Leptospira Kirschneri Serovar Grippotyphosa Research Articles

Levels of Cytokines in Leptospirosis Patients with Different Serovars and rfb Locus.

Leptospirosis has a wide spectrum of clinical manifestations ranging from mild to severe disease. The cytokine response is considered one of the key drivers for this varying manifestation. The different cytokine response observed in patients with leptospirosis could be due to the variation of infecting serovars. Since the rfb locus codes for the lipopolysaccharide synthesis of the bacterial cell wall, which also determines the serovar, this locus may play a role in driving a specific cytokine response in the host. We investigated 12 commonly used cytokine profiles in serum samples of culture, microscopic agglutination test (MAT), or polymerase chain reaction (PCR)-positive patients with leptospirosis. The sequences of the rfb locus in culture-positive samples were generated from whole genome sequencing and serovar status was drawn from original data published. Isolated cultures were subjected to whole genome sequencing using the PacBio RS II system, and the resulting data were used to determine the species. The recovered genomic data were annotated with the Rapid Annotation using Subsystem Technology (RAST) subsystem, and the rfb locus was extracted. The cytokine analysis was carried out using the Qiagen human ELISA kit. Eighteen samples were found to be positive by culture, while the other 7 samples were positive by PCR or MAT. Infections from Leptospira interrogans serovar Autumnalis (5), Pyrogens (3), Icterohaemorrhagiae (1) Leptospira borgpetersenii (all 7 samples clustered in same clonal group with serovar status not determined), Leptospira weilii (1 with serovar status not determined), and Leptospira kirschneri serovar Grippotyphosa (1) were included in the analysis. Three patients [infected with Leptospira interrogansserovar Autumnalis (2) and Pyrogens (1)] and 2 MAT-positive patients (highest titer against serovar Bratislava of L.interrognas) were reported to have severe clinical manifestations, while the rest had mild to moderate symptoms. Although the serum cytokine concentration of patients with severe clinical manifestation was comparatively higher, a statistically significant difference was observed only for interleukin (IL)-1β (P < 0.05). IL-10/tumor necrosis factor-alpha (TNF-α) ratio was high in patients with severe complications. In general, patients infected with L. interrogans showed higher concentration of cytokines compared to L. borgpetersenii.

Leptospira spp. in Cats in Estonia: Seroprevalence and Risk Factors for Seropositivity

Leptospirosis is a zoonotic bacterial disease that affects humans and animals worldwide. Recently, more attention has been paid to Leptospira spp. infections in domestic cats. In this seroepidemiological study, we estimated the prevalence of anti-Leptospira spp. antibodies in domestic cats in Estonia and evaluated risk factors for the seropositivity. A total of 546 surplus feline plasma and serum samples, collected in collaboration with small animal clinics and an animal shelter in 2013 and 2015, were included in this study. The samples were tested for antibodies against Leptospira spp. using a microscopic agglutination test. The panel included Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Bratislava, Canicola, and Hardjo, and Leptospira kirschneri serovar Grippotyphosa. Titers ≥100 (positive reactions at dilutions ≥1/100) were considered positive. Anti-Leptospira spp. antibodies were detected in 12.8% of the cats. The percentage of cats that tested positive for antibodies against L. interrogans serovars Pomona, Icterohaemorrhagiae, Bratislava, Canicola, and Hardjo were 9.3%, 3.5%, 2.4%, 0.4%, and 0.2%, respectively, and the percentage of cats that tested positive for antibodies against L. kirschneri serovar Grippotyphosa was 7.3%. Of the seropositive cats, 46.5%, 35.2%, 12.7%, 4.2%, and 1.4% tested positive for 1, 2, 3, 4, and 5 serovars, respectively. The prevalence of anti-Leptospira spp. antibodies was 11.2% in pet cats and 16.3% in shelter cats. Among pet cats, the seroprevalence was over four times higher in cats that had access to the outdoors (17.2%) than in indoor cats (3.9%). Multivariable models, one based on data on pet cats only and another including also data on shelter cats, identified having access to the outdoors, being a shelter cat, and being from Western Estonia as the risk factors for seropositivity. Cats could be better protected from exposure to Leptospira spp. by not allowing them to roam freely outdoors.

Variable-number tandem-repeat analysis of leptospiral DNA isolated from canine urine samples molecularly confirmed to contain pathogenic leptospires

OBJECTIVE To use variable-number tandem-repeat (VNTR) analysis to determine the infecting serovar and strain for leptospiral DNA isolated from canine urine samples confirmed through PCR testing to contain pathogenic leptospires and to evaluate the sensitivity and specificity of microscopic agglutination testing (MAT) for identifying the infecting serogroup. DESIGN Diagnostic survey and test evaluation. SAMPLE Leptospiral DNA isolated from urine samples from 98 dogs confirmed through PCR testing to have pathogenic leptospires in their urine. PROCEDURES VNTR analysis of DNA isolates was performed to identify the infecting leptospiral serovar and strain by use of primer pairs for the loci 4, 7, 10, and Lb5. Eighteen pathogenic and 2 saprophytic leptospiral serovars were used as reference strains for VNTR analysis. Results of MAT were compared with those of the PCR assay and VNTR analysis to determine the sensitivity and specificity of MAT for diagnosing leptospirosis and identifying the infecting serovar at various reciprocal titers. RESULTS VNTR analysis identified Leptospira kirschneri serovar Grippotyphosa strain DF as the most common infecting serovar in dogs (78/98 [80%]). Thirteen unique VNTR patterns could not be identified by comparison with the Leptospira reference strains used. The MAT had a maximum sensitivity of 41% and a specificity of 100% for identifying Grippotyphosa as the infecting serogroup. CONCLUSIONS AND CLINICAL RELEVANCE Findings confirmed the importance of Leptospira serovar Grippotyphosa among dogs in the United States. Serologic testing had poor sensitivity for identifying the infecting serogroup, and conclusions about emerging serogroups should be cautiously interpreted when serologic data are reported.

SEROLOGIC SURVEY AND RESULTS OF URINARY PCR TESTING FOR LEPTOSPIROSIS IN CAPTIVE BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS).

Leptospirosis is an important zoonotic disease occurring clinically and subclinically in humans and a wide variety of mammal species worldwide. Often, rodents and wild animals are identified as important reservoirs for the disease. Twenty-two captive black-tailed prairie dogs (Cynomys ludovicianus) housed within a zoo were examined as part of a routine census and preventive medicine program. During examinations, blood and urine were collected to screen for exposure to, or infection with, leptospirosis. All animals were apparently healthy at the time of examination. Leptospira microscopic agglutination test identified 12 of 22 (54.5%) prairie dogs with antibody titers ≥1 : 100 against Leptospira interrogans serovar bratislava on initial serologic examination. All prairie dogs within this collection were serologically negative for L. interrogans serovars canicola, hardjo, icterohaemorrhagiae, and pomona and Leptospira kirschneri serovar grippotyphosa. Leptospira polymerase chain reaction (PCR) testing of urine was negative in all animals tested. This report describes evidence that captive prairie dogs may be exposed to leptospirosis, most likely from wild rodent reservoirs; however, serum titers are low, and lack of leptospiral DNA detected by PCR indicates that these captive animals are unlikely to be important reservoirs for the disease.

Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes

Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis.

Effects of Recent Leptospira Vaccination on Whole Blood Real‐Time PCR Testing in Healthy Client‐Owned Dogs

Bacterin-based canine Leptospira vaccines could present a challenge for the use of whole blood real-time polymerase chain reaction (PCR) as a diagnostic tool. Recent vaccination could induce positive results if the targeted DNA fragment is present within the vaccine and in the blood of the recently vaccinated dog. The objective of this study was to assess whether 2 available 4-serovar vaccines induce a positive real-time PCR reaction in the blood of healthy recently vaccinated dogs. Twenty healthy dogs. This was a prospective study. Dogs were assigned to 1 of 2 vaccine groups. Both vaccines were culture-based and include Leptospira interrogans serovars Pomona, Canicola, and Icterohaemorrhagiae and Leptospira kirschneri serovar Grippotyphosa. Whole blood for real-time PCR and serum for the microscopic agglutination test (MAT) were collected prior to and 3 and 7 days after vaccination and weekly thereafter for 8 weeks. Two real-time PCR tests targeting 2 different genes were performed independently in a blinded fashion. Both Leptospira vaccines produced positive real-time PCR reactions when assayed undiluted or diluted 1 : 100 in canine blood. However, blood samples drawn from all dogs at all time points after vaccination were negative on PCR. All dogs developed MAT titers. Recent vaccination with 2 commercially available vaccines does not interfere with the use of real-time PCR for the identification of acute Leptospira infection in dogs.

Serovar-specific prevalence and risk factors for leptospirosis among dogs: 90 cases (1997-2002).

To estimate serovar-specific prevalence of leptospirosis by use of veterinary teaching hospital and laboratory submission data; describe annual and seasonal patterns of leptospirosis; and identify risk factors for age, sex, and breed. Retrospective study. 90 dogs with leptospirosis. Hospital records of dogs examined at Purdue University Veterinary Teaching Hospital with a diagnosis of leptospirosis and laboratory records of dogs from which sera were tested for antibodies against Leptospira spp at Purdue University Animal Disease Diagnostic Laboratory from 1997 through 2002 were reviewed. The likely infecting Leptospira serovar was identified. Seasonal and annual prevalences were calculated by use of hospital population at risk (hospital cases) or serologic testing submissions (diagnostic laboratory cases). Age-, sex-, and breed-specific risk factors for hospital cases were estimated by odds ratios. Of the 39 hospitalized dogs identified, 34 had been serologically tested, and 22 of those were infected with Leptospira kirschneri serovar grippotyphosa. Of the 51 diagnostic laboratory cases, 59% had a reciprocal titer > or = 800 against serovar grippotyphosa. Diagnostic laboratory cases were more common in summer, whereas hospital cases of leptospirosis were more common in fall. Male dogs were at significantly greater risk of leptospirosis than female dogs; and dogs 4 to 6.9 years old were at significantly greater risk than dogs < 1 year old. L kirschneri serovar grippotyphosa infection was associated with most cases of leptospirosis in dogs. Use of an effective vaccine that includes this serovar is advisable for dogs at risk of leptospirosis.

An improved immunohistochemical diagnostic technique for canine leptospirosis using antileptospiral antibodies on renal tissue.

The purpose of this study was to compare the immunoreactivity in canine renal tissues stained with antisera specific for 3 leptospiral antigens and those processed with traditional staining methods. In addition, immunoglobulin staining was done on tissues with immunoreactivity to leptospiral antigens. Formalin-fixed renal sections from 12 dogs with chronic interstitial nephritis suspected or proven to have leptospirosis (6 dogs with silver-stained leptospires and 6 dogs in which silver-stained leptospires were not detected) were used. Antibodies consisted of a monoclonal antibody to Leptospira kirschneri serovar grippotyphosa lipopolysaccharide (LPS) and 2 polyclonal antibodies to outer membrane proteins, including OmpL1, a leptospiral porin, and LipL41, an outer membrane lipoprotein. The murine monoclonal antisera against LPS (F71C2-1) had the most abundant and consistent immunoreactivity. Immunoreactive areas were present in 6 of 6 sections positive by silver staining and included extracellular granular debris in intertubular areas, debris in macrophages, organisms in tubular lumina, and cytoplasmic granules in tubular epithelia. Antisera with specificity for the outer membrane proteins OmpL1 and LipL41 detected only intact organisms in tubular lumina. Immunoreactivity to OmpL1 (polyclonal 338) occurred in 4 of 5 sections positive by silver staining, but immunoreactivity to LipL41 (polyclonal 813) occurred in only 1 of 6 silver-positive sections. Each of the kidney sections in which leptospiral antigens were detected by immunohistochemistry also was positive by silver staining. Sections negative by silver staining were also negative by immunostaining. Although immunohistochemistry did not enhance sensitivity, amplification of signal by secondary antibody and hematoxylin counterstaining improved the ease of diagnosis and allowed better evaluation of tissue morphology than did silver staining methods. IgG was the most abundant immunoglobulin. IgG immunoreactivity occurred predominantly in plasma cells within interstitial infiltrates. Interstitial infiltrates contained abundant immunoreactivity to LPS, but immunoreactivity to OmpL1 and LipL41 was not noted.

Novel 45-kilodalton leptospiral protein that is processed to a 31-kilodalton growth-phase-regulated peripheral membrane protein.

Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the alpha, beta, and beta' subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val(-4)-Phe(-3)-Asn(-2)-Ala(-1) (downward arrow)Cys(+1) was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31(LipL45). Membrane fractionation studies demonstrated that P31(LipL45) is a peripheral membrane protein. Finally, we found that P31(LipL45) levels increased as Leptospira entered the stationary phase, indicating that P31(LipL45) levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.