Moredun Scientificmagaz ineSUMMER 2012

A validated experimental model ofMycoplasma bovis pneumonia in calvesDavid Reddick and Cliff Ramage, Moredun Scientific.

Acute bovine respiratory disease(ABRD) continues to be a significanteconomic and welfare problem incalves. Severe cases can lead tomortality but more commonoutcomes include decreased growthrates, particularly where there ispermanent lung damage.

The disease syndrome can be caused by anumber of different viruses and bacteria.Themain viruses are: respiratory syncytial virus(RSV), parainfluenza type 3 (PI3) virus,infectious bovine rhinotracheitis (IBR) virus and less obviously bovine viral diarrhoea (BVD)virus. Mycoplasma bovis is one of severalbacterial species which can be involved, theothers being Mannheimia haemolytica, Pasteurellamultocida and Histophilus somni.

To meet the requirement of animal healthcompanies looking to test the efficacy of novelvaccines and therapeutics Moredun Scientificoffers a validated experimental model of M. bovis infection in calves for use in clientstudies. The model has been used successfullyin both vaccine and pharmaceutical trials.

Model Overview (Therapeutic efficacy studies)

Calves are screened (by ELISA) to ensure that M.bovis antibody levels are below a defined cut off prior to transportation to the study site. At between 5 and 8 weeks of age the animals are challenged with an M.bovis isolate over three days. For four days post final challenge, the animals are clinically observed and enrolled on the study once clinical signs of respiratory disease are observed. Post enrollment clinical observations are carried out once daily for 14 days at which point the animals are euthanased and the lungs assessed for the presence of M. bovis specific lesions.

Challenge ModelThe strain of M.bovis most commonly used

in the model was isolated from a calf withclinical respiratory disease in the UK. Thisisolate has been used in a number of differentclient studies with considerable success. Thegrowth of this and other M.bovis isolates hasbeen fully validated using defined growth mediaand conditions and the production of challengematerial to a defined level is reproduciblewithin tightly defined limits.

Clinical Signs A critical aspect of M.bovis studies is to

ensure a consistently high challenge successrate. The Moredun model, which utilises endo-bronchial deposition of a low volume/high titrechallenge on three consecutive days, has beenshown to be effective with up to 70% ofchallenge animals showing clear clinical signs of respiratory disease within a four day periodpost challenge.

The model has been validated to produceclear signs of clinical disease including increasedrectal temperature, increased respiratoryeffort/rate, abnormal demeanour and in somecases nasal discharge. These clinical signs allow

enrollment of animals on therapeutic trialsbased on clearly defined and validated criteria.The standard design for enrollment requiresanimals to have an increased rectaltemperature (≥39.5°C) and either abnormaldemeanour or respiration.

Lung Pathology The main determinant of the efficacy of the

challenge for the majority of studies which usethe Moredun model is the percentage of totallungs with lesions. The mean % lung damage inthe control groups is generally 15 to 20% andsignificant differences between treated andcontrol groups are routinely observed. Figure 1shows an example of lung lesion scoresfollowing challenge in a therapeutic study.

Bacteriology There are a number of options available to

confirm the presence of the challenge isolate inthe lungs of study animals. The most effectivemethod is recovery of the bacteria from lunglavage fluid samples collected at necropsy withsamples titrated onto Mycoplasma specific agarfor accurate colony counts.

www.moredun-scientific.com

0

2

4

6

8

10

12

14

16

18

20

Control Product 1 Product 2

Mea

n %

Lun

g w

ith

Lesi

on

Figure 1: Mean Group % Lung with Lesion

Mean

Median

MoredunResearch NewsParasitic Worm control consortiumScientists from the Moredun ResearchInstitute are involved in an exciting new EU project to develop new and sustainablestrategies to control parasitic worms in sheepand cattle.

The ‘GLOWORM’ project (an abbreviationfor “GLObal changes in parasitic WORMs”) willrun for 3 years and will harness the expertiseof 14 research groups throughout Europe,with the help of a 3 million euro grant fromthe EU 7th Research Framework Program.

Infection with parasitic worms represents a significant economic and welfare burden tothe European livestock industry.The increasingprevalence of anthelmintic (wormer)resistance means that current controlprogrammes, based on routine anthelmintictreatment, are costly and unsustainable in the longer-term. Recent changes in the

epidemiology, seasonality and geographicdistribution of parasitic worms have beenattributed to climate change, however, otherchanges in the environment (e.g. land-use)and in livestock farming (e.g. intensificationand changing management practices) will also have an impact.

The main objectives for the project are:• to design improved diagnostic tests to

improve surveillance of parasitic worm infections levels across Europe.

• to provide EU-wide modelling of infection risk in space and time with the aim of both supplying farmers with improved, up-to-date, information and designing new control strategies.

• to test new and innovative control strategies under field situations.

Ruminant Immunology ProjectResearchers at Moredun have secured nearly£1 million from the BBSRC and the ScottishGovernment to learn more about the immunesystems of livestock to aid the development of sheep and cattle vaccines. Scientists fromMoredun and the nearby Roslin Institute, with support from an industrial partner, AbDSerotec, will undertake detailed investigationsof the immune responses of sheep and cattle,vitally underpinning the development of futurevaccines. The team will be led by Moredun’sProfessor Gary Entrican.

Disease ModelsIn addition to the M.bovis model MoredunScientific offer validated experimental modelsof respiratory disease for a range of bovinepathogens which are available for client studies.The range includes models for bacterial andviral pathogens.

We have significant experience inconducting studies with Mannheimia haemolyticawith both vaccines and therapeutics and haveseveral recent publications in partnership withclients which refer to the use of the model:

ResearchPasteurella multocida causes major welfareproblems and economic losses in a range ofhosts and in many areas of the world. It isresponsible for both respiratory and systemicdisease in cattle and buffalo, but a detailedunderstanding of, and the ability to control,the underlying molecular mechanisms duringhost-pathogen interaction is lacking.

The organism is re-emerging as a primarycontributor to acute bovine respiratorydisease, accounting for up to 15% of

pneumonic pasteurellosis in the UK. There is no current test that differentiatesbetween dangerous and less harmful forms of the bacterium, nor any European vaccineand the industry needs new and effectivecontrol measures.

The current focus areas of the PasteurellaResearch Group at the Moredun ResearchInstitute are the molecular characterisation of bovine isolates of P. multocida duringcarriage and disease, what happens duringinfection and the involvement of biofilms indisease. The aim is to develop new diagnosticand control methods for diseases caused bythis bacterium.

Diagnostic testsThe Virus Surveillance Unit at Moredunprovides specialist virological support toScottish disease control centres. The unit has used the platform technology of real time polymerase chain reaction (PCR) todevelop a new generation of highly sensitive,quantitative tests to detect and identify,bovine respiratory viruses. A multiplex test isnow available for the detection of the virusesinvolved in bovine respiratory disease complex(calf pneumonia) in a single sample. The testdetects bovine herpesvirus type 1, bovinerespiratory syncytial virus and bovineparainfluenza virus 3.

For further information about how MoredunScientific can support your bovine respiratorydisease projects please visit our website or contact David Reddick: dreddick@moredun-scientific.com

Bovine Respiratory Expertise At Moredun

Moredun Scientific Magazine | Summer 2012

Liver fluke assaysLiver fluke is caused by Fasciola hepatica, a highlypathogenic flatworm parasite of ruminants. There is ongoing research at Moredun focussedon improving the diagnosis of active flukeinfection in the host animal, determining theefficacy of flukicidal treatment as a measure ofemerging resistance, monitoring fluke infectionlevels on farm as a predictor of disease risk andidentifying novel vaccine targets on the surfaceof the fluke gut.

Moredun Scientific offer the following testswhich can be used to study the efficacy offlukicidal drugs:

Faecal egg counting service –indicates thepresence of adult liver fluke in the faeces ofinfected animals.

Coproantigen ELISA test – detects thepresence of immature and mature flukethrough measurement of fluke coproantigensecreted from both immature and mature fluke in the faeces of infected animals.

The tests can be carried out on samplesfrom individual animals or on compositesamples (combined samples from 10 animals)which provides information on the presence of fluke within a flock or herd.

Animal HealthResearch ClubMoredun Scientific is a partner in a newlyformed Animal Health Research Club (ARC)which unites farmers, breeders andpharmaceutical companies in the UK in thefight against animal diseases. The club will investaround £9.5M of public and private money inresearch projects which aim to improve animalhealth and welfare through betterunderstanding, management and control ofpests and pathogens. The club is being led bythe Biotechnology and Biological SciencesResearch Council (BBSRC), with additionalfunding from 12 company members and theScottish Government.

The partners hope that by drawing on thecomplementary expertise of academia andindustry the club will spark new ideas forkeeping animals healthy and free from disease.The company members of the ARC will provideimportant input to the strategic direction of theClub's research. This ensures that the Club isbroadly directing its funding to areas where thecommercial sector sees scientific bottlenecks.

For further information visit:www. www.bbsrc.ac.uk

Biosafety testingThe biosafety testing business of MoredunScientific continues to develop and we haverecently recruited additional staff to meet thedemand from clients. Our focus is on in vivoand microbiology tests for the presence ofadventitious agents in cell lines and cell banksused in the development and manufacture ofhuman and veterinary biopharmaceuticals.Tests are conducted to GLP/GMP standards to meet international regulatory requirements.

For further information contact please contactJohn Murray: jmurray@moredun-scientific.com

Approval as French R&D SubcontractorMoredun Scientific has received notificationfrom the French Ministry of Research that it has successfully renewed its status as anapproved subcontractor for research anddevelopment projects. This approval enablesour clients who are private companies subjectto corporate tax in France to claim R&D taxcredits for work subcontracted to MoredunScientific. The process required us to present arecent development project that demonstratedour competence as a subcontractor.

RecentpublicationsThe following publications highlight recently published studies conducted in partnership with our clients:

The efficacy of a combined oral formulation of derquantel-abamectin against anthelmintic resistant gastro-intestinal nematodes of sheep in the UK. Geurden et al, Veterinary Parasitology April 2012.

Duration of efficacy of tulathromycin(Draxxin®) for the treatment ofexperimentally induced Mycoplasma bovisrespiratory infection in calves. Moyaert et alWorld Buiatrics Congress 2012.

Efficacy of a new 450mg/mL florfenicolformulation in the treatment of anexperimentally induced Actinobacilluspleuropneumoniae respiratory infection in pigs.Thomas et al, 4th European Symposium ofPorcine Health Management 2012.

Colostrum from cattle immunized with avaccine based on iron regulated proteins ofMannheimia haemolytica confers partial

protection. Makoschey B et al Vaccine 2012,30 pp969-973.

Cross protection of a Mannheimiahaemolytica A1 Lkt-/Pasteurella multocida ΔhyaEbovine respiratory disease vaccine againstexperimental challenge with Mannheimiahaemolytica A6 in calves. Crouch et al Vaccine2012, 30 pp2320-2328.

The efficacy of monepantel againstnaturally acquired inhibited and developingfourth-stage larvae of Teladorsagia circumcinctain sheep in the United Kingdom.Ramage et al,Veterinary Parasitology November 2011.

Receive your newsletter by emailIf you currently receive a print version of our newsletter and would prefer to receive a pdf version by email please contact Rhona Macdonald:

rmacdonald@moredun-scientific.com

Moredun Scientific Magazine | Summer 2012

Moredun Scientific is the commercial arm of the Moredun Group. To find out more about the Moredun group of companiesincluding the Moredun Research Institute visit www.moredun.org

Contact usMoredun ScientificPentlands Science ParkBush LoanPenicuikEH26 0PZScotland, UK

Tel: +44 (0) 131 445 6206Fax: +44 (0) 131 445 6205Email: info@moredun-scientific.comwww.moredun-scientific.com

Printed on recycled paper.Copyright of Moredun Scientific 2012.Liver fluke image © S.StammersMoredun Scientific Ltd is a company registered in ScotlandNo:SC107439Moredun is a trademark of the Moredun Foundation.

Disease ModelsOur disease models and areas of expertise are shown below:

Respiratory Diseases Bovine Virus Diarrhoea Virus (BVDV) Bovine Herpesvirus-Type 1 (BHV-1) Parainfluenza Virus Type 3 (PI3) Bovine Respiratory Syncytial Virus (BRSV) Mannheimia (Pasteurella) haemolytica (bovine,ovine)Pasteurella multocida (bovine)Mycoplasma bovisActinobacillus pleuropneumoniae (porcine)Mycoplasma hyopneumoniae (porcine) – under development

Mammary Gland DiseasesStaphylococcus aureus mastitis (bovine)Streptococcus uberis mastitis (bovine)Escherichia coli (bovine)

Reproductive DiseasesBovine Virus Diarrhoea Virus (BVDV) Neospora caninum (bovine)Chlamydophila abortus (ovine)Toxoplasma gondii (ovine)

Diseases of the GutEscherichia coli (bovine, ovine) – porcine under developmentRotavirus G6 & G10 (bovine)Coronavirus (bovine)Salmonella spp. (porcine)

Parasitic DiseasesEndo and ecto Parasitic Disease (Bovine, ovine, equine, avian natural and experimental infections)

Systemic DiseasesPasteurella trehalosi (ovine)Streptococcus suis (porcine)

Moredun Scientific Magazine | Summer 2012

Efficacy testing• Broad range of animal disease models.• New model and protocol development.• Short and long-term studies; experimental

or natural challenges. • Field studies.• MIC/MBC determination.• PK and PK/PD integration ex vivo or in vivo.• Potency testing.

Target animal safety testing• Safety and tolerance studies in compliance

with relevant guidelines for pharmaceuticals, biological and feed additives, including reproductive performance.

• Milk and tissue residue studies (portable milking parlours for dairy studies).

• Pharmacokinetic studies.

Biosafety testing• In vivo adventitious agent testing.• Microbiology testing services: Mycoplasma,

Mycobacteria, Sterility, Bioburden.• Tumorigenicity and Oncogenicity studies.

Biological Materials:• Production of viruses, antisera and other

biologicals to defined quality standards for diagnostic or research purposes.

Collaborative research Discovery and early phase developmentprojects in conjunction with the MoredunResearch Institute.

Rigorous quality systemsOur independent Quality Assurancedepartment has expertise in Good LaboratoryPractice (GLP), Good Manufacturing Practice(GMP),VICH-Good Clinical Practice (GCP), ISO9001:2000, ISO 17025 and ISO 14001. Allstudies are conducted to meet regulatoryguidelines.

State of the art animal facilities GLP accredited animal accommodation rangingfrom conventional farm accommodation, tospecific pathogen free (SPF), ContainmentLevel 3 or gnotobiotic units.

Laboratory facilitiesWe can support all aspects of safety andefficacy studies.

Moredun Scientific offer an unrivalled combination of research facilities, scientific expertise and commercial services:

Moredun Scientific Portfolio of Services