A Naked-Eye Visual Reverse Transcription Loop-Mediated Isothermal Amplification with Sharp Color Changes for Potential Pen-Side Test of Foot-and-Mouth Disease Virus.

Visual loop-mediated isothermal amplification (LAMP) is qualified to be applied in the field to detect pathogens due to its simplicity, rapidity and cost saving. However, the color changes in currently reported visual reverse transcription LAMP (RT-LAMP) for foot-and-mouth disease virus (FMDV) detection are not so obvious to the naked eye, so interpretation of results is troublesome. In this study, a new naked-eye visual RT-LAMP to detect all seven distinct serotypes of FMDV was established based on the 3D genes by using pH-sensitive neutral red as the indicator, rendering a sharp contrast of color changes between the negative (light orange) and the positive (pink). Analytical sensitivity tests showed that the detection limit of the visual RT-LAMP was 104 copies/µL while those were 103 and 104 copies/µL for the RT-qPCR and conventional RT-PCR methods, respectively. Specificity tests proved that the established visual RT-LAMP assay had no cross-reactivity with other common livestock viruses. Furthermore, the analysis of 59 clinical samples showed 98.31% and 100% concordance with the RT-qPCR and the RT-PCR, respectively. The pan-serotypic FMD visual RT-LAMP assay could be suitable for a pen-side test of all seven serotypes of FMDV because the results could be easily distinguished by the naked eye without the requirement of complicated instruments and professional technicians. Hence, the novel method may have a promising prospect in field tests which exert an important role in monitoring, preventing, and controlling FMD, especially in regions with no PCR or qPCR instrument available.

Infection Dynamics of Clostridium perfringens Fingerprinting in Buffalo and Cattle of Punjab Province, Pakistan.

Clostridium perfringens produces core virulence factors that are responsible for causing hemorrhagic abomasitis and enterotoxemia making food, animals, and humans susceptible to its infection. In this study, C. perfringens was isolated from necropsied intestinal content of buffalo and cattle belonging to four major bovine-producing regions in the Punjab Province of Pakistan for the purpose offind out the genetic variation. Out of total 160 bovine samples (n: 160), thirty-three (n: 33) isolates of C. perfringens were obtained from buffalo (Bubales bubalis) and cattle (Bos indicus) that were further subjected to biochemical tests; 16S rRNA based identification and toxinotyping was done using PCR (Polymerase Chain Reaction) and PFGE (Pulse Field Gel Electrophoresis) pulsotypesfor genetic diversity. Occurrence of C. perfringens was found to be maximum in zone-IV (Bhakkar and Dera Ghazi Khan) according to the heatmap. Correlation was found to be significant and positive among the toxinotypes (α-toxin, and ε-toxin). Response surface methodology (RSM) via central composite design (CCD) and Box-Behnken design (BBD) demonstrated substantial frequency of C. perfringens based toxinotypes in all sampling zones. PFGE distinguished all isolates into 26 different pulsotypes using SmaI subtyping. Co-clustering analysis based on PFGE further decoded a diversegenetic relationship among the collected isolates. This study could help us to advance toward disease array of C. perfringens and its probable transmission and control. This study demonstrates PFGE patterns from Pakistan, and typing of C. perfringens by PFGE helps illustrate and mitigate the incidence of running pulsotypes.

In Vitro Analysis of TGF-ß Signaling Modulation of Porcine Alveolar Macrophages in Porcine Circovirus Type 2b Infection.

Porcine circovirus 2 (PCV2) has been recognized as an immunosuppressive pathogen. However, the crosstalk between this virus and its host cells in related signaling pathways remains poorly understood. In this study, the expression profiles of 84 genes involved in transforming growth factor-beta (TGF-ß) signaling pathway were probed in PCV2b-infected primary porcine alveolar macrophages (PAMs) by using an RT2 profiler PCR array system. The protein expression levels of cytokines involved in the TGF-ß signaling pathway were determined with a RayBiotech fluorescent Quantibody® porcine cytokine array system. Results showed that 48, 30, and 42 genes were differentially expressed at 1, 24, and 48 h after infection, respectively. A large number of genes analyzed by a co-expression network and implicated in transcriptional regulation and apoptosis were differentially expressed in PCV2b-infected PAMs. Among these genes, TGF-ß, interleukin-10, CCAAT/enhancer-binding protein beta (C/EBPB), growth arrest, and DNA-damage-inducible 45 beta (GADD45B), and BCL2 were upregulated. By contrast, SMAD family member 1 (smad1) and smad3 were downregulated. These results suggested that the TGF-ß signaling pathway was repressed in PAMs at the early onset of PCV2 infection. The inhibited apoptosis was indicated by the upregulated C/EBPB, GADD45B, and BCL2, and by the downregulated smad1 and smad3, which possibly increased the duration of PCV2 replication-permissive conditions and caused a persistent infection. Our study may provide insights into the underlying antiviral functional changes in the immune system of PCV2-infected pigs.

First Report on Molecular Characterization of Taenia multiceps Isolates From Sheep and Goats in Faisalabad, Pakistan.

Coenurus cerebralis is the larval stage of Taenia multiceps commonly found in the brain (cerebral form), intramuscular and subcutaneous tissues (non-cerebral form) of ungulates. Globally, few reports exist on the molecular characterization and genetic diversity of C. cerebralis with none available for Pakistan. The current study molecularly characterized 12 C. cerebralis isolates surgically recovered from sheep (n = 4) and goats (n = 8) from a total of 3,040 small ruminants using a portion of the cytochrome c oxidase subunit 1 (cox1) mitochondrial (mt) gene. NCBI BLAST search confirmed the identity of each isolate. A high haplotype and a low nucleotide diversity with three haplotypes from the 12 isolates were observed. The findings suggest the existence of unique haplotypes of C. cerebralis in Pakistan. The negative value of Tajima's D and the positive value of Fu's Fs were inconsistent with population expansion, however, the sample size was small. Bayesian phylogeny revealed that all Pakistani isolates alongside the Chinese sequences (obtained from GenBank) constituted a cluster while sequences from other regions constituted another cluster. This is the first molecular study to determine the genetic diversity of C. cerebralis in Pakistan and serves as a foundation for prospective studies on the prevalence and population structure of C. cerebralis in the country. Furthermore, in this study, we amplified only a partial segment of the cox1 gene from a limited sample size. This could have implications on the interpretation of the actual population structure in reality. Thus, we recommend future studies to consider a larger sample size in a massive epidemiological survey for further insights.

Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots.

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10-7 and 1.35 × 10-8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.