The province of Bohol, located in the Visayas islands region in the Philippines has a human population of 1.13 million and was the 4th highest region for human rabies deaths in the country, averaging 10 per year, prior to the initiation of the Bohol Rabies Prevention and Elimination Project (BRPEP).The BRPEP was initiated in 2007 with the goal of building a sustainable program that would prevent human rabies by eliminating rabies at its source, in dogs, by 2010. This goal was in line with the Philippine National Rabies Program whose objective is to eliminate rabies by 2020.The intersectoral BRPEP was launched in 2007 and integrated the expertise and resources from the sectors of agriculture, public health and safety, education, environment, legal affairs, interior and local government. The program included: increasing local community involvement; implementing dog population control; conducting mass dog vaccination; improving dog bite management; instituting veterinary quarantine; and improving diagnostic capability, surveillance and monitoring. Funding was secured from the national government, provincial, municipal and village units, dog owners, NGOs, the regional office of the WHO, the UBS Optimus Foundation, and the Global Alliance for Rabies Control. The BRPEP was managed by the Bohol Rabies Prevention and Eradication Council (BRPEC) under the jurisdiction of the Governor of Bohol. Parallel organizations were created at the municipal level and village level. Community volunteers facilitated the institution of the program. Dog population surveys were conducted to plan for sufficient resources to vaccinate the required 70% of the dogs living in the province. Two island-wide mass vaccination campaigns were conducted followed by “catch up” vaccination campaigns. Registration of dogs was implemented including a small fee that was rolled back into the program to maintain sustainability. Children were educated by introducing rabies prevention modules into all elementary schools in Bohol. Existing public health legislation at the national, provincial, and municipal level strengthened the enforcement of activities. A Knowledge, Attitude and Practices (KAP) survey was conducted in 2009 to evaluate the educational knowledge of the population. Increased surveillance was instituted to ensure that dogs traveling into and out of the province were vaccinated against rabies. Human and animal cases of rabies were reported to provincial and national authorities.Within the first 18 months of the BRPEP, human rabies deaths had decreased annually from 0.77 to 0.37 to zero per 100,000 population from 2007–2009. Between October 2008 and November 2010 no human and animal cases were detected. Increased surveillance on the island detected one suspected human rabies case in November 2010 and one confirmed case of canine rabies in April 2011. Two mass vaccination campaigns conducted in 2007 and 2008 successfully registered and vaccinated 44% and 70% of the dogs on the island. The additional surveillance activities enabled a mobilization of mop up vaccination activities in the region where the human and canine case was located. Due to the increased effective and continuous surveillance activities, rabies was stopped before it could spread to other areas on the island. The program costs totaled USD 450,000. Registration fees collected to maintain the program amounted to USD 105,740 and were re-allocated back into the community to sustain the program. The Province of Bohol, Philippines has eliminated dog and human rabies in less than three years by empowering the community and implementing an intersectoral strategy. In 2006, Bohol ranked 4th highest in the Philippines for human rabies, averaging 10 deaths per year. Launched in 2007, the program utilized a social awareness campaign, dog population control, mass dog vaccination campaigns, improved dog bite management and veterinary quarantine, a new diagnostic laboratory, expanded surveillance, and the inclusion of education modules into the school curriculum. Improving community compliance to existing national and provincial rabies laws and engaging volunteers to help conduct the project was a key to success. The program, led by the Governor of Bohol, was administered through a group of departments working together at a provincial and local level, and supervised through the Office of the Provincial Veterinarian. Financial support came through the Governor and several NGOs including the Global Alliance for Rabies Control. The program is self-sustaining, through a small dog registration fee fed back into the program, through the continuing education of children in their classrooms, and through the dedicated efforts of over 15,000 staff and volunteers throughout the island. More than 99% of all global human rabies deaths occur as a result of being exposed to infected dogs. In the Philippines, as in most Asian countries, canine rabies is endemic and poses a particularly perilous risk for persons living in rural areas Program sustainability is a critically important issue for all public health programs, but especially for resource-poor countries with limited budgets and many problems to resolve. Thus, a successful rabies prevention and control program must be built around the strengthening of intersectoral collaboration and cooperation between several public health components The province of Bohol is located in central Visayas Region, Philippines and prior to 2007, was among the top ten provinces in the country for human rabies deaths, averaging 10 deaths  per year. Of the reported human rabies cases in 2007–2008, 25% occurred in children less than 15 years old after being bitten by an infected dog From the outset of the project, the BRPEP was developed and executed as an intersectoral program including: Local community involvement; communication management; dog population control; mass dog vaccination; dog bite management; veterinary quarantine; and improved diagnostic capability, surveillance and monitoring. Funding for the BRPEP program was secured through many avenues including: Cost-sharing activities from the national government, provincial, municipal and village (or barangay) local government units; dog owners; local non-government organizations; the regional World Health Organization (WHO) office, the UBS Optimus Foundation, and the Global Alliance for Rabies Control, a not-for-profit organization focused on rabies control and prevention that worked closely with the OPV to bring technical support and coordination to the project. Although securing external funding was a key component of eliminating canine rabies, it was also critical to build the program in a sustainable manner to ensure that after canine rabies elimination, the program would not fail due to dwindling resources as occurred in Bohol during a previous effort to eliminate canine rabies. In 2000, seven years prior to the launch of the BRPEP, the first province-wide rabies mass dog vaccination program had been conducted on Bohol in conjunction with a regional initiative. This earlier program had been directly supported by the national government and had utilized only the existing veterinary service staff. The following year, in 2001, the dog vaccination coverage on Bohol had dropped to 2% and no allocation had been made for the purchase of vaccine the succeeding years.This article focuses on how the BRPEP was developed and implemented over a three year period, from 2007–2010, in Bohol, Philippines and discusses specific intersectoral strategies applicable to establishing sustainable rabies control and prevention programs in resource-poor countries.The island province of Bohol is the 10th largest island in the Philippines . The totAt the provincial level, the BRPEP was managed by the Bohol Rabies Prevention and Eradication Council (BRPEC) under the jurisdiction of the Governor. Canine rabies prevention and elimination was coordinated by the Provincial Veterinarian while human rabies prevention and elimination was managed by the Provincial Health Officer. The BRPEC administered overall implementation, formulated proposals, measures and strategies that would ensure the implementation and sustainability of the BRPEP. This body also recommended the enactment of support legislation, policies and directives to strengthen the program and provided timely reports to program partners and the general public.Parallel organizations to the BRPEP were created at the municipal and barangay (villages) local government units (LGU) namely the Municipal Rabies Prevention and Elimination Council (MRPEC) and the Bantay Rabies sa Barangay (BRB) or the “Rabies Watchers”. The MRPEC assumed the same roles and functions at their areas of jurisdiction in accordance with the BRPEP. The BRB ensured implementation of the program at the community level, arranged mass vaccination campaigns in their areas and compiled a master list of dogs and dog owners.The legal framework for implementing a rabies prevention and control program in the Philippines was already in place at the inauguration of the BRPEP and included several national regulations: Republic Act No. 8485 known as the Animal Welfare Act was enacted in 1998; Philippine Republic Act No. 9482 was enacted on 25 May 2007 and is identified as the Anti-Rabies Act of 2007 The local legislation defined the roles and responsibilities of the councils including: The organization of dog and dog owner registration; collection of registration fees; elimination of stray dogs ; dog vaccination; surveillance of human and animal rabies and dog bite incidents; settlement of disputes/agreements between bite victims and dog owners; and promotion of responsible dog ownership. To ensure dissemination of information and understanding of the supporting legislation and to promote program advocacy throughout the entire province, orientation and paralegal training sessions were conducted for 7,763 BRB volunteers.All communications regarding the BRPEP were managed in a coordinated manner to ensure integration of all components of the program as it progressed through the development, initiation, implementation, maintenance and sustainability phases. It was deemed critically important from the beginning of the BRPEP to facilitate understanding, cooperation, and support among stakeholders including the citizens living on Bohol, medical professionals, volunteers and paid employees implementing the program as well as government officials and funding agencies. Information was therefore delivered to the beneficiaries at the grass roots level as well as to government agencies overseeing the project and funding agencies supporting the project. The Provincial as well as the Municipal/City Rabies Task Forces designated key spokespersons authorized to dispense information and answer questions relevant to their areas of jurisdiction.As part of the communications strategy, a BRPEP handbook was developed, published and distributed to every municipal rabies council to serve as a reference for all field units working within the program A two-pronged social mobilization plan, including a ‘Community-focused program’ and a ‘School-based education program’, was launched as a first step to increase awareness and enhance community participation and support. Components of the IEC included discussions on rabies as a disease, its epidemiology, and its prevention and control, the Bohol Program in general and related national and municipal rabies ordinances as they supported the program implementation and responsible pet ownership.The community program concentrated on campaigns using tri-media , display of posters and banners in strategic areas, distribution of flyers and other materials, public hearings of local ordinances and hosting of municipal and barangay symposia, meetings and seminars. Educational campaigns were also conducted at various government offices and in churches. Philippine National Rabies Awareness Month in March, and World Rabies Day, held annually on September 28 were both observed to remind people of the continual threat of rabies and the importance of the program to eliminate rabies on Bohol.The School-based educational program, designed to improve awareness about rabies prevention, was developed and implemented in close supervision with the Department of Education and in coordination with the Department of Health and other member agencies of the BRPEC. The integration of rabies education into the school curriculum was initially developed by the Department of Health's National Rabies Control Program in 2006. In 2008, this program was piloted in Bohol beginning with round table discussions with teachers, followed by intensive planning, a workshop to develop lesson plans, orientation/training of teachers, and testing of the developed lesson plans for 6 months in the municipality of Corella during the 2008–2009 school year. In 2009, they were integrated into the curriculum of all 962 public elementary schools in Bohol. Educational activities for the children included: Incorporation of rabies modules into various subjects in the public elementary school curriculum; creation of “Rabies Scouters” (boy and girl scouts who have successfully completed a rabies and responsible pet ownership training program); creation of a campaign slogan to encourage responsible pet ownership; conducting fun educational events to celebrate the bond between children and pets. Other forms of campaigns promoting responsible pet ownership were also adopted in the city/municipalities.Dog population data on Bohol was first estimated from the 2006 census published by the Bureau of Agriculture Statistics (BAS). To secure data on the number and location of dogs throughout Bohol, the BRB initially conducted a house-to-house inquiry using a master list of households. The collected data reflected dog owner's name, number of dogs owned, whether they were confined, leashed or free-roaming, sex of each dog and total number of households. Dog population data was updated annually. To further regulate the possession of dogs, establish dog ownership, and facilitate the traceability of dogs involved in bite cases, the mandatory registration of dogs, with a corresponding fee collection, was implemented at the barangay level in accordance with the Provincial Ordinance. Dogs from households that were not able to afford the fees were also registered and the dog owners were given a promissory note and allowed a staggered payment. The collected registration fees were shared in a manner specified in the Provincial Ordinance as follows: 50% was retained in the barangay and 50% divided between the municipal and provincial treasury to support the sustainability of the entire rabies control program.A parenteral mass dog vaccination program was initiated in August of 2007 with the Governor of Bohol proclaiming August 2007 as the synchronized rabies vaccination month. Vaccination teams were organized at the provincial and municipal levels. Provincial teams were assigned to oversee the vaccination activities and to ensure the presence and usage of cold chain equipment in every municipality. The Municipal Agricultural Officers (MAO) led the municipal vaccination teams composed of livestock technicians, Barangay Livestock Aides and other personnel duly designated by the MRPEC trained as dog vaccinators. Vaccination activities were supervised by the provincial and district veterinarians. The majority of the members of the vaccination team, , received pre-exposure rabies vaccination. Team members who handled registration and collection of fees, or prepared reports did not come in contact with animals, and were not given pre-exposure rabies vaccination. All team members were briefed on proper rabies vaccination activities, provided vaccination supplies and paraphernalia, registration/health certificates and dog tags, recording forms and education campaign materials.Mop-up dog vaccination campaigns targeting low coverage areas were conducted within six months following the initial mass vaccination campaign. Dogs not vaccinated during the scheduled synchronized mass rabies vaccination campaigns were accommodated upon special arrangement with the municipal vaccination teams.A uniform dog tag, indicating vaccination, was securely fastened on a dog collar with the help of the assistant dog vaccinator. The tag was valid for one year from the date of vaccination and was replaced annually upon renewal of registration at which time a booster rabies vaccination was administered. Standardized dog vaccination report forms were consolidated and submitted to the BRB, the MAO and the BRPEP. The community was also encouraged to bring their cats for vaccination during the mass vaccination campaign.Dog population and movement control was implemented as part of the BRPEP and in compliance with the PNRA and the Animal Welfare Act. The dog population was managed by selective elimination of captured stray dogs, impounded dogs unclaimed within 3 days, and unmanageable dogs voluntarily submitted by owners Municipal rabies ordinances in the Philippines include a section on dog population management and designated a task force to perform this function. At the barangay level, socially acceptable procedures were discussed and widely disseminated throughout the community. Euthanasia procedures were initially conducted in accordance with Administrative Order No. 21 of the Department of Agriculture on the Code of Conduct in the Euthanasia for Pets/Companion Animals. During the third year of the project (2010), the purchase of a mobile veterinary clinic was funded by one of the partners and provided the opportunity to improve neutering, spaying, and euthanasia procedures. Additionally, partnerships were established with animal welfare organizations to improve dog population management practices to comply with recommended international standards.In 2007, prior to the launch of the BRPEP, two government-operated Animal Bite Treatment Centers (ABTC) were located on Bohol. As part of the BRPEP, four additional ABTCs, all privately operated, were established on the island to increase accessibility of timely post-exposure prophylaxis (PEP). Medical personnel were trained to administer rabies vaccines intradermally (ID) in order to provide effective PEP as economically as possible. Rabies biologicals were acquired either through the support of the DOH National Rabies Program, the investment of the private ABTCs, or direct procurement by the provincial government. Additionally, the government subsidized the purchase of PEP for indigents without sufficient financial resources, and for persons bitten by the dogs of responsible pet owners  and responsible pet owners exposed to suspect rabid dogs. All exposed patients received PEP according to the WHO standard ID regimen and all adverse events were reported and managed accordingly When possible, animals involved in biting incidents were observed for 14 days. If the animal demonstrated clinical signs of rabies during the observation period, including behavioral change or illness, it was euthanized and submitted for testing at the Regional Animal Disease Diagnostic Laboratory (RADDL) located in the city of Cebu on the adjacent island province. Submitted specimens were analyzed using the direct fluorescent antibody test. Laboratory results usually available within 1–2 days were relayed to the OPV and the MRPEC who releases this to the victim or the immediate relatives. The sample testing was free of charge but the cost of transporting specimens was assumed by the BRPEP.Surveillance systems for both human and canine rabies cases were established in order to ensure immediate and reliable transfer of information and follow-up in the case of human or animal exposure to a confirmed rabid animal. The protocol for investigation and follow-up of all bite cases was established, and continues presently as follows: The reporting of bite cases is initiated at the BRB level when the bite victim presents at the Rural Health Center for consultation, first aid treatment and assessment; if referred, the patient attends the closest ABTC for PEP. In cases where multiple biting incidents have occurred or when the involved animal is suspected to be rabid, the MAO and the OPV are informed in order to monitor the status of the biting animal(s). In highly suspicious cases involving the death of a bite victim, the attending physician immediately reports the case to the Provincial Rabies Coordinator who in turn contacts the Quick Response Team (QRT) and the Provincial Surveillance Unit to initiate a field investigation. Contact tracing is conducted in coordination with the MRPEC, all human and animal contacts of the biting animal are assessed and, if deemed necessary, immediately given PEP (human patients) or a booster vaccination . Movement of all animals involved in biting incidents is strictly monitored, the dog vaccination record at the barangay level is reviewed and mop-up vaccination is conducted.For planning purposes, estimating dog vaccination coverage, and evaluating the outcome of the project, a household survey was conducted to collect data on the owned dog population, knowledge, attitudes and practices (KAP), and data regarding the dog-human relationship. The survey was designed using questionnaires and cluster sampling procedures and collected data among 300 households Sustainability was ensured through the BRPEP activities on advocacy, general public awareness, child education, legislation, dog registration with fees, 100% of which was re-invested back into the BRPEP to establish a self-sustaining funding stream for the program. Sustainability was strategically built into the program through an integrated program interface with LGU officials, community leaders and other stakeholders to ensure community participation; establishment of a rabies diagnostic laboratory on the island for increasing further disease surveillance; creation of volunteer quarantine aides in coastal barangays to continually monitor the entry of new dogs from other provinces; and provision of annual award system to motivate excellence in program execution.Approximately 25% of all reported human deaths reported prior to the launch of the BRPEP occurred in children less than 15 years of age. After the initiation of the campaign, the number of human deaths dropped dramatically . The numThe BPREP was strongly supported by the National Rabies Law and the local ordinances at the provincial and municipal level. There were 4,379 BRB members and 96 members of the MRPECs that received instruction through the paralegal training courses associated with the BRPEP. Local legal procedures and the utilization of paralegal forms reduced violations of the law and improved community compliance, resulting in fewer free roaming dogs to be eliminated.Guidelines at the provincial, municipal and barangay council levels ensured uniformity in the understanding of the program and resulted in synchronized efforts across the 47 municipalities, one city and 1,109 barangays of the province. The successful engagement of the local communities resulted in a community-focused program that increased the total workforce of the rabies elimination program from the initial 124 government paid staff prior to 2007, to about 15,021 in 2009. This included the participation of community volunteers and other local officials to augment the government employees. The KAP survey suggested that effective communications had promoted a sense of program ownership since 89% of the households surveyed believed that the BRPEP was good for their community and 73% of households no longer allowed their dogs to roam free.As a result of the extensive IEC, there was a dramatic increase in the number of residents that acknowledged ownership of and registered their dogs. The community focused program also directly increased the number of bite victims that sought medical treatment after exposure. Increased dog bite reporting is a function of awareness, and the KAP survey revealed a high level of rabies knowledge among the general public: 94% claimed they had heard about rabies disease, 82% had knowledge about the local rabies program and 85% were aware of the rabies ordinances.The school-based education program provided rabies lessons and modules that were integrated into the curriculum of all elementary schools throughout the province by year two of the program. This ensured early and continuous education for children considered most at risk. Rabies prevention education was expanded to all elementary schools, reaching over 182,000 children or 16% of the total provincial population. Additionally, 128 Rabies Scouters from three municipalities were mobilized as peer advocates for rabies prevention. Province-wide simultaneous World Rabies Day activities were observed in all elementary schools through the issuance of a directive from the Governor to the Education Department-Bohol and were highlighted by candle lighting ceremonies and offering of prayers for children that had died of rabies.The 2006 BAS data, reported prior to the initiation of the BRPEP, indicated that the dog population was 168,161 of which 2.8% were estimated to be vaccinated. The total dog population on Bohol in 2007, as more accurately reported by the house to house survey, was 100,752 dogs, a significantly lower number than the 2006 data. For 2007 and 2008, dogs registered and paid for by owners totaled 44,516 and 53,692 respectively.Vaccination coverage of the dog population increased from 2.6% in 2007 to 44% in 2008. In 2009, after a second BRB survey, the dog population was recorded to be 76,407, of which 70% were registered and vaccinated. This coverage was obtained despite the introduction of mandatory registration fees. Reasons why the remaining 30% of dogs were not vaccinated included: owner absent during the vaccination campaign (20%); owner unable to restrain dogs (18%); dogs were thought to be too young (18%); and unable to afford fees (5%).In 2008, the LGU task forces eliminated 2,677 free-roaming dogs and 705 in 2009. The same household survey revealed that unowned dogs were always present in the neighborhood, the median was 3.0 with a range of 1–18 Through the BRPEP, a specific protocol was established to ensure that all persons exposed to potentially rabid animals received appropriate and prompt PEP. As part of the program, the number of animal bite treatment centers/clinics (ABTCs) was increased from two to six. Between 2007 and 2009, 8,158 patients received PEP through the provincial and private ABTCs . Of thesA total of 148 dog head samples were tested by FAT between 2007 and 2010. Of the samples submitted, 4% were confirmed positive for rabies. All of the 134 samples submitted for testing in 2009 and 2010 were confirmed negative for rabies. In April 2011, one dog sample was confirmed positive in Ubay Municipality, the same area as where a suspected human case had been reported in November 2010. The dog was <1 yr old and unvaccinated and had bitten two people, including a 7 year old. All persons exposed to the dog received PEP and a mop-up vaccination campaign and a house-to-house educational program were both initiated within the week.One suspect human rabies case was reported in November 2010. The young girl had been bitten on the upper left thigh on 18 October 2010 by a nursing dog after she had provoked it with a stick. The wound had not been cleaned and the patient was taken to a traditional healer the same afternoon as the bite occurred. On 23 November, the patient experienced intermittent low-grade fever and was given paracetamol, on 25 November, the patient manifested hydrophobia. On 26 November the patient was brought to a hospital in Ubay and was prescribed paracetamol and sent home. The patient died at home the next day and was buried before samples could be secured for testing. Following the provincial program SOP, a joint investigation by the provincial health, veterinary and local government staff was conducted within 72 hours of the time of death. Contact tracing and interview of key informants including the family and neighbors was immediately conducted by the investigating team but revealed no possible related human and animal cases in the locality and neighboring villages. There was immediate initiation of a mop up dog vaccination, intensified public awareness campaign and dog catching and testing for rabies.In 2010, the Provincial Government allocated funds for the establishment of a Provincial Rabies Diagnostic Laboratory. In preparation for its actual operation, a training course for rabies diagnoses using the direct rapid Immunohistochemistry test (dRIT) was conducted for the veterinary staff by the US Centers for Disease Control and Prevention (CDC) and RITM. A total of 12 local government veterinarians were trained. The CDC subsequently signed a Material Transfer Agreement with the provincial government for the equipment, supplies and specimen sharing. Thereafter, testing tissue samples using the dRIT began with validation by RITM.Reporting of suspected dog rabies was not routinely conducted prior to the initiation and launch of the BRPEP. Between 2002 and 2006, sample submission and confirmation of rabies infection was minimal . During Two rounds of compulsory registration and the collection of yearly registration fee of a total of 95,167 dogs between 2007–2009 established dog ownership and traceability of biting incidents, and enabled the program to generate revolving funds in the amount of 105,740 US$.Annual data from the inter-provincial records of dog movement indicated that the number of dog entries recorded increased each year . RecordeThe three-year BRPEP, initially launched in 2007, successfully achieved the goal of establishing a sustainable program with the aim of eliminating human and canine rabies throughout the province of Bohol contrary to the program that was initiated in 2000. The lack of a sustainable program in 2000 inevitably allowed canine rabies to spread throughout Bohol and the number of human rabies cases began to steadily increase. As 25% of the rabies deaths were in children, there was a need to especially focus on increasing education in this age group. Support from several stakeholders, including the Governor of Bohol, resulted in an agreement on a new action plan to tackle the increasing rabies problem. From the early planning stages of the BRPEP, all stakeholders and partners committed to building an intersectoral rabies control program on Bohol that would be self-sustaining when outside funding channels were no longer available.The BRPEP was strongly supported by existing national and local laws and ordinances including the PNRA. However, actual implementation of the program required an understanding of the need to implement the existing laws. This was achieved by increasing educational awareness across all levels of society, particularly focusing on how enforcement of the laws would improve the daily lives and the public health of the community. From the beginning of the program it was understood that only acceptance and ownership of the program at the community level would achieve sustainable and effective field operation. Thus, the massive social mobilization component of the BRPEP, including increasing awareness and improving citizen involvement in rabies prevention activities, were major factors in creating a successful community-based rabies control and prevention program on Bohol.The support and involvement of the Department of Education was critical for the successful implementation of the early childhood education about rabies prevention. The full integration of lessons on rabies and responsible pet ownership into the curriculum of all elementary schools throughout the province was the first program of its kind in the country. It demonstrated that working with the education sector was an effective strategy to improve awareness in the young population who are at most risk to rabies exposure.The surveillance of animal rabies prior to the initiation of the BRPEP was very poor. There were no diagnostic facilities on the island and accurate data regarding the number and species of animals infected with rabies throughout the province was unavailable. The importance of submitting suspected dogs for laboratory examinations was not appreciated and sending these samples for testing was costly resulting in few samples being submitted. The program improved reporting of suspected canine rabies cases through the active participation of the BRBs, rural health centers and the ABTCs. Prior to the program, dogs were commonly raised without confinement and stray dogs were a part of daily life. Dogs were responsible for 98% of the human rabies cases. Therefore, controlling the dog population and dog movement to achieve a feasible regular vaccination program and prevent the spread of rabies were both deemed necessary. Although stipulated in the Anti-Rabies Act of 2007, that “LGU should enforce dog impounding activities and field control to eliminate stray dogs”, strong political will and sufficient financial resources required to comply with the law were lacking For planning and evaluation of control and vaccination activities and acceptable methods, accurate information on dog population density and dog-human relationships was extremely valuable. Data collected by the relatively simple field methods employed in this project sufficed for planning cost-effective dog rabies control campaigns throughout the province. Dog population and ecology data collected will also be useful in planned epidemiological analyses of canine rabies in Bohol.The dog population on Bohol was decreased over four years by various methods including: strengthening of the national and provincial rabies laws that supported the community task forces; owners voluntarily reducing the number of dogs they retained; tighter controls on animal movement; increased clinics for spaying and neutering; operation of dog pounds/cages; and strengthening of veterinary quarantine services that ensured only vaccinated dogs with registration cards entered and left the province. Province-wide paralegal trainings also reinforced responsible dog ownership and reduced the stray dog population. The component on dog population management and movement control was effectively implemented only during the second year as pressure mounted from the community itself, in response to the BRPEP.In any large rabies control program high operational costs become significant challenges not only for the provincial government but also for each partner community. To help defray the cost of the program and improve community involvement, the BRPEP enlisted volunteers throughout the province. Volunteers are well-known and respected by their neighbors, have a thorough knowledge of the local settings, particularly in remote areas, and are motivated to serve their own communities. Through volunteerism, the number of people involved in implementing the rabies control program was increased from 124 to over 15,000.The Bohol Rabies program implementation between 2007 and 2010, required a budget allocation of approximately USD 450,000 and therefore resource mobilization was essential. Funding was supplied by several different program stakeholders including local and national governments and partner NGOs. At the community level, funding was generated through the collection of dog registration fees. This was in compliance with the National Rabies Law and the Provincial Local Ordinance. Although the main purpose of the registration fee was to regulate the keeping of dogs at the household level, fees collected have also served as a resource for operational funds since registered dogs are entitled to a free rabies vaccination, a dog tag with collar and a registration card. The average program cost per vaccinated dog was USD 1.62. The total community funds generated since the initiation of the program was enough for the next annual program budget.This article reports the overall results from the model rabies eradication program in an island province in the Philippines. Human deaths and canine cases have been dramatically decreased and surveillance improved. The success of the program was achieved through empowerment of the local communities and the use of the one health intersectoral approach to rabies control. The program was achieved through the joint efforts and shared resources of local and national government, various sectors of public health, animal health and agriculture, environment, legislation and policy, as well as non-government partners.


The Tanzania Field Epidemiology and Laboratory Training Program (TFELTP) was established in 2008 as a partnership among the Ministry of Health and Social Welfare (MOHSW), Muhimbili University of Health and Allied Sciences, National Institute for Medical Research, and local and international partners. TFELTP was established to strengthen the capacity of MOHSW to conduct public health surveillance and response, manage national disease control and prevention programs, and to enhance public health laboratory support for surveillance, diagnosis, treatment and disease monitoring. TFELTP is a 2-year full-time training program with approximately 25% time spent in class, and 75% in the field. TFELTP offers two tracks leading to an MSc degree in either Applied Epidemiology or, Epidemiology and Laboratory Management. Since 2008, the program has enrolled a total of 33 trainees . Of these, 11 were enrolled in 2008 and 100% graduated in 2010. All 11 graduates of cohort 1 are currently employed in public health positions within the country. Demand for the program as measured by the number of applicants has grown from 28 in 2008 to 56 in 2011. While training the public health leaders of the country, TFELTP has also provided essential service to the country in responding to high-profile disease outbreaks, and evaluating and improving its public health surveillance systems and diseases control programs. TFELTP was involved in the country assessment of the revised International Health Regulations (IHR) core capabilities, development of the Tanzania IHR plan, and incorporation of IHR into the revised Tanzania Integrated Disease Surveillance and Response (IDSR) guidelines. TFELTP is training a competent core group of public health leaders for Tanzania, as well as providing much needed service to the MOHSW in the areas of routine surveillance, outbreak detection and response, and disease program management. However, the immediate challenges that the program must address include development of a full range of in-country teaching capacity for the program, as well as a career path for graduates. In 1998 Tanzania was the first country in Africa to launch the World Health Organization, Regional Office for Africa (WHO-AFRO)'s Integrated Disease Surveillance and Response (IDSR) strategy. An assessment of the strengths and weaknesses of the existing public health surveillance and response systems was conducted, and a plan to improve and integrate these systems was developed . In 2001Plans to establish a field epidemiology and laboratory training program (FELTP) in Tanzania were conceived in 2005 by the MOHSW, NIMR and CDC. At this time, three trainees from Tanzania were enrolled in the Kenya FELTP [Upon a MOHSW request in 2007, an assessment led by the CDC and the African Field Epidemiology Network (AFENET) was undertaken to identify the country's needs, describe the existing infrastructure and resources that could be leveraged to support the program, and define the elements and resources needed to implement a FELTP in Tanzania.The Tanzania FELTP (TFELTP) was established in October 2008 as a partnership among MOHSW, the Muhimbili University of Health and Allied Sciences (MUHAS), and NIMR, along with support of local and international partners, principally, AFENET, CDC, USAID, and WHO. The goals of the TFELTP are to strengthen MOHSW capacity to i) conduct public health surveillance and response through IDSR, ii) manage and evaluate national disease control and prevention programs, and iii) strengthen public health laboratory support for surveillance, diagnosis, treatment, and clinical monitoring.Tanzania continues to experience a high burden of endemic diseases, as well as recurrent outbreaks of epidemic prone diseases, including cholera, bacterial meningitis, measles, and Rift Valley Fever –10. SuccTanzania faces a shortage of qualified field epidemiologists at all levels. Similar needs exist for public health laboratorians trained in field epidemiology to be effective members of surveillance and outbreak investigation teams at the national and regional levels. Several factors can be attributed to this, including shortage of relevant training facilities . The MOHBefore TFELTP was established, there were three master-level courses addressing public health issues, a Master of Public Health (MPH), Master of Arts in Health Policy and Management, and a Master of Science (MSc) in Tropical Disease Control in Tanzania. However, MOHSW recognized that in order to achieve its vision of addressing the public health concerns of the population, the number and type of graduates produced at the time was inadequate to meet the growing needs of the country. The TFELTP will facilitate Tanzania's target of training at least 200 epidemiologists and 100 public health scientists as outlined in its Health Sector Strategic Plan III .The TFELTP program is housed within NIMR headquarters in Dar-es-Salaam, with additional office space for coordinating field activities being at the MOHSW building (Epidemiology and Disease Control Section). TFELTP is a 2-year full-time program involving approximately 25% class room contact, and 75% field placement. Didactic sessions are held within the NIMR building, and are facilitated by MUHAS, MoHSW and NIMR staff. Classroom time occurs across the two years and is interspersed with field placements and examinations. Residents are attached to field placements at the national level in the first year of training and then the regional level during the second year. During the field placement, residents are assigned to various positions to provide epidemiologic and laboratory service to the MOHSW under the supervision of a mentor. The trainees travel to various regions of Tanzania for supervised outbreak investigations and field projects.TFELTP offers two tracks, leading to either an MSc in Applied Epidemiology (Epi Track), or an MSc in Epidemiology and Laboratory Management (Lab Track). The degrees are awarded by MUHAS. The entry requirement for the MSc in Applied Epidemiology is a first degree (Bachelor's level) in any of the fields of Medicine, Dentistry, Laboratory Sciences, Veterinary Medicine, Pharmacy, Environmental Health Sciences, Nursing or any other health-related field of study. Other eligible disciplines include statistics, demography, biology, food science and public health. The entry requirement for the MSc in Epidemiology and Laboratory Management is a first degree in the field of Laboratory Sciences, Medical Sciences, Veterinary Medicine, Biological sciences or any other laboratory-related field of study. Applicants for both tracks should have a minimum of two years health related working experience within a government department.TFELTP conducts a number of short courses that are offered to health workers in Tanzania. Program trainees participate in some of these courses. The courses include: three-month short course in basic field epidemiology and outbreak investigation. Two weeks for didactics at the beginning, with remaining period for an applied field project; three-month short course in non-communicable disease (NCD) epidemiology and prevention. Two weeks for didactics at the beginning, with remaining period for an applied field project.The program is governed by a Steering Committee comprising all partners and stakeholders under the leadership of the Chief Medical Officer of the MOHSW. The MOHSW provides the overall leadership for the program including the day to day running of the program through the secretariat. The secretariat is comprised of three ministry of health staff working along with administrative support staff, and program resident advisors.MUHAS is the primary academic partner for TFELTP. TFELTP courses draw multidisciplinary expertise from within and outside the MUHAS. The university assures the qualification of teaching staff, provides faculty, organizes the assignment of academic supervisors for the residents’ thesis research, and awards the degree upon successful completion of the program. Additionally, MUHAS and program staff teach academic courses and oversee research activities for the students and provide expertise in abstract and manuscript writing.NIMR provides the base of operations for the FELTP, including office space and training rooms. NIMR staff also ssist with the academic and technical supervision of residents. NIMR through its centers and stations across the country provides facilities for laboratory, data management, and field site opportunities for the trainees.WHO provides technical and financial support to the program in areas of outbreak investigation and response, and in collaboration with program staff participate in the planning for outbreak and response activities that address the needs of Tanzania.CDC Tanzania and the programs it represents are the primary funding partners of the TFELTP through the Global AIDS Program (GAP). Additional support is provided by USAID through the President's Malaria Initiative (PMI). CDC Tanzania is the primary link to the MOHSW, MUHAS, and other partners in Tanzania. CDC Tanzania staffs also provide technical assistance through teaching and mentorship of trainees, as well as arranging opportunities for field work including evaluations and field investigations within its programmatic activities in Tanzania.CDC Atlanta through its Division of Public Health Systems and Workforce Development (DPHSWD) is the partner primarily responsible for providing technical assistance to the FELTP for training and IDSR implementation. With a 30 year history of implementing applied epidemiology training programs in dozens of countries, DPHSWD uses their experienced staff and links to other parts of the agency and partners to build the program. For the initial 3 years of the program DPHSWD managed the AFENET cooperative agreement through which the majority of TFELTP funding was channelled to conduct the day to day operations. CDC technical assistance included teaching, mentoring, and was also directed towards production and dissemination of IDSR bulletins.AFENET was involved in areas that assisted in preparing the country for the program including, disease surveillance and outbreak response short courses, identification of key partners for the program, and fundraising. AFENET provides technical assistance to TFELTP through teaching, and links TFELTP to other similar program across Africa, providing a platform for exchange of expertise and networking. AFENET is the primary partner responsible for the administrative and logistical implementation of the program.The World Bank through the East Africa Public Health Laboratory Network is providing support to the program for two slots in 2011-2012, as well as short courses in epidemiology and laboratory management. The International Association of National Public Health Institute (IANPHI) provides support for NCD field sites and has provided 2 slots in 2010-2012. Ifakara Health Institute provides field sites to the program, and its staff for mentorship. TFELTP is currently working with NIMR and Georgia State University to establish an NCD short course on research.Since 2008, the program has enrolled a total of 33  trainees. Of these, 11 were enrolled in 2008 and graduated in 2010 representing a completion rate of 100%. The 2009 cohort of 10 is expected to graduate at the end of 2011. Demand for the program as measured by the number of applicants has grown from 28 in 2008, to 40 in 2010, to 56 in 2011.All 11 graduates of cohort 1 have since found public health related employment within the country. Their distribution is as follows: one with a university, two at the National level of the MOHSW, three at regional level under the ministry of local government, two at TFELTP as faculty, one at Ministry of Health Zanzibar, one at Ministry of Defence, and one at an international NGO. The graduate working with the Zanzibar Ministry of Health currently heads the Epidemiology Unit. In 2010, the TFELTP staff and residents participated in the country assessment of the revised International Health Regulations (IHR) core capabilities, and played a key role in the development of the assessment report. Subsequently, program staff has been involved in development of the Tanzania IHR plan. The program staff and trainees have also been involved in the incorporation of the IHR 2005 into the revised Tanzania IDSR guidelines.TFELTP residents have been involved and led investigation and response to high profile outbreaks including cholera ,16, H1N1TFELTP staffs have developed an NCD short course curriculum for regional capacity building for chronic disease epidemiology in collaboration with CDC DPHSWD, University of Copenhagen, and NIMR. The program has identified additional field sites for NCD mentorship of trainees. In 2011, Tanzania was one of five countries selected for CDC's global NCD initiative and conducted the first NCD epidemiology, surveillance, and evaluation two-week short course for medical doctors, TFELTP residents, and other public health staff.TFELTP is working with the Ministry of Livestock Development on the introduction of special seminars for zoonotic diseases. Based on the one-health principle, the aim of establishing the special seminars on zoonotic diseases is to build a foundation for future development of a Veterinary track which can address priority disease conditions at the animal and human interface, and improve communication and flow of surveillance information from the animal and wildlife sectors to the MOHSW.The program seeks to expand enrolment to meet the needs of the public health system in Tanzania. In order to do this, the program will need to expand the available classroom and field facilities, and staff. The key priorities will be to better equip the field sites, and ensure that the field supervisors’ skills are continually improved. In order to address the shortage of competent mentors and supervisors for the trainees, one approach will be to explore the use of TFELTP graduates as mentors at the field sites.Regional and international linkages will be an important avenue to build on the program's early successes. The program will be seeking to open its doors to potential trainees from countries neighbouring Tanzania that do not have programs of their own, Burundi, Malawi, Zambia are examples. This will greatly facilitate progress to improve cross-border collaboration in infectious disease surveillance and implementation of the IHR. Additionally, the program would like to open the NCD short course for training opportunities for regional participants. TFELTP plans to take a leading role in advocacy of IHR 2005 at regional sites within Tanzania where trainees are placed.Over the next three to four years, the program will need to establish a sustainability plan that guarantees ongoing production of high quality graduates. The plan must not only look inward at the MoHSW as the driver, funder and consumer of TFELTP products, but must also look outwards to development partners, the private sector, and trainees meeting their own tuition as the program begins to mature. This plan development process will require input from the various stakeholders to establish which parts of the program they will be willing to pay for and would find attractive to their own needs and interests. The program will need to continue periodic internal and external reviews to ensure it is meeting its mandate.One of the immediate challenges that the program must address to assure sustainability includes development of a full range of local teaching capacity for the program. Currently the program depends on external support to teach some of the courses for which there is limited expertise within Tanzania. The program will also need to work with the Human Resource Department of the MOHSW, and other relevant stakeholders to develop a career path for graduates of the program. This career path will be critical for defining job placement and promotion within the government system, an important framework for graduate retention. Finally, the program leadership will need to build on the early success of using an all-inclusive strategy to ensure that old partners remain committed to the program, as new ones join the collaboration.The TFELTP was developed out of the Tanzania's need to address pressing public health systems and human resource gaps. As the country's lead agency responsible for health, the MOHSW has driven the development of the program and remained at the pinnacle of leadership to ensure key stakeholders are involved, and the country's needs are addressed.Over the last 3 years, the program has made a significant contribution to development of expertise for IDSR implementation, and other MOHSW programs and strategies. TFELTP has made important contributions to the training of Tanzania's field epidemiologists and public health laboratorians. While training the future public health leaders of the country, this program simultaneously provides a much needed service by responding to acute outbreaks, evaluating and improving public health surveillance systems, and strengthening disease control programs.The TFELTP will need to develop a clear sustainability plan that includes both internal and external support  to ensure that Tanzania's public health needs are addressed in the medium to long-term.


Objective. To assess the effectiveness of a commercial nutrition program in improving weight, blood lipids, and health-related quality of life (HRQOL). Methods. Prospective observational study with followup after 1, 3, 6, and 12 months with data from questionnaires and blood samples. Subjects. After 12 months, we had data from 524 subjects . 84.1% of the subjects were women. The average BMI at baseline was 30.3 (SD = 5.7). Results. After 12 months, the average weight loss was 6.8 kg (SD = 7.1 kg). Program adherence declined over time but was still high after 12 months and showed a positive linear correlation with weight loss. Relevant blood parameters as well as HRQOL improved significantly. Conclusion. After 12 months, nearly two thirds of the samples had achieved >5% reduction of their initial weights. The high degree of program adherence is probably due to personal counseling and individually designed nutrition plans provided by the program. There are numerous studies on effects of therapeutic measures for overweight and obese persons . NevertThe metabolic balance nutrition program aims at permanently changing the client's lifestyle . Key eleThe primary objective of this study was to measure the short-, medium-, and long-term outcome of weight loss achieved during participation as well as the improvement of the relevant blood lipids and health-related quality of life (HRQOL). Program adherence was evaluated at each time point. Particular attention was given to dropout analyses to assess to which extent results can be generalized.We chose a single-group pre-post observational design as we wished to gain knowledge about the degree of program adherence and the effects of participating in the program under the conditions of a “real-life situation.” That should allow us to generalize the study findings to normal participants in the program. With this study design, we could avoid the more or less artificial study conditions usually associated with randomized controlled trials . The focParticipants and advisors filled out questionnaires at a total of five time points: at the start of participation, 4 weeks after the start, and 3 months, 6 months, and one year after. In addition, blood samples were taken at all time points to determine the relevant metabolic parameters. Data compiled from clients included, in addition to sociodemographic parameters, baseline data, and psychological factors such as motivation to complete the program and adherence to the program. The latter was assessed by asking about adherence to the eight basic rules of the program  e.g., “. In ordeAdvisors were asked to provide information on height and weight of clients and on whether individual or group counseling sessions were attended. To record (co)morbidity of clients we presented a list of 14 illnesses. On this list all relevant illnesses of each client had to be marked. Advisors were furthermore asked to assess the client's motivation on a scale of six (from 1 = very high motivation to 6=not motivated). The advisor questionnaire had to be filled out not only for study participants, but also for clients who refused to participate. We also asked participants who dropped out of the program to fill out a dropout questionnaire to determine reasons for quitting and weight at the time of quitting. N = 46 advisors took part in the study. In the period from mid-August 2007 to the end of January 2008, each advisor should consecutively include a maximum of 70 clients. Basically all clients who were at least 18 years old and had a sufficient knowledge of German were to be enclosed in the study. The study was reviewed and approved by the ethics committee of the Albert Ludwig University Medical Center Freiburg. The study was explained to the clients, who gave their written informed consent for participation.t-tests or chi2-tests, respectively. At each time point, the same tests were used to determine systematic differences between dropout clients and clients who remained in the study. To compare these groups, we additionally employed the baseline levels of the IRES-24 sum score and the degree to which “individual goals” had been achieved at the previous assessment. The weight change between baseline and 6 months was included in the dropout analyses of the last time point. It was also checked whether the clients included in the study were representative of all clients who began the program in the second half of 2007 . Both groups were compared on the basis of the variables age, gender, and BMI.We compared clients who refused to participate in the study with study participants, examining the variables age, gender, BMI, motivation, and type of counseling. We used Weight changes were analyzed not only per protocol (as treated), but also according to the intention-to-treat principle (ITT), using methods like “last observation carried forward” (LOCF) and “return to baseline” (RTB). To estimate the outcome for nonresponders, we applied a procedure which imputes missing weight data using the expectation maximization algorithm . An “adhPotential predictors of outcome were studied using multiple linear regression analyses. The predictors we chose were initial weight, motivation, type of counseling, the characteristics age, gender, level of education, and marital status, as well as participants' baseline levels of HRQOL. Furthermore, weight changes and adherence to the program were integrated into the regression model.The alpha error was corrected by Bonferroni adjustment . All anaN = 472 clients. The mean age was 50 years (SD = 12.0) with a range from 19 to 81 years. 84.1% of the clients in the sample were women. 43.0% were employees, 18.2% self-employed, 12.5% housewives, and another 12.3% were retired. 65.5% of the subjects were married. 25.3% suffered from hypertension, 23.9% from muscular-skeletal diseases, and 6.1% from diabetes mellitus. 3.4% had a coronary heart disease, and 3.2% renal insufficiency. Compared with the normative sample, the participants of the study had much poorer baseline measurements on all dimensions of the IRES-24, particularly so on the dimension “mental health.” The weight data were taken mainly from advisors' records . We only fell back on data provided by the clients themselves if advisors' data were missing.The analyses of the treatment results at 12 months were based on the data of N = 30,364). With respect to age and gender, no significant differences were found between this sample and the study sample. The BMI at the start of participation showed significant differences (P < .001) between the two samples: there were more obese clients in the study population  than in the total sample . Although significant, the effect size of this difference is quite small (0.18).In order to check whether our study clients differed from all the clients who began the program in the second half of 2007, we used data routinely collected for each client in the course of compiling diet plans  than study clients , and their BMI  was significantly lower than the BMI of study clients .During the recruitment period, a total of P < .001) with their “individual goals” achieved  than dropout clients . The average age of dropouts was also significantly lower (P = .005) than that of responders . One year after the start of participation, we recorded similar differences regarding age and the degree of individual achievement of goals. On the parameters of initial weight and weight reduction, however, no significant differences were found.If one looks at the retention rates of all clients who consented to participate in the study, one finds a rate of 85.2% at 4 weeks, about ten percent less at 3 months (74.4%), 64.4% at 6 months, and finally a rate of 55.5% at 12 months. At 4 weeks as well as at 3 months, no significant differences were registered between dropouts and clients remaining in the study. At 6 months, however, study clients were significantly more satisfied (reason for quitting that participation in the program was not compatible with the demands of their jobs (49.2%) or their family obligations (40.5%). 29.4% were dissatisfied with the supervision by their advisors (multiple responses were possible). Almost half of those who dropped out during the study gave as As the retention rate of 55.5% was not really satisfactory after 12 months, a follow-up assessment was carried out in order to fill in missing weight data. Thus the rate could be increased to 61.6%.M = 30.3  (SD = 5.7) at baseline was reduced to M = 27.7  (SD = 4.8) after one year. The subjects' average BMI of M = 28.7, SD = 4.8 versus M = 31.0, SD = 5.3, P < .001). They also had an average adherence score of M = 20.4  (SD = 6.3) one year after the start of the program, indicating significantly lower program adherence than the successful clients . 62.5% of the subjects reduced their initial weight by at least five percent at 12 months, and 31.1% lost ten or more percent of their initial weight. Those clients who did not achieve a weight loss of at least five percent had a significantly lower baseline weight . Both ITT methods resulted in a lower mean weight reduction at the various follow-up times than the weight reductions reported “as treated” .P < .001). There was also a significant improvement for the total cholesterol and LDL cholesterol levels . The HDL cholesterol levels also improved during participation in the program. Statistically these changes were not significant, though. The percentage of clients whose ratio of total cholesterol to HDL cholesterol was within the optimal range (<3.5) [In the long term, the improvement in triglyceride levels was highly significant (e (<3.5)  increaseP < .001, resp.) in all dimensions of the IRES-24 and in the sum score at all time points. The treatment effects regarding mental health and somatic health were at the upper end of “medium” effect sizes at the one-year followup. For the sum score, “high” effects were reported. As treatment effects may be influenced by the baseline levels of participants, that is, by the overall available potential for improvement, There were significant changes .With respect to successful weight reduction, the degree of adherence to the program and initial weight were strong predictors. Equally important factors were initial weight reduction in the first 4 weeks of participation, and gender . There was a significant difference in initial weight, but the absolute difference was relatively small . Therefore it can be assumed that the study clients represent quite well the total of all clients who joined the program during the recruitment period of the study.With respect to age and gender, the sample of the clients included in the study corresponded to all new clients who joined the program in the second half of 2007 . The same program was the object of a recent effectiveness study including a total of >140 000 clients [Results from other commercial programs can be compared with findings of this study. A randomized controlled trial (RCT) comparing the Jenny Craig program with a control group reported a mean weight loss of 7.3 kg (SD = 10.4) at 12 months . These d clients . After 1 clients  reportedOverweight and obesity have a great influence on the HRQOL , 33. IntIn our study, program adherence turned out to be the most important factor for success. We found a linear positive correlation between the degree of program adherence and the outcome. Varying rates of success of dietary programs can probably not be attributed to the type of diet . They are most likely due to the degree of program adherence which particular diets evoke in their participants. Any evaluation of dietary programs would therefore shift the focus from the question “Which type of diet works best to reduce weight?” to the question “Which diet works best to evoke program adherence?”.As we have no comparative data from other studies, we cannot really determine whether the degree of program adherence found in our study is high or not. We assume, however, that an average of 68% of the participants following the eight basic rules of the program after 1 year within the categories “completely” or “mainly” can be considered as “good program adherence.” We think that this good result was achieved by the “individualization” of the program. Personal nutrition plans on the basis of individual metabolic parameters obviously convey the impression of a diet cut to personal measure, which in turn results in a high identification with the program itself. Personal counseling enhances this identification even more. Thus the diet turns into “my personal nutrition program.”N = 30,364), (4) the inclusion of the outcome parameters lipids and HRQOL, (5) the detailed measurement of adherence to the program, and (6) an assessment of the correlation between program adherence and weight reduction.Compared to other studies about weight reduction programs, we see the strengths of our study in (1) a relatively high retention rate after 12 months (61.6%), (2) meticulously carried out dropout analyses to determine the degree to which the results can be generalised, (3) a comparison of the study sample with all individuals who started the program during the time of recruitment  control group could be considered a serious limitation. We chose this study design with the intention to measure how the participants reacted to the program in a “real-life situation,” affected by the study itself as little as possible. We also wished to provide data concerning effectiveness which have been missing from the scientific literature despite their importance . One hasParticipation in the nutrition program led to long-term improvements in health status and HRQOL. The effectiveness of the program has probably to be attributed to the high degree of adherence to the program's basic rules. Comparing various diets, Sacks et al.  found thWe conclude that program adherence turns out to be a major factor of successful long-term weight reduction, which leads us to recommend a shift of focus. The emphasis of any dietary program should be set on both, the aspect of nutrition as well as the aspect of motivation. One should closely look at the link between motivation and highly individualized weight reduction programs. Psycho-social aspects of compliance will have to be given more consideration in future research.


We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM Rabbits are important laboratory animals, widely used in many areas of biomedical research, including the production of antibodies and recombinant proteins. Rabbit models have contributed to the understanding of human diseases and the development of therapeutic compounds, devices and techniques. However it has not been possible to engineer precise genetic alterations in rabbits because they have so far been refractory to the two key enabling technologies; (I) rabbit embryonic stem (ES) cells capable of contributing to the germ line have yet to be derived, and (II) rabbits are particularly difficult to produce by somatic cell nuclear transfer The power and facility of gene targeting in ES cells has made the mouse by far the most intensively studied mammal COL1A1 in sheep PRNP in sheep, cattle and goats GGTA1 in pigs IGH in cattle and pigs IGKC in pigs CFTR in pigs Nuclear transfer from cultured somatic cells (SCNT) was developed to circumvent the requirement for ES cells to generate gene-targeted animals. This is, however, technically difficult, and more than ten years since our first demonstration of targeting Zinc-finger nucleases (ZFNs) are new tools that promise to radically simplify gene knockout and targeted gene replacement. An appropriately designed ZFN can create a double-strand break at a single predetermined site in the genomic DNA of an organism. In eukaryotes, double-strand break repair pathways often create small insertions and deletions at the break site, a useful means of inactivating genes of interest , or Tris/EDTA buffer  into one of the two pronuclei or the cytoplasm of fertilized rabbit oocytes. Microinjection was verified in each oocyte by observing slight swelling of the pronucleus, or disturbance in the cytoplasm caused by solution streaming from the capillary. Of the oocytes injected into the pronucleus, only those injected with Tris/EDTA survived and developed further. Following cytoplasmic injection, survival and development was better in embryos injected with EDTA. Cytoplasmic microinjection of mRNA diluted in EDTA also led to earlier onset and higher EGFP expression at lower concentrations than pronuclear microinjection of mRNA in Tris/EDTA .in vitro polyadenylation of mRNA. Injected embryos were cultured in vitro and monitored visually for development up to blastocyst stage. Exons 1 or 2 of the IgM locus were then amplified from blastocysts by nested PCR. Results indicated that: the optimal mRNA concentration range was between 3 to 6 ng/µl; microinjection of non-polyadenylated mRNA did not lead to effective mutagenesis; and microinjection into the pronucleus did not lead to effective mutagenesis , and mRNA coding for ZFN pair 18255/18257 targeted to IgM exon 1 see  was injeOf the ten rabbits identified as carrying ZFN induced mutant IgM alleles, eight  survived to sexual maturity and were used as founders to produce a F1 generation. Seven F0 rabbits transmitted mutant IgM alleles at frequencies ranging from 1.3% to 50% . Among tIn the immune response, the first antibodies to appear are of the IgM class. IgG, IgA, or IgE are then generated from IgM via class-switch recombination. Lack of IgM should therefore result in the absence of other isotypes. To determine if this was the case in our rabbits, we generated IgM knockout rabbits by breeding ZFN-treated founders and measured both IgM and IgG in serum.The Δ1  and Δ7  mutations in exon 1 both result in frameshifts in the IgM coding region and were predicted to disrupt expression of full-length protein. The founder male 22826 was intercrossed with the founder female 22830 to generate Δ1/Δ7 compound heterozygous animals. Three mutated F1 animals were obtained, one carrying the Δ1 mutation, one carrying the Δ7 mutation, and one Δ1/Δ7 compound heterozygote. The Δ7 mutation was also bred to homozygosity in an F2 generation. Serum IgM and IgG levels in these animals were determined by ELISA at 10 weeks of age. IgM and IgG levels were similar to wild type in Δ1 and Δ7 heterozygotes, but were undetectable in Δ1/Δ7 and Δ7/Δ7 rabbits . The perThe founder 22826 (Δ1 mutation) was also crossed with founder 25035, which carries a Δ15 deletion in exon 2 predicted to result in an in-frame loss of 5 amino acids. Serum analysis of Δ1/Δ15 F1 offspring revealed normal levels of IgM and IgG . Thus, tThese results show that ZFN-induced knockout alleles of the rabbit IgM gene transmit normally through the germline, and in homozygous form result in animals that are IgM- and IgG-deficient, but otherwise do not display obvious phenotypic abnormalities. It remains to be elucidated what effect the loss of IgM and IgG has on overall immunological function in these rabbits.We then tested if ZFN-mediated targeted sequence replacement was feasible directly in the oocyte. A gene targeting vector, IgME1, was designed to replace IgM exon 1 with a PGK neo cassette, providing ∼1.9 kb of novel sequence. The strategy is illustrated in MluI linearised IgME1 DNA (concentrations ranging from 5 to 15 ng/µl) and ZFN 18255/18257 mRNAs (concentrations ranging from 3 to 9 ng/µl of each) were injected into pronuclear stage fertilized oocytes, see Mixtures of Oocytes were then injected with 9 ng/µl ZFN 18255/18257 mRNA plus 10 ng/µl IgME1 DNA, and transferred to recipients. Seventeen fetuses were removed at day 15 of gestation and analyzed. All showed normal development, and examination of the uterine horns showed no evidence of degenerate or absorbed conceptuses. DNA was prepared from each fetus and screened by PCR. Three fetuses  showed amplification of a 1.758 kb product across the 5′ junction and a 1.683 kb fragment across the 3′ junction of the target site, consistent with replacement of IgM exon 1 with the PGK neo cassette by homologous recombination, see In summary, our data show efficient ZFN-induced gene disruption and homology-directed sequence replacement through microinjection in rabbit oocytes. We have obtained the first rabbits genetically engineered at a specific endogenous locus, the immunoglobulin M locus.Gene targeting in ES cells has propelled the mouse to the forefront of biomedical research. This is despite the fact that its small size limits the scope for many physiological investigations and the low blood volume precludes its use as a source of serum factors. Metabolic and physiological differences also limit the relevance of mice to some human diseases, e.g. cardiomyopathy Like mice, rabbits have a short generation time, produce large numbers of offspring and can be raised under specific pathogen free conditions. They have long been an important research animal, and are an indispensible source of polyclonal and monoclonal antibodies Human polyclonal antisera produced by hyperimmunisation of animals with a humanized immune system are potentially an extremely useful new class of therapeutic agents. The inactivation of the IgM locus and loss of endogenous IgM and IgG expression on B cells and in serum that we describe is an essential step in the modification of rabbits to produce human immunoglobulins. The expression of immunoglobulin transgenes has been described for pigs, chicken, and rabbits ZFN-mediated mutagenesis of the IgM locus in rabbit oocytes was efficient, yielding up to 30% mutant animals in the founder generation. Seven of eight founders passed on mutant alleles to their progeny. We also assessed whether ZFN-mediated targeted sequence replacement could be carried out directly in the oocyte without subsequent selection for the desired event, as recently described in mice Rodents, rabbits and other mammals all have particular limitations and strengths for biomedical research and are best regarded as complementary. Parallel and comparative studies performed in more than one species will speed progress in disease research and provide more reliable evaluation of novel diagnostic and therapeutic strategies. Gene targeting in ES cells is established in mouse and now in rat All animal experiments were approved by the Government of Upper Bavaria (permit numbers 55.2-1-54-2531-26-04 and 55.2-1-54-2531-9-09) and performed according to the European Union Normative for Care and Use of Experimental Animals. Chemical reagents were obtained from Sigma unless otherwise indicated.The vector IgME1, used for ZFN-mediated targeted replacement, comprised a 0.758 kb homologous arm extending upstream of IgM exon 1 on the 5′ side, and a 1.035 kb arm extending downstream of IgM exon 1 to the end of exon 3 on the 3′ side, separated by a PGK neo polyA cassette see . Homologin vitro and purified using mMESSAGE mMACHINE T7 Ultra and MEGAclear Kits (Ambion). The quality and concentration of mRNAs were verified by denaturing gel analysis and spectrophotometry.Synthetic capped and poly A-tailed mRNAs were transcribed Animals used as oocyte donors for the gene inactivation experiments were homozogous Alicia/homozogous Basilea back-crossed for several generations onto the ZIKA background. Alicia (Ali) rabbits carry a natural deletion within the IgH locus Microinjected oocytes were either cultured to blastocyst stage, or allowed to rest for 1 hr then transferred laparoscopically 2, 10 mM Tris, pH 8.0, 0.5% NP40, 0.5% Tween20 and 100 µg proteinase K; or by lysis in 8 µl 50 mM NaOH ; and from fetal tissue or ear biopsies of offspring by standard methods. Rabbit Cµ was amplified by PCR using primer pairs E1.1f (TGG CAG GGA CAC AGG AAA ACA) and E1.1r (GGC TCA CCG GGA AAG GAC); or E1.2f (GGC TGC CTG GCG CGG GAC TTT CT) and E1.2r (CTG TTG CTG TTG CTG TGC TGG ACT TTG) for exon1 and E2.1f (GGC TGC CTG GCG CGG GAC TTT C) and E2.1r (CCT GGC CTG GGG ACT GGA CAC TCA CT) or E2.2f (TGC CAG GCC ACA GGT TTC) and E2.2r (CTC GGA GGA CAT GGA CAC GTT CTT ATC) for exon 2. Nested PCRs from blastocysts were performed using the two primer pairs specific for exon 1 or 2 consecutively. PCR products containing mutated sequences detected by DNA sequence chromatography were cloned into the TOPO TA cloning kit (Invitrogen) for DNA sequencing of ZFN-induced mutations.DNA was prepared from blastocysts by lysis in 50 mM KCl, 1.5 mM MgClGCT GTA CAG AGG TGA ACT GGG CTG GTC TTA) and targR4 (CCG CTG TAA GTC TGC AGA AAT TGA TGA TCT) and across the 3′ junction using primers targF2 (CGG AGA ACC TGC GTG CAA TCC ATC T) and targR2 (TGG GCA GAG AGA AGG TGG TGA GCC T). Southern hybridization of 10 µg samples of genomic DNA was performed with a digoxigenin dUTP labeled probe and detected by chemiluminescence using standard methods.Targeted insertion of the PGK neo gene was detected by PCR across the 5′ junction using primers targF4 . Capture was by anti-rabbit-IgM (μ-chain) or anti-rabbit-IgG (Fc) biotin ; detection used mouse anti-rabbit IgM or IgG horseradish peroxidase (BD Pharmingen).5 PBMCs were distributed as 50 µl aliquots in 1.5 ml tubes and kept on ice. Anti-rabbit IgG FITC was obtained from AbD Serotec . Anti-rabbit IgM biotin was from BD Pharmingen . FITC labelled streptavidin and anti-mouse IgM phycoerythrin (PE) conjugate were from Invitrogen (Germany). The monoclonal antibody RACT30A, which detects an unidentified rabbit B-cell surface marker PBMCs were isolated from whole blood samples using separation media  according to the manufacturer's instruction and stained and analysed immediately. For single colour flow cytometry, 5×10Figure S1IgM sequence polymorphisms in different rabbit strains. The DNA sequence of the genomic IgM locus in 14 rabbits was determined; 6 rabbits of the ZIKA strain, 2 of the Alicia strain (TIF)Click here for additional data file.Figure S2DNA sequence of IgME-1 targeted locus. The sequence of the IgME1 gene targeting vector is indicated by highlights; the 5′ and 3′ homologous arms in yellow and the PGK neo cassette in blue. The positions of primers used to amplify PCR fragments across the 5′ junction (targF4 and targR4) and across the 3′ junction (targF2 and targR2) are indicated. ApaI and MscI restriction sites used to confirm the identity of the 5′ PCR product Click here for additional data file.Figure S3Southern analysis of fetuses. Samples of genomic DNA from 9 of the 17 fetuses recovered (numbers indicated) were digested with BspHI and HinDIII and hybridized to a probe comprising IgM exon 3, intron 3 and exon 4. Arrows indicate positions of the diagnostic 5.911 kb HindIII-HindIII fragment, derived from the wild type allele, and the 3.943 kb HindIII-BspHI fragment, derived from the targeted allele.(TIF)Click here for additional data file.Table S1Microinjection of mRNA coding for EGFP. 1 Experiment.(DOC)Click here for additional data file.Table S2Microinjection of mRNAs coding for ZFN SBS 18257/18255. 4 Experiments (separated by double lines).(DOC)Click here for additional data file.