Search

This study was designed to characterize N gene sequences of peste des petits ruminants virus (PPRV) isolates circulating in Pakistan and to evaluate the efficacy of available diagnostic assays on local isolates. During the study period, a total of sixty PPR outbreaks were investigated. A total of 20 selected samples from these outbreaks were sequenced for N gene. The result analysis and the phylogenetic trees indicated two different viral groups in N gene: one was closer to China and Tajikistan, while other group was similar to isolates from Iran and Saudi Arabia. Efficacy of three commercially available tests for the antigen detection of PPR, that is, peste test, enzyme‐linked immunosorbent assay (ELISA) and reverse transcriptase‐polymerase chain reaction (RT‐PCR) was compared. Keeping PCR as gold standard, sensitivity was calculated as 85% and 57% and specificity was calculated as 83% and 79% for ELISA and peste test, respectively. Value of K for ELISA was 0.67 which indicates good agreement between ELISA and RT‐PCR. Value of K for peste test was 0.33 which indicates fair agreement between peste test and RT‐PCR. In conclusion, study provides premier information about the use of different diagnostic tests and molecular situation of PPRV in Pakistan. [ABSTRACT FROM AUTHOR]

Aflatoxin M1 contamination of milk in Pakistan, like many developing countries, is poorly understood. The present study was therefore conducted to determine AFM1 contamination of milk and its contributory factors in Pakistan. We sampled milk and feedstuffs from 450 peri-urban dairy farms in seven major cities following a cross-sectional study design. Analysis of milk using ELISA revealed high contamination with an overall average of 3164.5 ng of AFM1/L, and significant differences (p < 0.001) between cities. The milk sampled from Gilgit, in northern hilly areas, had an average AFM1 level of 92.5 ng/L. Milk from other cities had 3529.7 ng/L average contamination, with only 5.7% samples qualifying the maximum tolerable limit of 500 ng of AFM1/L. Heavy mean aflatoxin contamination was found in bakery waste (724.6 μg/kg), and cottonseed cake (600.8 μg/kg). Rest of the other feedstuffs had moderate to low mean aflatoxin contamination, ranging from 66.0 μg/kg in maize stover to 3.4 μg/kg in wheat bran. The mean aflatoxin level in commercial dairy concentrates was 32.7 µg/kg. About 80% of the total aflatoxin intake of dairy animals was contributed by cottonseed cake alone due to its high aflatoxin contamination and proportion in dairy rations. On-farm storage time of oilseed cakes varied (p < 0.01) in different cities but was not associated with aflatoxin contamination. The exceptionally high AFM1 contamination suggests that milk from peri-urban dairy farms is a serious public health threat in Pakistan. This situation can be mitigated by reducing aflatoxin contamination in cottonseed cake and promoting the use of commercial concentrates and other feedstuffs with low contamination. [ABSTRACT FROM AUTHOR]

Peste de petits ruminant (PPR) is a highly contagious viral disease affecting predominantly sheep and goats. The virus belongs to the genus Morbillivirus accounting for causing immunosuppression and severe lymphopenia in host lymphoid cells principally due to the presence of a glycoprotein known as Signaling Lymphocyte Activation Molecule (SLAM) acting as cellular receptor. Limitations regarding the efficiency of PPR virus isolation using a variety of cell lines paved the way to the use of a new cell line (BTS-34) expressing these receptors. In the present study, a total of 18 PPRV confirmed strains were inoculated onto low passage Vero- 76 and BTS-34 cell lines simultaneously to compare the susceptibilities of Vero and BTS cells for PPR virus isolation. The recovered viruses were then re-confirmed by Ic-ELISA and RT-PCR. In order to investigate the differences in titers of virus strains, serial tenfold dilutions were made separately using both cells at a density of 0.01x106 and tissue culture infective dose fifty (TCID50) was calculated. The results revealed that the characteristic pattern of the CPEs was more obvious in BTS-34 cell line marked by giant cells ultimately transforming into distinguished syncytia in 2 to 3 days. The average incubation time for virus isolation on Vero cells was about 10 days and syncytia formation were less marked. The growth curve indicated a one log increase in virus titer in case of BTS-34 cell line as compared to Vero-76. Statistically, there was significant difference between two types of cell lines in terms of number of days taken for PPR virus isolation as determined by single factor ANOVA (P<0.001). The study showed that BTS cells produced high virus titer with clearly distinct CPEs in short time due to the presence of SLAM receptors as compared to Vero cells suggesting the BTS cells to be more efficient and sensitive for PPRV isolation. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

The aim of the study was to determine the seroprevalence of foot-and-mouth disease (FMD) and isolation of the FMD virus (FMDV) in the milk of apparently healthy Asian buffaloes. A cross-sectional study was conducted in periurban dairy farms (n = 20) on =20 animals per farm in the suburbs of Islamabad, Pakistan. Sera samples (n = 200) were collected to monitor FMDV seroconversions. The analysis of serum samples using ELISA indicated a high seroprevalence of FMD (78%; 156/200) in Asian buffaloes. Milk samples were collected from FMDV seropositive animals only, and virus-specific signals were detected in 12.2% (19/156) of the animals by reverse transcriptase real-time PCR (rRT-PCR). Milk samples positive for FMDV using rRT-PCR were inoculated into LF-BK cells for virus isolation. Two FMDV isolates (serotypes O and Asia-1) were recovered from the milk samples. A high seroprevalence of FMD and isolation of FMDV from the milk of healthy Asian buffaloes may represent a possible role for healthy Asian buffaloes as an FMD reservoir. [ABSTRACT FROM AUTHOR]

An outbreak of Peste Des Petits Ruminants (PPR) in suburban area of Rawalpindi District of Punjab province Pakistan has been investigated. A total of 38 clinically affected animals out of 140 goats and sheep 10-18 months old with no history of PPR vaccination. Nasal and ocular swabs were analysed by RT-PCR for the presence of PPRV specific genome and their sera were analysed for PPR antibodies by competitive ELISA. Eight out of 10 swab samples were found positive for PPRV and all sera were positive for PPRV specific antibodies. It is thus speculated that a comparatively mild strain of PPR virus exists in the population. [ABSTRACT FROM AUTHOR]

The study evaluated the efficacy of three different Peste des petits ruminants (PPR) vaccines and assessed the appropriate age of vaccination in lambs born without maternal antibodies at Livestock Experimental Station, Rakh Ghulaman. The lambs (n=94) were divided into ten groups. The lambs in Group A1 (1-<2 month), A2 (2-3 months) and A3 (>3 months) were vaccinated with PPR vaccine from company A and the lambs in Group B1 (1-<2 month), B2 (2-3 months) and B3 (>3 months) were vaccinated with PPR vaccine from company B. However, PPR vaccine from company C was given to four groups of lambs (Group C1, C2, C3 and C4). Serum samples were collected from experimental animals at monthly intervals for five months. Sera were analyzed using c-ELISA. The results indicated that most appropriate age of vaccination against PPR in lambs without maternal antibodies is 2-3 months with any of three vaccines. However, vaccine C may also be used at the age of < 1 month to get protection against PPR. [ABSTRACT FROM AUTHOR]