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PLoS Biology Jun 2024CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new...
CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.
Topics: Gene Editing; CRISPR-Cas Systems; CRISPR-Associated Proteins; Alleles; Humans; Endodeoxyribonucleases; Animals; Protein Engineering; Bacterial Proteins; Polymorphism, Single Nucleotide; Mutation; DNA; HEK293 Cells
PubMed: 38865309
DOI: 10.1371/journal.pbio.3002680 -
PNAS Nexus Apr 2024Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly...
Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly killing bacterial invaders; however, many pathogens have evolved resistance evasion mechanisms such as cell surface modification, CAP sequestration, degradation, or efflux. We have discovered that several pathogenic and commensal proteobacteria, including the urgent human threat , secrete a protein (lactoferrin-binding protein B, LbpB) that contains a low-complexity anionic domain capable of inhibiting the antimicrobial activity of host CAPs. This study focuses on a cattle pathogen, , that expresses the largest anionic domain of the LbpB homologs. We used an exhaustive biophysical approach employing circular dichroism, biolayer interferometry, cross-linking mass spectrometry, microscopy, size-exclusion chromatography with multi-angle light scattering coupled to small-angle X-ray scattering (SEC-MALS-SAXS), and NMR to understand the mechanisms of LbpB-mediated protection against CAPs. We found that the anionic domain of this LbpB displays an α-helical secondary structure but lacks a rigid tertiary fold. The addition of antimicrobial peptides derived from lactoferrin (i.e. lactoferricin) to the anionic domain of LbpB or full-length LbpB results in the formation of phase-separated droplets of LbpB together with the antimicrobial peptides. The droplets displayed a low rate of diffusion, suggesting that CAPs become trapped inside and are no longer able to kill bacteria. Our data suggest that pathogens, like , leverage anionic intrinsically disordered domains for the broad recognition and neutralization of antimicrobials via the formation of biomolecular condensates.
PubMed: 38633880
DOI: 10.1093/pnasnexus/pgae139 -
International Journal of Systematic and... Feb 2024A novel species of the genus was isolated from an ocular swab from a cow with infectious bovine keratoconjunctivitis. 16S rRNA gene sequencing suggested this species...
A novel species of the genus was isolated from an ocular swab from a cow with infectious bovine keratoconjunctivitis. 16S rRNA gene sequencing suggested this species was (99.59 % nucleotide identity). Average nucleotide identity was calculated using a draft whole genome sequence of this strain compared with type strains of closely related species and results established that it represents a new species. The genome size was 2 006 474 nucleotides and the G+C content was 42.51 mol%. The species could not be identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry using a commercial database, confirming the novelty of the strain. We propose the name sp. nov. for this new species. The type strain is Tifton1 and has been deposited into the American Type Culture Collection (TSD-373) and the National Collection of Type Cultures (NCTC), UK Health Security Agency (NCTC 14942).
Topics: Cattle; Animals; Moraxella; RNA, Ribosomal, 16S; Keratoconjunctivitis, Infectious; Phylogeny; Cattle Diseases; DNA, Bacterial; Bacterial Typing Techniques; Base Composition; Sequence Analysis, DNA; Fatty Acids; Keratoconjunctivitis; Moraxellaceae Infections; Nucleotides
PubMed: 38415687
DOI: 10.1099/ijsem.0.006281 -
Revista Argentina de Microbiologia 2024Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that affects bovines and has significant economic and health effects worldwide. Gram negative bacteria...
Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that affects bovines and has significant economic and health effects worldwide. Gram negative bacteria Moraxella bovis and Moraxella bovoculi are its main etiological agents. Antimicrobial therapy against IBK is often difficult in beef and dairy herds and, although vaccines are commercially available, their efficacy is variable and dependent on local strains. The aim of this study was to analyze for the first time the genomes of Uruguayan clinical isolates of M. bovis and M. bovoculi. The genomes were de novo assembled and annotated; the genetic basis of fimbrial synthesis was analyzed and virulence factors were identified. A 94% coverage in the reference genomes of both species, and more than 80% similarity to the reference genomes were observed. The mechanism of fimbrial phase variation in M. bovis was detected, and the tfpQ orientation of these genes confirmed, in an inversion region of approximately 2.18kb. No phase variation was determined in the fimbrial gene of M. bovoculi. When virulence factors were compared between strains, it was observed that fimbrial genes have 36.2% sequence similarity. In contrast, the TonB-dependent lactoferrin/transferrin receptor exhibited the highest percentage of amino acid similarity (97.7%) between strains, followed by cytotoxins MbxA/MbvA and the ferric uptake regulator. The role of these virulence factors in the pathogenesis of IBK and their potential as vaccine components should be explored.
Topics: Animals; Moraxella; Cattle; Moraxella bovis; Keratoconjunctivitis, Infectious; Cattle Diseases; Genome, Bacterial; Moraxellaceae Infections; Uruguay; Virulence Factors
PubMed: 38403533
DOI: 10.1016/j.ram.2023.12.003 -
Veterinary World Dec 2023Infectious bovine keratoconjunctivitis (IBK) is a prevalent ocular disease that affects livestock, leading to substantial economic losses due to reduced production and...
BACKGROUND AND AIM
Infectious bovine keratoconjunctivitis (IBK) is a prevalent ocular disease that affects livestock, leading to substantial economic losses due to reduced production and culling of infected animals. spp. is common bacterial pathogens that can cause keratoconjunctivitis in livestock. Therefore, rapid and accurate diagnosis is crucial for effective treatment and disease control. This study aimed to develop a multiplex real-time polymerase chain reaction (mRT-PCR) assay for the detection and differentiation of , , and .
MATERIALS AND METHODS
Three reference strains of as positive controls and 36 lacrimal swab samples collected from cattle were used to evaluate the developed mRT-PCR assay DNA extraction that was performed using the RIBO-sorb DNA/RNA extraction kit. Primers and probes were designed using the SpeciesPrimer pipeline. The annealing temperature, primer and probe concentrations, and sensitivity and specificity of the assay were optimized.
RESULTS
An mRT-PCR assay was developed to detect pathogens associated with IBK in cattle on the basis of optimized parameters. The specificity and sensitivity of this assay were confirmed using samples containing individual pathogens (O - , B - , and BO - ), combinations of two pathogens (O-B, B-BO, and O-BO), and when the DNA of all three pathogens was present in a single reaction (O-B-BO). The analytical sensitivity of mRT-PCR for detecting and DNA was 21 copies or 50 fg per reaction, whereas that for . was 210 copies or 500 fg per reaction. In addition, this assay has been tested on samples isolated from the affected eyes of cattle in the Akmola region of the Republic of Kazakhstan.
CONCLUSION
For the first time in the Republic of Kazakhstan, the proposed mRT-PCR assay for the simultaneous detection of three spp. pathogens has been developed. This assay exhibits the required specificity and high sensitivity for m RT-PCR, facilitating the timely implementation of effective measures for disease control and the prevention of economic losses. These losses are linked to a reduction in livestock breeding value, a reduction in meat and milk production, a reduction in the reproductive performance of heifers, resulting in fewer offspring, as well as costs related to the treatment of affected animals.
PubMed: 38328358
DOI: 10.14202/vetworld.2023.2526-2532 -
Journal of Veterinary Diagnostic... Jan 2024Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of and . A third novel spp., designated tentatively as , has been identified from the eyes of...
Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of and . A third novel spp., designated tentatively as , has been identified from the eyes of cattle with and without pinkeye. These 3 spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing diversity found in IBK- and non-IBK-affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 spp. Targeted NGS with sequencing of spp. housekeeping genes appears to be a suitable method for speciation of directly from ocular swabs.
Topics: Cattle; Animals; Moraxella; Keratoconjunctivitis, Infectious; Mycoplasma Infections; Moraxellaceae Infections; Cattle Diseases; High-Throughput Nucleotide Sequencing
PubMed: 38018659
DOI: 10.1177/10406387231216698 -
Animal Microbiome Nov 2023Infectious bovine keratoconjunctivitis (IBK) is a common cause of morbidity in cattle, resulting in significant economic losses. This study aimed to characterize the...
BACKGROUND
Infectious bovine keratoconjunctivitis (IBK) is a common cause of morbidity in cattle, resulting in significant economic losses. This study aimed to characterize the bovine bacterial ocular surface microbiome (OSM) through conjunctival swab samples from Normal eyes and eyes with naturally acquired, active IBK across populations of cattle using a three-part approach, including bacterial culture, relative abundance (RA, 16 S rRNA gene sequencing), and semi-quantitative random forest modeling (real-time polymerase chain reaction (RT-PCR)).
RESULTS
Conjunctival swab samples were obtained from eyes individually classified as Normal (n = 376) or IBK (n = 228) based on clinical signs. Cattle unaffected by IBK and the unaffected eye in cattle with contralateral IBK were used to obtain Normal eye samples. Moraxella bovis was cultured from similar proportions of IBK (7/228, 3.07%) and Normal eyes (1/159, 0.63%) (p = 0.1481). Moraxella bovoculi was cultured more frequently (p < 0.0001) in IBK (59/228, 25.88%) than Normal (7/159, 4.40%) eyes. RA (via 16 S rRNA gene sequencing) of Actinobacteriota was significantly higher in Normal eyes (p = 0.0045). Corynebacterium variabile and Corynebacterium stationis (Actinobacteriota) were detected at significantly higher RA (p = 0.0008, p = 0.0025 respectively) in Normal eyes. Rothia nasimurium (Actinobacteriota) was detected at significantly higher RA in IBK eyes (p < 0.0001). Alpha-diversity index was not significantly different between IBK and Normal eyes (p > 0.05). Alpha-diversity indices for geographic location (p < 0.001), age (p < 0.0001), sex (p < 0.05) and breed (p < 0.01) and beta-diversity indices for geographic location (p < 0.001), disease status (p < 0.01), age (p < 0.001), sex (p < 0.001) and breed (p < 0.001) were significantly different between groups. Modeling of RT-PCR values reliably categorized the microbiome of IBK and Normal eyes; primers for Moraxella bovoculi, Moraxella bovis, and Staphylococcus spp. were consistently the most significant canonical variables in these models.
CONCLUSIONS
The results provide further evidence that multiple elements of the bovine bacterial OSM are altered in the context of IBK, indicating the involvement of a variety of bacteria in addition to Moraxella bovis, including Moraxella bovoculi and R. nasimurium, among others. Actinobacteriota RA is altered in IBK, providing possible opportunities for novel therapeutic interventions. While RT-PCR modeling provided limited further support for the involvement of Moraxella bovis in IBK, this was not overtly reflected in culture or RA results. Results also highlight the influence of geographic location and breed type (dairy or beef) on the bovine bacterial OSM. RT-PCR modeling reliably categorized samples as IBK or Normal.
PubMed: 37996960
DOI: 10.1186/s42523-023-00282-4 -
Veterinary World Sep 2023Infectious bovine keratoconjunctivitis (IBK) causes a significant economic loss to cattle industries in many countries, including Kazakhstan. Although is recognized as...
BACKGROUND AND AIM
Infectious bovine keratoconjunctivitis (IBK) causes a significant economic loss to cattle industries in many countries, including Kazakhstan. Although is recognized as an etiologic agent of IBK, other bacterial and viral agents have been suspected to play a role in the pathogenesis of this disease. This study aimed to evaluate samples collected from the eyes of IBK-affected cattle in Eastern Kazakhstan at different stages of IBK for the presence of , , , , and Bovine Herpes Virus Type 1 (BHV-1) and to characterize gene sequence diversity from positive samples.
MATERIALS AND METHODS
Individual ocular swabs (n = 168) were collected from cattle that had clinical signs of IBK during the summer of 2022 on farms in the Abay region of Kazakhstan. Eye lesion scores (1, 2, and 3) were assigned depending on the degree of ocular damage. Infectious bovine keratoconjunctivitis-associated organisms were detected using a multiplex real-time polymerase chain reaction assay. The gene was sequenced from positive samples.
RESULTS
and BHV-1 were not detected in any of the collected samples. was identified in the majority of samples overall, usually in mixed infection with spp. was detected in 76.2% of animals and predominated in animals with eye lesion scores 2 and 3. was detected only in association with and/or in animals with eye lesion scores 2 and 3. was found in 57.7% of animals and was always identified in association with another organism. Sequencing of the gene in 96 samples from positive samples identified five PilA groups. The majority belonged to PilA group A. However, three new PilA groups were identified and designated PilA groups N, O, and P.
CONCLUSION
The results indicate a high prevalence of and in eyes of cattle with IBK on livestock farms in Eastern Kazakhstan. Additional novel PilA groups were identified.
PubMed: 37859972
DOI: 10.14202/vetworld.2023.1833-1839 -
Vaccine: X Dec 2023Infectious bovine keratoconjunctivitis (IBK; pinkeye) is generally considered to be caused by corneal infections with . Previous studies demonstrated that...
BACKGROUND
Infectious bovine keratoconjunctivitis (IBK; pinkeye) is generally considered to be caused by corneal infections with . Previous studies demonstrated that cytotoxin-specific mucosal immune responses in the bovine eye can be stimulated by intranasal vaccination with a recombinant cytotoxin subunit adjuvanted with polyacrylic acid.
METHODS
A randomized controlled field trial (two-arm parallel design with blinding) was conducted in beef steers in Northern California to determine if this vaccine could prevent naturally occurring IBK and/or reduce morbidity rates associated with this disease. Beef steers were vaccinated intranasally on days 0 and 21 with either a recombinant cytotoxin subunit adjuvanted with polyacrylic acid (Vaccine group) or adjuvant alone (Control group). Eye examinations were performed on all steers every 7 days for 16 weeks to document the occurrence of IBK and to determine sizes of corneal ulcers. Serum and tear samples were collected on days 0, 42, and 112 from a subset of animals to measure changes in systemic and ocular immune responses to cytotoxin.
RESULTS
The cumulative proportion of steers that developed IBK after 16 weeks did not differ between groups. Variables related to disease severity were numerically lower in steers that received the experimental vaccine. IBK-affected Vaccine group steers had a significantly lower number of observation weeks with severe ulcers versus Control group steers. Cytotoxin-specific tear IgA was significantly higher in Vaccine group compared to Control group steers on day 112. Although the proportion of animals that developed corneal ulcers associated with IBK did not differ between groups, the lowered metrics of disease severity in vaccinated steers suggests that intranasal vaccination with recombinant cytotoxin can reduce the severity of IBK in cattle.
PubMed: 37693844
DOI: 10.1016/j.jvacx.2023.100378 -
Journal of Medical Entomology Nov 2023House flies (Musca domestica Linnaeus) are vectors of human and animal pathogens at livestock operations. Microbial communities in flies are acquired from, and correlate...
House flies (Musca domestica Linnaeus) are vectors of human and animal pathogens at livestock operations. Microbial communities in flies are acquired from, and correlate with, their local environment. However, variation among microbial communities carried by flies from farms in different geographical areas is not well understood. We characterized bacterial communities of female house flies collected from beef and dairy farms in Oklahoma, Kansas, and Nebraska using 16S rDNA amplicon sequencing and PCR. Bacterial community composition in house flies was affected by farm type and location. While the shared number of taxa between flies from beef or dairy farms was low, those taxa accounted >97% of the total bacterial community abundance. Bacterial species richness was 4% greater in flies collected from beef than in those collected from dairy farms and varied by farm type within states. Several potential pathogenic taxa were highly prevalent, comprising a core bacterial community in house flies from cattle farms. Prevalence of the pathogens Moraxella bovis and Moraxella bovoculi was greater in flies from beef farms relative to those collected on dairy cattle farms. House flies also carried bacteria with multiple tetracycline and florfenicol resistance genes. This study suggests that the house flies are significant reservoirs and disseminators of microbial threats to human and cattle health.
Topics: Humans; Cattle; Female; Animals; Diptera; Muscidae; Houseflies; Farms; Anti-Bacterial Agents; Prevalence; Bacteria; Drug Resistance, Microbial
PubMed: 37612042
DOI: 10.1093/jme/tjad112