Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus.

Single-domain antibody (sdAb) or nanobody possesses specific features non-accessible for conventional antibodies that make them suitable for research and biotechnological applications. Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets, resulting in great economic losses all over the world. To detect and isolate PEDV rapidly and accurately is important for the control and further research of the clinical PEDV strains. In this study, four sdAb fragments (sdAb-Mc19/29/30/37) targeting the membrane (M) protein of PEDV were selected from sdAb library that was constructed through M protein-immunized Camelus bactrianus. The selected sdAb-Mcs were solubly expressed in Escherichia coli. The functional characteristics analysis revealed that the recombinant sdAb-Mcs have excellent binding activity and specificity to M protein but have no neutralizing activity to PEDV. For further application, sdAb-Mc37 was conjugated with quantum dots to synthesize a nanoprobe for imaging PEDV in vero cells. The observed fluorescence in vero cells clearly reflects that PEDV virions can be reliably recognized and labeled by the nanoprobe. Furthermore, the sdAb-Mc29 was conjugated with superparamagnetic nanobeads to construct immunomagnetic nanobeads (IMNBs) used to isolate PEDV. One PEDV strain was successfully isolated from clinical fecal sample, suggesting IMNBs as a novel and efficient tool suitable for PEDV isolation from clinical samples. This study provided a novel application and substantiated the suitability of sdAb as a specific binder for the isolation of viruses.

Phylogeny and population structure of Echinococcus granulosus (sensu stricto) based on full-length cytb-nad2-atp6 mitochondrial genes - First report from Sialkot District of Pakistan.

Cystic echinococcosis is a zoonotic disease of livestock having serious economic setbacks. The etiological agents of the disease belong to Echinococcus granulosus sensu lato. Despite of worldwide distribution of the disease, the molecular studies mainly employ amplification of cox1, nad1 and nad5 genes. To further strengthen the knowledge about significance of other molecular markers and to investigate the genetic diversity and population structure of Echinococcus species in Pakistan, the current study was designed in which full length mitochondrial cytb, atp6 and nad2 genes were amplified. Based on BLAST searches of the generated cytb, atp6 and nad2 gene sequences from a total of 18 hydatid cysts collected from cattle, 12 isolates were identified as E. granulousus G3 and 6 as E. granulosus (G1). The phylogeny inferred by the Bayesian method using nucleotide sequences of cytb-atp6-nad2 further confirmed their identity. The diversity indices indicated a high haplotype and a low nucleotide diversity. The negative values of Tajima's D and Fu's Fs test demonstrated deviation from neutrality suggesting a recent population expansion. To the best of our knowledge, the present study described the genetic variation of E. granulosus population for the first time in Pakistan using full-length cytb, atp6 and nad2 mitochondrial genes. The findings on the genetic variation of E. granulosus in Pakistan will constitute useful baseline information for future studies on the prevalence and population structure of E. granulosus based on full-length cytb, atp6 and nad2.

Evaluation and Optimization of Antibiotics Resistance Profile against Clostridium perfringens from Buffalo and Cattle in Pakistan.

Clostridium perfringens is a serious threat to successful bovine farming. It causes severe damage to the buffalo and cattle health causing a drastic reduction in milk and meat production. In Pakistan, C. perfringens is a constant threat, and for its management, antibiotics are mostly used. Most bovine farmers use a single antibiotic to suppress the bacterial infection which in turn, increases the antimicrobial resistance (AMR) against the particular antibiotic. To reduce the resistance, the administration of multiple antibiotics in their standard doses at different times can be a possible remedy to manage the AMR and reduce their viability. This study aims to evaluate the effect of 11 commonly used antibiotics at their standard concentrations for inhibiting 33 strains of C. perfringens from five districts of Punjab province in Pakistan. Based on the zone of inhibition, ciprofloxacin, ampicillin, and cefotaxime (CAC) at their standard concentrations effectively inhibited the bacterium. These antibiotics showed appropriate significance statistically, i.e., correlation, Chi-square test, and cluster analysis. Optimization of these antibiotics using response surface methodology (RSM) revealed that the selected antibiotics from medium to high range not only reduce the bacterial propagation but also their population up to a considerable extent. Hence, the health of milk- and meat-producing large animals could be improved, which will be cost-effective and less harmful to the animal, human health, and the environment. Moreover, optimized administration of the selected antibiotics would reduce the impact of drug-resistant superbugs.

Occurrence and Toxicogenetic Profiling of Clostridium perfringens in Buffalo and Cattle: An Update from Pakistan.

Clostridium perfringens is a Gram-positive bacterium that possess seven toxinotypes (A, B, C, D, E, F, and G) that are responsible for the production of six major toxins, i.e., α, ß, ε, ι, CPE, and NetB. The aim of this study is to find out the occurrence of toxinotypes in buffalo and cattle of Punjab province in Pakistan and their corresponding toxin-encoding genes from the isolated toxinotypes. To accomplish this aim, six districts in Punjab province were selected (i.e., Lahore, Sahiwal, Cheecha Watni, Bhakkar, Dera Ghazi Khan, and Bahawalpur) and a total of 240 buffalo and 240 cattle were selected for the collection of samples. From isolation and molecular analysis (16S rRNA), it was observed that out of seven toxinotypes (A-G), two toxinotypes (A and D) were found at most, whereas other toxinotypes, i.e., B, C, E, F, and G, were not found. The most frequently occurring toxinotype was type A (buffalo: 149/240; cattle: 157/240) whereas type D (buffalo: 8/240 cattle: 7/240) was found to occur the least. Genes encoding toxinotypes A and D were cpa and etx, respectively, whereas genes encoding other toxinotypes were not observed. The occurrence of isolated toxinotypes was studied using response surface methodology, which suggested a considerable occurrence of the isolated toxinotypes (A and D) in both buffalo and cattle. Association between type A and type D was found to be significant among the isolated toxinotypes in both buffalo and cattle (p ≤ 0.05). Correlation was also found to be positive and significant between type A and type D. C. perfringens exhibits a range of toxinotypes that can be diagnosed via genotyping, which is more reliable than classical toxinotyping.

Porcine Circovirus Type 2 Induces Single Immunoglobulin Interleukin-1 Related Receptor (SIGIRR) Downregulation to Promote Interleukin-1ß Upregulation in Porcine Alveolar Macrophage.

Multisystemic inflammation in pigs affected by porcine circovirus type 2 (PCV2) indicates the disordered expression of inflammatory cytokines. However, the PCV2-induced expression profile of inflammation cytokines and its regulating mechanism remain poorly understood. In this study, inflammatory cytokines and receptors in porcine alveolar macrophages (PAMs) after PCV2 infection were profiled in vitro by an RT2 ProfilerTM PCR array assay. The regulatory mechanism of interleukin-1ß (IL-1ß) expression was investigated. Results showed that 49 of 84 inflammation cytokines and receptors were differentially expressed (p < 0.05, absolute fold change ≥2) in PAMs at different stages post-PCV2 infection. Moreover, the overexpression of single-immunoglobulin interleukin-1 related receptor (SIGIRR) or the blocking of NF-κB activation by its inhibitor markedly decreased IL-1ß secretion. This finding suggested that PCV2-induced overexpression of IL-1ß was associated with the downregulation of SIGIRR and the activation of NF-κB. Furthermore, the excessive activity of NF-κB in SIGIRR-knockout PAMs cell line, indicating that SIGIRR negatively regulated IL-1ß production by inhibiting the activation of NF-κB. Overall, PCV2-induced downregulation of SIGIRR induction of NF-κB activation is a critical process in enhancing IL-1ß production in PAMs. This study may provide insights into the underlying inflammatory response that occurs in pigs following PCV2 infection.