The Experts below are selected from a list of 1248 Experts worldwide ranked by ideXlab platform
Ben Adler - One of the best experts on this subject based on the ideXlab platform.
-
recombinant lipl32 stimulates interferon gamma production in cattle vaccinated with a monovalent Leptospira borgpetersenii serovar hardjo subtype hardjobovis vaccine
Veterinary Microbiology, 2014Co-Authors: Deanna Deveson Lucas, Dieter M. Bulach, Gerald L. Murray, Noelene Sheila Quinsey, Andrew G. Allen, Ben AdlerAbstract:Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjobovis) is the main causative agent of bovine leptospirosis in Australia, New Zealand, North America and elsewhere. Bovine leptospirosis can result in spontaneous abortion, stillbirth and reduced milk output. The organism is shed in the urine of infected animals and contact with contaminated materials can result in zoonotic infections in humans. Protective immunity in cattle against Hardjobovis involves stimulation of a Th1 cell mediated immune response, which can be characterized by the production of IFN-γ when blood from vaccinated animals is exposed to Hardjobovis antigens. However, the Leptospiral components involved in stimulating this response have yet to be identified. In this study, 238 recombinant Leptospiral proteins were evaluated for their ability to stimulate IFN-γ production in blood of cattle vaccinated with a commercial monovalent Hardjobovis vaccine. The conserved lipoprotein LipL32 is the major outer membrane protein of pathogenic Leptospira spp. A pool of soluble recombinant proteins which included LipL32, as well as LipL32 alone, stimulated significant IFN-γ production in blood of vaccinated cattle. A number of recombinant LipL32 fragments was generated, which identified the amino acids between 20 and 200 as containing the bovine T-cell reactive regions of LipL32. However, whether LipL32 plays a role in stimulating protective immunity in mammals has yet to be conclusively determined.
-
Evaluation of 238 antigens of Leptospira borgpetersenii serovar Hardjo for protection against kidney colonisation.
Vaccine, 2012Co-Authors: Gerald L. Murray, Dieter M. Bulach, Torsten Seemann, Amporn Srikram, Noelene Sheila Quinsey, Rasana W. Sermswan, Andrew G. Allen, Ben AdlerAbstract:Leptospirosis is a zoonotic disease affecting animals and humans worldwide. Leptospiral infection in cattle can cause reproductive failure and reduced weight gain, and importantly, infection represents a significant disease risk for farmers. Current bacterin vaccines offer protection that is short-lived and restricted at best to related serovars. The development of protective vaccines that stimulate immunity across multiple Leptospiral serovars would therefore be advantageous. This study used a reverse vaccinology approach to evaluate a set of Leptospira borgpetersenii proteins in the hamster infection model. The L. borgpetersenii serovar Hardjo strain L550 genome sequence was analysed and genes encoding 262 predicted outer membrane or secreted proteins were selected. From this list, 238 proteins or protein fragments were successfully expressed and purified; 28 proteins (12%) were soluble, while the remaining 210 proteins (88%) were insoluble and purified under denaturing conditions. Proteins were mixed into 48 pools of up to five each and tested for protection against infection as assessed by renal colonisation in the hamster model of infection. None of the pools of antigens protected the hamsters against infection, despite a detectable antibody response being mounted against the majority of proteins (71%). This study is the first large scale evaluation of individual Leptospiral proteins for ability to induce a protective immune response in the hamster infection model. It thus constitutes an important reference of protein immunogenicity and non-protective antigens that should be consulted before embarking on any future subunit vaccine experiments.
-
Genetic differences among the LPS biosynthetic loci of serovars of Leptospira interrogans and Leptospira borgpetersenii
FEMS immunology and medical microbiology, 2001Co-Authors: Alejandro M De La Pena-moctezuma, Dieter M. Bulach, Ben AdlerAbstract:The gene organization in the lipopolysaccharide biosynthetic (rfb) locus was analyzed in seven Leptospira interrogans serovars within serogroup Icterohemorrhagiae, seven non-Icterohemorrhagiae serovars and one Leptospira borgpetersenii serovar. Two groups of loci were delineated based on DNA hybridization and sequence analysis. Group 1 contained the two Hardjo subtypes, Hardjoprajitno and Hardjobovis. Group 2 (containing Copenhageni, Pomona, Naam, Mwogolo, Smithi, Lai, Canicola, Autumnalis, Pyrogenes, Australis and Icterohemorrhagiae) differed from Group 1 in its organization upstream of orf11, where five ORFs (32, 33, 34, 35, 37) were identified that were not contained in the Group 1 loci. These ORFs encoded a putative epimerase (orf32), a glycosyltransferase (orf33), two integral membrane proteins (orfs 34 and 35), and a galactosyltransferase (orf37). Serovars Australis, Pomona and Autumnalis did not contain orf37. Serovar Bataviae was excluded from the grouping because of its unique genetic organization upstream of orf13. In the Group 2 loci, comparison of the genetic layout at the 5′ end revealed differences which included mutations disrupting reading frames in either or both orf34 and orf35 and apparent allelic differences between orf33 homologs that may be sufficient to account for the genetic basis of serovar identity.
-
Functional analysis of genes in the rfb locus of Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis.
Infection and immunity, 2000Co-Authors: Dieter M. Bulach, Alejandro M De La Pena-moctezuma, Thareerat Kalambaheti, Ben AdlerAbstract:Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes for the biosynthesis and polymerization of nucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into loci; for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis, this locus, named rfb, spans 36.7 kb and contains 31 open reading frames, of which 28 have been assigned putative functions on the basis of sequence similarity. Characterization of the function of these genes is hindered by the fact that it is not possible to construct isogenic mutant strains in Leptospira. We used two approaches to circumvent this problem. The first was to clone the entire locus into a heterologous host system and determine if a “recombinant” LPS or polysaccharide was synthesized in the new host. The second approach used putative functions to identify mutants in other bacterial species whose mutations might be complemented by genes on the Leptospiral rfb locus. This approach was used to investigate the function of three genes in the Leptospiral rfb locus and demonstrated function for orfH10, which complemented a wbpM strain of Pseudomonas aeruginosa, and orfH13, which complemented an rfbW strain of Vibrio cholerae. However, despite the similarity of OrfH11 to WecC, a wecC strain of E. coli was not complemented by orfH11. The predicted protein encoded by orfH8 is similar to GalE from a number of organisms. A Salmonella enterica serovar Typhimurium strain producing no GalE was used as a background in which orfH8 produced detectable GalE enzyme activity.
-
comparative analysis of the lps biosynthetic loci of the genetic subtypes of serovar hardjo Leptospira interrogans subtype hardjoprajitno and Leptospira borgpetersenii subtype hardjobovis
Fems Microbiology Letters, 1999Co-Authors: Alejandro M De La Penamoctezuma, Thareerat Kalambaheti, Dieter M. Bulach, Ben AdlerAbstract:Although Leptospira borgpetersenii subtype Hardjobovis and L. interrogans subtype Hardjoprajitno belong to different species, they are serologically indistinguishable and are therefore classified as serovar Hardjo. Since LPS is the major antigen involved in serological classification, this implies that the LPS of these subtypes is identical. Comparison of the LPS biosynthetic loci (rfb) of the subtypes revealed remarkable similarity, with 32 and 31 origins of replication (orfs) in the Hardjoprajitno and Hardjobovis rfb loci, respectively. The order and orientation of these orfs were identical with the exception of an additional orf in Hardjoprajitno between orfs 4 and 5 and intergenic sequences differing between the subtypes. The Hardjoprajitno rfb locus has been divided into four intercalated regions based on sequence similarity to other Leptospiral rfb loci. orfJ1–orfJ14 as well as orfJ21–orfJ22 are more similar to regions of the rfb locus of L. borgpetersenii subtype Hardjobovis. orfJ15–orfJ20 as well as orfJ23–orfJ31 are almost identical to the corresponding orfs in L. interrogans serovar Copenhageni. We propose that the progenitor Hardjoprajitno strain, containing an rfb locus which closely resembled the Copenhageni locus, acquired orfs 1–14 and orfs 21–22 from subtype Hardjobovis resulting in two serologically indistinguishable subtypes of serovar Hardjo which in turn constituted the main bovine-adapted Leptospiral serovar.
David P. Alt - One of the best experts on this subject based on the ideXlab platform.
-
Bovine Immune Response to Vaccination and Infection with Leptospira borgpetersenii Serovar Hardjo.
mSphere, 2021Co-Authors: Jennifer H Wilson-welder, David P. Alt, Jarlath E Nally, Steven C. OlsenAbstract:This study examined the humoral and cellular response of cattle vaccinated with two commercial Leptospiral vaccines, Leptavoid and Spirovac, and a novel bacterin vaccine using Seppic Montanide oil emulsion adjuvant. Vaccination was followed by experimental challenge. All vaccinated cattle were protected from colonization of the kidney and shedding of Leptospira in urine, as detected by culture and immunofluorescence assay. Agglutinating antibody titers were detected in vaccinated cattle at 4 weeks following vaccination, with small anamnestic response detected following experimental challenge. Only animals vaccinated with the oil emulsion-adjuvanted bacterin produced significant IgG2 titers following vaccination, and nonvaccinated animals produced serum IgA titers after experimental challenge. CD4+ and γδ T cells from vaccinated cattle proliferated when cultured with antigen ex vivo Cellular responses included a marked proliferation of γδ T cells immediately following experimental challenge in vaccinated cattle and release of gamma interferon (IFN-γ), interleukin 17a (IL-17a), and IL-12p40 from stimulated cells. Proliferative and cytokine responses were found not just in peripheral mononuclear cells but also in lymphocytes isolated from renal lymph nodes at 10 weeks following experimental challenge. Overall, effects of leptospirosis vaccination and infection were subtle, resulting in only modest activation of CD4+ and γδ T cells. The use of Seppic Montanide oil emulsion adjuvants may shorten the initiation of response to vaccination, which could be useful during outbreaks or in areas where leptospirosis is endemic.IMPORTANCE Leptospirosis is an underdiagnosed, underreported zoonotic disease of which domestic livestock can be carriers. As a reservoir host for Leptospira borgpetersenii serovar Hardjo, cattle may present with reproductive issues, including abortion, birth of weak or infected calves, or failure to breed. Despite years of study and the availability of commercial vaccines, detailed analysis of the bovine immune response to vaccination and Leptospira challenge is lacking. This study evaluated immunologic responses to two efficacious commercial vaccines and a novel bacterin vaccine using an adjuvant chosen for enhanced cellular immune responses. Antigen-specific responsive CD4 and γδ T cells were detected following vaccination and were associated with release of inflammatory cytokines IFN-γ and IL-17a after stimulation. CD4 and γδ cells increased in the first week after infection and, combined with serum antibody, may play a role in clearance of bacteria from the blood and resident tissues. Additionally, these antigen-reactive T cells were found in the regional lymph nodes following infection, indicating that memory responses may not be circulating but are still present in regional lymph nodes. The information gained in this study expands knowledge of bovine immune response to leptospirosis vaccines and infection. The use of oil emulsion adjuvants may enhance early immune responses to Leptospiral bacterins, which could be useful in outbreaks or situations where leptospirosis is endemic.
-
Expansion of the in vitro assay for Leptospira potency testing to other serovars: case study with Leptospira Hardjo.
Biologicals : journal of the International Association of Biological Standardization, 2013Co-Authors: David P. Alt, Jennifer H Wilson-welderAbstract:Evaluation of Leptospiral vaccines for potency against Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Canicola, and Grippotyphosa is accomplished using the hamster potency test method described in 9 CFR 113.101-104. Applicability of this method to evaluation of bacterins developed for immunization against infection with L. interrogans serovar Hardjo or Leptospira borgpetersenii serovar Hardjo is complicated by several issues. Information from research on target host animal efficacy studies and evaluation of the immune response elicited using effective whole-cell bacterin formulations have revealed problems in relating these studies to either hamster-based or other potency testing methods. Future work on serovar Hardjo vaccines employing recombinant proteins will require preliminary testing methods in models other than the host animal. These models may also prove applicable to evaluation of potency for protein-based vaccines. Both an acute lethal infection model and a chronic infection model have been developed using two different strains of serovar Hardjo and will be described.
-
A Leptospira borgpetersenii Serovar Hardjo Vaccine Induces a Th1 Response, Activates NK Cells, and Reduces Renal Colonization
Clinical and vaccine immunology : CVI, 2011Co-Authors: Richard L Zuerner, David P. Alt, Mitchell V. Palmer, Tyler C. Thacker, Steven C. OlsenAbstract:Chronic infection of cattle with Leptospira borgpetersenii serovar Hardjo reduces animal production through reproductive failure and presents a persistent health threat to workers in the animal industry. Cattle are maintenance hosts for serovar Hardjo, and development of vaccines that establish long-term protective immunity has been problematic; induction of high titers of anti-serovar Hardjo antibody does not appear to be protective. Rather, development of an antigen-specific Th1 response appears to be critical for limiting renal colonization and urinary shedding of bacteria. In this study we compared two monovalent killed bacterial cell vaccines to assess long-term (12 months) protection against live serovar Hardjo challenge. Although neither vaccine prevented infection, renal colonization and urinary shedding of bacteria were reduced compared to those of control animals. Increased proliferation of CD4(+), CD8(+), and γδ T cells from vaccinated, but not control, animals was detected. In addition, NK cells from vaccinated animals and from all animals following infection, when exposed to antigen ex vivo, demonstrated a gamma interferon (IFN-γ) recall response. We propose that programming NK cells to respond quickly to L. borgpetersenii serovar Hardjo infection may be an important step toward developing protective immunity.
-
Genome reduction in Leptospira borgpetersenii reflects limited transmission potential.
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Dieter M. Bulach, Richard L Zuerner, Peter J. Wilson, Torsten Seemann, Annette Mcgrath, Paul Antony Cullen, John S. Davis, Matthew D. Johnson, Elizabeth Kuczek, David P. AltAbstract:Leptospirosis is one of the most common zoonotic diseases in the world, resulting in high morbidity and mortality in humans and affecting global livestock production. Most infections are caused by either Leptospira borgpetersenii or Leptospira interrogans, bacteria that vary in their distribution in nature and rely on different modes of transmission. We report the complete genomic sequences of two strains of L. borgpetersenii serovar Hardjo that have distinct phenotypes and virulence. These two strains have nearly identical genetic content, with subtle frameshift and point mutations being a common form of genetic variation. Starkly limited regions of synteny are shared between the large chromosomes of L. borgpetersenii and L. interrogans, probably the result of frequent recombination events between insertion sequences. The L. borgpetersenii genome is ≈700 kb smaller and has a lower coding density than L. interrogans, indicating it is decaying through a process of insertion sequence-mediated genome reduction. Loss of gene function is not random but is centered on impairment of environmental sensing and metabolite transport and utilization. These features distinguish L. borgpetersenii from L. interrogans, a species with minimal genetic decay and that survives extended passage in aquatic environments encountering a mammalian host. We conclude that L. borgpetersenii is evolving toward dependence on a strict host-to-host transmission cycle.
-
Evaluation of Type 1 Immune Response in Naïve and Vaccinated Animals following Challenge with Leptospira borgpetersenii Serovar Hardjo: Involvement of WC1+ γδ and CD4 T Cells
Infection and immunity, 2002Co-Authors: Brian M. Naiman, Richard L Zuerner, Carole A. Bolin, David P. Alt, Seth L. Blumerman, Rachel A. Brown, Cynthia L. BaldwinAbstract:The spirochete bacterium Leptospira spp. serovar Hardjo is a pathogen that causes disease in cattle and humans throughout the world. Infected cattle are the maintenance host for Leptospira borgpetersenii serovar Hardjo (subtype hardjobovis) (20) and Leptospira interrogans serovar Hardjo (subtype hardjo prajitno) and have a variety of clinical illnesses including abortion, infertility, and mastitis, while their calves may be stillborn, weak, or clinically normal but infected (see references 14-16, 21, 25, and 26). Infection is commonly transmitted by contact of urine or reproductive fluids from infected animals with the mucosal membranes of uninfected humans or animals, either directly or through fomites. Zoonotic infection of humans with leptospires including those of the serovar Hardjo group (1) poses a significant public health problem of increasing concern since leptospirosis in humans may be fatal due to involvement of multiple organs including liver, lungs, kidney, and brain (see reference 23). It was previously thought that protective immunity against leptospirosis was sufficiently provided by antibodies (20), since anti-Leptospiral lipopolysaccharide (LPS) antibodies have been shown elsewhere to provide passive immunity in some animal models, protecting against a number of strains and species of Leptospira (30, 36). However, Bolin et al. (4, 6) showed that high titers of anti-LPS antibody induced by conventional Leptospiral vaccines may not be protective against L. borgpetersenii serovar Hardjo. Moreover, recently developed vaccines that protect against L. borgpetersenii serovar Hardjo including renal colonization and urinary shedding (7, 41) and protect against transplacental infection of the fetus (D. Alt, R. Hornsby, and C. A. Bolin, submitted for publication) induce a type 1, or cell-mediated, immune response (18, 41). Cell-mediated or type 1 immunity is generally regarded as including production of gamma interferon (IFN-γ) and generation of cytotoxic CD8 T cells. While cytotoxic CD8 T cells and IFN-γ are both particularly important in control or clearance of infections with viruses and intracellular bacteria and protozoa, IFN-γ may also have a role in protection against extracellular microbes through its ability to activate macrophages and promote production of immunoglobulin G2 (IgG2) classes of antibodies, as has been suggested previously for immunity to extracellular stages of the protozoan parasite Babesia bigemina (9). While bovine IgG2 and IgG1 are both able to fix complement, which may be an important effector mechanism for control of leptospires in its own right, bovine IgG2 antibodies also act as opsonins (37), thereby potentially increasing the number of leptospires phagocytosed. Moreover, although leptospires are not considered to be intracellular pathogens that survive phagocytosis, IFN-γ activation of macrophages may increase the efficiency of killing. For example, Candida albicans, largely an extracellular organism that also targets the kidney, suppresses macrophage nitric oxide production through a soluble factor (10), and thus, IFN-γ is required for optimal production of nitric oxide by macrophages in vivo during a Candida infection (31). Because of the increasing incidence of infection in cattle and the zoonotic nature of human infections, it was of interest to examine the immune response of naive cattle following challenge with L. borgpetersenii serovar Hardjo during the periparturient period. This experimental design was chosen because of the effects that L. borgpetersenii serovar Hardjo infections have on events associated with pregnancy and transmission of the infection to the calves. Since natural infection is chronic in cattle, it was of interest to determine if it established itself due to the absence of a type 1 immune response or an insufficient one. While many bacterial infections induce type 1 immune responses, some are associated with induction of a type 2 response, such as that which occurs in patients with lepromatous leprosy (48), while other infections such as those with Brucella abortus may initially induce a type 1 response but after the first few weeks of infection show a hiatus of IFN-γ production (40). Here the cellular immune response of nonvaccinated cattle following challenge was compared with that of cattle that had been immunized prior to challenge with a vaccine protective against L. borgpetersenii serovar Hardjo infections (7) and which induces a type 1 cell-mediated immune response (41). Production of IFN-γ by peripheral blood mononuclear cells (PBMC) in response to antigen was measured by enzyme-linked immunosorbent assay (ELISA), and since any of the major T-cell populations including αβ CD4, αβ CD8, or γδ T cells may produce IFN-γ, two-color flow cytometry was used to determine the contribution by individual T-cell subpopulations. Generation of antigen-specific IgG1 and IgG2 antibodies in sera was also evaluated since the lack of IgG2 is correlated with a type 2 immune response.
Carole A. Bolin - One of the best experts on this subject based on the ideXlab platform.
-
Evaluation of Type 1 Immune Response in Naïve and Vaccinated Animals following Challenge with Leptospira borgpetersenii Serovar Hardjo: Involvement of WC1+ γδ and CD4 T Cells
Infection and immunity, 2002Co-Authors: Brian M. Naiman, Richard L Zuerner, Carole A. Bolin, David P. Alt, Seth L. Blumerman, Rachel A. Brown, Cynthia L. BaldwinAbstract:The spirochete bacterium Leptospira spp. serovar Hardjo is a pathogen that causes disease in cattle and humans throughout the world. Infected cattle are the maintenance host for Leptospira borgpetersenii serovar Hardjo (subtype hardjobovis) (20) and Leptospira interrogans serovar Hardjo (subtype hardjo prajitno) and have a variety of clinical illnesses including abortion, infertility, and mastitis, while their calves may be stillborn, weak, or clinically normal but infected (see references 14-16, 21, 25, and 26). Infection is commonly transmitted by contact of urine or reproductive fluids from infected animals with the mucosal membranes of uninfected humans or animals, either directly or through fomites. Zoonotic infection of humans with leptospires including those of the serovar Hardjo group (1) poses a significant public health problem of increasing concern since leptospirosis in humans may be fatal due to involvement of multiple organs including liver, lungs, kidney, and brain (see reference 23). It was previously thought that protective immunity against leptospirosis was sufficiently provided by antibodies (20), since anti-Leptospiral lipopolysaccharide (LPS) antibodies have been shown elsewhere to provide passive immunity in some animal models, protecting against a number of strains and species of Leptospira (30, 36). However, Bolin et al. (4, 6) showed that high titers of anti-LPS antibody induced by conventional Leptospiral vaccines may not be protective against L. borgpetersenii serovar Hardjo. Moreover, recently developed vaccines that protect against L. borgpetersenii serovar Hardjo including renal colonization and urinary shedding (7, 41) and protect against transplacental infection of the fetus (D. Alt, R. Hornsby, and C. A. Bolin, submitted for publication) induce a type 1, or cell-mediated, immune response (18, 41). Cell-mediated or type 1 immunity is generally regarded as including production of gamma interferon (IFN-γ) and generation of cytotoxic CD8 T cells. While cytotoxic CD8 T cells and IFN-γ are both particularly important in control or clearance of infections with viruses and intracellular bacteria and protozoa, IFN-γ may also have a role in protection against extracellular microbes through its ability to activate macrophages and promote production of immunoglobulin G2 (IgG2) classes of antibodies, as has been suggested previously for immunity to extracellular stages of the protozoan parasite Babesia bigemina (9). While bovine IgG2 and IgG1 are both able to fix complement, which may be an important effector mechanism for control of leptospires in its own right, bovine IgG2 antibodies also act as opsonins (37), thereby potentially increasing the number of leptospires phagocytosed. Moreover, although leptospires are not considered to be intracellular pathogens that survive phagocytosis, IFN-γ activation of macrophages may increase the efficiency of killing. For example, Candida albicans, largely an extracellular organism that also targets the kidney, suppresses macrophage nitric oxide production through a soluble factor (10), and thus, IFN-γ is required for optimal production of nitric oxide by macrophages in vivo during a Candida infection (31). Because of the increasing incidence of infection in cattle and the zoonotic nature of human infections, it was of interest to examine the immune response of naive cattle following challenge with L. borgpetersenii serovar Hardjo during the periparturient period. This experimental design was chosen because of the effects that L. borgpetersenii serovar Hardjo infections have on events associated with pregnancy and transmission of the infection to the calves. Since natural infection is chronic in cattle, it was of interest to determine if it established itself due to the absence of a type 1 immune response or an insufficient one. While many bacterial infections induce type 1 immune responses, some are associated with induction of a type 2 response, such as that which occurs in patients with lepromatous leprosy (48), while other infections such as those with Brucella abortus may initially induce a type 1 response but after the first few weeks of infection show a hiatus of IFN-γ production (40). Here the cellular immune response of nonvaccinated cattle following challenge was compared with that of cattle that had been immunized prior to challenge with a vaccine protective against L. borgpetersenii serovar Hardjo infections (7) and which induces a type 1 cell-mediated immune response (41). Production of IFN-γ by peripheral blood mononuclear cells (PBMC) in response to antigen was measured by enzyme-linked immunosorbent assay (ELISA), and since any of the major T-cell populations including αβ CD4, αβ CD8, or γδ T cells may produce IFN-γ, two-color flow cytometry was used to determine the contribution by individual T-cell subpopulations. Generation of antigen-specific IgG1 and IgG2 antibodies in sera was also evaluated since the lack of IgG2 is correlated with a type 2 immune response.
-
Protective killed Leptospira borgpetersenii vaccine induces potent Th1 immunity comprising responses by CD4 and γδ T lymphocytes
Infection and immunity, 2001Co-Authors: Brian M. Naiman, Richard L Zuerner, Carole A. Bolin, David P. Alt, Cynthia L. BaldwinAbstract:Leptospira borgpetersenii serovar hardjo is the most common cause of bovine leptospirosis and also causes zoonotic infections of humans. A protective killed vaccine against serovar hardjo was shown to induce strong antigen-specific proliferative responses by peripheral blood mononuclear cells (PBMC) from vaccinated cattle by 2 months after the first dose of vaccine. This response was absent from nonvaccinated control cattle. The mean response peaked by 2 months after completion of the two-dose vaccination regimen, and substantial proliferation was measured in in vitro cultures throughout the 7 months of the study period. Variations in magnitude of the response occurred among the vaccinated animals, but by 7 months postvaccination there was a substantial antigen-specific response with PBMC from all vaccinated animals. Up to one-third of the PBMC from vaccinated animals produced gamma interferon (IFN-gamma) after 7 days in culture with antigen, as ascertained by flow cytometric analysis, and significant levels of IFN-gamma were measured in culture supernatants by enzyme-linked immunosorbent assay. Two-color immunofluorescence revealed that one-third of the IFN-gamma-producing cells were gammadelta T cells, with the remaining cells being CD4(+) T cells. The significance of this study is the very potent Th1-type immune response induced and sustained following vaccination with a killed bacterial vaccine adjuvanted with aluminum hydroxide and the involvement of gammadelta T cells in the response. Moreover, induction of this Th1-type cellular immune response is associated with the protection afforded by the bovine Leptospiral vaccine against L. borgpetersenii serovar hardjo.
-
Evaluation of antibiotics for treatment of cattle infected with Leptospira borgpetersenii serovar hardjo.
Journal of the American Veterinary Medical Association, 2001Co-Authors: David P. Alt, Richard L Zuerner, Carole A. BolinAbstract:Objective—To evaluate antibiotics for treatment of cattle with leptospirosis caused by Leptospira borgpetersenii serovar hardjo. Design—Randomized controlled trial. Animals—42 healthy mixed-breed cattle. Procedure—Cattle were inoculated via conjunctival instillation with L borgpetersenii serovar hardjo. After infection and urinary shedding of L borgpetersenii were confirmed, cattle were treated with various antibiotics. To determine effectiveness of antibiotic treatment, urinary shedding of L borgpetersenii was monitored for 4 to 6 weeks after administration of antibiotics, using darkfield microscopic examination, microbial culture, immunofluorescence testing, and a polymerase chain reaction assay. Results—All inoculated cattle developed leptospirosis and shed leptospires in their urine. The following antibiotic treatments resulted in elimination of urinary shedding of leptospires: a single injection of oxytetracycline (20 mg/kg [9 mg/lb] of body weight, IM), tilmicosin (10 mg/kg [4.5 mg/lb], SC), or a co...
-
Use of a monovalent Leptospiral vaccine to prevent renal colonization and urinary shedding in cattle exposed to Leptospira borgpetersenii serovar hardjo.
American journal of veterinary research, 2001Co-Authors: Carole A. Bolin, David P. AltAbstract:Objective—To determine whether a monovalent Leptospira borgpetersenii serovar hardjo (type hardjobovis) vaccine commercially available in Australia, New Zealand, Ireland, and the United Kingdom would protect cattle from renal colonization and urinary shedding when exposed to a US strain of Leptospira borgpetersenii serovar hardjo. Animals—24 Hereford heifers that lacked detectable antibodies against serovar hardjo. Procedure—Heifers received 2 doses, 4 weeks apart, of the commercial hardjo vaccine (n = 8) or a monovalent US reference hardjo vaccine (8) or were not vaccinated (controls; 8). Heifers were challenged 16 weeks later by intraperitoneal inoculation or conjunctival instillation. Serum antibody titers were measured weekly, and urine samples were examined for leptospires. Heifers were euthanatized 11 to 14 weeks after challenge, and kidney tissue was examined for evidence of colonization. Results—All 8 heifers vaccinated with the reference vaccine were found to be shedding leptospires in their urin...
-
comparison of polymerase chain reaction assays with bacteriologic culture immunofluorescence and nucleic acid hybridization for detection of Leptospira borgpetersenii serovar hardjo in urine of cattle
American Journal of Veterinary Research, 2000Co-Authors: Jaap A Wagenaar, Richard L Zuerner, Carole A. BolinAbstract:OBJECTIVE: To compare sensitivity and specificity of various polymerase chain reaction (PCR) assays for detection of Leptospira borgpetersenii serovar hardjo in bovine urine and to compare results of the optimal PCR assay with results of immunofluorescence, nucleic acid hybridization, and bacteriologic culture. ANIMALS: 6 heifers. PROCEDURE: Heifers were exposed to serovar hardjo type hardjo-bovis by conjunctival instillation of 10(6) leptospires on 3 successive days. Urine samples were collected before and after infection. Sensitivity and specificity of 5 PCR assays were compared, to determine the optimal assay for use with bovine urine samples. The optimal PCR assay was then compared with results of bacteriologic culture, nucleic acid hybridization, and immunofluorescence. RESULTS: A PCR assay with the best combination of specificity (100%) and sensitivity (91%) was selected for comparison with the other diagnostic tests. Sensitivity for nucleic acid hybridization was 55%, whereas sensitivity for bacteriologic culture and immunofluorescence was 89 to 93%. CONCLUSIONS AND CLINICAL RELEVANCE: Bacteriologic culture, PCR, and immunofluorescence were sensitive for detection of L borgpetersenii serovar hardjo type hardjo-bovis in urine specimens of cattle, but a single technique was not the most sensitive for each animal tested. Therefore, the use of 2 techniques in combination is warranted for maximal sensitivity for diagnosis.
J. M. Montgomery - One of the best experts on this subject based on the ideXlab platform.
-
Development of an ELISA to detect antibodies to a protective lipopolysaccharide fraction of Leptospira borgpetersenii serovar hardjo in cattle.
Veterinary microbiology, 1999Co-Authors: K. T. Yan, S. W.j. Mcdowell, W A Ellis, M. J. Taylor, D.p. Mackie, J. M. MontgomeryAbstract:Abstract Monoclonal antibodies (Mabs) were produced against Leptospira borgpetersenii serovar hardjo -type Bovis antigens. A panel of 28 Mabs were characterised. Only the nine Mabs toward a lipopolysaccharide (LPS) fraction of 18, 24 kDa bands and a 26–28 kDa smear showed agglutinating, leptospiricidal and growth-inhibition activities, and passively protected hamsters against renal infection with hardjo . They also reacted strongly in the CH-ELISA, captured killed whole hardjo leptospires, gave good fluorescence in indirect FAT against smears of hardjo culture and exhibited no cross reactivity with strains in heterologous serogroups. On the basis of optimal activity in a range of tests, one IgG class Mab (designated 25) was selected for use in an antibody-capture ELISA system for the detection of bovine anti- hardjo antibodies. The system gave a wide separation of absorbance values between positive and negative sera at a 1 : 10 dilution. The antibodies detected by this assay are believed to be protective anti-LPS IgG.
-
Magnetic immuno capture PCR assay (MIPA): Detection of Leptospira borgpetersenii serovar hardjo
Veterinary Microbiology, 1997Co-Authors: M. J. Taylor, J. M. Montgomery, K. T. Yan, S. W.j. Mcdowell, W A Ellis, D.p. MackieAbstract:Magnetic immuno PCR assay (MIPA) was developed for the rapid detection of leptospires excreted in urine samples (n = 59) collected from 35 experimentally infected cattle. The immunomagnetic separation of leptospires from inhibitors in frozen formalin fixed bovine urine prior to PCR detection resulted in a marked improvement on previous detection methods. MIPA is a rapid 5 step protocol requiring 70 mins preparation time prior to amplification, which consistently detects 101 organisms. MIPA detected 76% (38/50) of culture positive urines and in addition three urines that were culture negative were shown to be positive by this method of detection. Consequently we conclude that whilst MIPA is an improvement on previously published PCR detection methods, the culture of the organism is still the standard against which other detection methods have to be compared.
-
Restriction fragment length polymorphisms distinguish Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates from different geographical locations.
Journal of clinical microbiology, 1993Co-Authors: R L Zuerner, Carole A. Bolin, W A Ellis, J. M. MontgomeryAbstract:Genetic variability among Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates representing several geographical regions was determined by restriction endonuclease analysis. Five previously unidentified EcoRI digestion patterns and one previously unidentified HhaI digestion pattern were seen with the various isolates. The copy number and genomic distribution of an L. borgpetersenii insertion sequence (IS1533) was determined. Hardjo-bovis isolate 033 (the type strain for hardjo-bovis) contained 40 well dispersed copies of IS1533. IS1533 probes were used to compare hardjo-bovis isolates by DNA blot hybridization analysis. Use of these probes showed the presence of additional genetic heterogeneity among hardjo-bovis isolates, which restriction endonuclease analysis did not show. Pulsed-field gel electrophoretic analysis of DNAs from several isolates suggested that some polymorphisms arose by genomic rearrangements. All hardjo-bovis isolates were categorized into 14 distinct groups on the basis of common hybridization and endonuclease digestion patterns. Most of these groups were isolated from distinct geographical regions, suggesting that several different clonal populations of hardjo-bovis exist. Images
Dieter M. Bulach - One of the best experts on this subject based on the ideXlab platform.
-
recombinant lipl32 stimulates interferon gamma production in cattle vaccinated with a monovalent Leptospira borgpetersenii serovar hardjo subtype hardjobovis vaccine
Veterinary Microbiology, 2014Co-Authors: Deanna Deveson Lucas, Dieter M. Bulach, Gerald L. Murray, Noelene Sheila Quinsey, Andrew G. Allen, Ben AdlerAbstract:Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjobovis) is the main causative agent of bovine leptospirosis in Australia, New Zealand, North America and elsewhere. Bovine leptospirosis can result in spontaneous abortion, stillbirth and reduced milk output. The organism is shed in the urine of infected animals and contact with contaminated materials can result in zoonotic infections in humans. Protective immunity in cattle against Hardjobovis involves stimulation of a Th1 cell mediated immune response, which can be characterized by the production of IFN-γ when blood from vaccinated animals is exposed to Hardjobovis antigens. However, the Leptospiral components involved in stimulating this response have yet to be identified. In this study, 238 recombinant Leptospiral proteins were evaluated for their ability to stimulate IFN-γ production in blood of cattle vaccinated with a commercial monovalent Hardjobovis vaccine. The conserved lipoprotein LipL32 is the major outer membrane protein of pathogenic Leptospira spp. A pool of soluble recombinant proteins which included LipL32, as well as LipL32 alone, stimulated significant IFN-γ production in blood of vaccinated cattle. A number of recombinant LipL32 fragments was generated, which identified the amino acids between 20 and 200 as containing the bovine T-cell reactive regions of LipL32. However, whether LipL32 plays a role in stimulating protective immunity in mammals has yet to be conclusively determined.
-
Evaluation of 238 antigens of Leptospira borgpetersenii serovar Hardjo for protection against kidney colonisation.
Vaccine, 2012Co-Authors: Gerald L. Murray, Dieter M. Bulach, Torsten Seemann, Amporn Srikram, Noelene Sheila Quinsey, Rasana W. Sermswan, Andrew G. Allen, Ben AdlerAbstract:Leptospirosis is a zoonotic disease affecting animals and humans worldwide. Leptospiral infection in cattle can cause reproductive failure and reduced weight gain, and importantly, infection represents a significant disease risk for farmers. Current bacterin vaccines offer protection that is short-lived and restricted at best to related serovars. The development of protective vaccines that stimulate immunity across multiple Leptospiral serovars would therefore be advantageous. This study used a reverse vaccinology approach to evaluate a set of Leptospira borgpetersenii proteins in the hamster infection model. The L. borgpetersenii serovar Hardjo strain L550 genome sequence was analysed and genes encoding 262 predicted outer membrane or secreted proteins were selected. From this list, 238 proteins or protein fragments were successfully expressed and purified; 28 proteins (12%) were soluble, while the remaining 210 proteins (88%) were insoluble and purified under denaturing conditions. Proteins were mixed into 48 pools of up to five each and tested for protection against infection as assessed by renal colonisation in the hamster model of infection. None of the pools of antigens protected the hamsters against infection, despite a detectable antibody response being mounted against the majority of proteins (71%). This study is the first large scale evaluation of individual Leptospiral proteins for ability to induce a protective immune response in the hamster infection model. It thus constitutes an important reference of protein immunogenicity and non-protective antigens that should be consulted before embarking on any future subunit vaccine experiments.
-
Genome reduction in Leptospira borgpetersenii reflects limited transmission potential.
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Dieter M. Bulach, Richard L Zuerner, Peter J. Wilson, Torsten Seemann, Annette Mcgrath, Paul Antony Cullen, John S. Davis, Matthew D. Johnson, Elizabeth Kuczek, David P. AltAbstract:Leptospirosis is one of the most common zoonotic diseases in the world, resulting in high morbidity and mortality in humans and affecting global livestock production. Most infections are caused by either Leptospira borgpetersenii or Leptospira interrogans, bacteria that vary in their distribution in nature and rely on different modes of transmission. We report the complete genomic sequences of two strains of L. borgpetersenii serovar Hardjo that have distinct phenotypes and virulence. These two strains have nearly identical genetic content, with subtle frameshift and point mutations being a common form of genetic variation. Starkly limited regions of synteny are shared between the large chromosomes of L. borgpetersenii and L. interrogans, probably the result of frequent recombination events between insertion sequences. The L. borgpetersenii genome is ≈700 kb smaller and has a lower coding density than L. interrogans, indicating it is decaying through a process of insertion sequence-mediated genome reduction. Loss of gene function is not random but is centered on impairment of environmental sensing and metabolite transport and utilization. These features distinguish L. borgpetersenii from L. interrogans, a species with minimal genetic decay and that survives extended passage in aquatic environments encountering a mammalian host. We conclude that L. borgpetersenii is evolving toward dependence on a strict host-to-host transmission cycle.
-
Genetic differences among the LPS biosynthetic loci of serovars of Leptospira interrogans and Leptospira borgpetersenii
FEMS immunology and medical microbiology, 2001Co-Authors: Alejandro M De La Pena-moctezuma, Dieter M. Bulach, Ben AdlerAbstract:The gene organization in the lipopolysaccharide biosynthetic (rfb) locus was analyzed in seven Leptospira interrogans serovars within serogroup Icterohemorrhagiae, seven non-Icterohemorrhagiae serovars and one Leptospira borgpetersenii serovar. Two groups of loci were delineated based on DNA hybridization and sequence analysis. Group 1 contained the two Hardjo subtypes, Hardjoprajitno and Hardjobovis. Group 2 (containing Copenhageni, Pomona, Naam, Mwogolo, Smithi, Lai, Canicola, Autumnalis, Pyrogenes, Australis and Icterohemorrhagiae) differed from Group 1 in its organization upstream of orf11, where five ORFs (32, 33, 34, 35, 37) were identified that were not contained in the Group 1 loci. These ORFs encoded a putative epimerase (orf32), a glycosyltransferase (orf33), two integral membrane proteins (orfs 34 and 35), and a galactosyltransferase (orf37). Serovars Australis, Pomona and Autumnalis did not contain orf37. Serovar Bataviae was excluded from the grouping because of its unique genetic organization upstream of orf13. In the Group 2 loci, comparison of the genetic layout at the 5′ end revealed differences which included mutations disrupting reading frames in either or both orf34 and orf35 and apparent allelic differences between orf33 homologs that may be sufficient to account for the genetic basis of serovar identity.
-
Functional analysis of genes in the rfb locus of Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis.
Infection and immunity, 2000Co-Authors: Dieter M. Bulach, Alejandro M De La Pena-moctezuma, Thareerat Kalambaheti, Ben AdlerAbstract:Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes for the biosynthesis and polymerization of nucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into loci; for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis, this locus, named rfb, spans 36.7 kb and contains 31 open reading frames, of which 28 have been assigned putative functions on the basis of sequence similarity. Characterization of the function of these genes is hindered by the fact that it is not possible to construct isogenic mutant strains in Leptospira. We used two approaches to circumvent this problem. The first was to clone the entire locus into a heterologous host system and determine if a “recombinant” LPS or polysaccharide was synthesized in the new host. The second approach used putative functions to identify mutants in other bacterial species whose mutations might be complemented by genes on the Leptospiral rfb locus. This approach was used to investigate the function of three genes in the Leptospiral rfb locus and demonstrated function for orfH10, which complemented a wbpM strain of Pseudomonas aeruginosa, and orfH13, which complemented an rfbW strain of Vibrio cholerae. However, despite the similarity of OrfH11 to WecC, a wecC strain of E. coli was not complemented by orfH11. The predicted protein encoded by orfH8 is similar to GalE from a number of organisms. A Salmonella enterica serovar Typhimurium strain producing no GalE was used as a background in which orfH8 produced detectable GalE enzyme activity.