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Abstract Background Foot-and-mouth disease (FMD) is endemic in Pakistan and causes huge economic losses. This work focus on the Landhi Dairy Colony (LDC), located in the suburbs of Karachi. LDC is the largest Buffalo colony in the world, with more than 300,000 animals (around 95% buffaloes and 5% cattle, as well as an unknown number of sheep and goats). Each month from April 2006 to April 2007 we collected mouth-swabs from apparently healthy buffaloes and cattle, applying a convenient sampling based on a two-stage random sampling scheme, in conjunction with participatory information from each selected farm. Furthermore, we also collected epithelium samples from animals with clinical disease, as well as mouth-swabs samples from those farms. In addition, we analysed a total of 180 serum samples randomly collecting 30 samples each month at the local slaughterhouse, from October 2006 to March 2007. Samples have been screened for FMDV by real-time RT-PCR and the partial or full 1D coding region of selected isolates has been sequenced. Serum samples have been analysed by applying serotype-specific antibody ELISA and non-structural proteins (NSP) antibody ELISA. Results FMDV infection prevalence at aggregate level shows an endemic occurrence of FMDV in the colony, with peaks in August 2006, December 2006 and February 2007 to March 2007. A significant association of prevalence peaks to the rainy seasons, which includes the coldest time of the year and the muslimic Eid-festival, has been demonstrated. Participatory information indicated that 88% of all questioned farmers vaccinate their animals. Analysis of the serum samples showed high levels of antibodies for serotypes O, A, Asia 1 and C. The median endpoint-titre for all tested serotypes, except serotype C, in VNT titration is at a serum dilution of equal or above 1/100. All 180 serum samples collected have been tested for antibodies against the non-structural proteins and all but four have been found positive. Out of the 106 swab-samples from apparently healthy and affected animals positive in real-time RT-PCR, we sequenced the partial or full 1D coding region from 58 samples. In addition we sequenced the full 1D coding region of 17 epithelium samples from animals with clinical signs of FMD. From all sequenced samples, swabs and epithelium, 19 belong to the regional PanAsia II lineage of serotype O and 56 to the A/Iran/2005 lineage of serotype A. Conclusion For an effective and realisable FMD control program in LDC, we suggest to introduce a twice annually mass vaccination of all buffaloes and cattle in the colony. These mass vaccinations should optimally take place shortly before the beginning of the two rainy periods, e.g. in June and September. Those vaccinations should, in our opinion, be in addition to the already individually performed vaccinations of single animals, as the latter usually targets only newly introduced animals. This suggested combination of mass vaccination of all large ruminants with the already performed individually vaccination should provide a continuous high level of herd immunity in the entire colony. Vaccines used for this purpose should contain the matching vaccine strains, i.e. as our results indicate antigens for A/Iran/2005 and the regional type of serotype O (PanAsia II), but also antigens of the, in this world region endemic, Asia 1 lineage should be included. In the long term it will be important to control the vaccine use, so that subclinical FMD will be avoided.

Abstract Background According to the World Reference Laboratory for FMD, a new subtype of FMDV serotype A was detected in Iran in 2005. This subtype was designated A/IRN/2005, and rapidly spread throughout Iran and moved westwards into Saudi Arabia and Turkey where it was initially detected from August 2005 and subsequently caused major disease problems in the spring of 2006. The same subtype reached Jordan in 2007. As part of an ongoing project we have also detected this subtype in Pakistan with the first positive samples detected in April 2006. To characterise this subtype in detail, we have sequenced and analysed the complete coding sequence of three subtype A/IRN/2005 isolates collected in Pakistan in 2006, the complete coding sequence of one subtype A/IRN/2005 isolate collected during the first outbreak in Turkey in 2005 and, in addition, the partial 1D coding sequence derived from 4 epithelium samples and 34 swab-samples from Asian buffaloes or cattle subsequently found to be infected with the A/IRN/2005 subtype. Results The phylogenies of the genome regions encoding for the structural proteins, displayed, with the exception of 1A, distinct, serotype-specific clustering and an evolutionary relationship of the A/IRN/2005 sublineage with the A22 sublineage. Potential recombination events have been detected in parts of the genome region coding for the non-structural proteins of FMDV. In addition, amino acid substitutions have been detected in the deduced VP1 protein sequence, potentially related to clinical or subclinical outcome of FMD. Indications of differential susceptibility for developing a subclinical course of disease between Asian buffaloes and cattle have been detected. Furthermore, hitherto unknown insertions of 2 amino acids before the second start codon, as well as sublineage specific amino acids have been detected in the genome region encoding for the leader proteinase of A/IRN/2005 sublineage. Conclusion Our findings indicate that the A/IRN/2005 sublineage has undergone two different paths of evolution for the structural and non-structural genome regions. The structural genome regions have had their evolutionary starting point in the A22 sublineage. It can be assumed that, due to the quasispecies structure of FMDV populations and the error-prone replication process, advantageous mutations in a changed environment have been fixed and lead to the occurrence of the new A/IRN/2005 sublineage. Together with this mechanism, recombination within the non-structural genome regions, potentially modifying the virulence of the virus, may be involved in the success of this new sublineage. The possible origin of this recombinant virus may be a co-infection with Asia1 and a serotype A precursor of the A/IRN/2005 sublineage potentially within Asian Buffaloes, as these appears to relatively easy become infected, but usually without developing clinical disease and consequently showing not a strong acute inflammatory immune response against a second FMDV infection.

Abstract Background Foot-and-Mouth Disease (FMD) causes significant economic losses in Turkish livestock. We have analysed the genetic diversity of the 1D sequences, encoding the hypervariable surface protein VP1, of Turkish isolates of serotype A and O collected from 1998 to 2004 in order to obtain epidemiological and immunological information. Results The 1D coding region of 33 serotype O and 20 serotype A isolates, obtained from outbreaks of FMD between 1998 and 2004, was sequenced. For serotype A, we confirmed the occurrence of the two subtypes IRN99 and IRN96. These subtypes are most divergent within the region encoding the immuno-dominant GH-loop. Also a close relationship to Foot-and-Mouth Disease virus (FMDV) serotype A isolates obtained from outbreaks in Iraq and Iran were detected and a clustering of isolates collected during the same period of time were found. The analysis of the deduced amino-acid sequences of these subtypes revealed evidence of positive selection in one site and one deletion, both within the GH-loop region. By inferring the ancestral history of the positively selected codon, two potential precursors were found. Furthermore, the structural alignment of IRN99 and IRN96 revealed differences between the tertiary structures of these subtypes. The similarity plot of the serotype O isolates suggested a more homogeneous group than the serotype A isolates. However, phylogenetic analysis revealed two major groups, each further divided in subgroups, of which some only consisted of Turkish isolates. Positively selected sites and structural differences of the Turkish isolates analysed, were not found. Conclusion The sequence and structural analysis of the IRN99 strains is indicative of positive selection suggesting an immunological advantage compared to IRN96. However, results of antigenic comparison reported elsewhere do not substantiate such a conclusion. There is evidence that IRN99 was introduced to Turkey, in all probability from Iran. Since, a member of the IRN96 lineage was included as a component of the FMDV vaccine produced since 2000, the outbreaks caused by IRN96 strains in 2004 could be due to incomplete vaccine coverage. The Turkish type O strains, all with a VP1 structure similar to the O1/Manisa/69 vaccine, appear in several sublineages. Whether these sublineages reflect multiple samplings from a limited number of outbreaks, or if they reflect cross-boundary introductions is not clear.

Abstract Background Two outbreaks of the disease contagious ecthyma were reported in 1999 and 2000 in Norwegian semi-domesticated reindeer (Rangifer tarandus tarandus). Contagious ecthyma is an epidermal disease of sheep and goats worldwide, which is caused by the zoonotic parapoxvirus orf virus. Characterisation of clinical samples from the two outbreaks in semi-domesticated reindeer in Norway by electron microscopy and PCR (B2L) revealed typical parapoxvirus particles and partial gene sequences corresponding to parapoxvirus, respectively. If contagious ecthyma in reindeer is caused by orf virus, the virus may be transferred from sheep and goats, via people, equipment and common use of pastures and corrals, to reindeer. Another possibility is that contagious ecthyma in reindeer is caused by a hitherto unclassified member of the parapoxvirus genus that circulates among reindeer herds and remains endemic in Norway. Results Genomic comparisons of one standard orf strain (orf NZ2) and the reindeer isolates, employing restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis, demonstrated high similarity between the reindeer viruses and known orf virus strains. Partial DNA sequences of two different viral genes were determined for the different isolates and compared with corresponding parapoxvirus genebank sequences. The comparison/alignment and construction of phylogenetic trees also point to an affiliation of the reindeer viruses to the species orf virus. Conclusion The results of this work imply that the parapoxvirus causing contagious ecthyma in Norwegian semi-domesticated reindeer belongs to the species orf virus and that the orf virus crosses the host species barrier from sheep and goat to semi-domesticated reindeer.