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Porcine circovirus type 2 (PCV2) is a causative agent of PCV2-associated disease, which is a growing problem in the swine industry worldwide. High nucleotide substitution occurs in the capsid (Cap) gene of PCV2, which allows the continuous evolution and the emergence of novel PCV2 strains. In this study, we sequenced 24 Chinese PCV2 strains collected from healthy and diseased pigs between 2013 and 2015. Analyses of the genome, Cap and phylogeny classified the 24 Chinese PCV2 strains as PCV-2a (four of 24), PCV-2b (five of 24) and PCV-2d (15 of 24). All strains shared 89.5%-100% and 87.2%-100% identities with the nucleotide and amino acid (aa) sequences of Cap, respectively. Selection pressure analysis showed that five sites at the epitope regions in Cap were under positive selection. Further analysis by Jameson-Wolf antigenic index indicated that aa substitutions occurring at the epitope regions contributed to the antigenic alterations of the different PCV2 strains. High genetic variation and genotype shift to PCV2d occurred in recent years, and different genotypes coexisted in Chinese pig herds. The data provide evidence for the increased genetic diversity and insights into the molecular epidemiology of PCV2.
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-1β (IL-1β) 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-1β secretion. This finding suggested that PCV2-induced overexpression of IL-1β 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-1β 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-1β production in PAMs. This study may provide insights into the underlying inflammatory response that occurs in pigs following PCV2 infection.
<i>Coenurus cerebralis</i> is the larval stage of <i>Taenia multiceps</i> 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 <i>C</i>. <i>cerebralis</i> with none available for Pakistan. The current study molecularly characterized 12 <i>C</i>. <i>cerebralis</i> isolates surgically recovered from sheep (<i>n</i> = 4) and goats (<i>n</i> = 8) from a total of 3,040 small ruminants using a portion of the cytochrome <i>c</i> oxidase subunit 1 (<i>cox</i>1) mitochondrial (<i>mt</i>) 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 <i>C. cerebralis</i> in Pakistan. The negative value of Tajima's <i>D</i> 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 <i>C</i>. <i>cerebralis</i> in Pakistan and serves as a foundation for prospective studies on the prevalence and population structure of <i>C. cerebralis</i> in the country. Furthermore, in this study, we amplified only a partial segment of the <i>cox</i>1 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.
Mucosal vaccination offer an advantage over systemic inoculation from the immunological viewpoint. The development of an efficient vaccine is now a priority for emerging diseases such as COVID-19, that was declared a pandemic in 2020 and caused millions of deaths globally. Lactic acid bacteria (LAB) especially Lactobacillus are the vital microbiota of the gut, which is observed as having valuable effects on animals’ and human health. LAB produce lactic acid as the major by-product of carbohydrate degradation and play a significant role in innate immunity enhancement. LAB have significant characteristics to mimic pathogen infections and intrinsically possess adjuvant properties to enhance mucosal immunity. Increasing demand and deliberations are being substantially focused on probiotic organisms that can enhance mucosal immunity against viral diseases. LAB can also strengthen their host’s antiviral defense system by producing antiviral peptides, and releasing metabolites that prevent viral infections and adhesion to mucosal surfaces. From the perspectives of “one health” and the use of probiotics, conventional belief has opened up a new horizon on the use of LAB as antivirals. The major interest of this review is to depict the beneficial use of LAB as antivirals and mucosal immunity enhancers against viral diseases.
Clostridium perfringens (C. perfringens) is widely distributed in broiler chickens causing clinical and subclinical enteritis and is especially known for causing necrotic enteritis (NE). There are numerous reports of NE outbreaks in Pakistan as well as China but there is a lack of information related to PFGE profile from both the countries. To close this gap, we designed this study and obtained samples from broiler chicken farms located in 3 different regions of Pakistan and 4 different regions of China. A total of 79 fecal swabs (Pakistan=29; China=50) were collected and grown on FTA media. Further, isolates were grown on TSE agar and black colonies were selected for DNA extraction. All 79 isolates were tested for toxin profiles by PCR (α-gene; beta-2; netB gene) and PFGE profiling (pulsotypes analysis). Toxinotyping results revealed that all the isolates (n=50) from China were type A (α-toxin positive) while 23 and 6 isolates (n=29) from Pakistan were type A (α-toxin positive) and type G (α-toxin, NetB positive), respectively. Toxinotyping revealed α-toxin is highly prevalent in both the countries while from Pakistani isolates, NetB toxin was also detected. PFGE discriminated 79 isolates into 45 different PFGE patterns (pulsotypes). The analysis further showed different pulsotypes originating from China and Pakistan and isolates were subtyped by SmaI. The results showed high genetic polymorphism in C. perfringens even within the same strain. These preliminary findings of genetic variations will further help to design control strategies
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.
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.
Background: Herbal preparations can be formed by combining several plant classes. One possible explanation for the effectiveness of combined medications is that the various mixtures with different mechanisms may add up to produce a more comprehensive therapeutic effect. Objective: This study aims to investigate the synergistic antibiotic potential of a cream containing three natural herbal extracts: Allium sativum, Moringa oleifera, and Thymus vulgaris. The efficacy of combining these plant extracts was compared to that of a standard antibiotic formulation (Polyfax). Methods: The herbal cream was formulated by using aqueous extracts of garlic (Allium sativum), moringa (Moringa oleifera) and essential oil of thyme (Thymus vulgaris). The study aimed to explore the therapeutic potential of these extracts against bacteria. P. aeruginosa, B. subtilis, E. coli, S. aureus, and S. pneumonia are commonly found in fresh wounds. Results: The results showed that garlic extract (5%) had the highest zone of inhibition, 14.26 ± 0.05 mm, and a combination of garlic (5%) and thyme (2%) exhibited a significant synergistic effect, with a 23.5 ± 0.05 mm zone of inhibition. High-performance liquid chromatography analysis revealed the presence of allicin, quercetin and thymol as potential therapeutic phytoconstituents. The formulated herbal cream had a soft texture, was easily spreadable, and had better stability and absorption than the standard polyfax. The topical application of the cream did not cause any skin reaction or allergy in mice. The in vivo wound healing effect of the herbal cream was investigated on an abrasion model of albino mice, and the results showed that the treatment group (46 ± 16.31%) had significant wound healing potential compared to the standard (64 ± 17.49%) and control groups (18 ± 3.74%). Conclusion: The formulated herbal cream was a better alternative to standard therapy, exhibiting promising healing and antimicrobial effects with significant compatibility and safety profile.
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-β) 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-β 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-β, 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-β 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.
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, β, 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.
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.
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.
Haemorrahgic Septiceamia (H.S) is a deadly, sub-acute livestock disease of cattle and buffalo in Southeast Asia including Pakistan. A whopping 98% case fatality rate renders vaccination a mandatory prophylactic control measure to counter HS. Very little information is available on serum biochemical profile of the HS vaccinated animals. This study was conducted to investigate the serum biochemistry of the vaccinated cattle and buffalo calves and adults alike. Three different experimental oil adjuvant vaccines were produced for Pasturella multocida serotype B:2 (locally prevalent). N=80 healthy cattle and buffalo (calves and adults) were divided into four groups. Each group consisted of 20 animals (10 calves and 10 adults). Group A was injected with vaccine prepared with Montanide ISA-50; Group B with vaccine prepared from Montanide ISA-206 while Group C with a vaccine produced from Liquid paraffin and Lanolin. Group D animals served as non-immunized control. Blood samples were collected from each animal at zero-day, after booster at 90 days, and then every quarter for one year. Twelve biochemical parameters were assessed by ANOVA and Kruskal Wallis Test. Post hoc multiple comparisons were done for glucose levels. It was concluded that vaccinating both cattle and buffalo calves and adults with H.S. vaccine formulated with three different adjuvants did not have a statistically significant effect on serum biochemistry of vaccinated animals compared to controls.
This research was focused to isolate and identify antagonistic bacteria from grapes against Penicillium verrucosum growth and Ochratoxin (OTA) yield, as well as to determine the substrate and conditions that promote P. verrucosum growth and OTA yield. For this purpose, 15 culture media and 5 levels of temperature, pH and water activity were selected at which fungus was checked for growth and OTA production. Maximum growth and toxin content was observed on MEA media at temperature 25°C, pH 5.5, 0.99 water activity. Optimization and correlation show a proper link between the circumstances that promote efficient growth and OTA output (r = 0.97). Lactobacillus brevis (strain 121A) significantly retard the growth and OTA production of P. verrucosum at 108 spores/mL concentration. This strain significantly reduced grapes spoilage and increased the shelf life. This approach not only promotes biological control of fungal diseases, but also overcomes certain food safety concerns.
Mastitis is an inflammatory disease that deteriorates the quality and quantity of milk, leading to poor performance and economic losses for the county's dairy farmers. Platelet-rich plasma (PRP) is a value-added product of blood that contains higher levels of platelet count in comparison to the blood. Platelets secrete growth factors that could positively impact clinical circumstances, needed speedy healing, and proper tissue regeneration. The extended usage of antibiotics is leading to serious health-associated concerns, such as drug residues in milk and milk products, and elevated trends of resistance to antibiotics. Considering the scenario, it is aimed to find a different management plan for mastitis using PRP as an alternative treatment option. Platelet Rich Plasma (PRP) is an alternative therapy for the treatment of Bubaline Mastitis. For this purpose, three treatment groups, i.e., the antibiotic group, the antibiotic and PRP group, and the PRP group, were evaluated. The data concluded that PRP, independently and along with judicious use of antibiotics, could be a helpful management option to address the inflammatory response and microbial adherence to the lining of udder tissues. Correlation and regression analysis also concluded a significant relationship between antibiotics and PRP. Optimization via response surface methodology also concludes that certain antibiotics at their recommended doses, along with PRP treatment, significantly reduce Bubaline mastitis in buffaloes. This study may be replicated in other domestic animals, such as sheep and goat, to evaluate their efficacy against mastitis to enhance the usage of PRP in different species.
Staphylococcus aureus is a common pathogen that can cause a wide range of infections, including endocarditis, severe bacteremia, and infections of the skin and soft tissues. Because of its resistance to β-lactam antibiotics, methicillin-resistant S. aureus (MRSA), which is caused by the mecA gene encoding penicillin-binding protein 2a (PBP2a), is a serious problem. This review describes the molecular mechanisms, regulatory pathways involving mecR1 and mecI, and the genetic context of mecA within the Staphylococcal Cassette Chromosome mec (SCCmec). Along with diagnostic techniques like PCR, CRISPR-based detection, and next-generation sequencing, we investigate the epidemiology of hospital-acquired (HA-MRSA), community-acquired (CA-MRSA), and livestock-associated (LA-MRSA) strains. Vancomycin resistance is one of the therapeutic limitations that are addressed; new strategies such as phage therapy, combination therapies, and anti-PBP2a inhibitors show promise. Emerging resistance mechanisms, such as mecC and biofilm formation, highlight the need for surveillance. In order to lessen MRSA's worldwide impact, future strategies will prioritize stewardship, new antibiotics, and quick diagnostics.
