Foot-and-mouth disease (FMD) is a highly contagious and economically important disease caused by foot-and-mouth disease virus (FMDV). Animals that can be affected include cattle, buffaloes, sheep, goats, pigs and wild ruminants . FMDV is a positive sense, single-stranded RNA virus (genus Aphthovirus, family Picornaviridae) occurring in seven serotypes, O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3, each with a wide spectrum of antigenic and epidemiological distinct subtypes. The wide diversity is considered a consequence of the high mutation rate, quasi-species dynamics and recombination [2, 3].
FMD is endemic in Pakistan  and causes huge economic losses to commercial cattle and buffalo owners. According to the Food and Agriculture Organization of the United Nations (FAO) there are no proper arrangements for providing vaccine to the farmers and the open market is flooded with uncontrolled vaccine of doubtful efficiency .
FMD is considered endemic with the serotypes O, A and Asia 1 in both Pakistan  and the neighbouring countries of India, Afghanistan, Iran and China [7–9] and those serotypes are a continued problem in Pakistan.
According to the OIE HandiSTATUS  Pakistan considers itself as having a seasonal, low-level, sporadic occurrence of FMD (Pakistan reported around 10–30 outbreaks per year until year 2000 after which no information is available). Animals are only vaccinated upon request and the yearly number of vaccine doses used varies between 12,000 to 95,000 doses for cattle and 7,000 to 60,000 for buffaloes in the years from 1997–2002 (no data available after 2002) . This amount of vaccine is likely in addition to an unknown amount of open market, uncontrolled vaccines, but is nevertheless not much considering that Pakistan has a population of 51,1 million cattle, 56,9 million buffaloes, 50,3 million sheep and 123,9 million goats .
The majority of commercial dairy farmers are vaccinating their animals against FMD, either with imported trivalent vaccine, e.g. Aftovax (Merial, France), or with a locally produced monovalent vaccine (serotype O) .
Major challenges to control FMD in Pakistan relate, in part, to the lack of sufficient resources for diagnosis and continuous FMD genotype surveillance, but also the difficulties of controlling the vaccine market, as well as the lack of basic biosecurity awareness and control of animal movements. The latter is also hampered by the annual religious festival Eid ul-Azza, where thousands of buffaloes, cattle and small ruminants are transported across the country.
The present work focuses on the Landhi Dairy Colony (LDC), located in the suburbs of Karachi in the Sindh province of South-Pakistan. LDC is the largest dairy colony in Pakistan and the largest Buffalo colony in the world. It was established in 1959 within an area of 752 acres (incl. 250 acres for roads, shops and other facilities) for 15,000 animals, but there are now more than 300,000 dairy animals (> 95% buffaloes) on approximately 2000 farms and an unknown number of sheep and goats, which are freely running around in the whole colony. This overload, and unclear land ownership leads to hygiene and environmental problems. The majority of the milking animals in LDC are kept only for one lactation phase and consequently approximately 10–12% of the population is replaced every month.
After the lactation period the majority of the animals are sold to breeders or for slaughter and only a few are kept by the dairy farmers for re-breeding. Most of the animals are brought to and from the animal rich districts of Punjab and Sindh provinces.
Previous studies employing participatory epidemiology indicated a relatively high annually FMD prevalence between 41% and 50% in the southern Sindh region around Karachi .
To develop an effective vaccination strategy it is crucial to understand the dynamic of the disease and thereby indicating the best time points of administering the vaccine. Thus, individually vaccination is already performed on the large ruminant population, but with vaccines of variable quality and efficiency, it is likely that the majority of potential FMDV infections are subclinical and therefore not recognised. From April 2006 to April 2007 we collected monthly a number of mouth-swabs from apparently healthy buffaloes and cattle, applying a convenient sampling scheme based on a two-stage random sampling setup, in conjunction with participatory information from each selected farm. The total number of collected samples was 960 mouth-swabs from 124 farms.
Furthermore, we collected epithelium samples from clinically affected animals as well as mouth-swab samples from farms with a recent FMD outbreak, and 180 serum samples collected from slaughtered animals in the period from October 2006 to March 2007. The collection of probang and blood samples from living cattle or buffaloes was considered not possible due to socio-religious reasons.
Samples have been screened for FMDV by real-time RT-PCR [11, 12] and the partial 1D coding region of selected, FMDV positive isolates, has been sequenced. In addition, the full 1D coding region of a locally produced monovalent vaccine (serotype O) has been sequenced to examine the relatedness of vaccine strain to the circulating serotype O lineages. Serum samples have been analysed by applying serotype O, A and Asia 1 specific antibody ELISA  and non-structural proteins (NSP) ELISA .
This work will help to develop an appropriate vaccination strategy for Pakistan's largest dairy colony, including the choice of the best matching vaccines, as well as helping to improve our understanding of the epidemiology of FMD.