The role of small ruminants in the epidemiology of leptospirosis.

Leptospirosis is a common global zoonotic disease of man and all farm animals. Although most leptospiral infections in sheep and goats are asymptomatic, they may play a role in the epidemiology of the disease by the spread of Leptospira through the urine. This study was carried out to evaluate the role of sheep and goats in the epidemiology of leptospirosis. Blood and urine samples were taken from 210 goats and 246 sheep. To detect antibodies, sera samples were tested with 8 live serovars of L. interrogans (Hardjo, Pomona, Grippotyphosa, Canicola, Ballum, Icterhemorrhagiae, Tarasovi, and Australis) by MAT. Then, urine samples were tested by Nested PCR targeting 16S rRNA gene for detection of pathogenic Leptospira. Results of MAT showed that 10.95% of goats and 8.53% of sheep had antibodies against at least one examined serovars. In both species, the highest reacting was L. i. Pomona with a rate of 68.18% and 56% in sheep and goats, respectively. Moreover, in PCR, 2 (0.95%) urine samples of goat and 12 (4.87%) urine samples of sheep were positive. All of the MAT positive studied animals were PCR negative and, statistical analysis showed that there was no relationship and agreement between the results of PCR and MAT in sheep (kappa = - 0.07, p > 0.05) and goats (kappa = - 0.02, p > 0.05). Finally, it is concluded that sheep and goats can excrete L. interrogans in the urine and thus transmit them to other animals and humans.

Serological and molecular detection of pathogenic Leptospira in domestic and stray cats on Reunion Island, French Indies.

Indian Ocean islands are endemic areas for human and animal leptospirosis. Maintenance host species for Leptospira spp. have still not been completely elucidated, and recently the role of cats (Felis catus) has been questioned. This cross-sectional study aims to determine whether cats are part of the maintenance community of different strains of Leptospira spp. in Reunion Island. The prevalence of Leptospira infection in an opportunistic sample of stray and domestic cats (n = 92) from Reunion Island has been studied using serological (microagglutination test) and molecular detection (polymerase chain reaction (PCR)). The results revealed a seroprevalence of 37.0% (34/92) (cut-off 1:40) without a significant difference in the living conditions of animals. The predominant serogroup was Icterohaemorrhagiae, but Ballum, Cynopteri and Australis were also detected. Using PCR, 28.6% (12/42) of stray cats were tested positive. Leptospiral DNA was detected in renal tissue, urine and blood of respectively 14.3% (6/42), 10.3% (4/39) and 11.9% (5/42) of stray cats, but 0% (0/3), 0% (0/50) and 0% (0/36) of domestic cats (P = non-applicable, P = 0,038, P = 0,058 respectively). Partial rrs gene (16S rRNA) sequencing identified Leptospira interrogans in all PCR-positive samples. Our study confirms that renal carriage and urinary shedding are possible, positioning cats, and especially stray cats as potential actors within the maintenance community of L. interrogans in Reunion Island.

Molecular and serological characterization of pathogenic Leptospira spp. isolated from symptomatic dogs in a highly endemic area, Brazil.

BACKGROUND: Leptospirosis is an endemic zoonosis in Brazil, with a great impact on human and animal health. Although dogs are frequently infected by pathogenic Leptospira, the current epidemiological understanding of canine leptospirosis is mainly based on serological tests that predict the infecting serogroup/serovar. Thus, the present study aimed at identifying the causative agent for severe cases of canine leptospirosis in a highly endemic area through the isolation and characterization of the isolated strains. RESULTS: Urine, serum and blood samples were collected from 31 dogs with suspected acute leptospirosis treated at the Veterinary Hospital Service of Santo Amaro University between 2018 and 2019. Acute infection was confirmed in 17 dogs (54.8%) by the associated use of Polymerase Chain Reaction (PCR), Microscopic Agglutination (MAT) and bacteriological culture. Eleven dogs (35.5%) had titers ≥800, with the most frequent serogroups being Autumnalis and Icterohaemorrhagiae (n = 4 each) and Canicola (n = 2). Leptospires were recovered from four dogs, and Multilocus Sequence Analysis (MLSA) revealed infection caused by L. interrogans, which were further characterized as serogroups Canicola (n = 1) and Icterohaemorrhagiae (n = 3). CONCLUSION: The identity of the isolates and serological pattern of MAT suggest that dogs are highly exposed to the serogroup Icterohaemorrhagiae and Canicola, also indicating possible circulation of serogroups not yet isolated in Brazil, notably serogroup Autumnalis. Our findings also reinforce the usefulness of using multiple diagnostic approaches to confirm acute canine leptospirosis.

Leptospirosis as a neglected burden at human-cattle interface in Mid-Delta of Egypt.

INTRODUCTION: Leptospirosis is a neglected zoonosis in developing countries including Egypt where its burden is underestimated. METHODOLOGY: A cross sectional study was carried out to estimate the seroprevalence and associated risk factors of Leptospira interrogans serovar Hardjo infection among cows and leptospirosis among human patients in Mid-Delta of Egypt. RESULTS: Out of 112 examined cows using ELISA, 3.6% were seropositive to L. interrogans serovar Hardjo infection. Seroconversion occurred in 5 animals (1 herd) of all examined animals in convalescent phase testing (5/112, 4.5%). Affected herd suffered acute outbreak with 43.3% within herd prevalence; signs of infection included abortions, bloody urine and sudden death of 2 cows. Highest risk for L. interrogans serovar Hardjo infection in cows was in animals drank from untreated surface water (6.7 times, p = 0.06). The seroprevalence of leptospirosis was 6.2% in all tested humans, 28.6% in nonspecific fever cases and 22.2% in non-viral hepatitis cases. The risk of leptospirosis among patients with nonspecific fever or non-viral hepatitis cases was 4 times higher than those with viral hepatitis (p = 0.01). Additionally, there was a significant association between leptospirosis and patients with livestock contact (Odds 8, p = 0.01). CONCLUSIONS: This is the first report of L. interrogans serovar Hardjo outbreak in cows in Egypt. The study also highlighted the role of leptospirosis as neglected cause of nonspecific fever/non-viral hepatitis in humans in study region.

CLASSIFICATION AND REGRESSION TREE ANALYSIS FOR PREDICTING PROGNOSIS IN WILDLIFE REHABILITATION: A CASE STUDY OF LEPTOSPIROSIS IN CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS).

The spirochete bacterium Leptospira interrogans serovar Pomona is enzootic to California sea lions (CSL; Zalophus californianus) and causes periodic epizootics. Leptospirosis in CSL is associated with a high fatality rate in rehabilitation. Evidence-based tools for estimating prognosis and guiding early euthanasia of animals with a low probability of survival are critical to reducing the severity and duration of animal suffering. Classification and regression tree (CART) analysis of clinical data was used to predict survival outcomes of CSL with leptospirosis in rehabilitation. Classification tree outputs are binary decision trees that can be readily interpreted and applied by a clinician. Models were trained using data from cases treated from 2017 to 2018 at The Marine Mammal Center in Sausalito, CA, and tested against data from cases treated from 2010 to 2012. Two separate classification tree analyses were performed, one including and one excluding data from euthanized animals. When data from natural deaths and euthanasias were included in model-building, the best classification tree predicted outcomes correctly for 84.7% of cases based on four variables: appetite over the first 3 days in care, and blood urea nitrogen (BUN), creatinine, and sodium at admission. When only natural deaths were included, the best model predicted outcomes correctly for 87.6% of cases based on BUN and creatinine at admission. This study illustrates that CART analysis can be successfully applied to wildlife in rehabilitation to establish evidence-based euthanasia criteria with the goal of minimizing animal suffering. In the context of a large epizootic that challenges the limits of a facility's capacity for care, the models can assist in maximizing allocation of resources to those animals with the highest predicted probability of survival. This technique may be a useful tool for other diseases seen in wildlife rehabilitation.

Leptospira interrogans serogroup Pomona strains isolated from river buffaloes.

At present, little is known regarding the prevalence of buffalo leptospirosis worldwide, especially with respect to which Leptospira strains may infect this animal species. Furthermore, most investigations into this disease in buffaloes have only been performed with serological studies. In Brazil, particularly in the Amazon, buffalo production is growing and is just as important as cattle production, although few studies have been performed on buffalo compared to cattle. Thus, the aim of this study was to isolate and characterise Leptospira strains from river buffaloes raised in the Brazilian Amazon region. We collected 109 kidney samples from slaughtered buffaloes raised in the Amazon Delta region of Brazil. The samples were analysed by bacteriological culture for the isolation of leptospires, and the obtained isolates were serologically and molecularly characterised by microscopic agglutination test (MAT), DNA sequencing and multiple locus variable-number tandem repeat analysis (MLVA). Five isolates were obtained, and in serogrouping analyses, these isolates were only reactive for the Pomona serogroup, with an observed titre of 25,600. The DNA sequencing results revealed that all the isolates belonged to the species Leptospira interrogans, and the MLVA results showed that the VNTR loci 4, 7 and 10 profile of all the isolates was 4-1-10. In this study, we observed that Pomona serogroup strains circulate in buffaloes in the Amazon, showing that in Brazil, buffaloes can be affected by Leptospira strains other than the Sejroe group, which are adapted to cattle.

Evaluation of the diagnostic potential of recombinant leptospiral OMP A-like protein (Loa22) and transmembrane (OmpL37) protein in latex agglutination test for serodiagnosis of leptospirosis in animals.

Leptospirosis is a re-emerging zoonotic disease of animals and humans caused by pathogenic Leptospira, which has major public health concerns. The study is aimed to express the recombinant outer membrane protein (OMP) A-like protein (rLoa22) and transmembrane (rOmpL37) protein of Leptospira interrogans serovar Hardjo in the Escherichia coli and their evaluation as a diagnostic antigen in the latex agglutination test (LAT) to detect anti-leptospiral antibodies in the sera of animals. The Loa22 and OmpL37 genes lacking signal peptide coding sequences were individually amplified (522 and 963 bp), by polymerase chain reaction, and directionally cloned into a pETite N-His Kan vector for expression. The expressed purified proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblot, which confirmed leptospiral specific reactive protein with a molecular weight of ~19 and 36 kDa, respectively. The sensitized latex beads coated with these OM proteins separately were evaluated in LAT using cattle sera of microscopic agglutination test (MAT) confirmed positive (n = 53) and negative (n = 52) cases of leptospirosis. The rLoa22 LAT and rOmpL37 LAT revealed the relative diagnostic sensitivity of 94·34 and 96·23%, diagnostic specificity of 92·31 and 96·15% and accuracy of 93·33 and 96·19%, with the excellent agreement of Cohen's kappa value of 0·87 and 0·92, respectively. After extensive evaluation, this rapid recombinant protein-based field diagnostic test can be applied as a screening test for the detection of anti-leptospiral antibodies in the sera of animals in the field conditions.

Serological study of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Tarassovi and Ballum in beef cattle, sheep and deer in New Zealand.

AIMS: To estimate animal-level seroprevalence of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Ballum and Tarassovi, in beef cattle, sheep and deer on New Zealand farms, and herd/flock-level seroprevalence of any serovar when existing same-sera data for serovars Hardjobovis and Pomona were included, and to determine associations between risk factors and animal-level seroprevalence. METHODS: Banked sera from sheep (n = 82), beef (n = 54) and deer (n = 62) herds/flocks (n = 3,878 animals) from seven regions were analysed using the microscopic agglutination test. Titres of ≥48 were designated positive. Herds/flocks were considered positive if either ≥1, ≥2 or ≥3 animals were positive. Existing same-sera data for serovars Hardjobovis and Pomona were included to establish farm-level any-serovar seropositivity. Factors associated with serological status were analysed using generalised estimating equations. RESULTS: Animal-level seroprevalence for serovars Ballum, Copenhageni, and Tarassovi, respectively, was 13.7 (95% CI = 11.7-16.0)%, 12.6 (95% CI = 10.6-14.7)% and 18.0 (95% CI = 15.7-20.5)% for beef cattle, 10.5 (95% CI = 9.0-12.1)%, 16.7 (95% CI = 14.9-18.6)% and 14.0 (95% CI = 12.4-15.8)% for sheep and 6.6 (95% CI = 5.3-8.2)%, 15.5 (95% CI = 13.5-17.7)% and 3.6 (95% CI = 2.7-4.8)% for deer, respectively. Herd/flock-level seroprevalence for Ballum was 86.6, 52.4 and 39.0% for sheep, 85.2, 52.7 and 33.3% for beef cattle and 50.8, 27.9 and 21.3% for deer at definitions ≥1, ≥2 and ≥3 seropositive animals per species, respectively. For Copenhageni, corresponding data were 95.1, 73.2 and 56.1% for sheep, 68.5, 48.2 and 29.6% for beef cattle and 73.8, 57.4 and 41.0% for deer, and for Tarassovi, 80.5, 59.7 and 45.1% for sheep, 83.3, 68.5 and 61.1% for beef cattle, and 42.6, 16.4 and 4.9% for deer. Seropositivity to all serovars was observed from all regions, with some differences in seroprevalence observed between species and regions, but not between islands. Combining with Hardjobovis and Pomona data, herd/flock-level seropositivity for all animal species and all five Leptospira serovars was 100% at definition ≥1 animal positive, and 97.5 and 96.3% for sheep flocks, 87.8 and 97.8% for beef cattle herds, and 89.3 and 75% for deer herds at ≥2 and ≥3 animals positive, respectively. CONCLUSIONS: Seropositivity to serovars Ballum, Copenhageni and Tarassovi is common in sheep, beef cattle and deer New Zealand and most, or all farms have ≥1 livestock species seropositive to ≥1 serovar. CLINICAL RELEVANCE: Serovars Ballum, Tarassovi and Copenhageni should be considered when clinical or subclinical signs of leptospirosis are observed in sheep, beef cattle or deer. Livestock sector workers are potentially at risk of exposure.

Presence of pathogenic Leptospira spp. in the reproductive system and fetuses of wild boars (Sus scrofa) in Italy.

Leptospirosis is a re-emerging and globally spread zoonosis caused by pathogenic genomospecies of Leptospira. Wild boar (Sus scrofa) are an important Leptospira host and are increasing in population all over Europe. The aim of this investigation was to evaluate Leptospira spp. infection in the reproductive systems of wild boar hunted in two Italian regions: Tuscany and Sardinia. From 231 animals, reproductive system tissue samples (testicles, epididymides, uteri) as well as placentas and fetuses were collected. Bacteriological examination and Real-Time PCR were performed to detect pathogenic Leptospira (lipL32 gene). Leptospires were isolated from the testicles and epididymides of one adult and two subadult wild boar. Four isolates from the two subadult males were identified as Leptospira interrogans serogroup Australis by MLST, whereas Leptospira kirschneri serogroup Grippotyphosa was identified from the adult testicles and epididymis. Using Real-Time PCR, 70 samples were positive: 22 testicles (23.16%) and 22 epididymides (23.16%), 10 uteri (7.35%), 3 placentas (6.66%), and 13 fetuses (28.88%). Amplification of the rrs2 gene identified L. interrogans and L. kirschneri species. The results from this investigation confirmed that wild boar represent a potential source of pathogenic Leptospira spp. Isolation of Leptospira serogroups Australis and Grippotyphosa from the male reproductive system and the positive Real-Time PCR results from both male and female samples could suggest venereal transmission, as already demonstrated in pigs. Furthermore, placentas and fetuses were positive for the lipL32 target, and this finding may be related to a possible vertical transmission of pathogenic Leptospira.

Development of lipL32 real-time PCR combined with an internal and extraction control for pathogenic Leptospira detection.

At least two real-time PCRs for the early diagnosis of leptospirosis have been described, evaluated and validated. However, at least one other report suggested adaptation and modification of primers and probes used in these assays since additional Leptospira species have been described and the primers and probe in use possess a serious mismatch to corresponding target sequence. In this study we developed a real-time PCR for detection of pathogenic Leptospira based on the lipL32 gene. The present method consists of generic primers and probes based on target sequence of 10 pathogenic Leptospira species including Leptospira interrogans. The hybridization, annealing and extension temperature (60°C) were optimized as the optimal temperature of the DNA polymerase enzyme which is used in the amplification reaction. The present assay has a high analytical sensitivity and specificity; the calculated diagnostic sensitivity and specificity were 93.0% and 98.3% respectively. Moreover, the present method includes an internal control which enables easy detection of false negative results and an optional extraction control which enables the estimation of the DNA extraction efficiency.

New insights on the infection of pathogenic Leptospira species in American mink (Neovison vison) in southern Chile.

Leptospirosis is a zoonosis of global distribution, caused by the infection of pathogenic Leptospira, a group of bacteria capable of infecting both domestic and wild animals. Mink (Neovison vison) in southern Chile is recognized as a wild and synanthropic rodent predator (among various other prey), and Leptospira infection in them can be acquired through contact with the pathogen in the environment or by eating infected prey. Thus, the aim of this study was to provide more specifics regarding the source of the infection for the American mink under the conditions of Southern Chile. Minks were captured in the Los Ríos region, southern Chile, in an area with well-developed dairy farming. Two areas were selected for mink trapping, one with a high degree of dairy farming and a second with a low degree of dairy farming. Within them, 16 study sites were visited, and 45 American mink were trapped and euthanized to obtain kidney tissue and blood serum samples for bacteria isolation and determination of antibodies titers, respectively. Molecular characterization of the isolated strains was performed. Three minks from sites of high-dairy farming industry and only one from sites with low-degree dairy farming were detected as infected through molecular confirmation. This study shows evidence that confirms previous findings made in southern Chile, regarding mink as host of Leptospira interrogans serovar Hardjo-prajitno associated to cattle-farming areas. However, typing information ( Leptospira interrogans Copenhageni and Icterohaemorrhagiae ) suggests that the consumption of rodents may also be a potential source of infection.

Extra-renal bovine leptospirosis: Molecular characterization of the Leptospira interrogans Sejroe serogroup on the uterus of non-pregnant cows.

Bovine genital leptospirosis is a chronic disease that causes reproductive disorders such as abortions, stillbirths, and estrus repetition, as well as economic losses. Despite clinical signs related to reproductive failure, the majority of studies have focused on the detection of Leptospira spp. in the urine, while few have considered the reproductive tract. Consequently, the aim of the present study was to investigate the uterus as an important extra-renal site of leptospiral infection in cows. A total of 42 non-pregnant cows were studied at a slaughterhouse. Blood samples and uterine fragments were collected for serology and molecular analysis, respectively. Concerning serologic results, 20.5 % presented as reactive, all of them against the Sejroe serogroup. Regarding lipL32 PCR, 26.2 % (11/42) of samples were positive for pathogenic Leptospira sp. Sequencing the secY gene short region enabled nine strains to be characterized, all of which were L. interrogans, with high identity (98.8 %-99.8 %) with serovar Hardjo. The use of molecular tools substantially improved the sensitivity of Leptospira sp. detection at species level and demonstrated that the uterus is an important site of bovine leptospiral infection. The findings of the present study reinforce our understanding that leptospiral uterine infection are associated to members of the Sejroe serogroup.

Seroprevalence of Antibodies against Borrelia burgdorferi s. l. and Leptospira interrogans s. l. in Cats in district of Brno and its environs, the Czech Republic.

OBJECTIVES: The aim of this study is to evaluate the seroprevalence of antibodies of Borrelia burgdorferi sensu lato (Bbsl) and Leptospira interrogans sensu lato (Lisl) and their possible concurrence in domestic cats living in variable conditions in South Moravia in the district of Brno and its environs. Additional objectives were to discover possible differences in seroprevalence between groups of cats living in different living conditions, and to determine the spectrum of Leptospira serogroups in cats in the same places. MATERIAL AND METHODS: A total of 360 blood sera from domestic cats of 3 different sets were collected during the period 2013-2015. All samples were examined using ELISA for the detection of IgM and IgG antibodies against Bbsl, and the microscopic agglutination test (MAT) for the detection of antibodies against 8 serogroups of Lisl. RESULTS: The ELISA method determined 15.8%, 4.8% and 10.3% IgM anti-Borrelia antibodies in the patient group, shelter cats and street cats, respectively. IgG anti-Borrelia antibodies were found in 6.2%, 9.5%, 5.2%, respectively. Antibodies specific for 5 Leptospira serogroups were detected by the use of MAT in 8.8%, 9.5% and 10.3% of cats from the investigated groups. The total positivity of all examined cats for anti-Borrelia antibodies was 18.0% and for anti-Leptospira - 9.2%. CONCLUSIONS: Cats can be infected with both Bbsl and Lisl. The obtained results are exclusive to the city of Brno and its environs, and are comparable to the limited previous studies. There is a need for further studies of clinical signs of both infections and the possible transmission of Leptospira by ticks.

[Isolation and identification of Leptospira in patients with fever of unknown origin in Guizhou province].

Objective: To understand the etiological characteristics of the patients with fever of unknown origin in Guizhou province through the isolation and identification of Leptospira interrogans and provide evidence for the control, prevention and treatment of human leptospirosis. Methods: Blood and urine samples were collected from patients with fever symptoms in Qiandongnan, an epidemic area, in Guizhou. The suspected Leptospira strains were primarily identified using pathogenic Leptospira specific G1/G2-PCR, and subsequently identified by using Leptospira serogroups specific PCR. The Leptospira strains were then genotyped with multiple locus sequence typing. MLST data based cluster analysis on the isolates and Leptospira reference strains of common serogroups were analyzed by using software NTsys 2.10e. Results: Three suspected strains of Leptospira were isolated from human blood samples, the isolation rate was 8.６%, which were designated as strain 17BX002, 17BX003 and 17AJX008. Strain 17BX002 was further identified as serogroup grippotyphosa by using Leptospira serogroup specific PCR, while the other two strains were negative (excluded as iterohaemorrhagiae, sejroe, canicola, autumnalis, grippotyphosa and hebdomadis). MLST genotyping showed that strain 17BX002 was typed as ST106, most closely clustered with Leptospira grippotyphosa, while strain 17BX003 and 17AJX008 were typed as ST96, the same as serogroup badaviae. Conclusion: There are leptospirosis cases in epidemic area of Guizhou in high incidence season, grippotyphosa and bataviae are the newly discovered serogroups of Leptospira in Guizhou.

Molecular Detection of Leptospira interrogans and Borrelia burgdorferi in Wild Rodents from Mexico.

Leptospirosis and Lyme borreliosis are zoonotic emerging diseases of global importance and wide distribution. The aim of this study was to detect by molecular testing to Leptospira interrogans and Borrelia burgdorferi sensu lato in wild rodents from Nuevo Leon, Quintana Roo, and Campeche, Mexico. This study is the first in report to Chaetodipus nelsoni, Dipodomys merriami, and Peromyscus eremicus infected with L. interrogans in Mexico. Besides, Chaetodipus hispidus, Heteromys gaumeri, Heteromys irroratus, Neotoma micropus, Peromyscus leucopus, Peromyscus maniculatus, and Sigmodon hispidus infected with B. burgdorferi s.l. in Mexico. Also, is the first report in identify coinfection of L. interrogans and B. burgdorferi s.l. in wild rodents such as H. irroratus and S. hispidus in Nuevo Leon, and H. gaumeri in Quintana Roo, Mexico. These wild rodent species infected represent a risk factor for the exposed population in these sylvatic and rural areas of Mexico.

Weil's disease in a young homeless man living in Lisbon.

Leptospirosis is a zoonotic disease of worldwide distribution caused by infection with Leptospira genus bacteria, a pathogenic spirochaete. We present the case of a 29-year-old man admitted to our hospital with fever and multiorgan failure. He provided poor information about his symptoms. No recent travel or occupational history was reported and his clinical presentation did not suggest any infectious foci. His relatives later disclosed that he had been homeless for 3 weeks in the context of behavioural changes, obtaining foodstuff from waste containers and water from rain puddles. In the setting of this epidemiology, his presentation of fever, jaundice, acute renal injury and thrombocytopaenia suggested leptospirosis. Prompt empirical antimicrobial coverage was started, alongside organ support therapy. The diagnosis was later confirmed through microscopical and molecular methods. The patient made a full recovery. Leptospirosis should be considered early in the diagnostic work-up of any patient with acute febrile illness with multiorgan system involvement, with the identification of risk factors being essential to treat early in development of the disease.

Isolation and propagation of leptospires at 37 °C directly from the mammalian host.

The causative agent of leptospirosis includes multiple serovars and species of pathogenic leptospires that are excreted via urine from reservoir hosts of infection. Primary isolation takes weeks to months, and is limited to semi-solid media at 28-30 °C. Here we present an alternative media formulation, HAN, compared to commercially available EMJH and the more specialized T80/40/LH media formulations, in semi-solid and liquid compositions, for the primary isolation of two diverse species and serovars of pathogenic leptospires directly from host kidney tissue. All three media types supported the isolation and propagation of L. interrogans serovar Copenhageni strain IC:20:001 in semi-solid media at 29 °C. However, only HAN and T80/40/LH supported the growth of L. borgpetersenii serovar Hardjo strain HB15B203 at 29 °C. In addition, HAN supported primary isolation at 37 °C. Both T80/40/LH and HAN supported primary isolation of strain IC:20:001 in liquid media at 29 °C but only HAN supported growth of strain HB15B203 in liquid media, at both 29 and 37 °C. HAN media supports the primary isolation of fastidious pathogenic leptospires directly from infected host tissue at either 29 or 37 °C: this formulation represents a more defined media for the continued optimization of growth factors required to support the primary isolation of the large and diverse range of species and serovars within the genus Leptospira circulating within domestic and wild animal populations.

Leptospira interrogans in wild Boa constrictor snakes from Northeast Brazil peri­urban rainforest fragments.

Leptospirosis, a disease that occurs worldwide, especially in tropical regions, is caused by bacteria of the genus Leptospira and affects mammals, amphibians, and reptiles. Boa constrictor snakes are commonly found in Atlantic rainforest fragments in peri­urban areas, which indicates a greater possibility of the contact of these animals with humans residing there. Therefore, the aim of this work was to detect Leptospira spp infection through molecular assays in wild B. constrictor snakes rescued in peri­urban areas and verify seroreactivity, by the microscopic agglutination test (MAT), as well as the most common serogroups. Among the 46 samples tested, 7 (15.21%) were positive according to PCR and confirmed as Leptospira interrogans through secY gene sequencing. In MAT, 37 (80.43%) of the 46 samples were classified as reactive. Panama was the serogroup with the highest occurrence. The results showed the presence of Leptospira spp DNA in asymptomatic snakes rescued in rainforest fragments located in peri­urban areas and support further investigations on the influence of these animals in the epidemiology of leptospirosis in tropical peri­urban areas.

Novel genotypes of Leptospira interrogans serogroup Sejroe isolated from human patients in Okinawa Prefecture, Japan.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of Leptospira species. It is a public health issue in the tropics, including Okinawa, the southernmost prefecture of Japan. This study reports the first isolation of L. interrogans serogroup Sejroe from two human patients in Japan, and describes its molecular characterization using multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). MLST on the two isolates, 168036 and 178129, showed that pfkB in 178129 is a novel allele, and that both isolates constitute novel sequence types (STs); ST286 for 168036 and ST287 for 178129. A minimum spanning tree based on seven alleles of L. interrogans indicates that both isolates are genetically close, but are distinct from known L. interrogans serogroup Sejroe strains. MLVA using 11 loci demonstrated that seven of the 11 loci were identical between the two isolates, whereas the identity between the isolates and the seven reference strains of L. interrogans serogroup Sejroe was zero to three loci. These results indicate that the isolates investigated in this study have novel genotypes, and are genetically closest to each other among the known L. interrogans serogroup Sejroe strains.

Leptospira interrogans and Leptospira kirschneri are the dominant Leptospira species causing human leptospirosis in Central Malaysia.

BACKGROUND: Leptospirosis, commonly known as rat-urine disease, is a global but endemic zoonotic disease in the tropics. Despite the historical report of leptospirosis in Malaysia, the information on human-infecting species is limited. Determining the circulating species is important to understand its epidemiology, thereby to strategize appropriate control measures through public health interventions, diagnostics, therapeutics and vaccine development. METHODOLOGY/PRINCIPLE FINDINGS: We investigated the human-infecting Leptospira species in blood and serum samples collected from clinically suspected leptospirosis patients admitted to three tertiary care hospitals in Malaysia. From a total of 165 patients, 92 (56%) were confirmed cases of leptospirosis through Microscopic Agglutination Test (MAT) (n = 43; 47%), Polymerase Chain Reaction (PCR) (n = 63; 68%) or both MAT and PCR (n = 14; 15%). The infecting Leptospira spp., determined by partial 16S rDNA (rrs) gene sequencing revealed two pathogenic species namely Leptospira interrogans (n = 44, 70%) and Leptospira kirschneri (n = 17, 27%) and one intermediate species Leptospira wolffii (n = 2, 3%). Multilocus sequence typing (MLST) identified an isolate of L. interrogans as a novel sequence type (ST 265), suggesting that this human-infecting strain has a unique genetic profile different from similar species isolated from rodents so far. CONCLUSIONS/SIGNIFICANCE: Leptospira interrogans and Leptospira kirschneri were identified as the dominant Leptospira species causing human leptospirosis in Central Malaysia. The existence of novel clinically important ST 265 (infecting human), that is different from rodent L. interrogans strains cautions reservoir(s) of these Leptospira lineages are yet to be identified.