High-throughput rapid amplicon sequencing for multilocus sequence typing of Mycoplasma ovipneumoniae from archived clinical DNA samples

IntroductionSpillover events of Mycoplasma ovipneumoniae have devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread of M. ovipneumoniae between the sheep populations. Most studies involving the typing of M. ovipneumoniae have used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing.MethodsOur study aimed to develop and validate an MLST workflow for typing of M. ovipneumoniae using Nanopore Rapid Barcoding sequencing and multiplex polymerase chain reaction (PCR). We compare the workflow with Nanopore Native Barcoding library preparation and Illumina MiSeq amplicon protocols to determine the most accurate and cost-effective method for sequencing multiplex amplicons. A multiplex PCR was optimized for four housekeeping genes of M. ovipneumoniae using archived DNA samples (N = 68) from nasal swabs.ResultsSequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time and lowest cost per sample when compared with Nanopore Native Barcoding and Illumina MiSeq methods.DiscussionOur proposed workflow is a convenient and effective method for strain typing of M. ovipneumoniae and can be applied to other bacterial MLST schemes. The workflow is suitable for diagnostic settings, where reduced hands-on time, cost, and multiplexing capabilities are important

Novel inflammatory cell infiltration scoring system to investigate healthy and footrot affected ovine interdigital skin

Ovine footrot is a degenerative disease of sheep feet leading to the separation of hoofhorn from the underlying skin and lameness. This study quantitatively examined histological features of the ovine interdigital skin as well as their relationship with pro-inflammatory cytokine (IL-1_) and virulent Dichelobacter nodosus in footrot. From 55 healthy and 30 footrot ovine feet, parallel biopsies (one fixed for histology) were collected post-slaughter and analysed for lesions and histopathological analysis using haematoxylin and eosin and Periodic Acid-Schiff. Histological lesions were similar in both conditions while inflammatory scores mirror IL-1_ expression levels. Increased inflammatory score corresponded with high virulent D. nodosus load and was significant (

The applied development of a tiered multilocus sequence typing (MLST) scheme for Dichelobacter nodosus

Dichelobacter nodosus (D. nodosus) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. Thescheme is publically available at https://pubmlst.org/dnodosus/

A new bovine conjunctiva model shows that Listeria monocytogenes invasion is associated with lysozyme resistance.

AbstractListerial keratoconjunctivitis (‘silage eye’) is a wide spread problem in ruminants causing economic losses to farmers and impacts negatively on animal welfare. It results from direct entry of Listeria monocytogenes into the eye, often following consumption of contaminated silage. An isolation protocol for bovine conjunctival swabbing was developed and used to sample both infected and healthy eyes bovine eyes (n=46). L. monocytogenes was only isolated from one healthy eye sample, and suggests that this organism can be present without causing disease. To initiate a study of this disease, an infection model was developed using isolated conjunctiva explants obtained from cattle eyes post slaughter. Conjunctiva were cultured and infected for 20h with a range of L. monocytogenes isolates (n=11), including the healthy bovine eye isolate and also strains isolated from other bovine sources, such as milk or clinical infections. Two L. monocytogenes isolates (one from a healthy eye and one from a cattle abortion) were markedly less able to invade conjunctiva explants, but one of those was able to efficiently infect Caco2 cells indicating that it was fully virulent. These two isolates were also significantly more sensitive to lysozyme compared to most other isolates tested, suggesting that lysozyme resistance is an important factor when infecting bovine conjunctiva. In conclusion, we present the first bovine conjunctiva explant model for infection studies and demonstrate that clinical L. monocytogenes isolates from cases of bovine keratoconjunctivitis are able to infect these tissues

RNA expression of TLR10 in normal equine tissues

Background: Toll like receptors are one of the major innate immune system pathogen recognition systems. There is little data on the expression of the TLR10 member of this family in the horse. Results: This paper describes the genetic structure of the Equine TLR10 gene and its RNA expression in a range of horse tissues. It describes the phylogenetic analysis of the Equine TLR1,6,10,2 annotations in the horse genome, firmly identifying them in their corresponding gene clades compared to other species and firmly placing the horse gene with other TLR10 genes from odd-toed ungulates. Additional 3’ transcript extensions to that annotated for TLR10 in the horse genome have been identified by analysis of RNAseq data. RNA expression of the equine TLR10 gene was highest in peripheral blood mononucleocytes and lymphoid tissue (lymph nodes and spleen), however some expression was detected in all tissues tested (jejunum, caudal mesenteric lymph nodes, bronchial lymph node, spleen, lung, colon, kidney and liver). Additional data on RNAseq expression of all equine TLR genes (1–4 and 6–10) demonstrate higher expression of TLR4 than other equine TLRs in all tissues. Conclusion: The equine TLR10 gene displays significant homology to other mammalian TLR10 genes and could be reasonably assumed to have similar fuctions. Its RNA level expression is higher in resting state PBMCs in horses than in other tissues

Defining the mechanisms of inflammation that underlie the pathogenesis of ovine footrot

Ovine footrot is a bacterial infection of the ovine interdigital skin with a significant welfare and economical concern for the sheep industry globally. Footrot is a major cause of lameness in sheep and it is characterised by two different clinical presentations, interdigital dermatitis (ID) and under-running footrot. The aetiology of this disease is complex with Dichelobacter nodosus as the essential pathogen initiating under-running footrot lesions, however the role of other bacteria, including Fusobacterium necrophorum and Treponema spp., and the roles of other bacterial populations in healthy, ID and footrot feet remain unclear. The severity of footrot is thought to be exacerbated by the intense inflammatory response against the infection; nevertheless, there is little information in the literature regarding the ovine immune response to this disease. In this context, the hypothesis of this study is that the pathology of footrot is a host mediated expression of local immune responses, in association with bacterial colonisation, leading to severe inflammation that can progress to under-running lesions. This hypothesis was investigated through the determination of bacterial prevalence and load using qPCR and host gene expression studies using RT-qPCR and RNA sequencing. Moreover, a 3D skin explant model was developed for in vitro infection studies. Key findings include that the highest prevalence and load of D. nodosus was on feet with ID and the vast majority of samples from UK sheep contained virulent D. nodosus strains; notably, the more pathogenic subspecies of F. necrophorum was found in these samples. In addition, a comparison of bacterial colonisation on the ovine interdigital skin surface and within the skin demonstrated that bacterial prevalence and load differed between those two locations. Gene expression analysis revealed that inflammation, as marked by mRNA expression levels of CXCL8 and IL1β, central mediators of immune response in the skin, were associated with virulent D. nodosus in footrot affected feet. This suggests that D. nodosus colonisation may impact on the inflammatory mechanisms involved in footrot. The global gene expression study (RNA sequencing) showed 34 significantly expressed genes in footrot samples indicating upregulation of the local inflammatory and innate immune responses, whilst little association of the local adaptive immune response. A 3D skin model culture was developed and infection with D. nodosus strains for 28h was demonstrated by qPCR and FISH and resulted in the release of IL1β and CXCL8 in the culture media. Hence, this confirms a role for IL1β and CXCL8 in the early local response to D. nodosus infection. Taken together, the data presented in this thesis may potentially endorse our hypothesis that the separation of the hoof horn from the sensitive underlying tissue is led by the host expression of local immune responses in association with bacterial colonisation

Defining the mechanisms of inflammation that underlie the pathogenesis of ovine footrot

Ovine footrot is a bacterial infection of the ovine interdigital skin with a significant welfare and economical concern for the sheep industry globally. Footrot is a major cause of lameness in sheep and it is characterised by two different clinical presentations, interdigital dermatitis (ID) and under-running footrot. The aetiology of this disease is complex with Dichelobacter nodosus as the essential pathogen initiating under-running footrot lesions, however the role of other bacteria, including Fusobacterium necrophorum and Treponema spp., and the roles of other bacterial populations in healthy, ID and footrot feet remain unclear. The severity of footrot is thought to be exacerbated by the intense inflammatory response against the infection; nevertheless, there is little information in the literature regarding the ovine immune response to this disease. In this context, the hypothesis of this study is that the pathology of footrot is a host mediated expression of local immune responses, in association with bacterial colonisation, leading to severe inflammation that can progress to under-running lesions. This hypothesis was investigated through the determination of bacterial prevalence and load using qPCR and host gene expression studies using RT-qPCR and RNA sequencing. Moreover, a 3D skin explant model was developed for in vitro infection studies. Key findings include that the highest prevalence and load of D. nodosus was on feet with ID and the vast majority of samples from UK sheep contained virulent D. nodosus strains; notably, the more pathogenic subspecies of F. necrophorum was found in these samples. In addition, a comparison of bacterial colonisation on the ovine interdigital skin surface and within the skin demonstrated that bacterial prevalence and load differed between those two locations. Gene expression analysis revealed that inflammation, as marked by mRNA expression levels of CXCL8 and IL1β, central mediators of immune response in the skin, were associated with virulent D. nodosus in footrot affected feet. This suggests that D. nodosus colonisation may impact on the inflammatory mechanisms involved in footrot. The global gene expression study (RNA sequencing) showed 34 significantly expressed genes in footrot samples indicating upregulation of the local inflammatory and innate immune responses, whilst little association of the local adaptive immune response. A 3D skin model culture was developed and infection with D. nodosus strains for 28h was demonstrated by qPCR and FISH and resulted in the release of IL1β and CXCL8 in the culture media. Hence, this confirms a role for IL1β and CXCL8 in the early local response to D. nodosus infection. Taken together, the data presented in this thesis may potentially endorse our hypothesis that the separation of the hoof horn from the sensitive underlying tissue is led by the host expression of local immune responses in association with bacterial colonisation

Canine infectious respiratory disease: New insights into the etiology and epidemiology of associated pathogens.

Canine infectious respiratory disease (CIRD) is a syndrome where multiple viral and bacterial pathogens are involved sequentially or synergistically to cause illness. There is limited information regarding the prevalence of pathogens related to CIRD in the United States as well as the role of co-infections in the pathogenesis of the syndrome. We aimed to conduct a comprehensive etiologic and epidemiologic study of multiple CIRD agents in a diverse dog population using molecular methods and statistical modeling analyses. In addition, a novel probe-based multiplex real-time PCR was developed to simultaneously detect and differentiate two species of Mycoplasma (M. canis and M. cynos). Canine adenovirus, canine distemper virus, canine parainfluenza virus, coronavirus, influenza A virus (H3N2 and H3N8), Bordetella bronchiseptica, M. canis, M. cynos and Streptococcus equi subsp. zooepidemicus were investigated in specimens from clinically ill and asymptomatic dogs received at the Athens Veterinary Diagnostic Laboratory. Results showed low occurrence of classical CIRD agents such as B. bronchiseptica, canine adenovirus and distemper virus, while highlighting the potential role of emerging bacteria such as M. canis and M. cynos. Statistical modeling analyses of CIRD pathogens emphasized the impact of co-infections on the severity of clinical presentation, and showed that host factors, such as animal age, are the most important predictors of disease severity. This study provides new insights into the current understanding of the prevalence and role of co-infections with selected viruses and bacteria in the etiology of CIRD, while underscoring the importance of molecular diagnosis and vaccination against this disease