Post-transcriptional control of the essential enzyme MurF by a small regulatory RNA in Brucella abortus.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.

Necrotizing hepatitis caused by Clostridium novyi type B in a dog with no predisposing liver lesions: a case report.

BACKGROUND: Infectious necrotic hepatitis (INH) is typically a disease of ruminants caused by Clostridium novyi type B. Growth of the causative agent is supported by development of an anaerobic environment within the liver. In dogs, C. novyi is rare and has only been previously reported as a post-mortem diagnosis. In one case, infection was secondary to metastatic pancreatic adenocarcinoma and the other was presumptively diagnosed on histopathology of a hepatic lesion in a dog initially presented for acute collapse. CASE PRESENTATION: An 8-year-old spayed, female mixed breed dog was presented for acute onset of hyporexia and vomiting. Serum biochemistry revealed elevated hepatocellular injury and cholestatic liver enzymes. Ultrasound revealed peritoneal fluid accumulation and multiple hepatic masses. Cytologic examination of liver aspirates and peritoneal fluid revealed frequent 4 × 1 µm bacilli with a terminal endospore. Anaerobic bacterial growth isolated from the fluid sample could not be identified using typical laboratory identification techniques. Long-read, whole genome sequencing was performed, and the organism was identified as Clostridium novyi type B. Antimicrobial and hepatic support treatment were initiated. The patient re-presented 27 days later, and the follow up liver aspirate with cytology revealed no appreciable bacteria and anaerobic culture was negative. The patient was presented four months later and a large hepatic mass and peritoneal fluid were again identified on abdominal ultrasound. Cytologic examination of the peritoneal fluid revealed bacilli similar to those identified on initial presentation. The patient was euthanized. The most significant finding on necropsy was necrotizing hepatitis with intralesional endospore-forming bacilli compatible with recurrence of Clostridium novyi type B. There was no identifiable cause of an anaerobic insult to the liver. CONCLUSIONS: This case demonstrates the diagnostic utility of using cytology as part of the initial diagnostic work up for infectious hepatitis. The cytologic findings coupled with whole genome sequencing and anaerobic culture were crucial for the identification and classification of the organism identified on fine needle aspirate. Clostridium novyi type B should be considered when bacilli organisms containing a terminal endospore are identified on liver aspirates collected from canine patients.

Complete Genome Sequence of Providencia stuartii CMC-4104, Isolated from a Human Splenic Abscess, Containing Multiple Copies of NDM-1 and PER-1 Carbapenem Resistance Genes.

We report the complete genome sequence of a clinical isolate of Providencia stuartii strain CMC-4104, isolated from a splenic abscess. Oxford Nanopore Technologies (ONT) and Illumina sequencing reads were assembled using Geneious to generate a 4,504,925-bp circular chromosome containing multiple copies of the NDM-1 and PER-1 genes in a genomic resistance island.

Alginate microencapsulation of an attenuated O-antigen mutant of Francisella tularensis LVS as a model for a vaccine delivery vehicle.

Francisella tularensis is the etiologic agent of tularemia and a Tier I Select Agent. Subspecies tularensis (Type A) is the most virulent of the four subspecies and inhalation of as few as 10 cells can cause severe disease in humans. Due to its niche as a facultative intracellular pathogen, a successful tularemia vaccine must induce a robust cellular immune response, which is best achieved by a live, attenuated strain. F. tularensis strains lacking lipopolysaccharide (LPS) O-antigen are highly attenuated, but do not persist in the host long enough to induce protective immunity. Increasing the persistence of an O-antigen mutant may help stimulate protective immunity. Alginate encapsulation is frequently used with probiotics to increase persistence of bacteria within the gastrointestinal system, and was used to encapsulate the highly attenuated LVS O-antigen mutant WbtIG191V. Encapsulation with alginate followed by a poly-L-lysine/alginate coating increased survival of WbtIG191V in complement-active serum. In addition, BALB/c mice immunized intraperitoneally with encapsulated WbtIG191V combined with purified LPS survived longer than mock-immunized mice following intranasal challenge. Alginate encapsulation of the bacteria also increased antibody titers compared to non-encapsulated bacteria. These data suggest that alginate encapsulation provides a slow-release vehicle for bacterial deposits, as evidenced by the increased antibody titer and increased persistence in serum compared to freely suspended cells. Survival of mice against high-dose intranasal challenge with the LVS wildtype was similar between mice immunized within alginate capsules or with LVS, possibly due to the low number of animals used, but bacterial loads in the liver and spleen were the lowest in mice immunized with WbtIG191V and LPS in beads. However, an analysis of the immune response of surviving mice indicated that those vaccinated with the alginate vehicle upregulated cell-mediated immune pathways to a lesser extent than LVS-vaccinated mice. In summary, this vehicle, as formulated, may be more effective for pathogens that require predominately antibody-mediated immunity.

Coinfection of cattle in Virginia with Theileria orientalis Ikeda genotype and Anaplasma marginale.

Theileria orientalis Ikeda is a newly identified agent of bovine infectious anemia in the United States. Although T. orientalis Ikeda is transmitted by ticks other than the tick that transmits Anaplasma marginale-a bacterial etiology of bovine infectious anemia-the geographic distributions of these 2 infectious organisms overlap, with coinfection reported in some cattle. Only anaplasmosis has an approved effective treatment in the United States. To provide rapid diagnostic information for producers with anemic animals, we developed a duplex real-time PCR (rtPCR) for A. marginale and T. orientalis. With a cutoff of 38 cycles, the duplex assay has a sensitivity of 97.0% and a specificity of 100% for A. marginale; with a cutoff of 45 cycles, the duplex assay has a sensitivity and a specificity of 100% for T. orientalis, compared to existing tests. In addition to providing a tool for improved clinical decision-making for veterinarians and producers, our rtPCR facilitates the study of coinfection of cattle in Virginia. Of 1,359 blood samples analyzed, 174 were positive for T. orientalis, 125 were positive for A. marginale, and 12 samples were positive for both T. orientalis and A. marginale. Hence, coinfection by these 2 agents of bovine infectious anemia does occur within Virginia. It is likely that this pattern of infection will be seen in other regions where T. orientalis and A. marginale infections are endemic, despite the difference in tick vectors.

Horses affected by EPM have increased sCD14 compared to healthy horses.

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease affecting horses across the Americas. Gaps in understanding the inflammatory immune response in EPM-affected horses create difficulties with diagnosis and treatment, subsequently negatively impacting the prognosis of affected horses. The purpose of the current study was to evaluate circulating levels of the inflammatory immune marker soluble CD14 (sCD14), in horses with EPM (n = 7) and determine if they differed from healthy neurologically normal horses (n = 6). Paired sera and cerebrospinal fluid (CSF) samples were analyzed for sCD14. Inclusion criteria for EPM horses consisted of the presence of neurologic signs consistent with EPM, Sarcocystis neurona surface antigens 2, 4/3 (SnSAG 2, 4/3) ELISA serum: CSF antibody ratio ≤ 100, and a postmortem diagnosis of EPM. Control horses were neurologically normal, healthy horses with SnSAG 2, 4/3 ELISA serum: CSF antibody ratios of > 100. Serum anti-Sarcocystis neurona antibodies indicate that healthy control horses were exposed to S. neurona but resistant to developing clinical EPM. EPM cases had significantly greater concentrations of sCD14 in CSF samples compared to control horses and increased serum sCD14 concentrations. A positive correlation between sCD14 serum and CSF concentrations was observed in EPM-affected horses but not healthy horses. Soluble CD14 is an inflammatory marker, and the study results suggest it is elevated in EPM patients. When performed in conjunction with clinical evaluation and standard antibody testing, there may be potential for sCD14 to be utilized as a correlate for EPM.

Stray Mexico origin cattle captured crossing into Southern Texas carry Babesia bovis and other tick-borne pathogens.

Cattle fever ticks, Rhipicephalus microplus and R. annulatus have been eradicated from the United States and inspectors from the U.S. Department of Agriculture (USDA), Animal Plant Health Inspection Service (APHIS), Cattle Fever Tick Eradication Program (CFTEP) monitor the quarantine zone along the Texas border to prevent the introduction of livestock carrying cattle fever ticks from Mexico. Stray livestock apprehended by CFTEP in the zone are checked for ticks and tested for infectious disease-causing pathogens but are not evaluated for evidence of infection with tick-borne pathogens. We tested blood samples collected from stray cattle by CFTEP inspectors for evidence of infection with tick-borne pathogens. As a comparison group representing U.S. resident cattle, we tested blood samples that had been sent to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) for unrelated testing. Both sets of blood samples were evaluated using the same specific and broad-spectrum PCR assays. For the border cattle the overall prevalence of infection with one or more tick-borne pathogen was 58.5 % (79/135) with many co-infections, including 30 cattle positive for Babesia bovis and/or Babesia bigemina (22.2 %) and 77 cattle positive for Anaplasma marginale (57 %), three of these animals were also positive for Borrelia theileri. No resident cattle represented by the TVMDL samples were infected with either of the Babesia spp., or with Borrelia theileri, but three were positive for Theileria orientalis and 7.3 % (7/96) were positive for A. marginale. These data show that cattle originating in Mexico have a higher prevalence of infection with tick-borne pathogens relative to resident U.S. cattle and specifically, a proportion are infected with bovine Babesia, which is absent from U.S. cattle populations. Consequently, these stray cattle may be a reservoir of tick-borne pathogens and if populations of Boophilus ticks become reestablished in areas where they had previously been eradicated, could pose a significant risk to the U.S. Cattle industry.

A U.S. isolate of Theileria orientalis, Ikeda genotype, is transmitted to cattle by the invasive Asian longhorned tick, Haemaphysalis longicornis.

BACKGROUND: Theileria orientalis is a tick-borne hemoparasite that causes anemia, ill thrift, and death in cattle globally. The Ikeda strain of T. orientalis is more virulent than other strains, leading to severe clinical signs and death of up to 5% of affected animals. Within the Asia-Pacific region, where it affects 25% of Australian cattle, T. orientalis Ikeda has a significant economic impact on the cattle industry. In 2017, T. orientalis Ikeda was detected in a cattle herd in Albermarle County, Virginia, United States. Months earlier, the U.S. was alerted to the invasion of the Asian longhorned tick, Haemaphysalis longicornis, throughout the eastern U.S. Abundant H. longicornis ticks were identified on cattle in the T. orientalis-affected herd in VA, and a subset of ticks from the environment were PCR-positive for T. orientalis Ikeda. A strain of T. orientalis from a previous U.S. outbreak was not transmissible by H. longicornis; however, H. longicornis is the primary tick vector of T. orientalis Ikeda in other regions of the world. Thus, the objective of this study was to determine whether invasive H. longicornis ticks in the U.S. are competent vectors of T. orientalis Ikeda. METHODS: Nymphal H. longicornis ticks were fed on a splenectomized calf infected with the VA-U.S.-T. orientalis Ikeda strain. After molting, a subset of adult ticks from this cohort were dissected, and salivary glands assayed for T. orientalis Ikeda via qPCR. The remaining adult ticks from the group were allowed to feed on three calves. Calves were subsequently monitored for T. orientalis Ikeda infection via blood smear cytology and PCR. RESULTS: After acquisition feeding on a VA-U.S.-T. orientalis Ikeda-infected calf as nymphs, a subset of molted adult tick salivary glands tested positive by qPCR for T. orientalis Ikeda. Adult ticks from the same cohort successfully transmitted T. orientalis Ikeda to 3/3 naïve calves, each of which developed parasitemia reaching 0.4-0.9%. CONCLUSIONS: Our findings demonstrate that U.S. H. longicornis ticks are competent vectors of the VA-U.S.-T. orientalis Ikeda strain. This data provides important information for the U.S. cattle industry regarding the potential spread of this parasite and the necessity of enhanced surveillance and control measures.

Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples.

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

Randomly primed, strand-switching, MinION-based sequencing for the detection and characterization of cultured RNA viruses.

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.

Detection of atypical porcine pestivirus genome in newborn piglets affected by congenital tremor and high preweaning mortality1.

Recently, piglets from a high-health status farm began exhibiting congenital tremors, high preweaning mortality and incidence of splayed legs. Postmortem histological examination identified a small number of scattered white matter vacuoles in the cerebellum and underlying brainstem of affected piglets. Presence of potential viral sources associated with this neurologic condition was initially infirmed using quantitative PCR for atypical porcine pestivirus (APPV), porcine teschovirus, and porcine sapelovirus. Using metagenomic analysis, APPV was identified as the main microbial species in serum obtained from piglets affected by congenital tremor. These piglets had higher preweaning mortality rates (46.4% vs. 15.3%) and incidence of splayed legs (33.0% vs. 0.8 %) compared to unaffected piglets. Piglets affected by congenital tremor had higher viral titer (P < 0.15) and larger birth weights (P < 0.05) compared to normal litter mates. Whole-genome sequencing and genome assembly of the novel APPV strain (MK728876) was carried out using Oxford Nanopore and related bioinformatics pipelines. Phylogenic analysis demonstrated that this strain along with other completely sequenced APPV strains were grouped into 2 clades, both including strains-inducing congenital tremor. Strains appear to cluster based on region but there were still significant differences within regions. Future research needs to address potential underdiagnosis due to genetic diversity but also to understand mode of transmission, variation in virulence, and the role of host genetics in APPV susceptibility.

Theileria orientalis Ikeda Genotype in Cattle, Virginia, USA.

Theileria orientalis Ikeda genotype is a parasite that causes a disease in cattle that results in major economic issues in Asia, New Zealand, and Australia. The parasite is transmitted by Haemaphysalis longicornis ticks, which have recently been reported in numerous states throughout the eastern United States. Concurrently, cattle in Virginia showed clinical signs consistent with a hemoprotozoan infection. We used amplicons specific for the major piroplasm surface protein and small subunit rDNA of piroplasms to test blood samples from the cattle by PCR. Bidirectional Sanger sequencing showed sequences with 100% identity with T. orientalis Ikeda genotype 2 sequences. We detected the parasite in 3 unrelated herds and from various animals sampled at 2 time points. Although other benign T. orientalis genotypes are endemic to the United States, detection of T. orientalis Ikeda genotype might represent a risk for the cattle industry in Virginia.

Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing.

BACKGROUND: Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs. METHODS: An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow. RESULTS: For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 101 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq. CONCLUSION: The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.

Fusobacterium Genomics Using MinION and Illumina Sequencing Enables Genome Completion and Correction.

Understanding the virulence mechanisms of human pathogens from the genus Fusobacterium has been hindered by a lack of properly assembled and annotated genomes. Here we report the first complete genomes for seven Fusobacterium strains, as well as resequencing of the reference strain Fusobacterium nucleatum subsp. nucleatum ATCC 25586 (total of seven species; total of eight genomes). A highly efficient and cost-effective sequencing pipeline was achieved using sample multiplexing for short-read Illumina (150 bp) and long-read Oxford Nanopore MinION (>80 kbp) platforms, coupled with genome assembly using the open-source software Unicycler. Compared to currently available draft assemblies (previously 24 to 67 contigs), these genomes are highly accurate and consist of only one complete chromosome. We present the complete genome sequence of F. nucleatum subsp. nucleatum ATCC 23726, a genetically tractable and biomedically important strain and, in addition, reveal that the previous F. nucleatum subsp. nucleatum ATCC 25586 genome assembly contains a 452-kb genomic inversion that has been corrected using our sequencing and assembly pipeline. To enable genomic analyses by the scientific community, we concurrently used these genomes to launch FusoPortal, a repository of interactive and downloadable genomic data, genome maps, gene annotations, and protein functional analyses and classifications. In summary, this report provides detailed methods for accurately sequencing, assembling, and annotating Fusobacterium genomes, while focusing on using open-source software to foster the availability of reproducible and open data. This resource will enhance efforts to properly identify virulence proteins that may contribute to a repertoire of diseases that includes periodontitis, preterm birth, and colorectal cancer.IMPORTANCEFusobacterium spp. are Gram-negative, oral bacteria that are increasingly associated with human pathologies as diverse as periodontitis, preterm birth, and colorectal cancer. While a recent surge in F. nucleatum research has increased our understanding of this human pathogen, a lack of complete genomes has hindered the identification and characterization of associated host-pathogen virulence factors. Here we report the first eight complete Fusobacterium genomes sequenced using an Oxford Nanopore MinION and Illumina sequencing pipeline and assembled using the open-source program Unicycler. These genomes are highly accurate, and seven of the genomes represent the first complete sequences for each strain. In summary, the FusoPortal resource provides a publicly available resource that will guide future genetic, bioinformatic, and biochemical experiments to characterize this genus of emerging human pathogens.

Frontal and caudal maxillary sinus lipoma in a horse.

An adult horse was diagnosed with a frontal and caudal maxillary sinus lipoma, which was surgically removed. This is the first known report of a sinus lipoma in a horse. Lipomas should be considered in the differential diagnoses of equine sinus masses; complete surgical excision appears to be curative.

Lipome du sinus maxillaire frontal et caudal chez un cheval. Un cheval adulte a été diagnostiqué avec un lipome du sinus maxillaire frontal et caudal qui a été enlevé par chirurgie. Il s'agit du premier rapport d'un lipome de sinus chez un cheval. Les lipomes devraient être considérés dans les diagnostics différentiels des masses des sinus chez les équidés; l'excision chirurgicale complète semble être curative.(Traduit par Isabelle Vallières).

Ventricular arrhythmias in Rhodesian Ridgebacks with a family history of sudden death and results of a pedigree analysis for potential inheritance patterns.

OBJECTIVE To evaluate a group of related Rhodesian Ridgebacks with a family history of sudden death for the presence of arrhythmia and to identify possible patterns of disease inheritance among these dogs. DESIGN Prospective case series and pedigree investigation. ANIMALS 25 Rhodesian Ridgebacks with shared bloodlines. PROCEDURES Pedigrees of 4 young dogs (1 female and 3 males; age, 7 to 12 months) that died suddenly were evaluated, and owners of closely related dogs were asked to participate in the study. Dogs were evaluated by 24-hour Holter monitoring, standard ECG, echocardiography, or some combination of these to assess cardiac status. Necropsy reports, if available, were reviewed. RESULTS 31 close relatives of the 4 deceased dogs were identified. Of 21 dogs available for examination, 8 (2 males and 6 females) had ventricular tachyarrhythmias (90 to 8,700 ventricular premature complexes [VPCs]/24 h). No dogs had clinical signs of cardiac disease reported. Echocardiographic or necropsy evaluation for 7 of 12 dogs deemed affected (ie, with frequent or complex VPCs or sudden death) did not identify structural lesions. Five of 6 screened parents of affected dogs had 0 to 5 VPCs/24 h (all singlets), consistent with a normal reading. Pedigree evaluation suggested an autosomal recessive pattern of inheritance, but autosomal dominant inheritance with incomplete penetrance could not be ruled out. CONCLUSIONS AND CLINICAL RELEVANCE Holter monitoring of Rhodesian Ridgebacks with a family history of an arrhythmia or sudden death is recommended for early diagnosis of disease. An autosomal recessive pattern of inheritance in the studied dogs was likely, and inbreeding should be strongly discouraged.

Potential mechanisms of attenuation for rifampicin-passaged strains of Flavobacterium psychrophilum.

BACKGROUND: Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease in salmonids. Earlier research showed that a rifampicin-passaged strain of F. psychrophilum (CSF 259-93B.17) caused no disease in rainbow trout (Oncorhynchus mykiss, Walbaum) while inducing a protective immune response against challenge with the virulent CSF 259-93 strain. We hypothesized that rifampicin passage leads to an accumulation of genomic mutations that, by chance, reduce virulence. To assess the pattern of phenotypic and genotypic changes associated with passage, we examined proteomic, LPS and single-nucleotide polymorphism (SNP) differences for two F. psychrophilum strains (CSF 259-93 and THC 02-90) that were passaged with and without rifampicin selection. RESULTS: Rifampicin resistance was conveyed by expected mutations in rpoB, although affecting different DNA bases depending on the strain. One rifampicin-passaged CSF 259-93 strain (CR) was attenuated (4 % mortality) in challenged fish, but only accumulated eight nonsynonymous SNPs compared to the parent strain. A CSF 259-93 strain passaged without rifampicin (CN) accumulated five nonsynonymous SNPs and was partially attenuated (28 % mortality) compared to the parent strain (54.5 % mortality). In contrast, there were no significant change in fish mortalities among THC 02-90 wild-type and passaged strains, despite numerous SNPs accumulated during passage with (n = 174) and without rifampicin (n = 126). While only three missense SNPs were associated with attenuation, a Ser492Phe rpoB mutation in the CR strain may contribute to further attenuation. All strains except CR retained a gliding motility phenotype. Few proteomic differences were observed by 2D SDS-PAGE and there were no apparent changes in LPS between strains. Comparative methylome analysis of two strains (CR and TR) identified no shared methylation motifs for these two strains. CONCLUSION: Multiple genomic changes arose during passage experiments with rifampicin selection pressure. Consistent with our hypothesis, unique strain-specific mutations were detected for the fully attenuated (CR), partially attenuated (CN) and another fully attenuated strain (B17).

Transcriptional Profiling of a Cross-Protective Salmonella enterica serovar Typhimurium UK-1 dam Mutant Identifies a Set of Genes More Transcriptionally Active Compared to Wild-Type, and Stably Transcribed across Biologically Relevant Microenvironments.

Vaccination with Salmonella enterica serovar Typhimurium lacking DNA adenine methyltransferase confers cross-protective immunity against multiple Salmonella serotypes. The mechanistic basis is thought to be associated with the de-repression of genes that are tightly regulated when transiting from one microenvironment to another. This de-repression provides a potential means for the production of a more highly expressed and stable antigenic repertoire capable of inducing cross-protective immune responses. To identify genes encoding proteins that may contribute to cross-protective immunity, we used a Salmonella Typhimurium DNA adenine methyltransferase mutant strain (UK-1 dam mutant) derived from the parental UK-1 strain, and assessed the transcriptional profile of the UK-1 dam mutant and UK-1 strain grown under conditions that simulate the intestinal or endosomal microenvironments encountered during the infective process. As expected, the transcriptional profile of the UK-1 dam mutant identified a set of genes more transcriptionally active when compared directly to UK-1, and stably transcribed in biologically relevant culture conditions. Further, 22% of these genes were more highly transcribed in comparison to two other clinically-relevant Salmonella serovars. The strategy employed here helps to identify potentially conserved proteins produced by the UK-1 dam mutant that stimulate and/or modulate the development of cross-protective immune responses toward multiple Salmonella serotypes.

Safety and Immunogenicity of a Mycoplasma ovipneumoniae bacterin for domestic sheep (Ovis aries).

BACKGROUND: Mortality from epizootic pneumonia is hindering re-establishment of bighorn sheep populations in western North America. Mycoplasma ovipneumoniae, a primary agent of this disease, is frequently carried asymptomatically by the domestic sheep and goats that constitute the reservoir of this agent for transmission to bighorn sheep. Our long-term objective is to reduce the risk of M. ovipneumoniae infection of bighorn sheep; one approach to this objective is to control the pathogen in its reservoir hosts. METHODS: The safety and immunogenicity of M. ovipneumoniae for domestic sheep was evaluated in three experimental immunization protocols: 1) live M. ovipneumoniae (50 ug protein); 2) killed M. ovipneumoniae (50 ug whole cell protein) in oil adjuvant; and 3) killed M. ovipneumoniae (250 ug whole cell protein) in oil adjuvant. Immunogenicity was assessed by two serum antibody measures: competitive enzyme-linked immunosorbent assay (cELISA) (experiments 1-3) and serum growth inhibition (Experiment 3). Passive immunogenicity was also assessed in the third experiment using the same assays applied to blood samples obtained from the lambs of immunized ewes. RESULTS AND CONCLUSIONS: Adverse reactions to immunization were generally minor, but local reactions were regularly observed at immunization sites with bacterins in oil adjuvants. No evidence of M. ovipneumoniae specific antibody responses were observed in the first or second experiments and no resistance to colonization was observed in the first experiment. However, the ewes in the third experiment developed strong cELISA serum antibody responses and significant serum M. ovipneumoniae inhibition activity, and these responses were passively transferred to their lambs. The results of these trials indicate that immunization with relatively large antigenic mass combined with an adjuvant is capable of inducing strong active antibody responses in ewes and passively immunizing lambs.

Regional immune response to immunization with Escherichia coli O157:H7-derived intimin in cattle.

Escherichia coli O157:H7 is an enteric pathogen of animals and humans that can result in deadly sequelae. Cattle are asymptomatic carriers and shedders of the bacteria and serve as an important reservoir of human infection. E. coli O157:H7 colonizes the gastrointestinal tract, most frequently at the rectoanal junction mucosa in cattle. Vaccination is a potentially highly effective means of decreasing cattle colonization and shedding and thereby decreasing human infections. Currently available vaccines are administered subcutaneously or intramuscularly, and immune responses have been evaluated solely by systemic immunoglobulin responses. This study evaluated local and systemic lymphoproliferative responses in addition to immunoglobulin responses following subcutaneous or mucosal (rectal) immunization with E. coli O157:H7 outer membrane protein intimin over three trials. In all three trials, significant local and systemic lymphoproliferative responses (P < 0.05) occurred following immunization in the majority of animals, as well as significant immunoglobulin responses (P < 0.001) in all animals. Surprisingly, local responses in the mesorectal lymph nodes were very similar between the subcutaneous and mucosal immunization groups. Moreover, the responses in mesorectal lymph nodes appeared targeted rather than generalized, as minimal or no significant responses were observed in the associated prescapular lymph nodes of subcutaneously immunized animals. The results indicate that both subcutaneous and mucosal immunizations are effective methods of inducing immune responses against E. coli O157:H7 in cattle.