Emerging Chlamydia-like organisms as novel causes of bovine reproductive failure

Lead Research Organisation: Royal Veterinary College


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Technical Summary

Incidents of bovine reproductive failure have been linked to Chlamydia-like organisms, a phylogenetically diverse group of emerging pathogens that have been detected by PCR and immunohistochemistry in aborted tissues with associated histopathology. As amoebal endosymbionts, Chlamydia-like organisms can persist in the environment, however, the disease incidence, immunopathogenesis and risk factors associated with infection are unknown.

Epidemiological surveys of the incidence of Chlamydia-like organisms and other infectious abortifacients will be performed to identify risk factors associated with reproductive failure. The role of Chlamydia-like organisms in reproductive failure will be specifically investigated in a cohort study, where immunohistochemistry and histopathology will be performed on tissue samples to investigate lesion development, localise pathogens and determine a causal role in disease pathogenesis. The phylogenetic similarity of Chlamydia-like organisms detected in environmental and tissue samples will be determined to identify genotypes linked to reproductive failure and potential environmental sources of transmission. The organisms will be isolated in amoebal co-culture and phenotypically characterised. They will be used in in vitro models of placental infection to study cytokine and cellular responses to infection. An in vivo pathogenesis model will be established to study the immunological and serological responses to infection, to define the pathologies associated with infection and determine a causal role in reproductive failure.

This project will determine the prevalence of bovine reproductive failure linked to Chlamydia-like organisms in the UK, identify genotypes implicated in the disease and define immunopathology associated with infection. This will substantially advance the understanding of these emerging pathogens and make a significant contribution to the development of detection, prevention and disease management strategies.

Planned Impact

Poor reproductive performance in cattle is of economic and welfare importance to the agricultural industry in the UK and worldwide. This project aims to generate knowledge about a group of emerging organisms that have been found to be associated with reproductive failure in cattle. In the process of this work we will analyse the impact of these organisms on cattle reproductive performance across the dairy industry, environmental contamination and provide evidence for a direct causal link between these organisms and abortion.

There will be multiple beneficiaries to the proposed work. Potential stakeholders include research scientists both national and international, academic and government research organisations, farmers, veterinary diagnostic agencies, the animal health industry and government. Ultimately the general public will benefit through improved animal health and welfare, animal production, as well as food security and safety.

This project will improve knowledge on the important abortifacients of cattle in the UK and will provide insight into the causal role of Chlamydia-like organisms on bovine reproductive failure, including information on the diversity and characterisation of the organisms and the development of in vivo model systems. The knowledge gained will open up pathways to impact at the farm level through improved farm management strategies and more effective diagnosis of cases of bovine abortion. Diagnostic tools and future vaccine development are the major potential benefits for the animal health industry, in particular farmers, veterinarians, other animal practitioners and commercial organisations. The outcomes of this research will be of interest to the farming industry, as well as government policy makers, allowing them to prioritise the management of important production diseases that impact negatively on the sustainability of the cattle industry and thus on the future security of our food. The wider public will not only benefit from this research though improved production, but there will also be a better understanding of the occurrence and role of these potentially zoonotic pathogens. The production of healthier animals, free of potential zoonotic infections will clearly benefit the public through a reduction in the possible zoonotic transmission to humans.

Knowledge exchange and outreach are important activities for communication and promotion of scientific outputs to a variety of audiences. For the scientific community this involves presentations at national and international meetings, and the production of peer reviewed publications in international journals. Communication with non-academic and non-research stakeholders will include articles for the farming and veterinary press, public engagement at science events, agricultural shows and liaising with government policy makers and potential industrial partners. We will engage and communicate with stakeholders to maximise the global impact of the proposed research. The industrial partner Pfizer will provide expertise in enhancing the outreach and impact of the project to the veterinary practitioners and will advise on possible commercial opportunities arising from the results of the project. The interaction of DairyCo whose mission statement is to 'promote world class knowledge to British dairy farmers so they can profit from a sustainable future' will further aid in the dissemination of project outputs to the agricultural community.

All the investigators have contacts and experience in these areas, providing an opportunity to promote the importance of this project. Staff working on the project will gain transferable scientific skills and will be given training that meets their personal and professional developmental needs. Transferable skills will include presentations and writing for communication with academic and non-academic audiences, time-management and project-management.


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Description With regards to the initial components of the project, data on herd management, health and production has been obtained by means of questionnaires from 219 dairy farms randomly selected across GB. A database has been established and data captured are currently being introduced in the database. From each of the 219 farms, a bulk milk tank sample has been collected and is currently being tested by our partners at Moredun Research Institute (MRI) and at the National Milk laboratories. The first component of the project is therefore pending laboratory results but all the field data and specimen collection has been completed. As for subsequent phases of the project, we have successfully engaged a number of veterinarians who have just started to obtain abortion materials and vaginal swabs from selected dairy herds. Testing of these samples for reproductive pathogens and Chlamydia-like organisms (CLO) will be conducted at MRI and, based on the results; a number of farms positive for CLO will be recruited to take part in the planned cohort study.

Update: the testing of milk samples collected as part of the first phase of the project (cross-sectional survey) has been completed. We now have a database including the frequency of a number of diseases in the study farms and their status with respect to a number of infectious agents infectious agents including Chlamydia-like organisms and major reproductive pathogens. The results of the cross sectional study have shown that 10.6% of study farms are positive to Chlamydiales on PCR conducted on bulk milk samples. Out of the positive samples, 46% were positive to Parachlamydia acanthamoeba on genome sequencing. This is the first large-scale probabilistic study of the frequency of CLO across UK dairy herds.
Regarding the second phase of the project, the cohort study, we originally planned to include farms found positive for Parachlaymida sp. However, due to the small numbers of positive farms it was decided to recruit farms with chronic undiagnosed reproductive issues. By August 2015 we recruited 20 farms in the study and 30 to 50 recently calved animals were recruited from these farms. Blood samples, placental samples and vaginal swabs (latter two depends on availability) were collected during recruitment from these animals. We are currently following-up these animals and recording relevant reproductive events. Animals will be re-sampled if they show any reproductive issues such as embryonic mortality or abortion. Analysis of some samples have already been done and results have been sent to the veterinarians.

Update II: Placental and vaginal swabs were not available due to logistical restrictions that required vets to be called out to sample each abortion or embryonic death event on the day. Therefore, only blood samples were collected from the animals recruited in the cohort study for serological analysis. In total 703 animals from 20 farms were recruited into the study. Most cows (93.9% n=660) were recruited within 30 days of calving and followed regularly to identify any reproductive problems. The remaining 43 cows (6.1%) were recruited due to them presenting reproductive problems. Second serum samples were taken from cows that presented a reproductive disorder after recruitment (n=70) and control cows (n=40). Control cows were selected if they had not experienced any reproductive problems and had calved around the same time as recruitment cows. Serum samples were tested for Neospora canium, Salmonella spp, Bovine Viral Diarrhoea (BVD), Infectious Bovine Rhinotracheitis (IBR), and Leptospira in accordance to the farm of origin's vaccination status. Vaccination status was available for 19/20 farms; coverage was varied with 10.5% (n=2/19) for salmonella, 68.4% (n=13/19) for leptospirosis, 73.7% (n=14/19) for IBR, and 94.7% (n=18/19) for BVD. Some test results are still to be received from laboratories. However, when using the available results and once vaccinated subjects were excluded, seropositivity against the studied pathogens at recruitment was: 7% (48/669) for neospora, 8% (50/633) for salmonella, 49% (33/67) for BDV, 33% (69/208) for IBR and 24% (58/238) for leptospira. Reproductive disorders were recorded for a total of 103 cows (43 at recruitment and 70 during follow-up); the most common reported disorder was anoestrus 41% (n=42/103) followed by abortion 28% (29/103), embryonic death 24% (25/103), repeat service 11% (11/103), and endometritis 6% (n=6/103). Serological results are currently still incomplete however, after using available results and excluding vaccinated animals, for all follow-up reproductive disorders (n=70) there was 11% (4/35) seropositivity for neospora, 0% (0/21) for salmonella, all vaccinated for BVD, 0% (0/16) for IBR, and 0% (0/1) for leptospira. Control cows (n=40) on the other hand presented 12% (3/26) seropositivity for neospora, 6% (1/18) for salmonella, all vaccinated for BVD, 0% (0/13) for IBR, and all vaccinated for leptospira. In conclusion, the study results suggests that CLO are endemic across GB dairy herds, with positive farms identified in the study distributed all over GB. When a cohort of farms with reproductive problems of unknown origin was followed-up in detail no association was found with exposure to known infectious causes of reproductive inefficiency, pointing at the potential role of non-infectious causes such as metabolic, management and nutritional factors.

Update III: The aim of this study was to explore the role of Chlamydia like organisms(CLOs) as a cause of reproductive disorders. At the time when the project was conceived there was concern that these novel organisms could be a cause of abortion in cattle and as a result of economic loss for dairy farmers. The evidence was limited but important enough e.g. for the industry to dedicate resources to ascertaining this potential association. The findings from the whole project including the results form our collaborators at Moredun Research Institute-MRI question the plausibility of such association. Publications from MRI showed the presence of CLOs in the environment and drinking waster samples suggesting CLOs are ubiquitous in distribution. It seems plausible that isolation of CLOs from aborted materials in previous studies (which was the reason why the were linked to reproductive disorders) could be a result of environmental contamination.
The final dataset with all tested samples' results includes a total of 656 cows recruited within 30 days of calving. Serological results at recruitment revealed 8%(49/653) seropositivity for Neospora canium; 3%(18/558) for Salmonella spp.; 47%(16/34) for BVD; 27%(48/175) for IBR; and 13%(26/197) for Leptospira. A total of 67 reproductive disorders were recorded out of the 656 recruited cows (11%) from 55% (11/20) of farms . Overall, the most commonly reported disorder was anoestrus (57%) followed by embryonic death (18%), repeat service (13%), endometritis (7%) and abortion (3%). For reproductive disorder cases, seropositivity was 12%(8/67) for Neospora canium; 2%(1/58) for Salmonella spp.; 50%(2/4) for BDV; 19%(4/21) for IBR; and 7%(1/14) for Leptospira. No statistically significant difference was found in seropositivity against any of the studied pathogens between control cows (n=40) and case cows (n=67). We suggest there is potential for other non-infectious causes such as metabolic conditions, nutritional deficiencies, lactation stress etc. to be affecting reproductive health in the study farms. This is further emphasised by the recording of only four seroconversions (from negative to positive) two control cows for Neospora , one anoestrus cow for Salmonella, and one embryonic death for BVD. This study exemplifies the challenges associated with establishing infectious pathogens as causative agents of reproductive disorders and the need for caution when such an association is proposed on the basis of identifying an infectious agent in aborted samples without contrasting whether the agent is also present in non-afected animals or the environment as appears to be the case for CLOs.
Exploitation Route Field and laboratory work is ongoing; once completed, we will calculate national-level prevalence and identify risk factors for the presence of reproductive pathogens and Chlamydia-like organisms in GB dairy herds. This information can be used to inform control policies for these pathogens at the national and individual herd level. More targeted and cost-effective control of these pathogens can minimize their impact on reproductive efficiency of dairy herds and as a result contribute to the financial sustainability of dairy farms and mitigate their environmental impact. The translation of research outputs into tangible impact will be facilitated by the involvement of DairyCo, the levy-funded, not-for-profit organisation working on behalf of Britain's dairy farmers, who are actively involved in this project, which overlaps with ongoing research they are currently funding. Through this involvement it will be possible for our results to reach individual farmers and veterinarians who may use them to make decisions on more cost-effective control of infectious diseases in British dairy herds.

Update: We have completed the testing and we have been communicating the results to vets and farmers. The results will inform the farm status regarding some major infectious diseases which will help them to decide control programmes and vaccination strategies. Currently the data collected are being analysed to identify risk factors of major reproductive pathogens in GB. This information may be crucial to design control policies for reproductive pathogens of dairy cows.

Update II: Results from the cohort study will be shared with vets and project partners. This will inform on the farm's disease status and can help improve current control programmes and vaccination strategies. The results will be further explored with the possibility of disseminating them through peer reviewed publications.

Update III: A publication presenting the methodology of the study and results is ready to be submitted to Veterinary record. Through this publication we highlight the challenges regarding the identification of a definite pathogenic agent causing reproductive disorders using our study as an example. These difficulties include study designs that fail to control for numerous potential confounders and the existence of potential unrecorded cases. From this we suggest that the extent of some established links between reproductive disorders and infectious pathogens may be questionable. As veterinarians are the main audience for veterinary record we hope our findings and interpretations will help them to improve reproductive efficiency in dairy farms in the UK.
Sectors Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology

Description The project is at the stage of data collection and testing of biological samples; databases are still being created. Update : 1. Cross-sectional study: the bulk milk samples collected as part of this study were tested for major infectious diseases including reproductive diseases. The samples were tested for IBR, Leptospira sp, Neospora sp, Salmonella sp, Coxiella burnetti, and Chalmydia - like - organisms. The results of the cross sectional study have shown that 10.6% of study farms are positive to Chlamydiales on PCR conducted on bulk milk samples. Out of the positive samples, 46% were positive to Parachlamydia acanthamoeba on genome sequencing. The results of the testing were communicated to farmers and vets and a large database has been created and is available for analyses that could inform prioritization of dairy cattle disease control programs. 2. Field study to identify potential farms for cohort study: 13 vets agreed to send vaginal swabs and placental samples from aborted animals to screen them for reproductive pathogens including Chlamydia-like organisms. The objective was to identify potential farms for the cohort study, but only one farm was positive for Parachlamydia sp, we communicated whole testing results to vets in order to help them with the diagnosis of reproductive issues. 3. Cohort study: Although we are still following up the animals recruited in the study, we have been testing samples simultaneously. The blood samples are tested for major reproductive pathogens and the results are communicated to vets. This helps them in the diagnosis of reproductive issues and aids them in developing vaccination and control strategies. Update:The main non-academic achievement of this project was engagement of farmers, vets and industry in the investigation of reproductive pathogens in UK dairy cows. The results from the cross sectional study were communicated to farmers and vets, and some analysis of the data has culminated in a publication being submitted for publication in a peer reviewed journal. The findings of the cross sectional study helped identify farms in which to conduct the cohort study. Throughout the cohort study regular communication was maintained between academic institutions and vets, whom received regular updates with preliminary results for each farm. The final results will be shared with project collaborators and vets to inform on the farm status regarding different reproductive pathogens and help guide prevention and control strategies. The results will be further explored with the aim to further disseminate to the wider scientific community through peer reviewed journals. UpdateII: A sort communication presenting the findings and discussing them is ready to be submitted to Veterinary Record which is journal frequently accessed by veterinarians in clinical practice. The aim is make sure our findings reach most of the farm animal practitioners.
First Year Of Impact 2015
Sector Agriculture, Food and Drink
Impact Types Economic

Description Influence on veterinary practice in the UK
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
Impact The results of all the testing carried out on the samples collected as part of this project have been regularly communicated to the vets. This helps them to understand the microbiological status of the farm for major reproductive diseases. In addition to assisting in diagnosing reproductive issues the results sent to them will also help them to advise farmers on the best vaccination strategies and reproductive health management of the animals.
Title Cohort study database 
Description A database has been created with detailed information from around 500 dairy cows recruited in the cohort study and their farms of origin. Farm-level data include data on vaccination, reproductive issues, reproductive history of the farms, details on animal purchase and herd size. Individual animal data cover dates of calving, artificial insemination, reproductive events, recruitment into the study and sampling. The database includes also the results of laboratory testing of blood samples for Neospora caninum, Leptospira hardjo, Salmonella sp and Chlamydia-like organisms. The database is currently being populated as and when testing results are received. All the laboratory tests are currently taking place at our partner Moredun Research Institute. Update: The database includes entries for 704 animals from 20 farms. These include both recruitment and follow-up sampling and laboratory results. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Once the database is complete it will be used to conduct survival analysis to determine the association between time to conception, time to calving and time to abortion in accordance with disease status of the animal. Update: Survival analysis was not possible due to the low seropositivity of animals and particularly in those presenting reproductive disorders. The database was used to carry out a descriptive analysis of the study pathogens as well as reproductive disorders. 
Title Dairy Herd Database 
Description This database includes management, production and health data for all dairy herds included as part of a nationwide cross-sectional study of British dairy herds. The database is currently being populated with data with data gathered by means of questionnaires and results from laboratory analysis of bulk tank milk samples; the analyses are currently taking place at Moredun Research Institute and National Milk Laboratories. Update: The database is finalized and has been used for extensive analysis. A total of 227 farms randomly selected using stratified (by region and herd size) random sampling across Great Britain are included in the database. Information available for individual farms includes herd and farm structure, management practices, estimated frequency of key production diseases and disease status with respect to 7 infectious agents ascertained by means of bulk tank testing. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Once data entry is completed, this database will allow baseline estimates for a number of key conditions affecting health and production of British dairy herds as well as the exploration of associations between management, health and production. Update: The database is finalized and has been used for extensive analysis including a detailed description of the spatial distribution of different reproductive pathogens and their risk factors in Great Britain. The database will also inform studies aimed at designing strategies to monitor the occurrence of major diseases and conditions affecting dairy farms in GB. 
Description Collaboration with DairyCo 
Organisation DairyCo
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Assistance was given for planning field work and data entry as part of the Cross-sectional study mentioned below. The RA working for the project participated in the organization of farm visits, conducted a number of field visits for data and sample collection and assisted with data entry into the electronic database. The project supported the costs of some of the laboratory testing - specifically those related to pathogens associated with reproductive inefficiency in cattle. Update: The RA working on the project also helped with monthly reports to be sent to Dairy Co and also was in charge of communicating test results to farmers and veterinarians.
Collaborator Contribution As part of a DairyCo-funded Research Partnership in Health, Welfare and Nutrition (led by Nottingham University, with Royal Veterinary College as one of the principal partners), a cross-sectional study of British Dairy herds has been carried out. DairyCo has allowed us to use this sample of dairy herds to estimate the prevalence of Chlamydia-like organisms in British dairy herds, and to recruit farms for a follow-up study. DairyCo provided the financial support representing time inputs for farm recruitment, staff-time to undertake farm visits, and travel and subsistence costs.
Impact As the project is still in the phases of data collection and laboratory analysis, the collaboration has mainly been limited to these initial stages of the project. The main outcome, so far, of the collaboration has been the successful completion of a nationwide cross-sectional study of British dairy herds. This involved the collection of herd health and production data and bulk milk samples from a random sample of around 220 herds. This large scale and challenging study has been greatly facilitated by the collaboration. The project has been mentioned in the BCVA (British Cattle Veterinary Association) newsletter to inform veterinarians of its aims and facilitate engagement and farm recruitment. Update: In addition to the outcomes above mentioned this collaboration has generated national-level estimates of the prevalence of chlamydia-like organisms and other major reproductive pathogens. These nationwide estimates are very valuable for the UK dairy industry as they will allow assessment of the economic and environmental impact of specific diseases compromising reproductive performance of dairy cows. This can help prioritizing disease control efforts and future research initiatives. This collaboration also facilitated engagement of veterinarians and farmers, which was essential for the implementation of the cohort study, the second phase of the project.
Start Year 2013
Description Collaboration with RAFT Solutions 
Organisation RAFT Solutions Ltd.
Country United Kingdom 
Sector Learned Society 
PI Contribution The results of the laboratory testing of the samples sent by veterinarians through the collaboration that has been established were sent back to them to help with the diagnosis of reproductive issues at the farms. We provided them with all the sampling materials, covered the financial costs of transporting the samples and testing of samples. An honorarium payment of £500 per farm was agreed to be given to vets once the study is completed.
Collaborator Contribution Raft solution agreed to recruit vets who in turn will recruit 15 farms (out of 20) for the study. They also agreed to be the contact point between the RA working in the project and vets thereby making the communication easier.
Impact As a result of the collaboration we were able to successfully recruit 15 farms (out of 20) in the study. We have recruited about 500 animals in the study Communication between the RA and vets have been easier, quicker and regular throughout the period so far which can be challenging because of the nature of the study. Update: In total 703 animals from 20 farms were recruited into the study. Most cows (93.9% n=660) were recruited within 30 days of calving and followed regularly to identify any reproductive problems. The remaining 43 cows (6.1%) were recruited due to them presenting reproductive problems. Second serum samples were taken from cows that presented a reproductive disorder (n=70) and control cows (n=40).
Start Year 2010