A reduced potential for lameness bacterial transmission by Lucilia sericata larvae and flies through metamorphosis.

Lameness in sheep is one of the most serious issues on farms in the UK and worldwide, affecting over 90% of all UK sheep flocks. Despite its severity and prevalence, there are knowledge gaps regarding transmission routes of bacterial pathogens associated with infectious lameness in sheep. As larvae of Lucilia sericata are commonly found on foot lesions on lame sheep, it was hypothesised that the flies or their larvae could harbour lameness associated bacteria. This study examined the gut contents of larvae obtained from the foot lesions of lame sheep and compared them to control larvae collected from infested cat food on the same farm. Of particular interest, were the presence of three different bacterial genera associated with lameness; Fusobacterium necrophorum, Dichelobacter nodosus and Treponema spp., for which viability was also investigated. Larvae were cultured In vitro and some allowed to metamorphose into flies before specific PCR assays were carried out on the gut contents. Results showed a significant association between the bacteria on the feet of the sheep and those within the larvae. Although the gut contents of all larvae found on sheep feet contained one or more of the lameness bacteria, none of the bacteria were recovered from the adult flies, suggesting a level of gut remodelling during metamorphosis. Interestingly, Treponema spp. and Fusobacterium spp. were viable when isolated from gut contents of larvae. Maintenance of infection from larvae to fly did not occur. However, it still remains important to control both disease and insect populations of farms to maintain animal welfare.

Epidemiology of protozoan and helminthic parasites in wild passerine birds of Britain and Ireland.

Avian endoparasites play important roles in conservation, biodiversity and host evolution. Currently, little is known about the epidemiology of intestinal helminths and protozoans infecting wild birds of Britain and Ireland. This study aimed to determine the rates of parasite prevalence, abundance and infection intensity in wild passerines. Fecal samples (n = 755) from 18 bird families were collected from 13 sites across England, Wales and Ireland from March 2020 to June 2021. A conventional sodium nitrate flotation method allowed morphological identification and abundance estimation of eggs/oocysts. Associations with host family and age were examined alongside spatiotemporal and ecological factors using Bayesian phylogenetically controlled models. Parasites were detected in 20.0% of samples, with corvids and finches having the highest prevalences and intensities, respectively. Syngamus (33%) and Isospora (32%) were the most prevalent genera observed. Parasite prevalence and abundance differed amongst avian families and seasons, while infection intensity varied between families and regions. Prevalence was affected by diet diversity, while abundance differed by host age and habitat diversity. Infection intensity was higher in birds using a wider range of habitats, and doubled in areas with feeders present. The elucidation of these patterns will increase the understanding of parasite fauna in British and Irish birds.

Contagious Ovine Digital Dermatitis: A Novel Bacterial Etiology and Lesion Pathogenesis.

Contagious ovine digital dermatitis (CODD) is a severe and common infectious foot disease of sheep and a significant animal welfare issue for the sheep industry in the UK and some European countries. The etiology and pathogenesis of the disease are incompletely understood. In this longitudinal, experimental study, CODD was induced in 18 sheep, and for the first time, the clinical lesion development and associated microbiological changes in CODD affected feet are described over time, resulting in a completely new understanding of the etiopathogenesis of CODD. The majority of CODD lesions (83.9%) arose from pre-existing interdigital dermatitis (ID) and/or footrot (FR) lesions. All stages of foot disease were associated with high levels of poly-bacterial colonization with five pathogens, which were detected by quantitative PCR (qPCR): Treponema medium, Treponema phagedenis, Treponema pedis, Dichelobacter nodosus, and Fusobacterium necrophorum. Temporal colonization patterns showed a trend for early colonization by T. phagedenis, followed by F. necrophorum and D. nodosus, T. medium, and then T. pedis, D. nodosus was present at significantly higher predicted mean log10 genome copy numbers in FR lesions compared to both ID and CODD, while Treponema species were significantly higher in CODD and FR lesions compared to ID lesions (p < 0.001). Treatment of CODD-affected sheep with two doses of 10 mg/kg long acting amoxicillin resulted in a 91.7% clinical cure rate by 3 weeks post-treatment; however, a bacteriological cure was not established for all CODD-affected feet. The study found that in an infected flock, healthy feet, healed CODD feet, and treated CODD feet can be colonized by some or all of the five pathogens associated with CODD and therefore could be a source of continued infection in flocks. The study is an experimental study, and the findings require validation in field CODD cases. However, it does provide a new understanding of the etiopathogenesis of CODD and further supportive evidence for the importance of current advice on the control of CODD; namely, ensuring optimum flock control of footrot and prompt isolation and effective treatment of clinical cases.

A survey of sheep dicrocoeliosis in Sulaymaniyah slaughterhouse, northern Iraq in 2013-2014.

Dicrocoelium dendriticum is a helminth which lives in the bile ducts and gall bladder of mammalian definitive hosts. Dicrocoeliosis is thought to be endemic in some countries and it has an increasing prevalence in Northern Iraq, potentially due to increased importation of infected animals, particularly sheep, in high numbers from neighboring countries. The parasite's ability to infect rodents, wild animals, livestock and humans means this parasite is of significant interest in veterinary and human medicine. While D. dendriticum causes relatively mild clinical disease in animals, infection leads to liver condemnation at slaughter and subsequent economic losses to farmers. In this study, the livers of 91,486 sheep slaughtered at Sulaymaniyah New Slaughterhouse (Northern Iraq) were visually inspected for D. dendriticum infection between November 2013 and March 2014, with 1269 livers rejected due to D. dendriticum in this period, representing 29.4% of all condemned livers. The highest rate of rejection due to D. dendriticum infection was seen in December, possibly linked to increased numbers of intermediate host snails during the wet season. Routine inspection of condemned livers revealed adult flukes of D. dendriticum and eggs were seen in the faeces of imported sheep, indicating these animals may be a vector for introduction of D. dendriticum to this region. Due to the complex life cycle and wide range of animals which it infects, we cannot establish the exact route of introduction into Iraq; however, our study suggests that slaughterhouse workers, farmers and local health authorities should be aware of the presence of D. dendriticum, and the potential risks it represents to both human and animal health. Our data also suggest that some level of quarantine or border checks may be useful to prevent further introduction of D. dendriticum or other pathogens into Iraq, although this may prove difficult until accurate diagnostic assays are developed.

Dissecting the molecular diversity and commonality of bovine and human treponemes identifies key survival and adhesion mechanisms.

Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention.

Multi-locus sequence typing of Ixodes ricinus and its symbiont Candidatus Midichloria mitochondrii across Europe reveals evidence of local co-cladogenesis in Scotland.

Ticks have relatively complex microbiomes, but only a small proportion of the bacterial symbionts recorded from ticks are vertically transmitted. Moreover, co-cladogenesis between ticks and their symbionts, indicating an intimate relationship over evolutionary history driven by a mutualistic association, is the exception rather than the rule. One of the most widespread tick symbionts is Candidatus Midichloria, which has been detected in all of the major tick genera of medical and veterinary importance. In some species of Ixodes, such as the sheep tick Ixodes ricinus (infected with Candidatus Midichloria mitochondrii), the symbiont is fixed in wild adult female ticks, suggesting an obligate mutualism. However, almost no information is available on genetic variation in Candidatus M. mitochondrii or possible co-cladogenesis with its host across its geographic range. Here, we report the first survey of Candidatus M. mitochondrii in I. ricinus in Great Britain and a multi-locus sequence typing (MLST) analysis of tick and symbiont between British ticks and those collected in continental Europe. We show that while the prevalence of the symbiont in nymphs collected in England is similar to that reported from the continent, a higher prevalence in nymphs and adult males is apparent in Wales. In general, Candidatus M. mitochondrii exhibits very low levels of sequence diversity, although a consistent signal of host-symbiont coevolution was apparent in Scotland. Moreover, the tick MLST scheme revealed that Scottish specimens form a clade that is partially separated from other British ticks, with almost no contribution of continental sequence types in this north-westerly border of the tick's natural range. The low diversity of Candidatus M. mitochondrii, in contrast with previously reported high rates of polymorphism in I. ricinus mitogenomes, suggests that the symbiont may have swept across Europe recently via a horizontal, rather than vertical, transmission route.

Treponema rectale sp. nov., a spirochete isolated from the bovine rectum.

A Gram-stain-negative, obligatory anaerobic spirochete, CHPAT, was isolated from the rectal tissue of a Holstein-Friesian cow. On the basis of 16S rRNA gene comparisons, CHPAT was most closely related to the human oral spirochete, Treponema parvum, with 88.8 % sequence identity. Further characterisation on the basis of recA gene sequence analysis, cell morphology, pattern of growth and physiological profiling identified marked differences with respect to other recognised species of the genus Treponema. Microscopically, the helical cells measured approximately 1-5 µm long and 0.15-0.25 µm wide, with two to five irregular spirals. Transmission electron microscopy identified four periplasmic flagella in a 2 : 4 : 2 arrangement. CHPAT grew independently of serum, demonstrated no evidence of haemolytic activity and possessed an in vitro enzyme activity profile that is unique amongst validly named species of the genus Treponema, exhibiting C4 esterase, α-galactosidase and ß-galactosidase activity. Taken together, these data indicate that CHPAT represents a novel species of the genus Treponema, for which the name Treponema rectale is proposed. The type strain of Treponema rectale is CHPAT (=DSM 103679T=NCTC 13848T).

Treponema ruminis sp. nov., a spirochaete isolated from the bovine rumen.

A novel bacterium, strain Ru1T, was encountered during a survey of spirochaetes living in the gastrointestinal tract of ruminants. Comparative analysis of 16S rRNA gene sequence data indicated that strain Ru1T clustered within the genus Treponemabut shared at most 86.1 % sequence similarity with other recognised species of the genus Treponema. Further phylogenetic analysis based on partial recombinase A (recA) gene sequence comparisons, together with phenotypic characterization, also demonstrated the divergence of strain Ru1T from other recognised species of the genus Treponema. Microscopically, strain Ru1T appeared as a very small, highly motile, helical spirochaete with eight periplasmic flagella in a 4 : 8 : 4 arrangement. It exhibited C8 esterase lipase, leucine arylamidase, ß-galactosidase and ß-glucosidase activity. A distinctive, serum-independent growth pattern was also observed, characterized by colonies with an absence of the local haemolysis that is typical of many pathogenic treponemes. On the basis of these data, strain Ru1T is considered to represent a novel species of the genus Treponema, for which the name Treponema ruminis sp. nov. is proposed. The type strain is Ru1T (=DSM 103462T=NCTC 13847T).

Multilocus Sequence Typing of Pathogenic Treponemes Isolated from Cloven-Hoofed Animals and Comparison to Treponemes Isolated from Humans.

UNLABELLED: Treponema species are implicated in many diseases of humans and animals. Digital dermatitis (DD) treponemes are reported to cause severe lesions in cattle, sheep, pigs, goats, and wild elk, causing substantial global animal welfare issues and economic losses. The fastidiousness of these spirochetes has previously precluded studies investigating within-phylogroup genetic diversity. An archive of treponemes that we isolated enabled multilocus sequence typing to quantify the diversity and population structure of DD treponemes. Isolates (n = 121) were obtained from different animal hosts in nine countries on three continents. The analyses herein of currently isolated DD treponemes at seven housekeeping gene loci confirm the classification of the three previously designated phylogroups: the Treponema medium, Treponema phagedenis, and Treponema pedis phylogroups. Sequence analysis of seven DD treponeme housekeeping genes revealed a generally low level of diversity among the strains within each phylogroup, removing the need for the previously used "-like" suffix. Surprisingly, all isolates within each phylogroup clustered together, regardless of host or geographic origin, suggesting that the same sequence types (STs) can infect different animals. Some STs were derived from multiple animals from the same farm, highlighting probable within-farm transmissions. Several STs infected multiple hosts from similar geographic regions, identifying probable frequent between-host transmissions. Interestingly, T. pedis appears to be evolving more quickly than the T. medium or T. phagedenis DD treponeme phylogroup, by forming two unique ST complexes. The lack of phylogenetic discrimination between treponemes isolated from different hosts or geographic regions substantially contrasts with the data for other clinically relevant spirochetes. IMPORTANCE: The recent expansion of the host range of digital dermatitis (DD) treponemes from cattle to sheep, goats, pigs, and wild elk, coupled with the high level of 16S rRNA gene sequence similarity across hosts and with human treponemes, suggests that the same bacterial species can cause disease in multiple different hosts. This multilocus sequence typing (MLST) study further demonstrates that these bacteria isolated from different hosts are indeed very similar, raising the potential for cross-species transmission. The study also shows that infection spread occurs frequently, both locally and globally, suggesting transmission by routes other than animal-animal transmission alone. These results indicate that on-farm biosecurity is important for controlling disease spread in domesticated species. Continued surveillance and vigilance are important for ascertaining the evolution and tracking any further host range expansion of these important pathogens.

Detection and isolation of digital dermatitis treponemes from skin and tail lesions in pigs.

Pig skin lesions are common significant welfare issues, and can cause large economic losses, due to culling of severely affected animals or carcass condemnation at slaughter. It was considered that the treponemal bacteria associated with digital dermatitis (DD) lesions in cattle, sheep and goats may have a role in these pig lesions. Specific diagnostic PCR assays for three cultivable DD Treponema phylogroups were used to survey relevant porcine lesion samples. Using these assays, DD treponemes were detected in 88% (22/25), 72% (8/11) and 82% (14/17) of tail, ear and flank lesions, respectively. Mouth swabs from animals kept in enclosures with high prevalence of skin lesions were positive for the DD treponemes, but not in enclosures with low lesion prevalence. Culture of treponemes from skin lesions resulted in pure isolates of all three DD-associated phylogroups. This study shows a strong association of DD treponemes with a range of pig skin lesions.

Isolation of digital dermatitis treponemes from cattle hock skin lesions.

BACKGROUND: Bovine hock lesions present a serious welfare and production issue on dairy farms worldwide. Current theories suggest that trauma is an important factor in the formation of hock lesions, although infection may also play a role in increasing their severity and duration. HYPOTHESIS: Digital dermatitis (DD) lesions in dairy cows are strongly associated with specific treponeme bacteria which are opportunistic invaders of other skin regions. Hock lesions were tested to ascertain if they too contained treponemes. ANIMALS: Swab and tissue samples were taken from hock lesions from two farms in South West England. METHODS: Hock lesions were classified into two categories: open lesions, which were often bleeding and ulcerated, or were encrusted; and closed lesions, which were classified as hair loss with no skin breakage. PCR assays and bacterial isolation were used to detect treponemes in hock lesions. RESULTS: All three phylogroups of digital dermatitis treponemes were detectable and isolated from open hock lesions only, with closed lesions showing no evidence of treponeme infection, either by PCR or bacterial culture. When analysed by 16S rRNA gene sequencing, the cultured treponeme DNA showed complete homology or was very similar to that found in foot lesions. Additionally, skin swabs from near the open hock wounds were also positive by PCR assay and isolation for the DD treponemes. CONCLUSIONS AND CLINICAL IMPORTANCE: Identification of the contribution of these infectious agents will allow for more optimal treatments to be developed that reduce the prevalence and healing times of both hock and DD lesions.

In vitro susceptibility of contagious ovine digital dermatitis associated Treponema spp. isolates to antimicrobial agents in the UK.

BACKGROUND: Contagious ovine digital dermatitis (CODD) is an important cause of infectious lameness in sheep in the UK and Ireland and has a severe impact on the welfare of affected individuals. The three treponemal phylogroups Treponema medium/Treponema vincentii-like, Treponema phagedenis-like and Treponema pedis spirochaetes have been associated with clinical CODD lesions and are considered to be a necessary cause of disease. There are scant data on the antimicrobial susceptibility of the treponemes cultured from CODD lesions. OBJECTIVE: The aim of this study was to determine in vitro the miniumum inhibitory concentration/ minimum bactericidal concentration (MIC/MBC) of antimicrobials used in the sheep industry for isolates of the three CODD associated treponeme phylogroups T. medium/T. vincentii-like, T. phagedenis-like and T. pedis. ANIMALS: Twenty treponeme isolates; from 19 sheep with clinical CODD lesions. METHODS: A microdilution method was used to determine in vitro the MIC/MBC of 10 antimicrobial agents for 20 treponeme isolates (five T. medium/T. vincentii-like, 10 T. phagedenis-like and five T. pedis). The antimicrobials tested were penicillin G, amoxicillin, oxytetracycline, tilmicosin, lincomycin, spectinomycin, tylosin, tildipirosin, tulathromycin and gamithromycin. RESULTS: The treponeme isolates tested showed low MICs and MBCs to all 10 antimicrobials tested. They were most susceptible to gamithromycin and tildipirosin (MIC90: 0.0469 mg/L), and were least susceptible to lincomycin, spectinomycin and oxytetracycline (MIC90: 48 mg/L, 24 mg/L and 3 mg/L, respectively). CONCLUSIONS: These data are comparable to in vitro antimicrobial susceptibility data for treponemes cultured from bovine digital dermatitis lesions. Dependent on local licensing, penicillin and tilmicosin appear to be the best candidates for future in vivo studies.