Biology subjects

Rajeev, S.

Publications and source records attributed to Rajeev, S..

8 recordsLinked to original sources

bioRxiv2026

Species Identification and Antimicrobial Susceptibility of Mycobacterial Isolates from Dogs and Cats

Nontuberculous Mycobacterium (NTM) species are increasingly recognized as opportunistic pathogens in companion animals, yet data on species distribution and antimicrobial resistance remain limited. The goal of this study was to evaluate species identification methods and determine antimicrobial susceptibility profiles of NTM isolates from cats and dogs submitted to the University of Tennessee veterinary diagnostic laboratory. Twenty-nine NTM isolates (25 feline, 4 canine) collected between 2016 and 2022 were cultured and identified using MALDI-TOF MS and 16S rRNA sequencing. Antimicrobial susceptibility testing (AST) was performed by broth microdilution. Most isolates (27/29) were rapidly growing mycobacteria from skin or wound samples. Combined MALDI-TOF and sequencing identified 11 isolates within the Mycobacterium fortuitum complex, 10 as M. smegmatis, 4 within the M. abscessus group, and one each as M. mucogenicum and M. farcinogenes. MALDI-TOF failed to identify four isolates, including two slow-growing strains. AST revealed consistent susceptibility to amikacin and moxifloxacin, while resistance was highest to doxycycline (44%), clarithromycin (37%), and tobramycin (33%). Rapidly growing NTM predominates in feline cutaneous infections, with species-specific resistance patterns underscoring the need for accurate identification and AST to guide therapy. Importance StatementNontuberculous mycobacteria (NTM) are emerging causes of skin infections in cats and dogs, yet little is known about which species are involved or how they respond to treatment. Knowing the specific species and their resistance patterns is critical for choosing effective treatments and improving outcomes for affected animals. This study provides critical baseline data by identifying NTM species and their antimicrobial resistance patterns in companion animals. These findings highlight the need for accurate diagnostic methods and targeted therapy to improve clinical outcomes. Understanding these trends is essential for guiding veterinary care and informing future research on antimicrobial resistance in animal health.

microbiology↗

bioRxiv2024

Leptospira seroprevalence in dogs, cats and horses: the effect of cross-reactivity between serovars and vaccination status on seroprevalence estimates

In this study, we estimated the Leptospira seroprevalence in dogs (n = 376), horses (n = 88), and cats (n = 169) using Microscopic Agglutination Test (MAT) against 12 Leptospira serovars. We observed a Leptospira seroprevalence of 29.41%, 47.73%, and 12.35% in dogs, horses, and cats, respectively. The highest seroprevalence was observed for serovar Autumnalis (74.55%) in dogs, and Bratislava in horses (95.24%) and cats (42.86%). We found a significant level of potential cross-reactivity between multiple Leptospira serovars tested, with highest cross-reactivity to serovar Autumnalis in dogs. The dogs that were vaccinated against Leptospira had a significantly higher seroprevalence (45.92%) compared to unvaccinated dogs (16.28%; p < 0.001). A significant difference in seroprevalence was observed in vaccinated and unvaccinated dogs to all four serovars included in the canine vaccine (p < 0.001). Among the diagnostic samples submitted from 2021-2023, 40% (103/252) of the canine serum samples were positive by MAT, with the highest positivity rate for the serovar Autumnalis. On Leptospira PCR, 10.7 % (35/325) urine samples and 5.8% (15/257) blood samples were positive. Our study findings show evidence of Leptospira exposure and potential disease in the study area. The cross-reactivity between the Leptospira serovars used in the MAT and vaccination status may also overestimate the levels of exposure.

immunology↗

bioRxiv2024

Enhancement of clinical signs in C3H/HeJ mice vaccinated with a highly immunogenic Leptospira methyl-accepting chemotaxis protein following challenge

Leptospirosis is the most widespread zoonosis and a life-threating disease of humans and animals. Licensed killed whole-cell vaccines are available for animals; however, they do not offer heterologous protection, do not induce a long-term protection, or prevent renal colonization. In this study, we characterized an immunogenic Leptospira methyl-accepting chemotaxis protein (MCP) identified through a reverse vaccinology approach, predicted its structure, and tested the protective efficacy of a recombinant MCP fragment in the C3H/HeJ mice model. The predicted structure of the full-length MCP revealed an architecture typical for topology class I MCPs. A single dose of MCP vaccine elicited a significant IgG antibody response in immunized mice compared to controls (P < 0.0001), especially the IgG1 and IgG2a subclasses. The vaccination with MCP despite eliciting a robust immune response, did not protect mice from disease and renal colonization. However, survival curves were significantly different between groups, and the MCP vaccinated group developed clinical signs faster than the control group. There were differences in gross and histopathological changes between the MCP vaccinated and control groups. The factors leading to enhanced disease process in vaccinated animals needs further investigation. We speculate that anti-MCP antibodies may block the MCP signaling cascade and may limit chemotaxis, preventing Leptospira from reaching its destination, but facilitating its maintenance and replication in the blood stream. Such a phenomenon may exist in endemic areas where humans are highly exposed to Leptospira antigens, and the presence of antibodies might lead to disease enhancement. The role of this protein in Leptospira pathogenesis should be further evaluated to comprehend the lack of protection and potential exacerbation of the disease process. The absence of immune correlates of protection from Leptospira infection is still a major limitation of this field and efforts to gather this knowledge is needed. Author summaryLeptospirosis is one of the underrecognized and neglected diseases of humans and animals. The presence of numerous Leptospira species/serovars infecting a broad range of animal reservoirs, and the resulting environmental contamination, makes control and prevention a cumbersome task. The bacterin-based vaccines available for animals do not offer protection against disease or renal colonization. A broader cross-protective vaccine is essentially needed to prevent Leptospira infections in humans and animals. Here we rationally selected a protein target based on its capacity to be recognized by antibodies of naturally infected animals and designed a recombinant vaccine. Our MCP vaccine was not effective in protecting mice from acute and chronic disease, and likely led to exacerbation of clinical signs in these animals. The development of an effective vaccine would contribute to control Leptospira infection in humans and animals and is important especially in low-income regions where leptospirosis is more prevalent and interventions to control the disease are not currently available.

microbiology↗

bioRxiv2024

Multisystemic inflammatory disease in Pheasantshell (Unionidae, Actinonaias pectorosa) associated with Yokenella regensburgei infection at sites experiencing seasonal mass mortality events

Freshwater mussels are integral components of riverine ecosystems, influencing water quality, nutrient cycling, and habitat characteristics. Enigmatic freshwater mussel declines, often characterized by sudden mass mortality events, pose significant challenges to conservation efforts. The Clinch River, a freshwater biodiversity hotspot, has experienced several enigmatic mass mortality events since 2016. Studies have reported bacteria associated with moribund Pheasantshell (Actinonaias pectorosa) during mortality events in the Clinch River, specifically Yokenella regensburgei. Despite reports of bacterial infection, little is known about their role as pathogens. Through a multiyear case-control study, combining in-situ experiments, field surveys, histology, bacterial isolation, and high-throughput sequencing, we assessed the role of bacteria in Pheasantshell (Actinonais pectorosa) mortality at two sites in the Clinch River. Between May 2021 and December 2023, we collected 29 wild moribund free-living A. pectorosa and 68 hatchery-reared A. pectorosa maintained in silos at the same sites and investigated differences in pathology and microbiology between groups. No silo mussels presented clinical signs of disease, or gross or microscopic lesions associated with pathological conditions leading to mortality. Our findings reveal a significant association between Yokenella regensburgei and severe multisystemic and multifocal infiltrative hemocytosis with necrosis, consistent with sepsis. Lesions associated with yokenellosis were of sufficient severity and physiological significance to explain mortality in infected hosts. Although our study does not explain the cause of these infections, it confirms that mussels at our study sites are ultimately dying from an infectious disease and that Y. regensburgei can be pathogenic in free-living mussels. Our results underscore the importance of considering bacterial diseases in wild mussel populations and emphasize the need for further research to elucidate the epidemiology and pathogenicity of Y. regensburgei. Overall, our study highlights the importance of integrated approaches combining pathology, microbiology, and epidemiology in freshwater mussel conservation efforts.

ecology↗

bioRxiv2023

Caenorhabditis elegans germ granules are present in distinct configurations that differentially associate with RNAi-targeted RNAs

RNA silencing pathways are complex, highly conserved, and perform widespread, critical regulatory roles. In C. elegans germlines, RNA surveillance occurs through a series of perinuclear germ granule compartments--P granules, Z granules, SIMR foci, and Mutator foci--multiple of which form via phase separation and exhibit liquid-like properties. The functions of individual proteins within germ granules are well-studied, but the spatial organization, physical interaction, and coordination of biomolecule exchange between compartments within germ granule "nuage" is less understood. Here we find that key proteins are sufficient for compartment separation, and that the boundary between compartments can be reestablished after perturbation. Using super-resolution microscopy, we discover a toroidal P granule morphology which encircles the other germ granule compartments in a consistent exterior-to-interior spatial organization. Combined with findings that nuclear pores primarily interact with P granules, this nuage compa rtment organization has broad implications for the trajectory of an RNA as it exits the nucleus and enters small RNA pathway compartments. Furthermore, we quantify the stoichiometric relationships between germ granule compartments and RNA to reveal discrete populations of nuage that differentially associate with RNAi-targeted transcripts, possibly suggesting functional differences between nuage configurations. Together, our work creates a more spatially and compositionally accurate model of C. elegans nuage which informs the conceptualization of RNA silencing through different germ granule compartments.

cell biology↗

bioRxiv2023

Leptospira transcriptome sequencing using long-read technology reveals unannotated transcripts and potential polyadenylation of mRNA molecules

Leptospirosis caused by the spirochete bacteria Leptospira is an emerging zoonosis that causes life-threatening disease in humans and animals. However, a detailed understanding of Leptospiras RNA profiles is limited. In this study, we sequenced and analyzed the transcriptome of multiple Leptospira serovars using Oxford Nanopore Technologies direct cDNA and direct RNA sequencing methods. We identified several operon transcriptional units, novel RNA coding regions, and evidence of potential posttranscriptional polyadenylation in the Leptospira transcriptome. Some coding regions of multiple RNA molecules that have not been previously annotated could be potential sRNA or ncRNA molecules that support gene expression regulation purposes in Leptospira. Many of the relative positions of these unannotated RNA coding regions were consistent in their neighboring coding regions across the reference genomes of two pathogenic Leptospira analyzed in this study. Besides, the majority of the unannotated coding regions and operon transcriptional units were not detected in the nonpathogenic Leptospira, suggesting potential virulence-related functions for these RNA molecules coding regions. Overall, our study confirms the utility of ONTs sequencing in studying prokaryotic transcriptome profiles and offers a tool to improve our understanding of the structural composition of RNA molecules and prokaryotic polyadenylation. However, the findings from our study also warrant that the presence of homopolymers of adenine bases in the transcripts may interfere with the interpretation of bacterial transcriptome profiles. Carefully designed experiments are needed to unravel the role of the features described in this study in Leptospira virulence and pathogenesis. Author SummaryLeptospirosis caused by the spirochete bacteria, Leptospira, is recognized as one of the most widespread zoonotic diseases. Leptospirosis is a neglected disease and has been estimated to cause over one million annual human clinical cases and over 60,000 deaths. Leptospira are maintained in the renal tubules of asymptomatic animal reservoirs, and in contaminated environments, such as soil and water. Studying gene expression profiles is an important component of bacterial pathogenesis studies, and a number of methods are available to do it. Our study compared various transcriptome sequencing methods using one of the latest next generation sequencing tools (Oxford Nanopore Technologies) and evaluated the compositions of RNA in different Leptospiras transcriptomes. We identified many previously undescribed and potentially significant features within the Leptospira transcriptome that may contribute to the virulence and pathogenesis of Leptospira sp. Our findings lead to new opportunities for researchers to sequence prokaryotic RNA molecules and to unravel many regulatory mechanisms.

microbiology↗

bioRxiv2022

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

BackgroundLeptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are the major transmission sources in humans and animals. Clusters of disease outbreaks are common during rainy seasons. Methodology/Principal FindingsIn this study, to detect the presence of Leptospira, we applied PCR, direct metagenomic sequencing, and enrichment culture followed by metagenomic sequencing on water and soil samples. Direct sequencing and enrichment cultures followed by PCR or sequencing effectively detected pathogenic and nonpathogenic Leptospira compared to direct PCR and 16S amplification-based metagenomic sequencing in soil or water samples. Among multiple culture media evaluated, Ellinghausen-McCullough-Johnson-Harris (EMJH) media containing antimicrobial agents was superior in recovering and detecting Leptospira from the environmental samples. Our results show that enrichment culture followed by PCR can be used to confirm the presence of pathogenic Leptospira in environmental samples. Metagenomic sequencing on enrichment cultures effectively detects the abundance and diversity of Leptospira spp from environmental samples. Conclusions/SignificanceThe selection of methodology is critical when testing environmental samples for the presence of Leptospira. Selective enrichment culture improves Leptospira detection efficacy by PCR or metagenomic sequencing and can be used successfully to understand the presence and diversity of pathogenic Leptospira during environmental surveillance. Author SummaryLeptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are major sources of transmission in humans and animals. For this reason, clusters of disease outbreaks are common during the rainy season. In this study, Leptospira enrichment cultures followed by PCR and sequencing detected pathogenic and nonpathogenic Leptospira in soil and water samples. The pathogenic and intermediate groups of Leptospira were more prevalent in soil samples tested. Metagenomic sequencing on enrichment culture is effective in detecting the abundance and diversity of various Leptospira spp. in environmental samples. Soil samples in proximity to water may be an ideal niche for Leptospira growth and survival and may be an appropriate sample of choice for testing.

microbiology↗

bioRxiv2020

Split-TurboID enables contact-dependent proximity labeling in cells

Proximity labeling (PL) catalyzed by promiscuous enzymes such as TurboID have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum (ER)-mitochondria contact sites, reconstituted TurboID catalyzed spatially-restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to ER-mitochondria contacts. We validated eight novel candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially-specific proximity labeling in cells.

cell biology↗