No abstract

The convalescent subclinical carrier state of foot-and-mouth disease virus (FMDV) infection has been thoroughly investigated; contrastingly, the subclinical form of new infections of vaccinated and naïve hosts is recognized, but poorly understood. To investigate the natural dynamics of subclinical FMDV infections, a prospective, 12-month, longitudinal study was conducted in vaccinated Asian buffalo (Bubalus bubalis) under natural conditions in Pakistan, where FMDV is hyperendemic. Oropharyngeal fluid (OPF) samples were obtained quarterly from 300 buffalo on 30 farms which reported no clinical FMD during the 12-month study period. At the start of the study, 77.7% of buffalo had FMDV anti-NSP antibodies, and all farms had at least one seropositive buffalo. Based upon the presence of viral RNA and viral VP1 sequences obtained, distinct subcategories of subclinical infections were documented, including new, persistent, and serial infections with different FMDV strains. Viral RNA was detected in at least one OPF sample from 180 (60%) of the 300 buffalo. Over the course of the study, FMDV was detected in OPF of 80 buffalo that had been FMDV-free in previous OPF samples, indicating the occurrence of new subclinical infections. Eight buffalo were confirmed to be persistently infected, and serial infection with different FMDVs was confirmed in 13 animals. The most prevalent serotype detected was Asia-1, followed by A, and O. Phylogenetic analysis indicated multiple distinct clusters of serotypes Asia-1 and A. This study indicates a high prevalence of subclinical FMDV infection in vaccinated buffalo in Pakistan and emphasizes the importance of clinically undetected infection in FMD dynamics in endemic regions.

This reported study is of Pakistan's indigenous Cholistani cattle breed, a native of the Cholistan desert where it has been reported as having high production potential for nomadic herders. The aim of the present study was to assess haematochemical profile of adult Cholistani service bulls (n = 16), being reared at the Semen Production Unit Karaniwala, Bahawalpur, Cholistan desert, Pakistan, during various seasons as an indicator of their health status. Keeping in view the prevailing climatic conditions of the study area, four seasons of three months duration each were defined as (i) cool dry winter (November, December, January) (ii) temperate spring (February, March, April) (iii) hot dry summer (May, June, July) and (iv) hot humid summer (August, September, October). Blood specimens were collected fortnightly during the study period for haematochemical analyses (automated). The present study revealed that Cholistani service bulls have a tendency to maintain most of their haematochemical parameters more or less at the same level during various seasons which is suggestive of their adaptability under stressful climates without showing any signs of stress.

Availability of a rapid and sensitive diagnostic technique is key in successful prevention and control of infectious diseases like foot-and-mouth disease (FMD). Existing conventional diagnostic tests for FMD are laborious and time-consuming with low sensitivity and specificity. Molecular-based techniques are costly and difficult, involving refined apparatus like a thermal cycler. In the present study, the technique of real-time loop-mediated isothermal amplification (RT-LAMP) was standardized for diagnosing FMDV and its serotypes, evaluated using field samples, and compared with the existing real-time PCR in Pakistan. RT-LAMP amplified the target 3D gene using specific primers at 65 °C for 60 min and the VP1 gene using serotype specific primers at 63 °C for 60 min. A total of 38 samples out of 50 were positive by RT-LAMP and identified serotypes were A (n = 15), O (n = 15), and Asia-1 (n = 8). The efficiency of RT-LAMP in this study was highest for serotype Asia-1 (80.9%) followed by serotype A (73.4%) and O (62.35).

We report the laboratory analysis of 125 clinical samples from suspected cases of foot-and-mouth disease (FMD) in cattle and Asian buffalo collected in Pakistan between 2008 and 2012. Of these samples, 89 were found to contain viral RNA by rRT-PCR, of which 88 were also found to contain infectious FMD virus (FMDV) by virus isolation (VI), with strong correlation between these tests (κ = 0.96). Samples that were VI-positive were serotyped by antigen detection ELISA (Ag-ELISA) and VP1 sequence acquisition and analysis. Sequence data identified FMDV serotypes A (n = 13), O (n = 36) and Asia-1 (n = 41), including three samples from which both serotypes Asia-1 and O were detected. Serotype A viruses were classified within three different Iran-05 sublineages: HER-10, FAR-11 and ESF-10. All serotype Asia-1 were within Group VII (Sindh-08 lineage), in a genetic clade that differs from viruses isolated prior to 2010. All serotypes O were classified as PanAsia-2 within two different sublineages: ANT-10 and BAL-09. Using VP1 sequencing as the gold standard for serotype determination, the overall sensitivity of Ag-ELISA to correctly determine serotype was 74%, and serotype-specific sensitivity was 8% for serotype A, 88% for Asia-1 and 89% for O. Serotype-specific specificity was 100% for serotype A, 93% for Asia-1 and 94% for O. Interestingly, 12 of 13 serotype A viruses were not detected by Ag-ELISA. This study confirms earlier accounts of regional genetic diversity of FMDV in Pakistan and highlights the importance of continued validation of diagnostic tests for rapidly evolving pathogens such as FMDV.