STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is SEQUENCE_LISTING_450120_401USPC.txt. The text file is 60.3 KB, was created on May 15, 2020, and is being submitted electronically via EFS-Web.

FIELD OF THE INVENTION

This invention relates to the diagnosis and prevention or treatment of ungulate diseases where treponeme spirochetes bacteria are involved, in particular for the prophylactic treatment of digital dermatitis in a mammal, more particularly for bovine digital dermatitis. In addition, the present invention is directed to developing immunogenic compositions, diagnostic, therapeutic and making and administering a vaccine against digital dermatitis in a mammal.

BACKGROUND OF THE INVENTION

Digital dermatitis (DD) is a bacterial disease that primarily affects the skin on the heels of cattle. Infection causes inflammation and skin damage, leading to pain and discomfort. It is a major cause of lameness in cattle and most commonly seen in intensive dairy cows. Hence it is a significant problem for the dairy industry in many countries, causing reduced animal welfare and economic loss. DD has been identified as an emerging issue in beef cattle in the UK and the bacteria believed to cause DD have been identified in similar lesions in sheep (known as Contagious Ovine Digital Dermatitis), dairy goats and even wild North American Elk. In addition to this, these DD associated bacteria have been detected in three types of severe bovine foot lesions which have emerged during the last 15 years; toe necrosis, non-healing white line disease and non-healing sole ulcer. These developments highlight the growing importance of DD for domestic and wild animals, and for farmers and veterinarians.

Despite the economic and welfare importance of this disease, many questions remain regarding its etiology, transmission, prevention and treatment. There are a number of reasons why the disease is proving difficult to deal with; firstly, the infection appears to be poly-microbial, with a variety of bacteria, particularly of the genusTreponema, isolated from lesions. In addition, most of bacteria involved in DD initially proved difficult to grow in culture, experimental infection models have been difficult to develop, and the mechanisms of disease transmission have thus remained rather mysterious as well as the high recurrence rate in antibiotic cured cases. Recent advances in laboratory methods have provided some progress in the identification of the most important pathogenic bacteria, and in the detection of these bacteria in the animals and in the environment of farms with endemic DD.

A wide range of infection levels has been found on infected farms, prompting investigations into both farm/herd level risk factors and animal level risk factors (for example parity and stage of lactation) for DD occurrence. Both the farm level and animal level risk factors can provide useful information when trying to minimize DD infection levels and understand when risks of infection are highest. An interesting, but less investigated, aspect of DD is that there appears to be individual variation between animals in susceptibility to the disease. In this sense, several scientific publications forming part of the prior art all found that some animals within a herd were infected repeatedly while others of the same breed and parity and kept under the same conditions were never infected. If the reasons for this individual variation in susceptibility could be identified, they could add to our understanding of the disease and contribute to the search for effective prevention and treatment methods.

Determination of treponeme types or species associated with DD lesions has been based on DNA sequence analysis and classification. Evans et al., (Evans N. J., Brown J. M., Demirkan I., Murray R. D., Vink W. D., Blowey R. W., Hart C. A., Carter S. D. Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle. Vet. Microbiol. 2008; 130:141-150), established the three most common phylotypes,T. vincentii/T. medium-like,T. phagedenis-like andT. denticola/T. putidum-like, clustered on 16S rDNA homology and flaB2 homology. Phylotypes (PT) are defined as clusters of treponemes in which the 16S rDNA sequence differs by ˜2% from known species and which are ≥99% similar to other members of their cluster (Klitgaard K., Foix Breto A., Boye M., Jensen T. K. Targeting the treponemal microbiome of digital dermatitis infections by high-resolution phylogenetic analyses and comparison with fluorescent in situ hybridization. J. Clin. Microbiol. 2013; 51:2212-2219). Others have expanded the number of phylotypes up to seven includingT. brennaborense, T. maltophilum-like (includingT. maltophilumandT. lecithinolyticum),T. refringens/T. calligyrum-like, withT. pedisclustering withT. denticola/T. putidum.

Many bacteria of different genera other thanTreponemaspp., such asFusobacterium necrophorum, Dichelobacter nodosus, Prevotellaspp. andPorphyromonasspp. have also been isolated from DD lesions.

Dichelobacter nodosus, formerly known asBacteroides, is a pathogenic, anaerobic, non-spore-forming Gram-negative bacteria. It has been detected in a number of DD lesions from different geographic locations. The findings ofD. nodosusin lesions suggest thatD. nodosushas a role in the pathogenesis of DD. It is also recognized as the primary agent involved in footrot in sheep.

Fusobacterium necrophorumis also a cause for lameness in cattle, sheep and other ruminants. It is an anaerobic bacteria. It has been mainly detected in the superficial keratinolyzed layers of the epidermis. It is suggested to be a secondary invader in DD lesions and footrot.

Several virulence factors have been described for pathogens involved in DD. For example, Nielsen M. W. et al., 2016 detectedTreponemaspp. virulence factors such as adhesins, surface antigens and proteins involved in motility. OtherTreponemaspp. virulence factors described are peptidases, proteases and haemolysins. Same type of virulence factors are described for other DD-associated pathogens, such asD. nodosus(Kennan, R. M., Dhungyel, O. P., Whittington, R. J., Egerton, J. R., & Rood, J. I. The Type IV Fimbrial Subunit Gene (fimA) ofDichelobacter nodosusIs Essential for Virulence, Protease Secretion, and Natural Competence.Journal of bacteriology,2001, 183.15: 4451-4458).

Treatments for DD include systemic and topical antibiotics. In herds where a high proportion of animals are infected with DD individual treatment is very time-consuming, so many farmers use instead footbaths to treat the entire herd. Unfortunately, elimination of DD is rarely seen, so repeated application of treatments is required to prevent recurrence of infection. An antibody response is produced by dairy cows when they are infected with DD, however this response does not seem sufficient to prevent further infections, as some animals are infected repeatedly. One reason suggested for the difficulties encountered when trying to treat DD and prevent recurrence is the fact that a number of the treponemes thought to be involved in DD have been shown to have encysted as well as spiral forms. It is possible that these encysted forms of the bacteria could persist deep within the lesions and cause a recurrence of clinical disease at a later date, though more research is required to determine the significance of the encysted form of the bacteria and its response to DD treatments.

There have been a number of efforts over the last years to develop a vaccine against DD, however none of these vaccines are currently available. In this sense, Berry et al. (Berry S L, Ertze R A, Read D H, Hird D W. Field evaluation of prophylactic and therapeutic effects of a vaccine against (papillomatous) digital dermatitis of dairy cattle in 2 California dairies. Proc 16th Intl Symp Lameness in Ruminants, Slovenia, 2004) reported a study in two dairy herds in Nebraska where a differentTreponemabacterin marketed as TrepShield® (Novartis Animal Health) was tested. The study concluded that theTreponemabacterin did not provide significant phophylactic or therapeutic effects in vaccinated animals. The vaccine (TrepShield®) has since been withdrawn.

Fiddler et al. (Fidler A. P., Alley M. L., Smith G. W. Evaluation of aSerpensspecies bacterin for treatment of digital dermatitis in dairy cattle. Res. Vet. Sci. 2012; 93:1258-1260. doi: 10.1016/j.rvsc.2012.07.002) reported the findings of a study which evaluated a vaccine containingSerpenssp. bacterin, developed by a commercial company. They found that, although the dairy cows involved in the trial did develop an immune response to the bacterin, there was no reduction in the prevalence or severity of DD infections among vaccinated cows when compared to controls. They therefore concluded that the vaccine did not show any clinical efficacy in terms of DD prevention.

In contrast and for the first time, the present invention provides new vaccine compositions comprisingTreponemaspp. bacterins, preferably supplemented with antigens fromTreponemaspp. or other digital dermatitis causative pathological agents such asD. nodosusorF. necrophorum, that effectively immunize susceptible mammals, preferably ungulates, against DD, in particular against bovine digital dermatitis.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides new vaccine compositions comprisingTreponemaspp. bacterins, preferably but not limited toTreponema pedisbacterins, optionally supplemented with antigens fromTreponemaspp. or other digital dermatitis causative pathological agents such as but not limited toD. nodosusorF. necrophorum, for effectively immunizing susceptible mammals, preferably ungulates, against DD, in particular against bovine digital dermatitis.

The present invention also identifiesTreponema pedisas one of the etiologic agents of digital dermatitis (DD) in mammals, in particular ungulate digital dermatitis. The invention therefore also provides isolated cultures ofTreponema pedisfor effectively immunizing susceptible mammals, preferably ungulates, against DD, in particular against bovine digital dermatitis. In addition, the present invention provides methods of diagnosing DD by detecting infection with a series of specificTreponemaantigens.

Thus in one aspect, the invention provides a biological, preferably a biologically pure, culture ofTreponemaspp. bacterins, preferablyTreponema pedisbacterins, preferably supplemented with antigens fromTreponemaspp. or other digital dermatitis causative pathological agents such asD. nodosusorF. necrophorum, for effectively immunizing susceptible mammals, preferably ungulates, against DD, in particular against bovine digital dermatitis.

In another aspect, the invention provides a, preferably biologically pure culture of, preferably ungulate,Treponema pedisbacterins.

In another aspect, the invention provides a pharmaceutical composition comprising an immunogenically effective amount ofTreponemaspp. bacterins, preferablyTreponema pedisbacterins, optionally supplemented with antigens, preferably recombinant antigens, fromTreponemaspp. or other digital dermatitis causative pathological agents such asD. nodosusorF. necrophorum, for effectively immunizing susceptible mammals, preferably ungulates, against DD, in particular against bovine digital dermatitis. Preferably, said antigens areTreponemaantigens selected from the list consisting of MSP (Major Outer Sheath Protein), PrtP (Serine-protease Complex PrtP-like Protein), TlyC (Hemolysin C), OrfC (Surface Antigen OrfC Lipoprotein), and Hemolysin III. It is noted that all of these antigens can be obtained fromTreponemaspp. such as but not limited toT. pedis, T. phagedenis, orT. vincentii. Additional antigens useful in the present invention can be selected from other digital dermatitis causative pathological agents such asD. nodosusorF. necrophorum. All of these antigens can be used in combination in the pharmaceutical composition of the invention and are preferably recombinantly produced.

More preferably but not limited to, said proteins, polypeptides or antigens useful in the present invention are selected from virulence factors such as adhesins, peptidases, proteases, surface antigens, proteins involved in motility and haemolysins. Even more preferably said proteins, polypeptides or antigens useful in the present invention are selected from any of the following list:MSP (Major Outer Sheath Protein);PrtP (Serine-protease Complex PrtP-like Protein) and PrtPM (PrtP Mature Protein);TylC (Hemolysin C);OrfC (Surface Antigen OrfC Lipoprotein);Haemolysin III;Apr2 (Acidic Extracellular Subtilisin-like Protease Apr2) and Apr2BM (Apr2 Benign Mature Protein);Apr5 (Acidic Extracellular Subtilisin-like Protease Apr5) and Apr5M (Apr5 Mature Protein);Bpr (Basic Extracellular Subtilisin-like Protease Bpr) and BprM (Bpr Mature Protein);PrcB (Serine-protease Complex PrcB-like protein);PrcA (Serine-protease Complex PrcA-like protein);Cys peptidase;Hemolysin erythrocyte lysis protein 2;Surface antigen BspA;Filament protein;Flagellar hook protein;Prolyl endopeptidase;Oligopeptidase;Oligopeptidase B; andOligopeptidase F.

Still more preferably, said antigens are antigens fromTreponema pedisselected from the list consisting of MSP, PrtP, preferably PrtPM, TlyC, and OrfC; and/or antigens MSP and Haemolysin III fromTreponema phagedenis; and/or antigens PrtP, preferably PrtPM, fromTreponema vincentii; and/or antigens Apr2, preferably Apr2BM, Apr5, preferably Apr5M, and Bpr, preferably BprM, fromDichelobacter nodosus, or any combination thereof, wherein optionally said antigens are recombinantly produced.

In addition, said pharmaceutical composition might further comprise an immunogenically effective amount of a bacterin selected from the group consisting of:Treponema medium, Treponema vincentii, Treponema phagedenis, Treponema refringens, Treponema brennaborense, Treponema calligyrum, andTreponema maltophilum.

In another aspect, the invention provides a method for inducing an immune response against the etiologic agents of digital dermatitis (DD) in mammals, in particular ungulate digital dermatitis. This method includes the step of administering to a mammal, preferably to an ungulate animal, the pharmaceutical composition as defined in the above paragraphs.

In one embodiment, the pharmaceutical composition further includes an antigen from an organism that causes ungulate footrot selected from the group consisting ofFusobacterium necrophorum, Porphyromonas levii, andDichelobacter nodosus. In another embodiment, the pharmaceutical composition further comprises additional antigens such as a bovine respiratory syncytial virus antigen, a bovine herpes virus antigen, a leptospiral antigen, a bovine diarrhea virus antigen, a bovine parainfluenza virus antigen, a vesicular stomatitis virus antigen, a malignant catarrhal fever virus antigen, a blue tongue virus antigen, a pseudorabies virus antigen, a rabies virus antigen, a rinderpest virus antigen, aFusobacterium necrophorumantigen, aDichelobacter nodosusantigen,Guggenheimellaantigen,Porphyromonasantigen,Bacteroidesantigen,Prevotellaantigen, Peptosteptococcus antigen,Campylobacterantigen,Mycoplasmaantigen,Corynebacterium/Actinomycesantigen,Cryptosporidiumantigen, Rotavirus antigen, Coronavirus antigen or aClostridiumspp. antigen.

In a still further aspect theTreponemaspp. bacterins of the pharmaceutical composition are in the form of a suspension of killed or attenuated bacteria. Preferably, theTreponemabacterin isT. pedisbacterin in the form of a suspension of killed or attenuated bacteria. Preferably, theT. pedisbacterin is in the form of a killed bacteria suspension.

In another embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier.

In another embodiment, the pharmaceutical composition of the invention is a vaccine.

In another embodiment, the pharmaceutical composition or the vaccine of the invention is administered parenterally.

In another embodiment the pharmaceutical composition or the vaccine of the invention is for use in a method of treatment or prevention of digital dermatitis; preferably, bovine digital dermatitis, contagious ovine digital dermatitis and/or footrot; more preferably, bovine digital dermatitis.

In another aspect, the invention provides a vaccination kit characterized in that it comprises a container comprising the pharmaceutical composition or the vaccine of the invention.

In another aspect, the invention provides a vaccination kit characterized in that it comprises an informative manual or leaflet which contains the information on administering said pharmaceutical composition or vaccine of the invention.

In another aspect, the invention provides a method for detecting the presence of antibodies specifically immunoreactive with aTreponemaantigen, or with other digital dermatitis causative pathological agents such asD. nodosusorF. necrophorum, in a biological sample, the method comprising contacting the sample with an antigen selected fromTreponemaspp. antigens: MSP (Major Outer Sheath Protein), PrtP (Serine-protease Complex PrtP-like Protein), TlyC (Hemolysin C), OrfC (Surface Antigen OrfC Lipoprotein), or Hemolysin III. It is noted that all of these antigens can be obtained fromTreponemaspp. such as but not limited toT. pedis, T. phagedenis, orT. vincentii; and/or with antigens selected from other digital dermatitis causative pathological agents such asD. nodosusorF. necrophorum, thereby forming an antigen/antibody complex; and detecting the presence or absence of the complex. More preferably but not limited said proteins, polypeptides or antigens useful in the present methodology are selected from virulence factors such as adhesins, peptidases, proteases, surface antigens, proteins involved in motility and haemolysins. Even more preferably said antigens useful in the present methodology are selected from the following list:MSP (Major Outer Sheath Protein);PrtP (Serine-protease Complex PrtP-like Protein) and PrtPM (PrtP Mature Protein);TylC (Hemolysin C);OrfC (Surface Antigen OrfC Lipoprotein);Haemolysin III;Apr2 (Acidic Extracellular Subtilisin-like Protease Apr2) and Apr2BM (Apr2 Benign Mature Protein);Apr5 (Acidic Extracellular Subtilisin-like Protease Apr5) and Apr5M (Apr5 Mature Protein);Bpr (Basic Extracellular Subtilisin-like Protease Bpr) and BprM (Bpr Mature Protein);PrcB (Serine-protease Complex PrcB-like protein);PrcA (Serine-protease Complex PrcA-like protein);Cys peptidase;Hemolysin erythrocyte lysis protein 2;Surface antigen BspA;Filament protein;Flagellar hook protein;Prolyl endopeptidase;Oligopeptidase;Oligopeptidase B; andOligopeptidase F.

Still more preferably, said antigens are antigens fromTreponema pedisselected from the list consisting of MSP, PrtP, preferably PrtPM, TlyC, and OrfC; and/or antigens MSP and Haemolysin III fromTreponema phagedenis; and/or antigens PrtP, preferably PrtPM, fromTreponema vincentii; and/or antigens Apr2, preferably Apr2BM, Apr5, preferably Apr5M, and Bpr, preferably BprM, fromDichelobacter nodosus, or any combination thereof; all of these antigens are particularly useful for forming an antigen/antibody complex; and thus for detecting the presence or absence of the complex.

In one embodiment, the biological sample is bovine serum. In another embodiment, the antigen is immobilized on a solid surface. In another embodiment, the complex is detected using a labeled anti-bovine antibody.

In another aspect, the invention provides a method of detecting the presence of ungulateTreponemain a biological sample or the presence of other ungulate digital dermatitis causative pathological agents such asD. nodosusorF. necrophorum. This method includes the steps of contacting the sample with an antibody specifically immunoreactive with an antigen selected from any of the above identified antigens, thereby forming an antigen/antibody complex; and detecting the presence or absence of the complex. Said method of detecting the presence of ungulateTreponemain a biological sample is useful for diagnosis or prognosis purposes, such as for diagnosis DD, particularly for diagnosing bovine digital dermatitis.

In another embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is immobilized on a solid surface. In another embodiment, the complex is detected using a second labeled antibody. In another embodiment, the biological sample is ungulate foot tissue.

