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Brucella abortus strain RB51 is the commercial cattle vaccine used in the United States (US) and many parts of the world against bovine brucellosis. RB51 was licensed for use in 1996, and it has been shown to be safe and efficacious in cattle, eliciting humoral and cellular responses in calves and adult animals. In 2017, an epidemiological trace-back investigation performed by the Centers for Disease Control and Prevention (CDC) identified human cases of brucellosis caused by infection with RB51. These infections resulted from the consumption of unpasteurized dairy products, which were traced back to otherwise healthy animals that were shedding RB51 in their milk. At the current time, six adult Jersey cows have been identified in the U.S. that are shedding RB51 in milk. One of the RB51 shedding cattle was obtained and housed at the National Animal Disease Center (NADC) for further study. Improved understanding of host cellular and humoral immune responses to RB51 in persistently colonized cattle may be achieved by the characterization of responses in shedding animals. We hypothesized, based on the lack of RB51 clearance, that the RB51 shedder animal has a diminished adaptive cellular immune response to RB51. Our data demonstrate that in the presence of persistent RB51 infection, there is a lack of peripheral anti-RB51 CD4+ T cell responses and a concurrently high anti-RB51 IgG humoral response. By understanding the mechanisms that result in RB51 persistence, the development of improved interventions or vaccinations for brucellosis may be facilitated, which would provide public health benefits, including reducing the risks associated with the consumption of non-pasteurized milk products.
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Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.
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[This corrects the article DOI: 10.3389/fvets.2024.1334858.].
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Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.
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Introduction: Brucella abortus is the causative agent of brucellosis in cattle and in humans, resulting in economic losses in the agricultural sector and representing a major threat to public health. Elk populations in the American Northwest are reservoirs for this bacterium and transmit the agent to domestic cattle herds. One potential strategy to mitigate the transmission of brucellosis by elk is vaccination of elk populations against B. abortus; however, elk appear to be immunologically distinct from cattle in their responses to current vaccination strategies. The differences in host response to B. abortus between cattle and elk could be attributed to differences between the cattle and elk innate and adaptive immune responses. Because species-specific interactions between the host microbiome and the immune system are also known to affect immunity, we sought to investigate interactions between the elk microbiome and B. abortus infection and vaccination. Methods: We analyzed the fecal and vaginal microbial communities of B. abortus-vaccinated and unvaccinated elk which were challenged with B. abortus during the periparturient period. Results: We observed that the elk fecal and vaginal microbiota are similar to those of other ruminants, and these microbial communities were affected both by time of sampling and by vaccination status. Notably, we observed that taxa representing ruminant reproductive tract pathogens tended to increase in abundance in the elk vaginal microbiome following parturition. Furthermore, many of these taxa differed significantly in abundance depending on vaccination status, indicating that vaccination against B. abortus affects the elk vaginal microbiota with potential implications for animal reproductive health. Discussion: This study is the first to analyze the vaginal microbiota of any species of the genus Cervus and is also the first to assess the effects of B. abortus vaccination and challenge on the vaginal microbiome.
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Leptospirosis is a global zoonotic disease affecting humans, domestic, and wild animals. Leptospira are typically shed in the urine of reservoir hosts which persist in suitable environments where incidental host transmission occurs after direct contact with infected urine or contaminated environments. Interestingly, serologically identical L. borgpetersenii serovar Hardjo strains JB197 and HB203 show divergent disease severity in the hamster model; JB197 causes severe acute infection while HB203 causes persistent chronic infection. Historically, serovar Hardjo was limited to culture at 29 °C, but utilization of HAN media allows propagation from host tissues at 37 °C. Here, the proteome of strains JB197 and HB203 were characterized after culture from experimentally challenged hamsters at 29 °C and 37 °C. Comparative analyses of JB197 and HB203 samples cultured at 29 °C yielded 425 significantly differentially expressed (DE) proteins, while strains at 37 °C yielded 613 DE proteins including prominent outer membrane proteins and known virulence factors. In agreement, membrane protein GO terms were identified by STRING network analyses along with numerous metabolic KEGG pathways consistent with condition differences. Within strain, JB197 cultured at 29 °C vs 37 °C identified 529 DE proteins, while HB203 identified 524 DE proteins. Investigating differential protein profiles provide insights into strain specific behaviors with implications for better understanding host-pathogen interactions, disease transmission, and response to environmental conditions which can contribute to vaccine development, diagnostic improvement, and ultimately leptospirosis control. SIGNIFICANCE: Leptospirosis is a devastating zoonotic disease affecting humans, wild and domestic animals around the globe. Different species and serovars of Leptospira can affect various animal host species differently; for instance, a serovar that is asymptomatic in the rat may cause severe disease in a dog or human. These differences in host response are not only found at the species and serovar level for Leptospira, but also at the strain level. A prime example comes from strains JB197 and HB203, both species L. borgpetersenii, both serovar Hardjo. Interestingly, JB197 causes a severe acute infection in the hamster while HB203 causes an asymptomatic chronic infection. Understanding these unique relationships between pathogen and host species is important, especially in the context of prevention technologies such as vaccine design, where the strain of Leptospira used as a bacterin might have different efficiencies in different hosts. In this study, proteomic profiles of strains JB197 and HB203 were analyzed, and results revealed diverse protein expression profiles of outer membrane proteins, as well as proteins functioning in motility and growth.
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Doenças dos Bovinos , Leptospira , Leptospirose , Cricetinae , Animais , Humanos , Ratos , Cães , Bovinos , Sorogrupo , Infecção Persistente , Proteômica , Temperatura , Zoonoses , Proteínas de MembranaRESUMO
Genetic selection for milk production traits in US Holsteins has affected numerous genes associated with reproduction and immunity. This study compares the transcriptomic response of peripheral blood mononuclear cells to an in vitro Brucella abortus strain RB51 (RB51) bacterial challenge between contemporary Holsteins and Holsteins that have not been selected for milk production traits since the mid-1960s. Total RNA was extracted from peripheral blood mononuclear cells from four contemporary and four unselected lactating, primiparous cows following 24-h incubation with or without stimulation with RB51 bacteria. RNA was sequenced and reads analyzed using tools from galaxy.scinet.usda.gov. A total of 412 differentially expressed genes (false discovery rate p < 0.05, log fold change > |1|) were identified. The upregulated genes (genes with higher expression in contemporary than unselected cattle) were enriched for 19 terms/pathways, including alanine, aspartate, and glutamate metabolism, indicating a cellular stress response. Downregulated genes (genes with higher expression in unselected than contemporary cows) were enriched for 37 terms/pathways, representing diverse immune responses, including natural killer cell-mediated immunity, interferon-γ production, negative regulation of interleukin-10 production, and cytokine receptor activity indicating a broad immune response with an emphasis on immune defense. These results provide evidence that differences exist between the two genotypes in response to in vitro bacterial challenge. This suggests that contemporary cows, genetically selected for milk production, may have reduced immune function, including limitations in response to intracellular bacteria.
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Brucella abortus , Leucócitos Mononucleares , Feminino , Bovinos/genética , Animais , Brucella abortus/genética , Lactação , Genótipo , RNA , ImunidadeRESUMO
Bovine tuberculosis (bTB) is a zoonotic bacterial disease presenting public health, veterinary, and economic threats around the globe. Although cattle producers rely on regular testing and management practices to minimize domestic herd exposure, wildlife species around the world continue to be the main reservoirs for disease. Wildlife reservoirs for bTB include the Eurasian badger (Meles meles) in Great Britain and Ireland, the brushtail possum (Trichosurus vulpecula) in New Zealand, wild boar (Sus scrofa) in Spain, as well as white-tailed deer (Odocoileus virginianus) in the United States and red deer (Cervus elaphus) in Spain. Although all reservoir species share the ability to infect cattle, they differ in transmission capability, disease pathogenesis, diagnostic detection, and vaccination strategies. In this review, bTB interactions with these wildlife reservoirs are discussed, illustrating the need to address bTB disease in wildlife hosts to achieve eradication in domestic livestock.
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Cervos , Mycobacterium bovis , Tuberculose Bovina , Bovinos , Animais , Animais Selvagens , Cervos/microbiologia , Reservatórios de Doenças/microbiologia , Reservatórios de Doenças/veterináriaRESUMO
In many parts of the world, bovine tuberculosis eradication efforts are hampered by wildlife reservoirs of Mycobacterium bovis, which serve as a constant source of M. bovis for nearby cattle. The human tuberculosis vaccine, M. bovis BCG has been investigated for use in several wildlife species, including deer. In the US, white-tailed deer in Michigan have been the source of infection for over 82 cattle herds since M. bovis was discovered in free-ranging deer in 1995. The efficacy of BCG may be influenced by many factors, including prior exposure or infection with non-tuberculous mycobacteria, that is, species other than members of the M. tuberculosis complex. M. avium subspecies paratuberculosis (Map) infection is not uncommon in ruminants such as deer. Using natural exposure to Map and experimental infection with M. bovis, we demonstrate that Map infection increased BCG vaccine efficacy as measured by lesion severity scores.
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Leptospirosis is a global zoonotic bacterial disease which is a threat for humans and most mammals. Bacterin vaccines for leptospirosis are available however they are severely limited in cross protection between serogroups. Leptospira typically colonize the kidneys of reservoir hosts where they are subsequently shed in the urine and persist in the environment and can thus be indirectly or directly transmitted to incidental hosts. Leptospira borgpetersenii serovar Hardjo is the primary cause of leptospirosis in cattle which can result in abortion, unhealthy calves, and rebreed problems. This dataset comprises proteomic profiles of four strains of L. borgpetersenii serovar Hardjo propagated at the routinely utilized culture temperature of 29 °C, and a newly achieved culture temperature of 37 °C, which more closely emulates the temperature of an infected host. The strains analyzed include JB197 (established strain that causes Hardjo atypical acute disease in the hamster model of leptospirosis), HB203 (established strain, causes typical chronic disease in hamsters), as well as TC129 and TC273 (recently isolated strains from the central United States). Differential expression profiles were detected not only between strains but also within strains between culture temperatures. Mass spectrometry data are available via ProteomeXchange with identifier PXD032831.
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Leptospirosis is a global zoonotic disease affecting humans and livestock species. Bacterin vaccines lack cross protection between serogroups, and include multiple serovars propagated at 29 °C. Recent work demonstrated substantial variation in the transcriptome of identical species and serovars of Leptospira. Here, substantial differences in protein abundance profiles were identified in Leptospira borgpetersenii serovar Hardjo; strain HB203, which was isolated in the 1980s, compared to newer strains TC129 and TC273 isolated in 2016, and whether they were propagated at the routine temperature of 29 °C, compared to 37 °C which more closely emulates host infection. While 388 and 385 significantly differentially expressed (DE) proteins (FDR of 0.01) were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 29 °C respectively, only 66 and 4 DE proteins were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 37 °C respectively. Within each strain comparing temperatures, HB203 had 524 significantly DE proteins, TC129 had 347 DE proteins, and TC273 had 569 DE proteins. Data are available via ProteomeXchange with identifier PXD032831. Results highlight significant differential protein expression among identical serovars of L. borgpetersenii suggesting that bacterin vaccine design can benefit from consideration of strains employed and effects of temperature on growth. SIGNIFICANCE: Leptospirosis is a zoonotic disease caused by spirochete bacteria of the genus Leptospira. While leptospirosis affects over one million people per year, symptoms range vastly in severity from completely asymptomatic, to flu-like, to multi-organ failure and death in severe cases. Incidental hosts become infected after encountering pathogens directly from contact with another host, including domestic or wildlife animals, or indirectly from contaminated environments. Though animal vaccines exist, they lack cross protection across serogroups, and instead rely on inclusion of multiple carefully selected serovars from laboratory strains prepared at ~29 °C. Recent interest in gene expression at the Leptospira strain level, along with a newly achieved culture temperature of 37 °C (which more closely resembles host body temperature), led us to investigate the proteomic profiles of an older, established challenge strain HB203 in comparison to TC129 and TC273, two strains isolated in 2016 from abattoir cattle in the central United States. Herein, we identify substantial proteomic differences not only between strains of the same species and serovar, but notably between growth temperatures, collectively suggesting that bacterin vaccine composition may benefit from investigating strain selection and the temperature employed for growth of the bacteria used in bacterin preparation.
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Doenças dos Bovinos , Leptospira , Leptospirose , Animais , Vacinas Bacterianas , Bovinos , Humanos , Proteoma/genética , Proteômica , Sorogrupo , Temperatura , ZoonosesRESUMO
Selective breeding of US dairy cows since the mid-1960s has contributed to remarkable gains in milk yield per cow. This increased milk yield has been associated with an increase in health issues. Since 1964, the University of Minnesota has selectively bred a Holstein herd to maintain genetically static, unselected Holsteins (UH). Comparison of these UH cows with contemporary Holsteins (CH) has demonstrated that the UH cows not only produce less milk but also have fewer health concerns than their CH herdmates. The objective of this study was to determine the effects of Holstein genotype on innate immune response in an experimental intramammary Escherichia coli challenge model. Primiparous UH (n = 5) and CH (n = 7) cows received 430 cfu of E. coli strain P4 in 1 quarter. Blood and affected quarter milk samples were collected at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 7, 9, and 11 d relative to E. coli infusion. Rectal temperatures were recorded at each milking through d 4 of the experiment. Milk bacterial counts, somatic cell count and BSA concentrations, complete blood cell counts, rectal temperature, and serum and milk whey cytokine (IL-1ß and IL-6) concentrations were used as metrics to determine infection severity. Longitudinal (repeated) data were analyzed using general linear models with PROC MIXED with day of study as the repeated effect. Whole blood transcriptomes were generated by RNA sequencing. Transcripts with a false discovery rate of P < 0.05 and a delta log2 expression value greater than 0.7 or less than -0.7 were used for functional enrichment analysis. Bacterial counts were consistently greater in milk from CH than UH cows from d 0.25 through d 2.5. Milk somatic cell count increased within 6 h (d 0.25) after E. coli administration in CH and UH cows but did not differ between genotypes after d 1. Rectal body temperature peaked at d 1 in CH and UH cows but was greater in CH cows. Milk BSA, IL-1ß, and IL-6 concentrations were greater in CH than UH cows after E. coli administration. Blood lymphocyte and neutrophil counts were decreased at 0.5 and 1 d in CH but not in UH cows. The number of differentially expressed transcripts at each of the postinfusion sampling times was consistently greater (4- to 90-fold) in CH than in UH cows. A key difference between the immune reaction of the 2 genotypes was that the immune response to E. coli was largely contained within the mammary gland of the UH cows but became more systemic in the CH cows. These data demonstrate that UH cows exerted more effective control of E. coli infused into the mammary gland and thus support the hypothesis that selection practices since the mid-1960s have resulted in CH cows with an immune system that is less effective in fighting intramammary infections. Identification of genetic factors associated with enhanced immune functions that differ between the UH and CH cows could contribute to efforts to reintroduce or enhance beneficial components that have been lost or reduced in the CH cows since the mid-1960s.
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Doenças dos Bovinos , Infecções por Escherichia coli , Mastite Bovina , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Escherichia coli , Infecções por Escherichia coli/veterinária , Feminino , Genótipo , Imunidade Inata/genética , Interleucina-6/metabolismo , Glândulas Mamárias Animais/metabolismo , Mastite Bovina/microbiologia , Leite/metabolismoRESUMO
Leptospirosis is a global zoonotic disease that causes significant morbidity and mortality in human and animal populations. Leptospira interrogans is a leading cause of human disease, and L. borgpetersenii is a leading cause of animal disease. Cattle are reservoir hosts of L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges resulting in abortion and poor reproductive performance. Bovine bacterin vaccines can only protect against those serovars included in vaccine formulations and typically include serovar Hardjo among others. Genotyping and serotyping represent two different and unique methods for classifying leptospires that do not always correlate well; comprehensive characterization using either method requires recovery of isolates from infected animals. In this study, we report for the first time, isolation of L. borgpetersenii serovar Tarassovi from the urine of a dairy cow in the U.S. The classification of the isolate, designated strain MN900, was confirmed by whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, Matrix Assisted Laser Desorption/Ionization (MALDI), and immunoblotting with reference antisera. Strain MN900 was excreted in urine samples for 18 weeks even as the cow was seronegative for serovar Tarassovi. Strain MN900 has an unusual morphology since it is not as motile as other leptospires and lacks hooked ends. Serovar Tarassovi is not included in U.S. bacterin vaccines. These results demonstrate the importance of culture and concomitant genotyping and serotyping to accurately classify leptospires, and as required to design efficacious vaccine and diagnostic strategies to not only limit animal disease but reduce zoonotic risk.
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Pathogenic species of Leptospira cause leptospirosis, a global zoonotic disease affecting humans and all major livestock species. Cattle act as a reservoir host for L. borgpetersenii serovar Hardjo which colonize the kidneys and reproductive tract from which they are excreted and transmitted to other cattle via urine, semen or uterine discharges. Bovine leptospirosis results in reproductive failure, abortion, stillbirth and loss of milk production, and is an occupational risk for those working with infected animals. A recent study determined that 7.2% of cattle from an abattoir in the central United States were actively shedding pathogenic Leptospira. Here, we report and compare the complete genome sequence of four recent isolates of L. borgpetersenii serovar Hardjo designated strain TC112, TC147, TC129, and TC273.
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Leptospirosis is a world-wide zoonotic disease caused by pathogenic Leptospira and can be asymptomatic or can cause clinical signs ranging from influenza-like to multi-organ failure and death in severe cases. While species and strain specificity can play a major role in disease presentation, the hamster is susceptible to most leptospiral infections and is the model of choice for vaccine efficacy testing. During evaluation of blood smears from hamsters challenged with different species and strains of Leptospira, a circulating population of large, mononuclear, lipid-filled cells, most similar to foamy macrophages (FMs), was detected. Circulating FMs were identified by Giemsa staining and verified by scanning and transmission electron microscopy. FMs were found in the circulating blood of all Leptospira-challenged hamsters, indicating that the finding was not species or strain specific, although higher numbers of FMs tended to correlate with severity of disease. The unique finding of circulating FMs in the hamster model of leptospirosis can yield additional insights into the pathogenesis of leptospirosis and other diseases that induce circulating FMs.
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Leptospirose , Doenças dos Roedores , Animais , Cricetinae , Modelos Animais de Doenças , Leptospirose/veterinária , Macrófagos , Mesocricetus , Eficácia de VacinasRESUMO
BACKGROUND: Leptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C). METHODOLOGY/PRINCIPAL FINDINGS: L. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent differential expression of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203. CONCLUSIONS/SIGNIFICANCE: Investigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets.
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Leptospira/genética , Sorogrupo , Temperatura , Transcriptoma , Animais , Cricetinae , Rim/microbiologia , Leptospira/isolamento & purificação , Leptospirose/microbiologiaRESUMO
Leptospirosis is a worldwide zoonosis caused by pathogenic species of the genus Leptospira. The recent application of CRISPR interference (CRISPRi) to Leptospira facilitates targeted gene silencing and provides a new tool to investigate pathogenic mechanisms of leptospirosis. CRISPRi relies on the expression of a catalytically "dead" Cas9 (dCas9) and a single-guide RNA (sgRNA). Previously, our group generated a LipL32 and a double LigA/LigB (LigAB) mutant, which, in the current study, are characterized by whole-cell proteomics in comparison with control leptospires harboring plasmid expressing dCas9 alone. Comparison of control and LigAB mutant leptospires identified 46 significantly differentially expressed (DE) proteins, including 27 proteins that were less abundant and 19 proteins that were more abundant in the LigAB mutant compared with the control. Comparison of the control and LipL32 mutant leptospires identified 243 DE proteins, of which 84 proteins were more abundant and 159 were less abundant in the LipL32 mutant strain. Significantly increased amounts of known virulence impactors and surface membrane receptors, including LipL45, LipL31, LigB, and LipL41, were identified. The virulence of LipL32 and LigAB mutants were evaluated in the hamster model of leptospirosis; the LigAB mutant was unable to cause acute disease although mutant leptospires could still be recovered from target organs, albeit at a significantly lower bacterial burden (<850 and <16-fold in liver and kidney, respectively, in comparison with control), indicating attenuation of virulence and a shift to chronic bacterial persistence. Notably, the LipL32 mutant displayed augmented virulence as evidenced by early onset of clinical symptoms and increased numbers of circulating foamy macrophages. Validation of LipL32 and LigAB mutants recovered from liver and kidney in the presence or absence of antibiotic selection revealed high plasmid stability and, by extension, gene silencing in vivo. Collectively, this work emphasizes the advantages and feasibility of using CRISPRi technology to evaluate and characterize virulence factors of leptospires and their respective host-pathogen interactions in animal models of leptospirosis. Importantly, it also provides insight into the requirements of LigA and LigB for acute disease and explores the impact of silencing expression of lipL32, which resulted in substantial changes in amounts of outer membrane proteins.
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Leptospirosis is a devastating zoonotic disease affecting people and animals across the globe. Pathogenic leptospires are excreted in urine of reservoir hosts which directly or indirectly leads to continued disease transmission, via contact with mucous membranes or a breach of the skin barrier of another host. Human fatalities approach 60,000 deaths per annum; though most vertebrates are susceptible to leptospirosis, complex interactions between host species and serovars of Leptospira can yield disease phenotypes that vary from asymptomatic shedding in reservoir hosts, to multi-organ failure in incidental hosts. Clinical symptoms of acute leptospirosis reflect the diverse range of pathogenic species and serovars that cause infection, the level of exposure, and the relationship of the pathogen with the given host. However, in all cases, pathogenic Leptospira are excreted into the environment via urine from reservoir hosts which are uniformly recognized as asymptomatic carriers. Therefore, the reservoir host serves as the cornerstone of persistent disease transmission. Although bacterin vaccines can be used to abate renal carriage and excretion in domestic animal species, there is an urgent need to advance our understanding of immune-mediated host-pathogen interactions that facilitate persistent asymptomatic carriage. This review summarizes the current understanding of host-pathogen interactions in the reservoir host and prioritizes research to unravel mechanisms that allow for colonization but not destruction of the host. This information is required to understand, and ultimately control, the transmission of pathogenic Leptospira.
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BACKGROUND: Mastitis is the most common health concern plaguing the modern dairy cow and costs dairy producers estimates of two billion dollars annually. Staphylococcus aureus infections are prevalent, displaying varied disease presentation and markedly low cure rates. Neutrophils are considered the first line of defense against mastitis causing bacteria and are frequently targeted in the development of treatment and prevention technologies. We describe a case of naturally occurring, chronic mastitis in a Holstein cow (1428), caused by a novel strain of S. aureus that was not able to be cleared by antibiotic treatment. CASE PRESENTATION: The infection was identified in a single quarter, 2 months into the cow's first lactation. The infection persisted for the following 20 months, including through dry off, and a second calving and lactation. This case of mastitis was associated with a consistently high somatic cell count, however presented with no other clinical signs. This cow was unsuccessfully treated with antibiotics commonly used to treat mastitis, consisting of two rounds of treatment during lactation and an additional round at the beginning of dry off. The chronic infection was also unchanged through an experimental mid-lactation treatment with pegylated granulocyte-colony stimulating factor (PEG-gCSF) and an additional periparturient treatment with PEG-gCSF. We isolated milk neutrophils from 1428 and compared them to two cows challenged with experimental S. aureus, strain Newbould 305. Neutrophils from 1428's milk had higher surface expression of myeloperoxidase compared to experimental Newbould challenged animals, as well as increased presence of Neutrophil Extracellular Traps. This suggests a heightened activation state of neutrophils sourced from 1428's naturally occurring infection. Upon postmortem examination, the affected quarter revealed multifocal abscesses separated by fibrous connective tissues. Abscesses were most common in the gland cistern and collecting duct region. Microscopically, the inflammatory reaction was pyogranulomatous to granulomatous and consistent with botryomycosis. Colonies of Gram-positive cocci were found within the eosinophilic matrix of the Splendore-Hoeppli reaction within granulomas and intracellularly within the acinar epithelium. CONCLUSIONS: Collectively, we describe a unique case of chronic mastitis, the characterization of which provides valuable insight into the mechanics of S. aureus treatment resistance and immune escape.
Assuntos
Mastite Bovina/microbiologia , Neutrófilos/enzimologia , Infecções Estafilocócicas/veterinária , Abscesso/microbiologia , Abscesso/veterinária , Animais , Antibacterianos/uso terapêutico , Bovinos , Doença Crônica/veterinária , Feminino , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Mastite Bovina/tratamento farmacológico , Leite/citologia , Peroxidase/metabolismo , Polietilenoglicóis/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacosRESUMO
Cattle are susceptible to infection with multiple serovars of pathogenic leptospires, resulting in abortion, stillbirth, premature birth, reproductive failure and milk drop syndrome. Cattle also act as a reservoir host for L. borgpetersenii serovar Hardjo which is excreted from renal tubules via urine into the environment where it persists in suitable moist conditions. Our previous work demonstrated that 7% of urine samples from beef cattle were positive for L. borgpetersenii serovar Hardjo by culture and/or the fluorescent antibody test (FAT). In this study, a real-time PCR (rtPCR) assay was applied to determine the relative performance of rtPCR based detection of L. borgpetersenii serovar Hardjo compared to previously reported culture and FAT techniques. Of 42 bovine urine samples positive for leptospires by culture and/or FAT, 60% (25/42) were positive by rtPCR. Of 22 culture-positive samples, 91% (20/22) were rtPCR-positive. Of 32 FAT-positive samples, 50% (16/32) were rtPCR-positive. For 10 samples that were culture-positive but FAT-negative, 90% (9/10) were rtPCR-positive. For 20 samples that were FAT-positive but culture-negative, 25% (5/20) were rtPCR-positive. Collectively, these results indicate that no single assay is optimal, and the use of more than one assay to detect leptospires in urine from naturally infected cattle is recommended.