MicroRNA profiles for different tissues from calves challenged with Mycoplasma bovis or challenged with Mycoplasma bovis and bovine viral diarrhea virus.

The objective was to determine differences in microRNAs (miRNAs) counts in several tissues of calves challenged with Mycoplasma bovis (M. bovis) or with M. bovis and bovine viral diarrhea virus (BVDV). Eight calves approximately 2 months of age were randomly assigned to three groups: Control (CT; n = 2), M. bovis (MB; n = 3), and Coinfection (CO; n = 3). On day 0, calves in CO were intranasally challenged with BVDV and calves in MB with M. bovis. On day 6, CO calves were challenged with M. bovis. Calves were euthanized 17 days post-challenge and serum (SER), white blood cells (WBC), liver (LIV), mesenteric (MLN) and tracheal-bronchial (TBLN) lymph nodes, spleen (SPL), and thymus (THY), were collected at necropsy. MiRNAs were extracted from each tissue from each calf. Significant (P< 0.01) differences in miRNAs expression were observed in SER, LIV, MLN, TBLN, SPL, and THY. There were no significant (P> 0.05) miRNAs in WBC. In SER, the CO group had levels of miR-1343-3p significantly higher than the CT and MB groups (P = 0.0071). In LIV and SPL, the CO group had the lowest counts for all significant miRNAs compared to CT and MB. In TBLN, the CT group had the highest counts of miRNAs, compared to MB and CO, in 14 of the 21 significant miRNAs. In THY, the CO group had the highest counts, in 4 of the 6 significant miRNAs compared to CT and MB. BVDV was associated with reduction of miRNAs in LIV, SPL, MLN, and TBLN, and M. bovis reduced counts of miRNAs in only TBLN. Measuring circulating miRNAs to assess disease condition or to develop intervention strategies to minimize respiratory diseases in cattle caused by BVDV or M. bovis will be of limited use unless an alternative approach is developed to use them as indicators of disease.

Protection against Mycoplasma bovis infection in calves following intranasal vaccination with modified-live Mannheimia haemolytica expressing Mycoplasma antigens.

Novel live vaccine strains of Mannheimia haemolytica serotypes (St)1 and St6, expressing and secreting inactive yet immunogenic leukotoxin (leukotoxoid) fused to antigenic domains of Mycoplasma bovis Elongation Factor Tu (EFTu) and Heat shock protein (Hsp) 70 were constructed and tested for efficacy in cattle. Control calves were administered an intranasal mixture of M. haemolytica St1 and St6 mutants (ΔlktCAV4) expressing and secreting leukotoxoid while vaccinated calves were administered an intranasal mixture of like M. haemolytica St1 and St6 leukotoxoid mutants coupled to M. bovis antigens (EFTu-Hsp70-ΔlktCAV4). Both M. haemolytica strains were recovered from palatine tonsils up to 34 days post intranasal exposure. On day 35 all calves were exposed to bovine herpes virus-1, four days later lung challenged with virulent M. bovis, then euthanized up to 20 days post-challenge. Results showed all cattle produced systemic antibody responses against M. haemolytica. The vaccinates also produced systemic antibody responses to M. bovis antigen, and concurrent reductions in temperatures, middle ear infections, joint infection and lung lesions versus the control group. Notably, dramatically decreased lung loads of M. bovis were detected in the vaccinated cattle. These observations indicate that the attenuated M. haemolytica vaccine strains expressing Mycoplasma antigens can control M. bovis infection and disease symptoms in a controlled setting.

Prevalence of Mycoplasma spp. in the Respiratory Tract of Healthy North American Bison (Bison bison) and Comparison with Serum Antibody Status.

Mycoplasma bovis is a primary cause of respiratory and reproductive diseases in North American bison (Bison bison), with significant morbidity and mortality. The epidemiology of M. bovis in bison is poorly understood, hindering efforts to develop effective control measures. Our study considered whether healthy bison might be carriers of M. bovis, potentially serving as unrecognized sources of exposure. We used culture and PCR to identify mycoplasmas in the nasal cavity or tonsil of 499 healthy bison from 13 herds and two abattoirs in the US and Canada. Mycobacterium bovis was detected in 15 bison (3.0%) representing two herds in the US and one in Canada, while M. bovirhinis, M. bovoculi, M. arginini, or M. dispar was identified from an additional 155 bison (31.1%). Mycoplasma bovirhinis was identified most frequently, in 142 bison (28.5%) representing at least 10 herds. Of the 381 bison for which serum was available, only 6/13 positive for M. bovis (46.2%) tested positively with an M. bovis ELISA, as did 19/368 negative for M. bovis (5.2%). Our data reveal that M. bovis can be carried in the upper respiratory tract of healthy bison with no prior history or clinical signs of mycoplasmosis and that a large proportion of carriers may not produce detectable antibodies. Whether carriage of other mycoplasmas can trigger cross-reactive antibodies that may confound M. bovis serology requires further study.

Serological evidence for historical and present-day exposure of North American bison to Mycoplasma bovis.

BACKGROUND: Mycoplasma bovis causes mastitis, otitis, pneumonia and arthritis in cattle and is a major contributor to bovine respiratory disease complex. Around the year 2000, it emerged as a significant threat to the health of North American bison. Whether healthy bison are carriers of M. bovis and when they were first exposed is not known. To investigate these questions we used a commercially available ELISA that detects antibodies to M. bovis to test 3295 sera collected from 1984 through 2019 from bison in the United States and Canada. RESULTS: We identified moderately to strongly seropositive bison from as long ago as the late 1980s. Average seroprevalence over the past 36 years is similar in the United States and Canada, but country-specific differences are evident when data are sorted by the era of collection. Seroprevalence in the United States during the pre-disease era (1999 and prior) was significantly higher than in Canada, but was significantly lower than in Canada during the years 2000-2019. Considering individual countries, seroprevalence in the United States since the year 2000 dropped significantly as compared to the years 1985-1999. In Canada the trend is reversed, with seroprevalence increasing significantly since the year 2000. ELISA scores for sera collected from free-ranging bison do not differ significantly from scores for sera from more intensively managed animals, regardless of the era in which they were collected. However, seroprevalence among intensively raised Canadian bison has nearly doubled since the year 2000 and average ELISA scores rose significantly. CONCLUSIONS: Our data provide the first evidence that North American bison were exposed to M. bovis many years prior to the emergence of M. bovis-related disease. Patterns of exposure inferred from these results differ in the United States and Canada, depending on the era under consideration. Our data further suggest that M. bovis may colonize healthy bison at a level sufficient to trigger antibody responses but without causing overt disease. These findings provide novel insights as to the history of M. bovis in bison and will be of value in formulating strategies to minimize the impact of mycoplasmosis on bison health and production.

Mycoplasma bovis Infections in Free-Ranging Pronghorn, Wyoming, USA.

Mycoplasma bovis is 1 of several bacterial pathogens associated with pneumonia in cattle. Its role in pneumonia of free-ranging ungulates has not been established. Over a 3-month period in early 2019, ¼60 free-ranging pronghorn with signs of respiratory disease died in northeast Wyoming, USA. A consistent finding in submitted carcasses was severe fibrinosuppurative pleuropneumonia and detection of M. bovis by PCR and immunohistochemical analysis. Multilocus sequence typing of isolates from 4 animals revealed that all have a deletion in 1 of the target genes, adh-1. A retrospective survey by PCR and immunohistochemical analysis of paraffin-embedded lung from 20 pronghorn that died with and without pneumonia during 2007-2018 yielded negative results. These findings indicate that a distinct strain of M. bovis was associated with fatal pneumonia in this group of pronghorn.

Complete Genome Sequences of 16 Mycoplasma bovis Isolates from Canadian Bison and Cattle.

Here, we report the complete genome sequences of 12 Mycoplasma bovis isolates cultured from Canadian bison and 4 cultured from Canadian cattle. The sequences are of value for understanding the phylogenetic relationship between cattle and bison isolates and will aid in elucidating the genetic basis for virulence and host specificity.

Comparison of Two Multilocus Sequence Typing Schemes for Mycoplasma bovis and Revision of the PubMLST Reference Method.

Mycoplasma bovis causes pneumonia, pharyngitis, otitis, arthritis, mastitis, and reproductive disorders in cattle and bison. Two multilocus sequence typing (MLST) schemes have been developed for M. bovis, with one serving as the PubMLST reference method, but no comparison of the schemes has been undertaken. Although the PubMLST scheme has proven to be highly discriminatory and informative, the recent discovery of isolates missing one of the typing loci, adh-1, raises concern about its suitability for continued use. The goal of our study was to compare the performance of the two MLST schemes and identify a new reference scheme capable of fully typing all isolates. We evaluated 448 isolates from diverse geographic and anatomic sites that collectively represent cattle, bison, deer, and a goat. The discrimination indexes (DIs) for the PubMLST and the alternative scheme are 0.909 (91 sequence types [STs]) and 0.842 (77 STs), respectively. Although the PubMLST scheme outperformed the alternative scheme, the adh-1 locus must be retired from the PubMLST scheme if it is to be retained as a reference method. The DI obtained using the six remaining PubMLST loci (0.897, 79 STs) fails to reach the benchmark recommended for a reference method (0.900), mandating the addition of a seventh locus. Comparative analysis of genome sequences from the isolates used here identified the dnaA locus from the alternative scheme as the optimal replacement for adh-1 This revised scheme, which will be implemented as the new PubMLST reference method, has a DI of 0.914 and distinguishes 88 STs from the 448 isolates evaluated.

Comparison of multilocus sequence types found among North American isolates of Mycoplasma bovis from cattle, bison, and deer, 2007-2017.

A prior multilocus sequence typing (MLST) study reported that Mycoplasma bovis isolates from North American bison possess sequence types (STs) different from those found among cattle. The 42 bison isolates evaluated were obtained in 2007 or later, whereas only 19 of 94 (~20%) of the available cattle isolates, with only 1 from North America, were from that same time. We compared STs of additional, contemporary, North American cattle isolates with those from bison, as well as isolates from 2 North American deer, all originating during the same timeframe, to more definitively assess potential strain-related host specificity and expand our understanding of the genetic diversity of M. bovis. From 307 isolates obtained between 2007 and 2017 (209 from cattle, 96 from bison, 2 from deer), we identified 49 STs, with 39 found exclusively in cattle and 5 exclusively in bison. Four STs were shared between bison and cattle isolates; one ST was found in cattle and in a deer. There was no clear association between ST and the health status of the animal of origin. An MLST-based phylogeny including 41 novel STs identified in our study reveals that STs found in bison fall within several divergent lineages that include STs found exclusively in cattle.

Relative virulence in bison and cattle of bison-associated genotypes of Mycoplasma bovis.

Mycoplasma bovis, a frequent contributor to polymicrobial respiratory disease in cattle, has recently emerged as a major health problem in North American bison. Strong circumstantial evidence suggests it can be the sole pathogen causing disease manifestations in outbreaks of mortality in bison, but direct evidence is lacking. The goal of this study was to compare clinical signs and lesions in bison and cattle experimentally infected with field isolates of M. bovis recovered from bison. Bison (n = 7) and cattle (n = 6), seronegative for anti-M. bovis IgG, were exposed intranasally to M. bovis and necropsied 4-6 weeks later. Blood and nasal swabs were collected on day 0 (before exposure), day 11 and at necropsy. Samples of lung, lymph node, liver and spleen were also collected at necropsy. The only clinical sign observed was an elevation in the core body temperature of bison during the first few weeks post-exposure. Grossly visible lesions were apparent at necropsy in the lungs of five bison and the lymph node of one bison, while none were evident in cattle. Histologic evaluation revealed moderate to severe pulmonary lesions in four bison but none in cattle. M. bovis was recovered from tissues demonstrating gross lesions and from the lymph nodes of one additional bison and two cattle. All animals seroconverted by the time of necropsy. These data provide the first direct evidence that M. bovis can be a sole or primary cause of respiratory disease in healthy bison, although the isolates used were unable to cause disease in healthy cattle.

Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis.

Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobial activity against various bacterial pathogens, including several involved in bovine respiratory disease complex (BRDC) in cattle; however, such studies are yet to be performed with one important contributor to the BRDC, Mycoplasma bovis. Therefore, the goal of this study was to assess the antimicrobial activity of bovine NK-lysin-derived peptides on M. bovis. Thirty-mer synthetic peptides corresponding to the functional region helices 2 and 3 of bovine NK-lysins NK1, NK2A, NK2B, and NK2C were evaluated for killing activity on M. bovis isolates. Among four peptides, NK2A and NK2C showed the highest antimicrobial activity against the M. bovis isolates tested. All four NK-lysin peptides induced rapid plasma membrane depolarization in M. bovis at two concentrations tested. However, based on propidium iodide uptake, only NK2A and NK2C appeared capable of causing structural damage to M. bovis plasma membrane. Confocal microscopy, flow cytometry, and transmission electron microscopy further suggested NK-lysin-induced damage to the plasma membrane. Taken together, the findings in this study suggest that plasma membrane depolarization alone was insufficient to induce lethality, but disruption/permeabilization of the M. bovis plasma membrane was the cause of lethality.

Association of Circulating Transfer RNA fragments with antibody response to Mycoplasma bovis in beef cattle.

BACKGROUND: High throughput sequencing allows identification of small non-coding RNAs. Transfer RNA Fragments are a class of small non-coding RNAs, and have been identified as being involved in inhibition of gene expression. Given their role, it is possible they may be involved in mediating the infection-induced defense response in the host. Therefore, the objective of this study was to identify 5' transfer RNA fragments (tRF5s) associated with a serum antibody response to M. bovis in beef cattle. RESULTS: The tRF5s encoding alanine, glutamic acid, glycine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with antibody response against M. bovis. tRF5s encoding alanine, glutamine, glutamic acid, glycine, histidine, lysine, proline, selenocysteine, threonine, and valine were associated (P < 0.05) with season, which could be attributed to calf growth. There were interactions (P < 0.05) between antibody response to M. bovis and season for tRF5 encoding selenocysteine (anticodon UGA), proline (anticodon CGG), and glutamine (anticodon TTG). Selenocysteine is a rarely used amino acid that is incorporated into proteins by the opal stop codon (UGA), and its function is not well understood. CONCLUSIONS: Differential expression of tRF5s was identified between ELISA-positive and negative animals. Production of tRF5s may be associated with a host defense mechanism triggered by bacterial infection, or it may provide some advantage to a pathogen during infection of a host. Further studies are needed to establish if tRF5s could be used as a diagnostic marker of chronic exposure.

Mistaken identity of an open reading frame proposed for PCR-based identification of Mycoplasma bovis and the effect of polymorphisms and insertions on assay performance.

Mycoplasma bovis is an important cause of disease in cattle and bison. Because the bacterium requires specialized growth conditions, many diagnostic laboratories routinely use PCR to replace or complement conventional isolation and identification methods. A frequently used target of such assays is the uvrC gene, which has been shown to be highly conserved among isolates. We discovered that a previously described PCR putatively targeting the uvrC gene amplifies a fragment from an adjacent gene predicted to encode a lipoprotein. Comparison of the lipoprotein gene sequence from 211 isolates revealed several single nucleotide polymorphisms, 1 of which falls within a primer-binding sequence. Additionally, 3 isolates from this group were found to have a 1,658-bp transposase gene insertion within the amplified region that leads to a false-negative result. The insertion was not detected in a further 164 isolates. We found no evidence that the nucleotide substitution within the primer-binding region affects the assay sensitivity, performance, or limit of detection. Nonetheless, laboratories utilizing this method for identification of M. bovis should be aware that the region amplified may be prone to nucleotide substitutions and/or insertions relative to the sequence used for its design and that occasional false-negative results may be obtained.

Acquisition and loss of virulence-associated factors during genome evolution and speciation in three clades of Bordetella species.

BACKGROUND: The genus Bordetella consists of nine species that include important respiratory pathogens such as the 'classical' species B. bronchiseptica, B. pertussis and B. parapertussis and six more distantly related and less extensively studied species. Here we analyze sequence diversity and gene content of 128 genome sequences from all nine species with focus on the evolution of virulence-associated factors. RESULTS: Both genome-wide sequence-based and gene content-based phylogenetic trees divide the genus into three species clades. The phylogenies are congruent between species suggesting genus-wide co-evolution of sequence diversity and gene content, but less correlated within species, mainly because of strain-specific presence of many different prophages. We compared the genomes with focus on virulence-associated genes and identified multiple clade-specific, species-specific and strain-specific events of gene acquisition and gene loss, including genes encoding O-antigens, protein secretion systems and bacterial toxins. Gene loss was more frequent than gene gain throughout the evolution, and loss of hundreds of genes was associated with the origin of several species, including the recently evolved human-restricted B. pertussis and B. holmesii, B. parapertussis and the avian pathogen B. avium. CONCLUSIONS: Acquisition and loss of multiple genes drive the evolution and speciation in the genus Bordetella, including large scale gene loss associated with the origin of several species. Recent loss and functional inactivation of genes, including those encoding pertussis vaccine components and bacterial toxins, in individual strains emphasize ongoing evolution.

Identification and taxonomic characterization of Bordetella pseudohinzii sp. nov. isolated from laboratory-raised mice.

Bordetella hinzii is known to cause respiratory disease in poultry and has been associated with a variety of infections in immunocompromised humans. In addition, there are several reports of B. hinzii infections in laboratory-raised mice. Here we sequenced and analysed the complete genome sequences of multiple B. hinzii-like isolates, obtained from vendor-supplied C57BL/6 mice in animal research facilities on different continents, and we determined their taxonomic relationship to other Bordetella species. The whole-genome based and 16S rRNA gene based phylogenies each identified two separate clades in B. hinzii, one was composed of strains isolated from poultry, humans and a rabbit whereas the other clade was restricted to isolates from mice. Distinctly different estimated DNA-DNA hybridization values, average nucleotide identity scores, gene content, metabolic profiles and host specificity all provide compelling evidence for delineation of the two species, B. hinzii - from poultry, humans and rabbit - and Bordetella pseudohinzii sp. nov. type strain 8-296-03T (=NRRL B-59942T=NCTC 13808T) that infect mice.

Association of MicroRNAs with Antibody Response to Mycoplasma bovis in Beef Cattle.

The objective of this study was to identify microRNAs associated with a serum antibody response to Mycoplasma bovis in beef cattle. Serum from sixteen beef calves was collected at three points: in summer after calves were born, in fall at weaning, and in the following spring. All sera collected in the summer were ELISA-negative for anti-M. bovis. By the fall, eight animals were seropositive for IgG (positive group), while eight remained negative (negative group). By spring, all animals in both groups were seropositive. MicroRNAs were extracted from sera and sequenced on the Illumina HiSeq next-generation sequencer. A total of 1,374,697 sequences mapped to microRNAs in the bovine genome. Of these, 82% of the sequences corresponded to 27 microRNAs, each represented by a minimum of 10,000 sequences. There was a statistically significant interaction between ELISA response and season for bta-miR-24-3p (P = 0.0268). All sera collected at the initial summer had a similar number of copies of this microRNA (P = 0.773). In the fall, the positive group had an increased number of copies when compared to the negative group (P = 0.021), and this grew more significant by the following spring (P = 0.0001). There were 21 microRNAs associated (P< 0.05) with season. These microRNAs could be evaluated further as candidates to potentially improve productivity in cattle. The microRNAs bta-let-7b, bta-miR- 24-3p, bta-miR- 92a, and bta-miR-423-5p, were significatly associated with ELISA status (P< 0.05). These microRNAs have been recognized as playing a role in the host defense against bacteria in humans, mice, and dairy cattle. Further studies are needed to establish if these microRNAs could be used as diagnostic marker or indicator of exposure, or whether intervention strategies could be developed as an alternative to antibiotics for controlling disease due to M. bovis.

Comparison of ribotyping and sequence-based typing for discriminating among isolates of Bordetella bronchiseptica.

PvuII ribotyping and MLST are each highly discriminatory methods for genotyping Bordetella bronchiseptica, but a direct comparison between these approaches has not been undertaken. The goal of this study was to directly compare the discriminatory power of PvuII ribotyping and MLST, using a single set of geographically and genetically diverse strains, and to determine whether subtyping based on repeat region sequences of the pertactin gene (prn) provides additional resolution. One hundred twenty-two isolates were analyzed, representing 11 mammalian or avian hosts, sourced from the United States, Europe, Israel and Australia. Thirty-two ribotype patterns were identified; one isolate could not be typed. In comparison, all isolates were typeable by MLST and a total of 30 sequence types was identified. An analysis based on Simpson's Index of Diversity (SID) revealed that ribotyping and MLST are nearly equally discriminatory, with SIDs of 0.920 for ribotyping and 0.919 for MLST. Nonetheless, for ten ribotypes and eight MLST sequence types, the alternative method discriminates among isolates that otherwise type identically. Pairing prn repeat region typing with ribotyping yielded 54 genotypes and increased the SID to 0.954. Repeat region typing combined with MLST resulted in 47 genotypes and an SID of 0.944. Given the technical and practical advantages of MLST over ribotyping, and the nominal difference in their SIDs, we conclude MLST is the preferred primary typing tool. We recommend the combination of MLST and prn repeat region typing as a high-resolution, objective and standardized approach valuable for investigating the population structure and epidemiology of B. bronchiseptica.

Cilia-associated bacteria in fatal Bordetella bronchiseptica pneumonia of dogs and cats.

Bordetella bronchiseptica frequently causes nonfatal tracheobronchitis, but its role in fatal pneumonia is less recognized. Our study evaluated histologic identification of cilia-associated bacteria as a method for diagnosis of B. bronchiseptica pneumonia. Cases of fatal bronchopneumonia were studied retrospectively, excluding neonates and cases of aspiration pneumonia, minor lung lesions, or autolysis. The study population comprised 36 canine and 31 feline cases of bronchopneumonia. B. bronchiseptica was identified in 8 of 36 canine and 14 of 31 feline cases based on immunohistochemistry (IHC) using serum from a rabbit hyperimmunized with pertactin, PCR testing (Fla2/Fla12), and/or bacterial culture data when available. Of these, IHC was positive in 4 canine and 7 feline cases, PCR was positive in 8 canine and 14 feline cases, and B. bronchiseptica was isolated in 2 of 5 canine and 3 of 9 feline cases tested. Examination of histologic sections stained with hematoxylin and eosin revealed bronchial cilia-associated bacteria in 4 of 36 canine and 5 of 31 feline cases; these were all positive by IHC and PCR. The presence of cilia-associated bacteria had been noted in the pathology report for only 2 of these 9 cases. Thus, the presence of cilia-associated bacteria seems frequently overlooked by pathologists, but is a diagnostically significant feature of B. bronchiseptica pneumonia. A specific diagnosis of B. bronchiseptica pneumonia is important because it suggests primary or opportunistic bacterial pneumonia rather than aspiration pneumonia, and because of the risk of animal-to-animal transmission of B. bronchiseptica, the availability of vaccines for disease prevention, and the potential zoonotic risk to immunocompromised pet owners.

Comparative genomic analysis of the swine pathogen Bordetella bronchisepticastrain KM22.

The well-characterized Bordetella bronchiseptica strain KM22, originally isolated from a pig with atrophic rhinitis, has been used to develop a reproducible swine respiratory disease model. The goal of this study was to identify genetic features unique to KM22 by comparing the genome sequence of KM22 to the laboratory reference strain RB50. To gain a broader perspective of the genetic relationship of KM22 among other B. bronchiseptica strains, selected genes of KM22 were then compared to five other B. bronchiseptica strains isolated from different hosts. Overall, the KM22 genome sequence is more similar to the genome sequences of the strains isolated from animals than the strains isolated from humans. The majority of virulence gene expression in Bordetella is positively regulated by the two-component sensory transduction system BvgAS. bopN, bvgA, fimB, and fimC were the most highly conserved BvgAS-regulated genes present in all seven strains analyzed. In contrast, the BvgAS-regulated genes present in all seven strains with the highest sequence divergence werefimN, fim2, fhaL, andfhaS. A total of eight major fimbrial subunit genes were identified in KM22. Quantitative real-time PCR data demonstrated that seven of the eight fimbrial subunit genes identified in KM22 are expressed and regulated by BvgAS. The annotation of the KM22 genome sequence, coupled with the comparative genomic analyses reported in this study, can be used to facilitate the development of vaccines with improved efficacy towards B. bronchiseptica in swine to decrease the prevalence and disease burden caused by this pathogen.

A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease.

BACKGROUND: Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. METHODS: The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. RESULTS: Here we describe a novel Type II-C CRISPR and its associated genes-cas1, cas2, and cas9-in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. CONCLUSIONS: Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.