Variation in ovine KRTAP8-1 affects mean staple length and opacity of wool fiber.

In this study, keratin-associated proteins gene (KRTAP8-1) from five different sheep breeds and breed-crosses (n = 310) was genotyped using a Polymerase Chain Reaction-Single Strand confirmation Polymorphism (PCR-SSCP). Six unique genotypes were observed: AA, AC, AD, AE, DD and EE, with AA being the most common in the different breeds and crosses. Twelve wool characteristics: yield, mean staple length (MSL), bulk, mean fiber diameter (MFD), fiber diameter standard deviation (FDSD), coefficient of variation of fiber diameter (CVFD), medullation, standard deviation of medullation (MeSD), coefficient of variation of medullation (CVMed), opacity, standard deviation of opacity (OpSD), and coefficient of variation of opacity (CVOp) were measured on wool derived from the sheep. Variation in KRTAP8-1 was found to have strong association with MSL, OpSD and CVOp (p ≤ 0.027). The MSL of sheep of genotype AE was greater (p = 0.027) than for sheep of genotype AA. The OpSD of sheep of genotype AA was less (p = 0.017) than sheep with the AE genotype, and the CVOp of sheep with genotype AA was less (p = 0.018) than sheep with genotype AE. Further studies are required to confirm the role of variation in KRTAP8-1 in improving quality wool production.

Variation in the KRTAP6-3 gene and its association with wool characteristics in Pakistani sheep breeds and breed-crosses.

This study investigated variation in the keratin-associated proteins gene, KRTAP6-3, in 5 Pakistani sheep breeds/crosses using polymerase chain reaction-single strand confirmation polymorphism (PCR-SSCP) analysis. Different banding patterns were revealed, including previously described patterns and a novel pattern (named variant H). The amplified PCR product of the novel banding pattern was directly sequenced, and a synonymous nucleotide variation c.51T>C was revealed. Among the wool traits assessed, a strong correlation (r = 0.929; P < 0.001) was observed between fibre diameter standard deviation (FDSD) and coefficient of variation of fibre diameter (CVFD), between FDSD and medullation (r = 0.720; P < 0.001), between FDSD and medullation standard deviation (MeSD) (r = 0.734; P < 0.001), between MeSD and coefficient of variance of medullation (CVMed), (r = 0.903, P < 0.001), and between CVFD and medullation (r = 0.660), CVFD and MeSD (r = 0.786; P < 0.001), CVFD and CVMed (r = 0.701; P < 0.001) and medullation and MeSD (r = 0.771; P < 0.001). Variant B was found to be associated (P = 0.018) with CVFD; the presence of B being associated with a higher CVFD, than in its absence (41.08 ± 3.98 versus 36.34 ± 3.08). Variant C was associated with CVMed (P = 0.040), where sheep with C had a lower CVMed than sheep where it was absent. Variation in KRTAP6-3 was found to affect fibre diameter related traits of wool.

Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR.

Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n = 5), aborted fetuses (n = 13), and vaginal swabs (n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.

Foot-and-mouth disease: past, present and future.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals including cattle, pigs, sheep and many wildlife species. It can cause enormous economic losses when incursions occur into countries which are normally disease free. In addition, it has long-term effects within countries where the disease is endemic due to reduced animal productivity and the restrictions on international trade in animal products. The disease is caused by infection with foot-and-mouth disease virus (FMDV), a picornavirus. Seven different serotypes (and numerous variants) of FMDV have been identified. Some serotypes have a restricted geographical distribution, e.g. Asia-1, whereas others, notably serotype O, occur in many different regions. There is no cross-protection between serotypes and sometimes protection conferred by vaccines even of the same serotype can be limited. Thus it is important to characterize the viruses that are circulating if vaccination is being used for disease control. This review describes current methods for the detection and characterization of FMDVs. Sequence information is increasingly being used for identifying the source of outbreaks. In addition such information can be used to understand antigenic change within virus strains. The challenges and opportunities for improving the control of the disease within endemic settings, with a focus on Eurasia, are discussed, including the role of the FAO/EuFMD/OIE Progressive Control Pathway. Better control of the disease in endemic areas reduces the risk of incursions into disease-free regions.

Prevalence and risk factors of bluetongue in small and large ruminants maintained on Government farms in North-western Pakistan.

This study reports prevalence of antibodies against bluetongue virus (BTV) in animals kept on Government farms/research stations in North-western Pakistan and its association with different risk factors. In total, 1257 blood samples were collected, at random, from animals on 12 separate Government farms/research stations. The prevalence of antibodies against BTV was evaluated using a competitive ELISA. Mixed effects univariate and multivariate logistic regressions were applied to ascertain different risk factors associated with the prevalence of the infection using farm as random effect variable. The overall weighted seroprevalence was recorded as 52%. In univariate analysis, a significant association between sero-conversion to BTV infection and species (P < 0.0001), sex (P < 0.0001), herd size (P = 0.0295) and age of animal (P < 0.0001) was recorded. In multivariate mixed effects logistic regression analysis, prevalence of the infection was found to be 7 (95% CI =2-28) times higher in goats and buffalo than in sheep. Prevalence of the infection was found to be 2.5 (95% CI =1.7-3.3) times higher in female than male animals. However, no significant association was found between sero-conversion of BTV and herd size in multivariate mixed effects logistic regression. Age was found to be a risk factor for the sero-conversion; odds of sero-conversion to BTV increased by 1.29, 1.4, 1.32 and 1.6 times per year increase in age of sheep, goats, buffalo and cattle, respectively. Prevalence of bluetongue was found higher in animals maintained on Government owned farms than that in individual holdings, as previously reported in Pakistan.

Evolutionary analysis of serotype A foot-and-mouth disease viruses circulating in Pakistan and Afghanistan during 2002-2009.

Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. Three different serotypes of the virus, namely O, A and Asia-1, are responsible for the outbreaks of this disease in these countries. In the present study, the nucleotide-coding sequences for the VP1 capsid protein (69 samples) or for all four capsid proteins (P1, seven representative samples) of the serotype A FMD viruses circulating in Pakistan and Afghanistan were determined. Phylogenetic analysis of the foot-and-mouth disease virus (FMDV) VP1-coding sequences from these countries collected between 2002 and 2009 revealed the presence of at least four lineages within two distinct genotypes, all belonging to the Asia topotype, within serotype A. The predominant lineage observed was A-Iran05 but three other lineages (a new one is named here A-Pak09) were also identified. The A-Iran05 lineage is still evolving as revealed by the presence of seven distinct variants, the dominant being the A-Iran05AFG-07 and A-Iran05BAR-08 sublineages. The rate of evolution of the A-Iran05 lineage was found to be about 1.2×10(-2) substitutions per nucleotide per year. This high rate of change is consistent with the rapid appearance of new variants of FMDV serotype A in the region. The A22/Iraq FMDV vaccine is antigenically distinct from the A-Iran05BAR-08 viruses. Mapping of the amino acid changes between the capsid proteins of the A22/Iraq vaccine strain and the A-Iran05BAR-08 viruses onto the A22/Iraq capsid structure identified candidate amino acid substitutions, exposed on the virus surface, which may explain this antigenic difference.

Foot-and-mouth disease viruses of the O/ME-SA/Ind-2001e sublineage in Pakistan.

The presence of foot-and-mouth disease virus (FMDV) of the O/ME-SA/Ind-2001e sublineage within Pakistan was initially detected in two samples collected during 2019. Analysis of further serotype O FMDVs responsible for disease outbreaks in 2019-2020 in the country has now identified the spread of this sublineage to 10 districts within two separate provinces in North-Eastern and North-Western Pakistan. Phylogenetic analysis indicates that these viruses are closely related to those circulating in Bhutan, Nepal and India. The VP1 coding sequences of these viruses from Pakistan belong to three distinct clusters, which may indicate multiple introductions of this virus sublineage, although the routes of introduction are unknown. Vaccine matching studies against O1 Manisa, O 3039 and O TUR/5/2009 support the suitability of existing vaccine strains to control current field outbreaks, but further studies are warranted to monitor the spread and evolution of the O/ME-SA/Ind-2001e sublineage in the region. (145 words).

Status of foot-and-mouth disease in Pakistan.

The present study reports the distribution of different serotypes of foot-and-mouth disease virus (FMDV) in Pakistan during the period 1952-2007. During this time, a total of 1,543 epithelial samples out of 2,484 tested were found positive for various serotypes of FMDV. Serotype O was found to be the most prevalent (p < 0.001) followed by serotype Asia-1 and A. Serotype C was detected only in 1954, 1963 and 1995. The disease was found to be more prevalent (p < 0.0001) in cattle than buffaloes. The geographical distribution of 153 laboratory confirmed FMD outbreaks from 2002 to 2007 and the serotypes of the virus involved has been mapped. Higher number of outbreaks of the disease was noted between the months of January to March during this period, which may be attributed to the livestock movement in the country particularly due to religious festival, Eidul Azha, in which the animals are sacrificed.

Evidence for multiple recombination events within foot-and-mouth disease viruses circulating in West Eurasia.

Phylogenetic studies on foot-and-mouth disease viruses (FMDVs) circulating in the West Eurasian region have largely focused on the genomic sequences encoding the structural proteins that determine the serotype. The present study has compared near-complete genome sequences of FMDVs representative of the viruses that circulate in this region. The near-complete genome sequences (ca. 7,600 nt) were generated from multiple overlapping RT-PCR products. These amplicons were from FMDVs belonging to serotypes O, A and Asia-1, including members of the O-PanAsia-II and the A-Iran05 lineages, and of Group-II and Group-VII (Sindh-08) within serotype Asia-1, which are currently predominant and widespread in West Eurasia. These new sequences were analysed together with other sequences obtained from GenBank. Comparison of different regions of the FMDVs genomes revealed evidence for multiple, inter-serotypic, recombination events between FMDVs belonging to the serotypes O, A and Asia-1. It is concluded from the present study that dramatic changes in virus sequences can occur in the field through recombination between different FMDV genomes. These analyses provide information about the ancestry of the serotype O, A and Asia-1 FMDVs that are currently circulating within the West Eurasian region.

Polymorphism in the ovine keratin-associated protein gene KRTAP7-1 and its association with wool characteristics.

The keratin-associated proteins (KAPs) are structural components of wool fibers and variation in the genes encoding the KAPs can affect wool traits. In this study, sequence variation in the ovine KAP7-1 gene (KRTAP7-1) was investigated in 222 sheep across 5 different Pakistani breeds and breed crosses. Two previously identified variants (A and B) of the KRTAP7-1 coding sequence were identified. The frequency of the genotypes AA and AB was 76% and 23%, respectively, and that of BB was 1%. The association of sequence variation with various wool traits and measurements included yield (the proportion of greasy fleece weight that is clean fleece), mean staple length (MSL), wool bulk, mean fiber diameter, fiber diameter SD, the coefficient of variation of fiber diameter, medullation, the SD of medullation, the coefficient of variation of medullation, fiber opacity, the SD of opacity, and the coefficient of variation of opacity. Variation in KRTAP7-1 was found to be associated with yield (P = 0.017). The adjusted mean yield of sheep of genotype AA (n = 169) was 79.9 ±â€…2.72%, while that of genotype AB (n = 51) was 81.9 ±â€…3.37%. There was also an association between variation in KRTAP7-1 and MSL (P = 0.024), with sheep of genotype AA (n = 169) having an adjusted mean MSL of 47.3 ±â€…0.57 mm compared with sheep of genotype AB (n = 51, 50.9 ±â€…0.65 mm). Yield and MSL are both important wool production traits, hence variation in KRTAP7-1 needs to be further investigated in more sheep of differing breed.

Molecular epidemiology, evolution and phylogeny of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) is responsible for one of the most economically important infectious diseases of livestock. The virus spreads very easily and continues to affect many countries (mainly in Africa and Asia). The risks associated with the introduction of FMDV result in major barriers to trade in animals and their products. Seven antigenically distinct forms of the virus are known, called serotypes, but serotype C has not been detected anywhere for many years and may now be extinct. The serotypes have been further divided into topotypes (except for serotype Asia-1 viruses, which comprise a single topotype), genotypes, lineages and sub-lineages, which are usually restricted to specific geographical regions. However, sometimes, trans-regional spread of some strains occurs. Due to the error-prone replication of the RNA genome, the virus continuously evolves and new strains frequently arise (e.g. with modified antigenicity). Using nucleotide sequencing technologies, this rapid evolution of the viral genome can be followed. This allows the tracing of virus transmission pathways within an outbreak of disease if (near) full-length genome sequences can be generated. Furthermore, the movement of distinct virus lineages, from one country to another can be analyzed. Some important examples of the spread of new strains of FMD virus are described.

Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

Rapid and accurate diagnosis of foot-and-mouth disease (FMD) and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT) values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples) and specificity (100% each for serotypes O, A and Asia-1 positive samples) for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor recognition of some of the viruses by the earlier assays. These new assays should help in the early detection and typing of serotype O, A and Asia-1 FMDVs circulating in West Eurasia to enable improved disease control.

Rescue of foot-and-mouth disease viruses that are pathogenic for cattle from preserved viral RNA samples.

BACKGROUND: Foot and mouth disease is an economically important disease of cloven-hoofed animals including cattle, sheep and pigs. It is caused by a picornavirus, foot-and-mouth disease virus (FMDV), which has a positive sense RNA genome which, when introduced into cells, can initiate virus replication. PRINCIPAL FINDINGS: A system has been developed to rescue infectious FMDV from RNA preparations generated from clinical samples obtained under experimental conditions and then applied to samples collected in the "field". Clinical samples from suspect cases of foot-and-mouth disease (FMD) were obtained from within Pakistan and Afghanistan. The samples were treated to preserve the RNA and then transported to National Veterinary Institute, Lindholm, Denmark. Following RNA extraction, FMDV RNA was quantified by real-time RT-PCR and samples containing significant levels of FMDV RNA were introduced into susceptible cells using electroporation. Progeny viruses were amplified in primary bovine thyroid cells and characterized using antigen ELISA and also by RT-PCR plus sequencing. FMD viruses of three different serotypes and multiple lineages have been successfully rescued from the RNA samples. Two of the rescued viruses (of serotype O and Asia 1) were inoculated into bull calves under high containment conditions. Acute clinical disease was observed in each case which spread rapidly from the inoculated calves to in-contact animals. Thus the rescued viruses were highly pathogenic. The availability of the rescued viruses enabled serotyping by antigen ELISA and facilitated genome sequencing. CONCLUSIONS: The procedure described here should improve the characterization of FMDVs circulating in countries where the disease is endemic and thus enhance disease control globally.

Molecular characterization of serotype Asia-1 foot-and-mouth disease viruses in Pakistan and Afghanistan; emergence of a new genetic Group and evidence for a novel recombinant virus.

Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. The FMD virus serotypes O, A and Asia-1 are responsible for the outbreaks in these countries. Diverse strains of FMDV, even within the same serotype, co-circulate. Characterization of the viruses in circulation can facilitate appropriate vaccine selection and tracing of outbreaks. The present study characterized foot-and-mouth disease serotype Asia-1 viruses circulating in Pakistan and Afghanistan during the period 1998-2009. Phylogenetic analysis of FMDV type Asia-1 revealed that three different genetic Groups of serotype Asia-1 have circulated in Pakistan during this time. These are Group-II, -VI and, recently, a novel Group (designated here as Group-VII). This new Group has not been detected in neighbouring Afghanistan during the study period but viruses from Groups I and -II are in circulation there. Using near complete genome sequences, from FMD viruses of serotypes Asia-1 and A that are currently circulating in Pakistan, we have identified an interserotypic recombinant virus, which has the VP2-VP3-VP1-2A coding sequences derived from a Group-VII Asia-1 virus and the remainder of the genome from a serotype A virus of the A-Iran05(AFG-07) sub-lineage. The Asia-1 FMDVs currently circulating in Pakistan and Afghanistan are not efficiently neutralized by antisera raised against the Asia-1/Shamir vaccine strain. Thus, new Asia-1 vaccine strains may be required to block the spread of the current Asia-1 viruses.

Genetic diversity of foot-and-mouth disease virus serotype O in Pakistan and Afghanistan, 1997-2009.

Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan; serotypes O, A and Asia-1 of the virus are responsible for the outbreaks in these countries with FMDV type O usually being the most common. In the present study, the nucleotide sequences encoding the FMDV capsid protein VP1 from virus samples were determined. Phylogenetic analysis of the serotype O FMD viruses circulating in Pakistan and Afghanistan between 1997 and 2009 revealed the presence of at least three different lineages within the ME-SA (Middle East South Asia) topotype. The three lineages detected in this study are Pak98, Iran2001 and PanAsia. The PanAsia lineage is currently dominant in the area and is evolving with time as revealed by the appearance of distinct variants e.g. PanAsia-II and a new variant designated here as PanAsia-III. The rates of evolution of the O-PanAsia-II and III sublineages prevalent in the region were found to be 6.65 × 10(-3) (95% CI=5.49-7.80 × 10(-3)) and 7.80 × 10(-3) (95% CI=6.72-8.89 × 10(-3)) substitutions per nucleotide per year, respectively. The present study reveals the presence of multiple (sub-)lineages of FMDV serotype O co-circulating in the region and that significant new variants are frequently emerging.

Foot-and-mouth disease vaccine potency testing: the influence of serotype, type of adjuvant, valency, fractionation method, and virus culture on the dose-response curve in cattle.

The aim of this study was to determine a relationship between vaccine potency (amount of PD50 per dose) and fraction of clinically protected cattle following homologous challenge with infectious foot-and-mouth disease (FMD) virus, and to determine the effect of method of fractionation, serotype, type of adjuvant, valency and type of virus culture on the dose-response curve. Data from 297 potency tests of FMD vaccines, comprising 4004 vaccinated cattle, performed at the FMD vaccine production facility in the Netherlands, were used for the present study. A generalised linear mixed effect model was used to analyse the results. Our study showed that the relation between FMD vaccine potency and fraction protected was also affected by the serotype and type of adjuvant. No common level of protection could be assigned to all FMD vaccines with the same amount of PD50 per dose, this information is essential when designing a new standard FMD vaccines control.