Ellinghausen-McCullough-Johnson-Harris Research Articles

Ketone body oxidation and susceptibility to ethyl acetoacetate in a novel hemolytic multidrug-resistant strain Leptospira interrogans KeTo originated from sewage water

Terrestrial and aquatic environments contaminated with animal urine may contribute to the transmission of Leptospira, a causative agent of leptospirosis in humans and wild/domesticated animals. Although enormous amounts of work have been done decoding the ecophysiology, the factors governing the cell growth and virulence in Leptospires derived from environmental samples still remain elusive. Here, we show oxidation of a wide array of organic acids including acetoacetate by a new strain of Leptospira interrogans designated as KeTo, isolated from a sewage sample originating from a wildlife enclosure located at Mangalore, India. We further demonstrate the susceptibility of KeTo to ethyl ester of acetoacetate (ethyl acetoacetate, EA). A 4.7 Mbp genome of KeTo shared the highest relatedness to pathogenic L. interrogans RGAT (99.3%), followed by L. kirschneri 3522CT (91.3%) and other related species of Leptospira (80.8‒74.3%), and harbored genes encoding acetoacetyl-CoA synthetase and acetoacetate decarboxylase respectively involved in the acetoacetate utilization and acetone formation. In line with this, strain KeTo oxidized acetoacetate when supplied as a sole carbon. Aqueous EA suppressed biofilm formation (p < 0.0001) of KeTo in basal Ellinghausen–McCullough–Johnson–Harris (EMJH) medium. Similarly, significant inhibition in the growth/biofilm of Leptospira was recorded in semisolid EMJH with/without blood supplementation when exposed to volatile EA. The extent of ketone body oxidation and susceptibility to EA was found to vary with strain as evident through the analysis of L. interrogans serogroup Australis sv. Australis strain Ballico and L. interrogans serogroup Icterohaemorrhagiae sv. Lai Like strain AF61. In conclusion, our study demonstrated the ketone body metabolic ability and susceptibility to an esterified derivative of a major ketone body in the tested strains of L. interrogans. Molecular aspects governing EA-driven growth inhibition warrant further investigations to develop optimal therapeutics for leptospirosis.

Leptospira enrichment culture followed by ONT metagenomic sequencing allows better detection of Leptospira presence and diversity in water and soil samples.

Leptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are the major transmission sources in humans and animals. Clusters of disease outbreaks are common during rainy seasons. In this study, to detect the presence of Leptospira, we applied PCR, direct metagenomic sequencing, and enrichment culture followed by PCR and metagenomic sequencing on water and soil samples. Direct sequencing and enrichment cultures followed by PCR or sequencing effectively detected pathogenic and nonpathogenic Leptospira compared to direct PCR and 16S amplification-based metagenomic sequencing in soil or water samples. Among multiple culture media evaluated, Ellinghausen-McCullough-Johnson-Harris (EMJH) media containing antimicrobial agents was superior in recovering and detecting Leptospira from the environmental samples. Our results show that enrichment culture followed by PCR can be used to confirm the presence of pathogenic Leptospira in environmental samples. Additionally, metagenomic sequencing on enrichment cultures effectively detects the abundance and diversity of Leptospira spp. from environmental samples. The selection of methodology is critical when testing environmental samples for the presence of Leptospira. Selective enrichment culture improves Leptospira detection efficacy by PCR or metagenomic sequencing and can be used successfully to understand the presence and diversity of pathogenic Leptospira during environmental surveillance.

Seroprevalence of anti-Leptospira antibodies in equines and associated workers-Isolation of Leptospira interrogans serogroup Canicola from equine urine.

To investigate seroprevalence of anti-Leptospira antibodies in equines and associated workers in Uruguay, 891 equine and 150 human sera were drawn; 212 equine urine samples were also taken for culture. Environmental conditions and equine raising or managing practices were recorded in all 72 visited establishments; epidemiological information was obtained from each worker. Microscopic agglutination technique (MAT) was performed with 10 Leptospira strains for equines and 18 for human sera, that were also studied with IgM indirect immunofluorescence (IgM-IIF). Equine titres ≥100 were considered positive, and human sera titres ≥200 suggested probable recent or past infection. Urines were cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) media; local identification of one obtained isolate with lipL32 PCR, Multiple Locus Variable number tandem repeat Analysis and partial rrs gene sequencing, were completed at Institut Pasteur, Paris. Estimated reactivity was 61.3% for equines, which was higher than the studied bovine national levels (21%) and mainly observed with Icterohaemorrhagiae serogroup (40.3%), Sejroe, Canicola, Pomona or Ballum. Aged animals from slaughterhouses and cattle farms were the most frequently positive. Multiple regression analysis confirmed a significant association between seropositivity and equine age. Only one positive culture could be fully studied, and confirmed to be Leptospira interrogans serogroup Canicola; it was added to the MAT antigen panel and revealed fairly frequent reaction with equine and human sera. Three workers (2%) showed titres=200 with Icterohaemorrhagiae or Canicola serogroups, without recent clinical manifestations. Their attended equines reacted with the same serogroups, suggesting common source infections or infection transmitted by equines. Three other humans yielded titres=100, and none of the 150 showed an IgM-IIF-positive result. Equines seem not to be an important origin of regional human leptospirosis, except perhaps during acute animal infection. More culture work is required to study intensity and lapses of leptospiruria, as well as to further identify circulating strains.

Multi-locus sequence typing for species/serovar identification of clinical isolates of Leptospira spp

Leptospirosis is an emerging zoonotic disease endemic in Kerala and close monitoring of emerging serovars is essential to adopt appropriate control strategies. Multi-Locus Sequence Typing (MLST) was reported to be less expensive compared to other cumbersome methods like whole genome sequencing. The present study was conducted to obtain isolates of Leptospira from infected animals and rats and for the identification of serovars using MLST. A total of 205 blood samples (dog, cat, cattle, goat), 43 urine samples (dog, cattle) and post-mortem kidney samples from various animals such as dog (n=12), cattle (n=2) and rat (n=25) were collected and subjected to polymerase chain reaction (PCR) using G1/G2 primers to identify the pathogenic Leptospira. Fifteen samples were found to be positive, these samples when inoculated in the Ellinghausen- McCullough-Johnson-Harris (EMJH) semi-solid medium to obtain ten isolates. These ten isolates were further subjected to secY, icdA and GyraseB PCR and sequenced. The obtained sequences were analysed using BLAST and were fed into specified MLST database of Leptospira scheme-3, the allelic profile and sequence type were generated. The MLST results obtained in the study indicated that the isolates S24 and S33 belonged to serovar Canicola, S40 and 47 were Sejroe and S19, S27, S55, S69 and S71 were Bataviae, Autumnalis, Pomona, Icterohaemorraghiae and Australis, respectively. It was concluded that MLST is a convenient method for identifying leptospiral serovars.

LEPTOSPIRA SPECIES STATUS OF CAPTIVE NONHUMAN PRIMATES AND FREE-RANGING RODENTS AT THE BARRANQUILLA ZOO, COLOMBIA, 2013.

Leptospirosis is a zoonotic disease with worldwide distribution caused by pathogenic Leptospira spp. Pathogenic Leptospira spp. are shed in urine of infected hosts and transmitted via ingestion of contaminated food or water, inoculation, inhalation of aerosolized urine, and absorption through mucous membranes. Leptospirosis is of particular concern in tropical and subtropical regions such as Barranquilla, Colombia. Recent reports indicate that in Barranquilla, rodents, dogs, and humans have a high leptospiral seroprevalence; and amongst zoo mammals, nonhuman primates have a high prevalence of Leptospira spp. infection. We therefore sought to determine whether primates in captivity at the Barranquilla Zoo were exposed to Leptospira spp. and whether there was a probable causal transmission link between the primates and peridomestic rodents. Samples were collected from 29 captive nonhuman primates, 15 free-ranging rats (Rattus rattus), and 10 free-ranging squirrels (Sciurus granatensis). Serum samples from primates, rats, and squirrels were evaluated via microagglutination test (MAT) vs 24 reference Leptospira serovars. Blood and urine from the primates and kidney tissue from the rats and squirrels were cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and polymerase chain reaction (PCR) of lipL32 was performed to determine whether active infection was present. Leptospiral seroprevalence was found to be 66.7% (10/15) in rats, 60% (6/10) in squirrels, and 6.9% (2/29) in neotropical primates. Ateles hybridus and Ateles fusciceps had positive titers to serogroups Cynopteri and Ictohaemorrhagiae, respectively. Of the rodents that had antibodies against Leptospira spp., 90% of the rats and 66.7% of the squirrels corresponded to the serovar australis. Interestingly, all animals were culture and PCR negative, indicating Leptospira spp. exposure in the absence of current infection. While their status as maintenance hosts needs to be investigated further, this is the first study to show leptospiral seropositivity in red-tailed squirrels (S. granatensis).

Prevalence of Leptospira interrogans in wild rats (Rattus norvegicus and Cricetomys gambianus) in Zaria, Nigeria

Leptospirosis is a neglected disease of zoonotic importance and rodents have a known role in epidemiology of Leptospira globally. Paucity of information on the prevalence of leptospirosis in wild rats used as games in Zaria, Nigeria informed the study. The study aimed to detect Leptospira interrogans in wild rats in Zaria, Nigeria. A total of 71 wild rats comprising 57 Rattus norvegicus and 14 Cricetomys gambianus were sampled over a period of 3 months (April–June 2019). Fisher exact test was used with confidence interval set at 0.05 to ascertain associations between positive cases and species. Blood was collected from 56 rats and harvested sera screened for Leptospira interrogans antibody using rat IgG competitive enzyme linked immunosorbent assay (c-ELISA). Following humane euthanasia of rats, 71 samples (62 kidney tissues and 9 urine samples) were collected in sterile labeled tubes and cultured using Ellinghausen Mc-cullough Johnson Harris (EMJH) enrichment and basal medium. Results indicated over all Leptospira spp antibody detection of 73.2 % (41/56) in Rattus norvegicus (60.7 %) and Cricetomys gambianus (12.5 %). No significant difference (P > 0.05) existed for the prevalence of Leptospira interrogans antibody in the species of wild rats. Over all occurrence of Leptospira interrogans were 74.2 % (46/62) in kidneys and 55.6 % (5/9) in urine samples. Based on species of rats, Rattus norvegicus recorded prevalence of 76.9 % (40/52) and 40.0 % (2/5) in kidney and urine samples respectively. Prevalence of 60.0 % (6/10) and 75.0 % (3/4) in kidney and urine samples respectively were recorded for Cricetomys gambianus. There was significant difference (P < 0.05) in the prevalence of Leptospira interrogans in kidney samples of both wild rats. These species of rats could be reservoirs of Leptospira interrogans. The result showed high prevalence of Leptospira spp in the wild rats and the possibility of domestic animals and humans contracting the disease. This study is the first documentation of evidence of pathogenic Leptospira species in wildlife used as games in Zaria, Nigeria.

Detection of Pathogenic Leptospires in Water and Soil in Areas Endemic to Leptospirosis in Nicaragua.

In Nicaragua, there are ideal environmental conditions for leptospirosis. The objective of this investigation was to detect pathogenic and saprophytic leptospires in water and soil samples from leptospirosis-endemic areas in Nicaragua. Seventy-eight water and 42 soil samples were collected from houses and rivers close to confirmed human cases. Leptospira spp was isolated in Ellinghausen–McCullough–Johnson–Harris (EMJH) culture medium with 5-fluororacil and positive samples were analyzed through PCR for the LipL32 gene, specific for pathogenic leptospires (P1 clade). There were 73 positive cultures from 120 samples, however only six of these (5% of all collected samples) were confirmed to be pathogenic, based on the presence of the LipL32 gene (P1 clade). Of these six pathogenic isolates, four were from Leon and two from Chinandega. Four pathogenic isolates were obtained from water and two from soil. This study proved the contamination of water and soil with pathogenic leptospires, which represents a potential risk for public health.

Leptospira infection and shedding in dogs in Thailand

BackgroundLeptospirosis is a widespread zoonosis and has been recognized as a re-emerging infectious disease in humans and dogs, but prevalence of Leptospira shedding in dogs in Thailand is unknown. The aim of this study was to determine urinary shedding of Leptospira in dogs in Thailand, to evaluate antibody prevalence by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA), and to assess risk factors for Leptospira infection.In Northern, Northeastern, and Central Thailand, 273 stray (n = 119) or client-owned (n = 154) dogs from rural (n = 139) or urban (n = 134) areas were randomly included. Dogs that had received antibiotics within 4 weeks prior to sampling were excluded. No dog had received vaccination against Leptospira. Urine was evaluated by real-time polymerase chain reaction (PCR) specific for lipL32 gene of pathogenic Leptospira. Additionally, urine was cultured for 6 months in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. Antibodies were measured by ELISA and MAT against 24 serovars belonging to 15 serogroups and 1 undesignated serogroup. Risk factor analysis was performed with backwards stepwise selection based on Wald.ResultsTwelve of 273 (4.4%; 95% confidence interval (CI): 2.0–6.8%) urine samples were PCR-positive. In 1/273 dogs (0.4%; 95% CI: 0.01–1.1%) Leptospira could be cultured from urine. MAT detected antibodies in 33/273 dogs (12.1%; 95% CI: 8.2–16.0%) against 19 different serovars (Anhoa, Australis, Ballum, Bataviae, Bratislava, Broomi, Canicola, Copenhageni, Coxi, Grippotyphosa, Haemolytica, Icterohaemorrhagiae, Khorat, Paidjan, Patoc, Pyrogenes, Rachmati, Saxkoebing, Sejroe). In 111/252 dogs (44.0%; 95% CI: 37.9–50.2%) immunoglobulin M (IgM) and/or immunoglobulin G (IgG) antibodies were found by ELISA. Female dogs had a significantly higher risk for Leptospira infection (p = 0.023).ConclusionsLeptospira shedding occurs in randomly sampled dogs in Thailand, with infection rates comparable to those of Europe and the USA. Therefore, the potential zoonotic risk should not be underestimated and use of Leptospira vaccines are recommended.

Leptospiral Culture without 5’-Fluorouracil Revealed Improved Leptospira Isolation from Febrile Patients in North-Eastern Malaysia

Objectives: Isolation of Leptospira by culture represents a definitive growth and confirmation of the disease, yet it is hampered with its nature of slow growth. With slight modification of culture method, the study aims to isolate and characterize Leptospira spp. from patients with acute febrile illness. Methods: A total of 109 blood samples were collected from patients with acute febrile illness that presented at the Emergency Department of Hospital Universiti Sains Malaysia, Malaysia. Clinical samples were subjected to Leptospira IgM Rapid test, microscopic agglutination test (MAT), isolation by culture method, and direct real-time PCR test. For leptospiral isolation, the samples (whole blood and deposit from spun plasma) were cultured into modified Ellinghausen McCullough Johnson Harris (EMJH) media with and without 5’-fluorouracil (5-FU). In every culture positive sample, partial 16S rRNA gene sequencing was performed for molecular identification of the isolates. Phylogenetic analysis was carried out to determine the genetic relatedness among the isolates. An inhibition of 5-FU study was performed on Leptospira interrogans serovar Canicola with different concentrations to compare the growth detection of the tested Leptospira with or without 5-FU within 7 days of incubation. Results: Leptospirosis was diagnosed in 14.7% of patients with acute febrile illness. Two Leptospira spp. (n = 2/109, 1.85%) were successfully isolated from whole blood and deposit from spun plasma samples. B004 and B208 samples were positive at day 11 and day 7, respectively, in EMJH media without addition of 5-FU. Sample B004 was identified as Leptospira interrogans and B208 as Leptospira weilli. Phylogenetic analysis confirmed that both of them were within pathogenic group and they were not related. The 5-FU inhibition study revealed that additional of 5-FU at final concentration of 200 µg/mL to EMJH media demonstrated an inhibitory effect on the growth of the tested strain Conclusion: Isolation of Leptospira spp. using EMJH media without addition of 5’-fluorouracil resulted in a better outcome. Two pathogenic Leptospira isolates were successfully cultivated from patients with acute febrile illness that were genetically not related.

Study protocol: characterising the clinical, epidemiological and aetiological aspects of leptospirosis in Sri Lanka: a hospital based clinico-epidemiological study

IntroductionSri Lanka has one of the highest incidences of leptospirosis worldwide. We hypothesised that different geographical locations and patient context will have a distinct molecular epidemiology of leptospirosis, based on...

Occurrence of Leptospira Species from Rodents, Soil and Water from an Oil Palm Plantation in Northern Sarawak

Leptospirosis is a death-causing disease caused by corkscrew-shaped bacteria, Leptospira especially in tropical countries. Current study was aimed to detect pathogenic, intermediate and saprophytic Leptospira species using polymerase chain reaction (PCR) assay from an oil palm plantation in Borneo, specifically in Miri, Malaysia. Materials and Methods: A total of 63 samples from rodents (n = 3), water (n = 30) and soil (n = 30) were isolated from an oil palm estate in Northern Sarawak, Borneo. All samples were inoculated into modified semisolid Ellinghausen-McCullough-Johnson-Harris (EMJH) broth with 5-fluorouracil and incubated for a month. Polymerase chain reaction (PCR) was performed using primer targeting lipL32 (423 bp) for pathogenic, 16S rRNA (331 bp) for intermediate and rrs (240 bp) for saprophytic species. Results: pathogenic Leptospira was found in 33.3% rodents (1/3) Rattus tiomanicus, 23.3% soil samples (7/30) and 16.7% water samples (5/30). Intermediate species were demonstrated in the other 66.7% rodents (2/3), Sundamys muelleri and Rattus exulans and 10% soil samples (3/30). Saprophytic species was found in only 3.3% soil sample (1/30). Results from DNA sequencing analysis indicated that the most dominant pathogenic Leptospira species discovered in the study was Leptospira interrogans, followed by Leptospira noguchii and Leptospira weilii. Conclusion: These preliminary findings provide baseline data on the occurrence of Leptospira species in captured rodents and the environment. These findings could assist in control and prevention of leptospirosis among oil palm estate workers in Sarawak. Awareness and knowledge on leptospirosis should be promoted among oil palm workers for prevention and mitigation

Detection of Leptospire spp. in animals and in environmental samples from peridomestic areas in NicaraguaDetecção de Leptospira spp. em animais e em amostras ambientais de áreas peridomiciliares na Nicarágua

RESUMENObjetivoEl objetivo de este estudio fue conocer las características epidemiológicas de la leptospirosis en animales domésticos y en los casos de leptospirosis humana en áreas peridomésticas en Nicaragua entre 2014 y 2016.MétodosLas muestras se extrajeron en áreas donde se confirmaron casos en humanos utilizando un muestreo no probabilístico en 10 de los 17 departamentos del país. Se incluyeron 112 muestras de orina de animales domésticos, 129 muestras de agua y 69 de tierra para aislar leptospiras en medio Ellinghausen-McCullough-Johnson-Harris (EMJH). Además, se aplicó la prueba de microaglutinación (MAT) en 263 muestras de suero de animales y 88 aislados se analizaron mediante PCR.ResultadosEn 32,6% (101/310) de las muestras se aislaron espiroquetas, 23,2% (26/112) se aislaron en la orina de animales domésticos, 47,3% (61/129), en las muestras de agua y 20,3 % (14/69), en las de tierra. El análisis de aislamiento mostró diferencias significativas (P < 0,05) entre los departamentos para los diferentes tipos de muestras, y el aislamiento fue más frecuente en agua que en tierra (OR = 3,49; IC95%: 1,56-7,80). El 14,1% (37/263) de los animales fueron reactores en la prueba de microaglutinación. El serogrupo más frecuente fue Icterohaemorrhagiae (40%). En el análisis con la PCR para identificar leptospiras de las especies patógenas 10,2% (9/88) de los aislamientos fueron positivos.ConclusionesEsta investigación demuestra que los animales domésticos y el ambiente desempeñan un papel importante en la aparición de brotes de la leptospirosis y confirma el comportamiento endémico de la enfermedad en Nicaragua.

Anti-microbial Activity of Aqueous Quercus infectoria Gall Extract against Pathogenic Leptospira.

BackgroundQuercus infectoria gall extract is known to have broad spectrum anti-microbial activity in vitro. This study was conducted to determine the anti-microbial activity of Q. infectoria gall extract against pathogenic Leptospira and to evaluate the morphological changes of extract-treated cells using a scanning electron microscope (SEM).MethodsA two-fold serial microdilution broth assay was used to determine the minimum inhibitory concentration (MIC) of aqueous Q. infectoria gall extract against the L. interrogans serovar Javanica and the L. interrogans serovar Icterohaemorrhagiae, at concentrations ranging from 4.00 mg/mL to 0.0078 mg/mL. The minimum bactericidal concentration (MBC) was determined by sub-culturing the broth from the microtiter plate wells that showed no apparent growth or turbidity to the freshly prepared Ellinghausen-McCullough-Johnson-Harris (EMJH) broth media, and it was subsequently observed under a dark field microscope following three weeks of incubation for purposes of growth detection. The cell morphology of both extract-treated and untreated L. interrogans serovar Icterohaemorhagiae was analysed using the SEM.ResultsThe results of the broth microdilution assay demonstrate that the aqueous Q. infectoria gall extract possesses anti-microbial activity against both of the L. interrogans serovars with MIC values of 0.125 mg/mL. The MBC values for the L. interrogans serovar Javanica and the L. interrogans serovar Icterohaemorhagiae are 0.125 mg/mL and 0.250 mg/mL, respectively. The SEM micrograph shows changes in shape and size of the extract-treated cells (at 8× MIC) in comparison to the untreated cells.ConclusionThe Q. infectoria gall extract displays anti-microbial inhibition and killing activity against the pathogenic Leptospira isolates, and thus has the potential for further exploration of its efficacy and use in the treatment of leptospirosis.

Prevalence and renal pathology of pathogenic Leptospira spp. in wildlife in Abeokuta, Ogun State, Nigeria.

There is paucity of information on the prevalence of leptospirosis in wildlife in Nigeria. This study investigated the prevalence and renal pathology of leptospirosis in wild animals in Southwest Nigeria. One hundred and five kidney samples were examined from 10 different wildlife species (antelope) greater cane rat (GCR), hare, African giant rat (AGR), tree hyrax, civet cat, monitor lizard, python, bushbuck and partridge) using a combination of Ellinghausen McCullough Johnson Harris (EMJH) medium, microscopic agglutination test (MAT), Warthin–Starry silver stain (WSss) and immunohistochemistry. Chi-square test was used with confidence level set at 0.05 to ascertain associations between positive cases and sex and species. Eighty-two (78.1%) samples were culturally positive, while 67.7% (63/93), 57.0% (16/28) and 66.7% (8/12) were WSss, MAT and immunohistochemically positive, respectively. Interstitial nephritis (41.0%) and tubular nephrosis (81.0%) were the most prominent histopathological changes. Pathogenic Leptospira organisms were highest in GCR (32.1%) and antelope (14.3%). Serovars hardjo (11.54%), bratislava (3.9%), canicola (3.9%), icterohaemorrhagiae (15.4%), pomona (7.14%) gripptotyphosa (19.2%) and undetermined isolates were also detected in other animals. The result showed high prevalence of Leptospira infection in the wild and the possibility of domestic animals and humans contracting the disease. This study is the first documentation of evidence of pathogenic Leptospira species in wildlife in Nigeria.

Isolation of a Seawater Tolerant Leptospira spp. from a Southern Right Whale (Eubalaena australis).

Leptospirosis is the most widespread zoonotic disease in the world. It is caused by pathogenic spirochetes of the genus Leptospira spp. and is maintained in nature through chronic renal infection of carrier animals. Rodents and other small mammals are the main reservoirs. Information on leptospirosis in marine mammals is scarce; however, cases of leptospirosis have been documented in pinniped populations from the Pacific coast of North America from southern California to British Columbia. We report the isolation of a Leptospira spp. strain, here named Manara, from a kidney sample obtained from a Southern Right Whale (Eubalaena australis) calf, which stranded dead in Playa Manara, Península Valdés, Argentina. This strain showed motility and morphology typical of the genus Leptospira spp. under dark-field microscopy; and grew in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and Fletcher medium after 90 days of incubation at 28°C. Considering the source of this bacterium, we tested its ability to grow in Fletcher medium diluted with seawater at different percentages (1%, 3%, 5%, 7% and 10% v/v). Bacterial growth was detected 48 h after inoculation of Fletcher medium supplemented with 5% sea water, demonstrating the halophilic nature of the strain Manara. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the pathogenic species of the genus Leptospira spp., with sequence similarities within the range 97–100%, and closely related to L. interrogans. Two different PCR protocols targeting genus-specific pathogenic genes (G1-G2, B64I-B64II and LigB) gave positive results, which indicates that the strain Manara is likely pathogenic. Further studies are needed to confirm this possibility as well as determine its serogroup. These results could modify our understanding of the epidemiology of this zoonosis. Until now, the resistance and ability to grow in seawater for long periods of time had been proven for the strain Muggia of L. biflexa, a saprophytic species. To the best of our knowledge, this is the first isolation of a Leptospira sp. from cetaceans. Our phenotypic data indicate that strain Manara represents a novel species of the genus Leptospira, for which the name Leptospira brihuegai sp. nov. is proposed.

Pesquisa de leptospiras e de anticorpos contra leptospiras em animais e humanos de propriedades rurais nos biomas brasileiros Pantanal e Caatinga

&lt;p&gt;Foi investigada a ocorrência de leptospiras e de sororreatividade para leptospiras em animais e seres humanos de seis propriedades rurais localizadas em dois biomas brasileiros que apresentam condições geoclimáticas distintas: Pantanal – municípios de Miranda (MS), Itiquira (MT) e Poconé (MT) e Caatinga – municípios de Sobradinho (CE), Garanhuns (PE) e Sobral (BA). Em cada uma das propriedades, foram realizadas colheitas de sangue e de urina de animais selvagens de vida livre, animais domésticos e de seres humanos. As colheitas de materiais foram realizadas no período de fevereiro a abril de 2012 no bioma Caatinga e no período de julho a setembro de 2012 no bioma Pantanal. A reatividade sorológica contra &lt;em&gt;Leptospira &lt;/em&gt;spp. foi verificada pela técnica de soroaglutinação microscópica (SAM) efetuada com uma coleção de antígenos constituída por 24 sorovares de &lt;em&gt;Leptospira &lt;/em&gt;spp. A pesquisa de leptospiras foi efetuada por cultivos de amostras de urina semeadas nos meios Fletcher e de Ellinghausen – McCullough – Johnson – Harris (EMJH). Os cultivos em que houve crescimento de leptospiras foram encaminhados ao Laboratório de Leptospirose do Instituto de Patobiologia, Instituto Nacional de Tecnologia Agropecuária, Buenos Aires, Argentina e as estirpes de leptospiras isoladas foram genotipadas com o emprego da técnica de &lt;em&gt;Multiple Locus Variable Number Tandem Repeat Analysis &lt;/em&gt;(MLVA). O procedimento de tipificação empregou os VNTR 4, 7, 9, 10, 19, 23, 31, Lb4 e Lb5, que discriminam estirpes de &lt;em&gt;L. interrogans &lt;/em&gt;e &lt;em&gt;L. borgpetersenii. &lt;/em&gt;No Pantanal, foram examinados 17 animais selvagens, 65 animais domésticos e dois humanos. Na Caatinga, foram examinados sete animais selvagens, 100 animais domésticos e 26 humanos. Das 84 amostras de sangue examinadas no Pantanal, 47 (55,95%) foram reagentes e, das 133 da Caatinga, 59 (44,36%) foram reagentes. Pelo teste exato de Fisher, considerando-se um nível de significância de 0,05, não houve diferença entre as proporções de animais sororreagentes para &lt;em&gt;Leptospira &lt;/em&gt;spp. nos dois biomas avaliados (p = 0,063). Os sorovares predominantes nas reações de SAM foram: 1) Pantanal – Bratislava (animais selvagens, cães e humanos); Grippotyphosa (equinos e bovinos); 2) Caatinga – Copenhageni (humanos e cães), Patoc (equinos e bovinos), Panama (ovinos e caprinos), Patoc, Copenhageni e Australis (animais selvagens). Houve isolamento de quatro estirpes de leptospiras: duas em Sobradinho, BA, &lt;em&gt;L. interrogans &lt;/em&gt;sorogrupo Pomona em &lt;em&gt;Cavea aperea &lt;/em&gt;e &lt;em&gt;L. interrogans &lt;/em&gt;em &lt;em&gt;Euphractus sexcinctus&lt;/em&gt;; e duas em Sobral, CE, &lt;em&gt;L. interrogans &lt;/em&gt;em &lt;em&gt;Cerdocyon thous &lt;/em&gt;e &lt;em&gt;L. interrogans &lt;/em&gt;sorogrupo Pomona em &lt;em&gt;Euphractus sexcinctus.&lt;/em&gt;&lt;/p&gt;

Isolation of Leptospira spp. from a man living in a rural area of the Municipality of Cruz Alta, RS, Brazil

The aim of this study was to describe the isolation of a pathogenic strain of Leptospira interrogans from the urine sample of a male human living in the rural area of the County of Cruz Alta, Rio Grande do Sul. An aliquot of each urine sample was sown in a Fletcher and Ellinghausen - McCullough - Johnson - Harris (EMJH) media. Samples in which there was growth of spirochetes were sent to the Leptospirosis Laboratory of the Institute of Pathobiology in the National Institute of Agricultural Technology, Buenos Aires, Argentina and were typified by the Multiple Locus of Variable Number Tandem Repeat technique (MLVA). Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 was isolated, and this is a very important finding that serves as a warning to characterize risk situation of leptospirosis epidemic by a pathogenic strain. Health professionals need to be more committed to the primary health care in Brazil and routinely apply actions of preventive medicine in rural communities in order to get success in the control of leptospirosis and other important zoonoses

Isolamento de Leptospira borgpetersenii em Didelphis albiventris sinantrópicos em Jaboticabal, São Paulo, Brasil

Leptospirosis is a waterborne disease and, therefore, stands out for the possibility of environmental contamination, the cross transmission between domestic and wild animals and humans. Opossum species are important reservoirs of this disease making them potential pathogen spreaders. Aiming to verify the presence of Leptospira spp. and the antibodies against Leptospira spp. in the Campus of Sao Paulo State University, in Jaboticabal, Sao Paulo, Brazil, freeliving wild life opossum (Didelphis albiventris) were captured for blood and urine sampling. Serological analysis was performed Microscopic Agglutination Test (MAT). Aliquots of urine were seeded in media Ellinghausen-McCullough- Johnson-Harris (EMJH) and Fletcher without antibiotics. The samples in which there was growth of leptospires were forwarded to the Leptospirosis Laboratory of the Institute of Pathobiology in the National Institute of Agricultural Technology, Buenos Aires, Argentina and were genotyped using Multiple Locus Variable number tandem repeat Analysis (MLVA). Of the 15 analyzed animals, nine (60.0%) were reactant to Patoc serovar. The pathogenic specie Leptospira borgpetersenii was isolated and identified in three Didelphis albiventris. The isolation findings of pathogenic specie Leptopsira borgpetersenii in the urine culture of three Didelphis albiventris in a university campus are a major discovery in the area of preventive veterinary medicine and public health and open a discussion about the important role of free-living wild animals as reservoirs of this agent to domestic animals and humans, a condition that serves as a warning for the improvement of health practices.

A simple technique for long-term preservation of leptospires

The viability of six serovars of Leptospira spp. was studied after long storage at -70°C. The bacteria were either preserved in Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid growth medium or in sheep blood added as a cryoprotectant. The viability of the strains was observed on a monthly basis by dark-ground microscopy over a period of 20 months at -70°C. Addition of sheep blood was not significantly advantageous, since leptospires that were stored in EMJH showed a slight increase in number after recovery. The results suggest a very simple and useful technique for long-term preservation of such Leptospira.

Investigation of localisation of Leptospira spp. in uterine and fetal tissues of non-pregnant and pregnant farmed deer

AIM: To find evidence for localisation in the uterus, and fetal infection, of Leptospira spp. in farmed deer in the lower North Island of New Zealand during and shortly after the breeding season. METHODS: Between February and July 2008, 116 blood samples, 120 kidneys, 120 uteri and 27 fetuses were collected from 120 mixed-age hinds from lines from nine farms, at a deer slaughter premises. Serum samples were tested for antibodies against Leptospira borgpetersenii serovar Hardjo-bovis and Leptospira interrogans serovar Pomona, using the microscopic agglutination test (MAT). For both serovars, a titre of >1:48 was considered positive. Samples from kidneys, uteri and fetal tissue were subjected to bacterial culture, using Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, and real-time PCR, using DNA gyrase subunit B gene primers. RESULTS: Thirty-four of 116 (29.3%) serum samples were positive for serovar Hardjo-bovis, and 13 (11.2%) for serovar Pomona. Seven of 120 kidneys were positive for serovar Hardjo-bovis by culture, and five of these, but no others, were positive by real-time PCR. Of 120 uteri, none was culture- or PCR-positive. None of 27 fetal samples was culture-positive but one was positive by real-time PCR. The dam of the PCR-positive fetus was culture-negative from the kidney, but had an MAT titre of 1:192 for Hardjo-bovis. CONCLUSIONS: Attempts to isolate Leptospira spp. from the genital tracts and early fetuses of farmed deer were unsuccessful. However, molecular evidence suggested fetal infection in one case. This finding justifies further study of the role of leptospires in the genital tract and fetus and its association with reproductive loss in farmed deer.