Tularemia is a bacterial disease of humans, wild, and domestic animals. Francisella tularensis, which is a Gram-negative coccobacillus-shaped bacterium, is the causative agent of tularemia. Recently, an increase in the number of human tularemia cases has been noticed in several countries around the world. It has been reported mostly from North America, several Scandinavian countries, and certain Asian countries. The disease spreads through vectors such as mosquitoes, horseflies, deer flies, and ticks. Humans can acquire the disease through direct contact of sick animals, consumption of infected animals, drinking or direct contact of contaminated water, and inhalation of bacteria-loaded aerosols. Low infectious dose, aerosol route of infection, and its ability to induce fatal disease make it a potential agent of biological warfare. Tularemia leads to several clinical forms, such as glandular, ulceroglandular, oculoglandular, oropharyngeal, respiratory, and typhoidal forms. The disease is diagnosed through the use of culture, serology, or molecular methods. Quinolones, tetracyclines, or aminoglycosides are frequently used in the treatment of tularemia. No licensed vaccine is available in the prophylaxis of tularemia and this is need of the time and high-priority research area. This review mostly focuses on general features, importance, current status, and preventive measures of this disease.

Foot and mouth disease is an economically devastating disease of livestock that mainly effect cloven-hoofed animals i.e. sheep, goat, cattle, pig, buffalo, deer etc. The aim of this study was to determine the serotypes circulating in the region during 2016. Sampling was done from different outbreaks initially on the basis of clinical signs and later reverse transcriptase-polymerase chain reaction (RT-PCR) was employed for the confirmation of FMDV genome. Out of total 72 samples, 65 were found positive which were then serotyped into type O (n=30), Asia1 (n=19) and A (n=5). Some samples (n=5) were found positive for more than one serotype that were subjected to reverse transcriptase loop-mediated isothermal amplification assay (RT-LAMP) for serotype determination.

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This study was undertaken to develop and validate direct competitive ELISA for the determination of chloramphenicol residues in bovine milk. Antisera and an enzyme-tracer for chloramphenicol were prepared and used to develop an ELISA with inhibition concentrations, IC and IC, of 0.09 and 0.44 ng mL, respectively. Milk samples were spiked with standards equivalent to 0, 0.2, 0.3, 0.5, 1.0 & 1.5 ng mL and extracted in methanol. The mean recoveries were found to be 73-100% with coefficient of variance 7-11%. The decision limit (CCα) and detection capability (CCβ) were calculated as 0.10 and 0.12 ng mL, respectively. The results were found comparable with the commercial ELISA, having recoveries of 87 to 100%, CCα 0.09 ng mL and CCβ 0.12 ng mL. As per Commission Decision 2002/657/EC, in-house ELISA was further validated by using LC-MS/MS. Mass spectral acquisition was done by using electrospray ionization in the negative ion mode applying single reaction monitoring of the diagnostic transition reaction for CAP (m/z 152, 194 and 257). The calibration curve showed good linearity in concentrations from 0.025 to 1.6 ng mL with correction coefficient 0.9902. The mean recoveries were found to be 88 to 100%. The CCα was calculated as 0.057 ng mL and CCβ 0.10 ng mL. Since CCα and CCβ are less than half of the MRPL (0.15 ng mL), the test was found suitable for screening and quantification of CAP residues in bovine milk samples. Results of surveillance studies indicated that out of 31 analyzed milk samples, 12.9% samples were found with CAP residues but only 3.2% samples were declared positive with maximum concentration 0.31 ng mL, slightly above the MRPL.

This study reports the molecular characterization of foot-and-mouth disease virus (FMDV) in the provinces of Punjab and Sindh, Pakistan during 2014-17. FMDV genome was detected in 42 and 41 out of 46 samples (epithelial tissue and saliva) by reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Sequences of the complete VP1 coding region of the samples (n = 33) was achieved showing that 10, 4 and 19 samples belonged to serotype O, A and Asia1 respectively. Phylogenetic analysis of serotype O revealed that at least one novel sublineage within the ME-SA topotype is circulating in the region, named here as PAK-14. This sublineage showed similarity with the viruses circulating in Turkey and Pakistan during 2010 indicating that viruses circulating in these countries have common origin. Analysis of serotype A viruses revealed a new lineage is circulating in the region, reported here as A-PAK14 showing close identity with the strain prevalent in Pakistan during 2007. Circulation of these new linages in the region shows continuous evolution of the viruses. Two of the undisclosed serotype A sublineages within the Iran-05 lineage were also found circulating in the region. In addition, molecular investigation of the VP1 coding region sequences of serotype Asia1 strains revealed that they belong to Group-VII (Sindh-08). Interestingly some of the serotype Asia1 isolates (n = 6) showed 99.9% similarity (among themselves) although they were collected from different districts more than 100 Km apart from one another. This unusual conservation among serotype Asia1 over long distances can be explored by studying the role of wild animals, slaughter houses and milk collection centres in the spread the disease.

Infectious bursal disease (IBD) is an immunosuppressive disease of young, growing chickens which results in impaired growth or mortality of rearing flocks. In the current era there is a re-emergence of very virulent Infectious Bursal Disease Viruses (vvIBDV) and classical variant (cv) IBDV strains which increased the financial losses of poultry industry worldwide. Recent studies were conducted to characterize the existing vvIBDVs prevailing in Pakistan. The suspected samples were collected from the field outbreaks during the period from 2014-2017. IBDV was detected by RT-PCR. The sequences of VP2 gene (hyper variable region) were determined and available details were aligned with sequences submitted inGenBank. Phylogenetic analysis reveals that both vvIBDV and classical variant strains were circulating in different regions of Pakistan. In Indo-Pak isolates, the presence of virulent markers, amino acids (A222, I242, Q253, I256 and S299) and "Serine rich-heptapeptide" indicated the presence of very virulent viruses. The presence of T284A isan indicator of vvIBDVs in local poultry farms. More than 99% similarity of Pakistani isolates with Indian sequences reflects the trans-boundary spread of IBD. In recent studies amino acid, Glutamine (Q) is present at position 221 (as reported in previous studies) rather than Histidine (H) in Pakistani sequences. It is investigated that Glutamic acid (E) is located at position 300 in minor hydrophilic region III of VP2 protein in all reported Pakistani isolates. It is the unique feature of indigenous strains. This study will be useful in understanding the origin and pathotypes of IBDV circulating in Pakistan.

Frequently, a sequence from a single strain of a virus is used to design primers for PCR-based virus detection. However, high mutation rates in RNA viruses lead to failure of microorganism detection in clinical samples. Therefore, it is essential to find conserved regions within sequences to design primers for the detection of pathogen genomes in suspected clinical samples. The aim of the study was to find conserved regions of the genome for improved serotype detection. In the present study, primers were designed for serotype detection based on the VP2 coding region of foot-and-mouth disease virus (FMDV) after alignment of full genome (n = 375) sequences. These primer pairs and previously reported primer pairs were then compared using positive samples (n = 91) collected during 2010-2016 from Faisalabad District, Pakistan. The detection rate of newly designed primer pairs O-Wqs-F/Rev-Wqs (96.5%), As-Wqs-F/Rev-Wqs (90.4%), and A-Wqs-F/Rev-Wqs (100%) was better compared with previously reported primer pairs P38/P33 (81%), P74-77/P33 (47.6%), and P87-92/P33 (41.6%) in detecting FMDV serotypes O, Asia1, and A, respectively. The higher detection rates of the newly designed primer pairs appeared to be due to the selection of highly conserved sequences within the serotypes for designing primers. To the best of the authors' knowledge, this is the first study that describes serotype diagnosis of FMDV based on primers targeting the VP2 region. In conclusion, this new method offers an improved approach for the serotyping of FMDV compared to previous methods.

In this study, the capsid protein coding region of serotype Asia-1 viruses (n=131) were analyzed, giving importance to the viruses circulating since 2011 within the Group VII (Sind-08). The isolates recovered during 2011-2017 were found to group within the re-emerging cluster of Group VII (Sind-08). The time of the most recent common ancestor for this cluster was estimated to be approximately 2004. In comparison to the older isolates of Group VII (2001-2004), the re-emerging viruses showed variation at fourteen amino acid positions, including substitutions at the antigenically critical residues VP1140, VP1142 and VP277. In Group VII (Sind-08), all three major antigenic sites have mutations (Site I and II had four consensus changes at positions 140, 141 and 77, 79 respectively, while site IV had a replacement at position 59) relative to the internationally recommended vaccine strain (Shamir 89). This study also explains the development and optimization of a new RT-PCR method that may be employed to amplify and sequence a 2901 base pair (bp) section covering entire capsid coding region (P1) of the FMDV genome. This method offers a tool that can be employed for antigenic profiling and phylogenetic analyses of FMDV to help vaccine matching or strain selection in the episode of outbreaks.