigens representing the following Leptospira interrogans serogroups: Australis, Autumnalis, Ballum, Canicola, Grippotyphosa, Icterohaemorrhagiae, Louisiana, Pomona, Sejroe, Shermani and Tarassovi. Eighty-one of the 442 cows were positive against one or more serogroups (P=18.33%). Serologically, L. bratislava and L. grippotyphosa were detected as the most prevalent serovars (P=7.92 and 7.69%, respectively) and as the serovars against which the probability of seroconversion was highest (P=0.27 and 0.25, for a 12-month period, respectively) among those studied. The proportional hazards regression method was used for evaluating the seasonal trend of seroconversions against these two serovars. The risk of seroconversion against L. grippotyphosa was significantly higher during spring. The risk of seroconversion against L. bratislava did not differ significantly among seasons. Our results suggest that infections by L. bratislava did not follow, among the study animals and during the study period, the pattern typically described for non-adapted serovars, pointing out the possibility that some strains of this serovar could behave as adapted serovars.

Competitive enzyme-linked immunosorbent assay for detection of Leptospira interrogans serovar pomona antibodies in bovine sera.

A competitive enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (M898) was developed for detection of bovine antibodies to Leptospira interrogans serovar pomona. This assay was evaluated using field sera (n = 190) with serovar pomona microscopic agglutination test (MAT) titers of > or =100 as the positive population (group A); field sera (n = 1,445) which were negative in the MAT (1:100 dilution) for serovar pomona (group B); and sera (from a specific-pathogen-free cattle herd [n = 210]) which were negative in the MAT (1:100 dilution) for serovars canicola, copenhageni, grippotyphosa, hardjo, pomona, and sejroe (group C). At the cutoff point recommended by receiver operating characteristic (ROC) curve analysis of the combined ELISA results of serum groups A, B, and C, the sensitivity and specificity values were 93.7 and 96.3%, respectively. The value for the area under this ROC curve was 0.977, indicating a high level of accuracy for the ELISA. Similar results were obtained from the analysis of the combined results of serum groups A and B and from the analysis of the combined results of serum groups A and C.

A rapid latex agglutination test for detection of leptospiral antibodies.

A rapid semi-quantitative latex agglutination test (LAT) has been standardized for the detection of leptospiral antibodies in serum samples of man and animals. The efficacy of the LAT was compared with the plate enzyme linked immunosorbent assay (ELISA). A total of 276 human serum samples were analyzed by both LAT and ELISA and percentage positives were 84.8 and 85.9%, respectively. Similarly, of 65 animal samples tested, 63.1 and 69.2% positivity were observed in LAT and ELISA, respectively. Even though the ELISA test was slightly more sensitive than LAT, the rapidity, simplicity and economics of the LAT were found to fulfill the requirements of a screening test for leptospiral antibodies.

Infertility and abortion among first-lactation dairy cows seropositive or seronegative for Leptospira interrogans serovar hardjo.

OBJECTIVE: To estimate the extent to which exposure to Leptospira hardjo before or at the time of first parturition was associated with infertility and abortion during the first lactation among dairy cows that had not been vaccinated for > or = 12 months. ANIMALS: 207 first-lactation cows from a herd of 2,000 lactating cows. PROCEDURE: Cows were tested for antibodies to L hardjo within 40 days after calving. Time from calving to first breeding, time from calving to conception, number of breedings per conception, and risk of abortion were compared between cows seropositive for L hardjo and cows that were seronegative. RESULTS: For the 9 (4.3%) cows that were seropositive for L hardjo, median time from calving to conception (132.6 days) was significantly longer than time for seronegative cows (95.4 days). Cows that were seropositive were twice as likely (relative risk, 2.07) to fail to conceive as seronegative cows. Mean number of breedings required per conception for seropositive cows (3.4) was significantly higher than that for seronegative cows (2.1). The proportion of seropositive cows that aborted was not significantly different from the proportion of seronegative cows that aborted. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure of nonvaccinated dairy cows to L hardjo can be associated with a subsequent reduction in fertility, as indicated by a greater time from calving to conception and higher number of breedings required per conception. The efficacy of leptospiral vaccines should be assessed to determine whether vaccination will minimize herd infertility associated with L hardjo infection.

Incidence of leptospiral abortion in Brazilian dairy cattle.

We examined dams' paired serum samples and foetal kidneys (histopathologically and attempting leptospiral cultures) from 120 Brazilian Holstein abortions from 10 herds near Sao Paulo, Brazil, in 1996. Leptospiras was isolated from 15 foetuses. The Leptospira interrogans serovar hardjo was obtained from four, pomona from three and wolffi from eight foetuses. Ten of these 15 foetuses showed a four-fold rise in titres between the day of abortion and the second samples taken 15 days later. Fifty-seven other foetuses had histologic evidence of leptospires; 27 of their dams (47%) had four-fold rises in titres. In total, 72 of 120 aborted foetuses had evidence of leptospiral infection.

Three cases of canine leptospirosis in Quebec.

Three dogs from different locations with acute renal failure were hospitalized in autumn 1996 and 1997. Leptospira interrogans serovar pomona was detected by the microscopic agglutination test. All dogs recovered after antibiotic treatment. The importance of the development of vaccines adapted to emerging serovars in dogs should be addressed.

Production and characterization of monoclonal antibodies specific for Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno.

Murine monoclonal antibodies were produced by immunizing BALB/c mice with a killed whole-cell antigen prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis. Six of these antibodies recognized epitopes on the homologous antigen and on whole-cell antigen prepared from Leptospira interrogans serovar hardjo type hardjoprajitno. These antibodies did not cross-react with whole-cell antigens prepared from L. borgpetersenii serovar sejroe, 10 other pathogenic Leptospira serovars, or the saprophytic Leptospira biflexa serovar patoc. Three other monoclonal antibodies reacted with antigens prepared from the 2 hardjo serovars and serovar sejroe but not with antigens from the 10 other pathogenic serovars, or serovar patoc. The epitopes recognized by all of the hardjo-specific antibodies and 2 of the 3 hardjo/sejroe-specific antibodies were susceptible to sodium meta-periodate oxidation. All of the antibodies were characterized by Western blots with the hardjobovis whole-cell antigen. Each of the 9 monoclonal antibodies was inhibited from binding to the hardjobovis antigen by bovine sera which were obtained from cattle experimentally infected with hardjobovis and from field cattle, with anti-serovar hardjo microscopic agglutination test antibody titres ranging from 100 to 12800. Some of these antibodies may be suitable for incorporation into competitive enzyme immunoassays for the specific detection of antibodies to either of the hardjo serovars.

Leptospira interrogans antibodies in feral pigs from New South Wales.

The sera of 195 hunter-killed feral pigs (Sus scrofa), collected in New South Wales (Australia) from April to November 1995, were screened against a reference panel of 14 Leptospira interrogans serovars using a microscopic agglutination test (MAT). The panel represented those serovars previously isolated from wild and domestic mammals in mainland Australia. Antileptospiral agglutinins were detected in 20% of the sera tested and included nine L. interrogans serovars. The majority of serological reactors (63%) were to L. interrogans serovar pomona. Sera from 26% of immunoreactors cross reacted with antigens from one or more serovars. No differences were noted in the prevalence of L. interrogans antibodies between the sexes, or between pigs from areas of low and high rainfall. The implications of leptospirosis in feral pigs on the transmission of leptospires to wildlife, livestock, and humans are discussed.

Prevalence and geographic origin of pigs with serological evidence of infection with Leptospira interrogans serovar pomona slaughtered in abattoirs in Victoria, Australia.

A set of 10,440 sera was collected from pigs slaughtered at Victorian abattoirs. These sera were subjected to the microscopic agglutination test for antibodies to Leptospira interrogans serovar pomona. Identification of the herd of origin was possible for 6511 pigs, and these were derived from 167 herds in Victoria (84% of sera), from 32 herds in New South Wales (8% of sera) and 29 herds in South Australia (8% of sera). The overall prevalence of titres of 512 and above was 3.7%. This was higher (5.3%) among pigs for which the property of origin was unknown than among pigs with identified properties of origin. Among the latter the prevalence was 2.7% (Victoria 0.6%, New South Wales 1.3%, South Australia 25.2%.) Most of the pigs with unknown properties of origin were derived from market groups and were probably typically from smaller herds. Within Victoria a comparison of results with the known pig populations of the 12 statistical divisions indicated that infection was spread throughout the State. Of the 228 identified herds of origin sampled, 32 (14%) had at least one pig with a high titre. However, this may underestimate the proportion of infected herds, as in many cases only a few serum samples were obtained. Of 73 herds from which 25 or more serum samples were obtained, serological evidence of infection was obtained in 18 herds (25%).

Epidemiologic features of equine Leptospira interrogans of human significance.

Leptospirosis is a zoonotic bacterial disease caused by Leptospira interrogans. There is a serologic evidence that horses are exposed to L. interrogans and, as a shedder of these organisms, can be a threat to humans. We examined risk factors associated with the risk of testing seropositive to three L. interrogans serovars (L. icterohaemorrhagiae, L. grippotyphosa, and L. canicola) in the horses of New York State, in order to understand the epidemiology of the disease and suggest strategies to control and prevent equine leptospirosis. To carry out this study, blood samples were collected from a random sample of 2551 horses and tested for the presence of antibodies to the above serovars using the microscopic agglutination test. Samples with a titer $100 were considered positive. Clinical and demographic data were collected on each horse, the farms' management practices and ecology. Logistic regression analysis was used to develop a multivariate indexing system and to identify factors significantly associated with the risk of leptospirosis. Four indices were developed based on the possible sources of exposure: rodent exposure index; wildlife exposure index; soil and water index; and management index. The soil and water index was significantly associated with the risk of exposure to all three serovars. Management was positively associated with L. icterohaemorrhagiae and L. canicola. Density of horses turned out together was positively associated with the risk of exposure to L. grippotyphosa. We concluded that indirect exposure of horses to L. interrogans through contaminated soil and water appears to be significantly associated with the risk of exposure to all three serovars. Management appears to play an important role in the exposure to L. interrogans. Modification of management practices might reduce the horses' risk of exposure and hopefully minimize the human hazards.

Transmission of Leptospira interrogans serovar Balcanica infection among socially housed brushtail possums in New Zealand.

Leptospira interrogans serovar balcanica is a potential vector being investigated for spreading a biological control agent among introduced brushtail possums (Trichosurus vulpecula) in New Zealand. As previous studies have shown that possums are unlikely to contract leptospirosis through a contaminated environment alone, the objective was to determine whether L. interrogans serovar balcanica could be transmitted between sexually mature, socially housed possums. Possums were infected experimentally with L. interrogans serovar balcanica and housed in pairs or groups with uninfected possums for either 70 or 140 days, during the breeding or non-breeding seasons. No transmission occurred between any infected and uninfected possums during the non-breeding season. However, transmission occurred between females that had been socially housed in pairs or groups in the breeding season. Mixed sex transmission also occurred in pairs and groups, both from males to females and from females to males. Mixed sex transmission usually occurred rapidly (< 44 days) and was not associated with the production of offspring. No transmission occurred between males during the breeding or the non-breeding seasons. Transmission probably occurs as a result of affiliative or sexual behaviour, but is unlikely to occur through fighting. The social transmission pathways determined in this study suggest that L. interrogans serovar balcanica may have the transmission attributes desired in a vector for biological control.

Leptospirosis in rehabilitated Pacific harbor seals from California.

Renal disease was observed in two rehabilitated Pacific harbor seals (Phoca vitulina richardsii) from a facility in California (USA). The seals had leukocytosis and high serum phosphorus, blood urea nitrogen and creatinine concentrations. A retrospective study of leptospiral antibody serum titers indicated both seals had elevated titers to Leptospira interrogans serovar grippotyphosa. A third seal, which died about the time when the index cases occurred, also had elevated titers to L. interrogans serovar grippotyphosa. Post mortem histopathologic examination of all three seals showed tubular necrosis consistent with interstitial nephritis; spirochetes were seen within the kidney parenchyma of the third seal. Sea lions (Zalophus californianus) or elephant seals (Mirounga angustirostris) housed near the harbor seals were possible sources of exposure, but local wildlife also could have been responsible.

Retinal immunopathology in horses with uveitis.

BACKGROUND: Equine uveitis is a spontaneous disorder of horses that can serve as a model for the study of human uveitis. Although the initial presentation is that of an anterior uveitis, retinal involvement has been noted in some cases. We report here the immunohistopathology of retinas from horses with uveitis. METHODS: Sections of eyes recovered from horses with naturally occurring uveitis and from Shetland ponies with experimental leptospira-induced uveitis were stained by hematoxylin and eosin for histopathological evaluation. Immunohistochemistry was used to evaluate retinas for MHC Class II antigen expression and infiltration of T and B lymphocytes. RESULTS: Histopathological abnormalities in retinas from horses with uveitis ranged from minimal to total loss of retinal tissue. MHC Class II antigen-positive round and dendritiform cells were seen in these retinas, but were not seen in retinas from horses without uveitis. There was no significant reactivity noted in the retinal pigment epithelial cells or Muller cells. Numbers of MHC Class II antigen-expressing cells and T lymphocytes correlated with the extent of retinal histopathology. B lymphocytes were seen primarily in retinas from horses that were seroreactive for Leptospira interrogans serovar pomona. Retinas from ponies with experimental uveitis had changes similar to those from horses with spontaneous uveitis. CONCLUSIONS: These results suggest that retinal pathology may be a primary immunological event in equine uveitis, provide evidence that leptospira-associated uveitis may be a distinct subset of equine uveitides, underscore the relevance of the study of equine uveitis to human uveitis, and support the plausibility of a post-infectious immunopathogenesis of some naturally occurring uveitides in both humans and horses.

Leptospira interrogans serovar icterohaemorrhagiae seropositivity and the reproductive performance of sows.

The reproductive performance of 28 sows seropositive to Leptospira interrogans serovar icterohaemorrhagiae was compared with that of 87 Leptospira sp. seronegative dams belonging to the same herd. Sows were sampled during 1988 to 1993. During this period the herd was not submitted to any kind of intervention (antibiotic therapy, immunoprophylaxis or rodent control). Relative risks (RR) of return to heat, mummified fetuses, stillbirth, and weak newborn piglets for infected sows were assessed and the differences in means of total piglets born per litter, piglets born alive, piglets effectively housed, weaned piglets, stillbirths, mummified fetuses, weak newborn piglets, weight at birth of the piglets effectively housed, weight at 21 days of life and weight at weaning were evaluated. Seropositive dams had a greater risk of having weak newborn piglets (RR = 1.67, 1.02 < or = CI 95% < or = 2.72) and also of having more weak newborn piglets per litter (P = 0.01). Other variables examined were not different (P > 0.05).