Identification, molecular characterization, and pathological features of orf virus in sheep and goats in Punjab province, Pakistan.

Orf virus (ORFV) causes an acute, contagious, skin disease of sheep and goats which is economically important. The objectives of this study were to identify ORFV and to explore its pathological and phylogenetic profiles in 350 goats and 91 sheep of 14 districts of Punjab, Pakistan, from July 2020 to July 2021. Skin scrapings (total no. of samples = 441) of suspected animals were subjected to polymerase chain reactions, phylogenetic analysis, and pathological observations. The partial length of GIF/IL-2 gene (408 bp) was successfully amplified in 58/441 samples. Phylogenetic analysis of GIF/IL2 gene showed that the study isolates belonged to ORFV-cluster I, together with the viruses reported in India and China. Pakistan ORFV isolates were shared 97.6-98.7% nucleotide and 97.6-100% amino acid identities with the reference strain (NC_005336). Moreover, Chinese ORFV-isolates were detected unique multiple amino acid substitutions (F11L, Q21H, D27N, I46V, N49S, N82D, D103N, S129G) with study isolates. Naturally infected animals were anorexic, emaciated, dull, and depressed. The macroscopic lesions included multifocal to coalescing, ulceration followed by proliferative papules, pustules, and crust formation on the epidermis of gums, lips, mouth commissure, muzzles, nose, and udder. Histopathological examination revealed hyperplasia, anastomosing rete ridges formation and degenerative changes, including spongiosis and vacuolation of epidermal cells. Keratinocytes exhibited eosinophilic intracytoplasmic inclusion bodies with pyknotic and karyorrhexis nuclei. This is the first report on molecular characterization of ORFV from Pakistan, with insight into its pathogenesis and comparative analysis of pathological alterations and genetic diversity between ORFV strains reported in different geographical areas.

Effect of Selenium Nanoparticles and Mannan Oligosaccharide Supplementation on Growth Performance, Stress Indicators, and Intestinal Microarchitecture of Broilers Reared under High Stocking Density.

The current study investigated the potential of selenium nanoparticles (SeNPs) and mannan-oligosaccharide (MOS) supplementation in ameliorating high stocking density (HSD) stress in broilers. A total of 392 day-old male chicks were divided into seven groups with eight replicates (n = 7): NSD [basal diet (BD) + normal stocking density: 10 bird/m2], HSD [BD + high stocking density: 16 bird/m2], Se−HSD [BD + Selenium (Se) 0.15 mg/kg], MOS−HSD (BD + MOS 5 gm/kg), Se−MOS−HSD (BD + Se 0.15 mg/kg and MOS 5 gm/kg), SeNPs−HSD (BD + SeNPs 0.15 mg/kg) and SeNPs−MOS−HSD (BD + SeNPs 0.15 mg/kg and MOS-5 gm/kg). HSD stress decreased (p < 0.05) weekly body weight and body weight gain and increased (p < 0.05) FCR compared to the NSD group. Supplementation with SeNPs and the SeNPs−MOS combination improved (p < 0.05) the weekly body weight and FCR in HSD-stressed broilers during the 5th and 6th weeks. On day 21, HSD stress decreased (p < 0.05) duodenal villus height (VH) and villus surface area (VSA) and increased (p < 0.05) serum corticosterone and cholesterol compared to the NSD group. Supplementation with the SeNPs−MOS combination increased (p < 0.05) duodenal VH and VH:CD, and jejunal total goblet cell (TGC) density and decreased (p < 0.05) serum corticosterone and cholesterol and ileal intra-epithelial lymphocyte (IEL) density in HSD-stressed broilers. On day 42, HSD stress decreased (p < 0.05) duodenal and jejunal VH, VSA, VH:CD, PCNA positive cell density and TGC density, Ileal VSA and TGC density, and increased (p < 0.05) serum cholesterol and ileal IEL density compared to the NSD group. Supplementation with the SeNPs−MOS combination increased (p < 0.05) spleen and bursa absolute weights, duodenal VH, VSA, VH:CD, PCNA positive cell density and jejunal VH, VH:CD, and decreased (p < 0.05) serum cholesterol and ileal IEL density in HSD-stressed broilers. Our findings signify that HSD is stressful for broilers particularly during the finishing phase. Supplementation with the SeNPs−MOS combination mitigated HSD stress by partially improving the gut microarchitecture, gut barrier function and performance indicators.

Supplementation of Selenium Nanoparticles-Loaded Chitosan Improves Production Performance, Intestinal Morphology, and Gut Microflora in Broiler Chickens.

The current study aimed to evaluate the efficacy of selenium nanoparticles (SeNPs), combined or loaded with chitosan (COS), in broiler chickens reared under standard management protocols. The parameters under investigation were production performance, organ development, components of the intestinal barrier, and ileal microbial count. Two hundred and forty day-old chicks were raised in five groups, with each group containing eight replicates (n=6/replicate). The control group received a basal diet whereas the other four groups received basal diets supplemented with SeNPs (0.5 mg/kg), COS (200 mg/kg), SeNPs+COS (0.5 mg/kg SeNPs + 200 mg/kg COS), and SeNPs-loaded COS (SeNPs-L-COS) (200 mg/kg) respectively. On day 35, two birds/replicate were sampled to collect the viscera under investigation. The results revealed that dietary inclusion of SeNPs-L-COS increased (p<0.05) the body weight gain and improved (p<0.05) feed conversion ratio. Similarly, SeNPs-L-COS supplementation increased (p<0.05) the small intestinal villus surface area as well as the count of acidic goblet cells and intraepithelial lymphocytes when compared with the control group. Whereas the total goblet cell count was higher (p<0.05) in the small intestines of both the SeNPs+COS and SeNPs-L-COS groups. Microbial analysis of ileal contents also revealed an increase (p<0.05) in Lactobacilli species count with a concurrent decrease (p<0.05) in Escherichia coli count in the SeNPs-L-COS group when compared with the COS and control groups. Based on the results of the current trial, we can conclude that supplementation with SeNPs-L-COS is a superior combination for promoting the gut health and performance of broilers.

Comparative Study to Characterise the Pharmaceutical Potential of Synthesised Snake Venom Bradykinin-Potentiating Peptides In Vivo.

BACKGROUND: Bradykinin-potentiating peptides (BPPs) are snake venom peptides inhibiting the angiotensin-converting enzyme (ACE). ACE plays an important role in the regulation of blood pressure. BPPs lead to the development of ACE inhibitors for the treatment of hypertension. OBJECTIVE: The objective of the present work was to carry out a comprehensive comparative study of four synthesised snake venom BPPs in vivo. METHODS: Four synthesised snake venom BPPs were administered to rats via the intraperitoneal route for 15 days at a fixed dose. Lisinopril was used as a comparative standard. Thirty male albino rats were divided into six groups: A, B, C, D, E (lisinopril), and F (control). Group F was maintained as the control group and given only saline. After 15 days, blood samples and tissues were removed for the study of selective biochemical parameters and histomorphometric analysis. Statistical evaluation of all results was also performed. RESULTS: The results indicated that peptide I, with the sequence ZSAPGNEAIPP, was highly toxic and adversely affected all the biochemical and histological parameters studied in this work. Peptide II (ZNWPHPQIPP) and peptide IV (ZQWAQGRAPHPP) showed lower toxicity. None of the BPPs raised the serum creatinine level and exhibited nephroprotective effects. Although lisinopril raised the creatinine level, it showed a protective role towards the pancreas and lungs in parallel. CONCLUSION: The present work shows that although there is a high sequence similarity between the four BPPs, their in vivo activity varies. The sequences of peptide II and peptide IV can be used to improve the design of current ACE inhibitors used for hypertension treatment.

Effect of Bacillus cereus and Phytase on the Expression of Musculoskeletal Strength and Gut Health in Japanese Quail (Coturnix japonica).

We conducted a 28-day trial to evaluate the influence of Bacillus cereus and phytase supplementation on the expression of musculoskeletal strength and intestinal histological features in Japanese quail. Two-hundred day-old quail chicks were divided into four groups with five replicates (n=10): the first group served as a control and was fed only a basal diet (BD); the second group was fed BD + 0.1% B. cereus; the third group was fed BD + 0.01% microbial phytase; and the fourth group was fed BD + 0.01% microbial phytase + 0.1% B. cereus. Compared to the control, individual and combined supplementation of probiotic and phytase increased (P<0.05) the tibial weight, length, outside diameter, and weight of bone ash, but decreased (P<0.05) the weight-to-length index. The water-holding capacity, fiber diameter, fiber cross-sectional area, number of fibers per unit area, fascicle diameter, and fascicle cross-sectional area increased significantly (P<0.05) in birds fed on the combination of phytase and B. cereus. The villus height, width, depth, height-to-crypt depth, and surface area increased significantly (P<0.05) in the B. cereus and phytase groups on an individual basis. The strength of the musculoskeletal system was fully expressed when B. cereus and phytase were given synergistically. However, the histological features of the intestines improved in birds fed on an individual basis.

Single or Combined Applications of Zinc and Multi-strain Probiotic on Intestinal Histomorphology of Broilers Under Cyclic Heat Stress.

Two-hundred-eighty-day-old broiler chicks were divided into seven groups. The groups were designated as T1, thermoneutral zone; T2, heat stressed (HS); T3, HS + zinc (Zn) supplementation (30 mg/kg); T4, HS + Zn (60 mg/kg); T5, HS + probiotic (0.1 g/kg); T6, HS + probiotic (0.1 g/kg) + Zn (30 mg/kg); and T7, HS + Zn (60 mg/kg) + probiotic (0.1 g/kg). Significant decrease (p < 0.05) was observed in villus height (VH), VH to crypt depth ratio, and villus surface area of all intestinal segments in the T2 group when compared with the T1 group. The same parameters had significantly higher (p < 0.05) values in the jejunum and ileum of the Zn- and probiotic-supplemented groups (alone + combination) when compared with the T2 group. The birds exposed to HS showed fewer (p < 0.05) intraepithelial lymphocytes (IELs) in the jejunum and ileum than the T1 group, while their count increased in the jejunum and ileum with dietary treatments. In conclusion, Zn and probiotic positively modulated the intestinal microstructures of broilers kept under high environmental temperature.

Effect of Dietary Supplementation of Zinc and Multi-Microbe Probiotic on Growth Traits and Alteration of Intestinal Architecture in Broiler.

This experiment aimed to investigate the effect of different levels of zinc (Zn) and a probiotic on the growth and intestinal health of broiler chickens. One hundred ninety-two-day-old broiler chicks were randomly assigned to 6 groups. The birds were supplemented with Zn (30 and 60 mg/kg) and a probiotic mixture alone or in combination with Zn. The results revealed that the broilers receiving a probiotic mixture alone or in combination with Zn (30 and 60 mg) increased (P < 0.05) final body weight, feed conversion ratio (FCR), and total goblet cell count in duodenum compared with the control group. Morphometry of the duodenum also revealed an increase (P < 0.05) in villus height (VH) and the ratio of VH and crypt depth in all the supplemented groups, whereas ileal VH increased (P < 0.05) only in Zn60 group when compared with control. Based on our results, we concluded that compared to the individual supplementation, the combined effect of Zn and probiotic mixture was superior in improving the growth performance and histomorphology of the intestine; however, the effect of two levels of Zn did not vary when combined with probiotic.

A comparative study of ammonia transport across ruminal epithelia from Bos indicus crossbreds versus Bos taurus.

Absorption of ammonia from the rumen of cattle decreases nitrogen availability for fermentational protein synthesis, leading to increased competition of cattle with humans for protein and enhancing the release of toxic nitrogenous compounds into the environment. Given that differences in feeding and breeding might induce differences in ruminal ammonia transport, we compared electrophysiological, histological, and molecular biological characteristics of ruminal epithelia of Bos indicus crossbreds (Sahiwal-Mix, SWM) with those of Bos taurus (Holstein-Friesian, HF). As in HF, the stratified cornified epithelium of SWM expressed claudin 1 and 4. Measurements of ammonia flux (HF) and serosal pH (both breeds) suggested that at a mucosal pH of 6.4, net transport primarily occurred as NH4 + . As shown previously for HF, NH4 + induced a concentration-dependent rise in short circuit current (Isc ) in SWM that could be further stimulated by the TRP channel agonist menthol. Relative mRNA expression levels for TRPV3, TRPV4, TRPM6, and TRPM7 were significantly lower in SWM than in HF, with TRPA1 expression near the limit of detection. We conclude that uptake of ammonia from the rumen of both breeds occurs electrogenically as NH4 + with functional and molecular biological evidence pointing towards involvement of TRPV3 and TRPV4.

Salmonella infection may alter the expression of toll like receptor 4 and immune related cells in chicken bursa of Fabricius.

Toll like receptor 4 (TLR4), eosinophils and mast cells play significant role in host immunity during several pathogenic infections. However in vivo tissue expression of TLR4 and distribution pattern of eosinophils and mast cells in chicken bursa of Fabricius (BF) during Salmonella enterica serovar Typhimurium (STm) infection is poorly studied. Therefore, herein, following immunostaining, we found localization of TLR4 in follicular cortex and medulla and its expression was statistical increased after 36 h and 72 h of STm stimulation. Chromotrope 2R staining revealed that eosinophils were mostly distributed in follicular cortex, inter-follicular spaces and in or around blood vessels and their number in BF were statistical increased after 72 h of STm stimulation. The presence of eosinophils was confirmed using immunostaining with anti-rabbit eosinophil cationic protein antibody. Toluidine blue stained mast cells were mostly distributed in connective tissues between inter-follicular spaces while some were also present in follicular cortex of BF. However, STm stimulation illustrated non-significant effect on the number of mast cells or their de-granulation, instead their number were gradually decreased in BF with advancement in age of chickens. Hence, this study provided novel information about in vivo tissue distribution of TLR4, eosinophils and mast cells in BF during STm infection.

Anti-avian influenza virus H9N2 activity of aqueous extracts of Zingiber officinalis (Ginger) and Allium sativum (Garlic) in chick embryos.

In the present study, anti-Avian influenza virus H9N2 activity of aqueous extracts (5, 10, 15, 20, 25%) of Zingiber officinalis and Allium sativum was evaluated. Embryo-toxicity was evaluated by histopathological scoring of Chorio-allantoic membrane of chick embryos. Cytotoxicity of extracts was determined by MTT assay on Vero cells. Aqueous extract of ginger had antiviral activity at 10, 15, 20 and 25% while garlic had activity at 15, 20 and 25%. Histopathological scoring of chorio-allantoic membrane for aqueous extracts (5, 10, 15, 20, 25%) of ginger (0.66±0.57, 1.33±0.57, 1.66±0.57, 2.66±0.57, 3.66±0.57, respectively) and garlic (1.00±0.00, 1.33±0.57, 2.00±0.00, 2.33±0.57, 3.66±0.57, respectively) was concentration dependant. MTT assay revealed cytotoxicity of both plants was also concentration dependent. Extracts of ginger (5, 10, 15, 20, 25%) had lower cytotoxicity (71, 59, 28, 22, 0 % cell survival, respectively) as compared to garlic (61, 36. 20, 11, 3% cell survival, respectively). Overall results revealed that concentration of aqueous extract of ginger (10%), showing antiviral activity against H9N2, was less toxic to vero cells (> 50% cell survival). It is insinuated that ginger may have anti- Avian influenza virus H9N2 potential and its active compounds needs further investigations.

Effect of sodium butyrate on performance, immune status, microarchitecture of small intestinal mucosa and lymphoid organs in broiler chickens.

OBJECTIVE: This study aimed to examine the effect of sodium butyrate (SB) on growth performance, immune status, organs weights, and microarchitecture of lymphoid organs and small intestine. METHODS: A total of 120, 1-d-old broiler chicks were distributed into the following four treatment groups: corn-soy based basal diet (BD) without supplement (control), or the same BD supplemented with 0.1 g/kg zinc bacitracin (ZnB), 0.5 g/kg SB (SB-0.5), or 1.0 g/kg SB (SB-1), respectively. Six birds/group were killed on d-21 and d-35, and samples were collected. RESULTS: Cell-mediated immune response at 48 h post-Phytohemagglutinin-P injection, and antibody titer against Newcastle disease vaccine and sheep red blood cells on d-35 was noted higher (p<0.05) in SB-1 compared to ZnB and control. Lower (p<0.05) feed conversion ratio (FCR) was attained by the supplemented groups. Thymus and spleen weighed more (p<0.05) in SB-1, and bursa registered more (p<0.05) weight in both SB groups compared to control. On d-21, areas of thymus medulla and spleen germinal centers were noted higher (p<0.05) in SB-1 group. The villus height and villus surface area increased (p<0.05) in duodenum and jejunum in both SB groups on d-21, and in SB-1 on d-35, respectively compared to ZnB and control. On d-21, number of goblet cells containing mucins of acidic nature increased (p<0.05) in all the segments of small intestines in SB-1 group compared to control, and on d-35 in ileum compared to other groups. CONCLUSION: In conclusion, SB improved growth performance and immunity as well as modulated morphology of lymphoid organs and gut mucosa in broiler chickens.

Effect of Prebiotic Galacto-Oligosaccharides on Serum Lipid Profile of Hypercholesterolemics.

The present study focused on the role of galacto-oligosaccharides (GOS) against the development of hypercholesterolemia. In the current research, GOS synthesized from lactose solution were fed to hypercholesterolemic female Sprague-Dawley rats. Negative control group (G1) was fed on standard basal diet alone. Positive control group (G5) was fed on inulin (154 mg/250 g body weight), while treatment groups G2, G3 and G4 received 110 mg, 154 mg and 198.4 mg/250 g body weight, respectively, of GOS along with high-fat diet for a period of 60 days. Findings from this study revealed that animals belonging to prebiotic GOS (G2, G3 and G4)-fed group showed significantly decreased serum triglycerides, total cholesterol, LDL cholesterol and VLDL cholesterol as compared to control group (G0). The groups which were fed on different doses of GOS revealed a significant reduction in TC, TG, LDL, and VLDL levels and an increase in HDL level corresponding to the reference group that was fed on inulin, while G1 negative control group revealed increased levels of TC, TG, LDL and VLDL. In contrast to positive control group G5 (154 mg inulin), all doses of GOS lowered serum TC, TG and LDL-C and raised HDL-C; however, G4 (198.4 mg) proved to be more effective. Hence, GOS proved to be supportive in preventing hypercholesterolemia leading cause of cardiovascular disease and atherosclerosis. This study reported a significant reduction of serum TC, TG and LDL-C in female rats for 60 days as compared to control. Conclusively, GOS were found to be worthless against hypercholesterolemia.

Report: antioxidant and nutraceutical value of wild medicinal Rubus berries.

Nutritional quality and antioxidant capacity of three edible wild berries (Rubus ellipticus Smith, Rubus niveus Thunb, Rubus ulmifolius L.) from Lesser Himalayan Range (LHR) were evaluated. Their edible portion was assayed for moisture, fats, ash, carbohydrates, proteins, fibers, essential minerals (Ca, P, Mg, K, Na, Cl, S, Mn, Zn, Fe, Cu, Se, Co, Ni) and DPPH free radical scavenging activity was applied to determine the antioxidant potential. The fruit of Rubus ulmifolius L. (blackberry) possessed the highest values of energy (403.29 Kcal), total protein (6.56g/100 g), Nitrogen (N) content (1500mg/100g), K (860.17mg/100g), Ca (620.56mg/100g), Zn (17.509mg/100g) and the strongest antioxidant activity (98.89% inhibition). While the raspberries (Rubus ellipticus Smith, Rubus niveus Thunb.) exhibited more significant contents of dietary fiber (5.90g/100g), carbohydrates (86.4 g/100 g) and Fe (4.249mg/100g). Significant variation was observed among the tested samples in all the investigated features. The combination of bio elements and active antioxidants clearly showed the applicability of these berries as a nutraceutical supplement.

Ex vivo expansion of functional human UCB-HSCs/HPCs by coculture with AFT024-hkirre cells.

Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34(+) cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF- ß with other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34(+)CD38(-) cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs.

Feasibility and safety of transabdominal chorionic villus sampling.

BACKGROUND: Chorionic Villus Sampling (CVS) is the technique of choice for prenatal diagnosis prior to 12 weeks gestation. The objective of this study was to determine the feasibility, and pattern of complications following first trimester Trans-abdominal Chorionic Villus Sampling (TA-CVS). METHODS: This was a descriptive study conducted in the Obstetrics and Gynaecology Department Military Hospital (MH) Rawalpindi from Jan 2007 to July 2008. Couples at risk of giving birth to a child with genetic disorder were identified and counselled. Trans-abdominal Chorionic Villus Sampling was done using double needle technique under ultrasound guidance. Immediate and late complications were followed up. Data was analysed using SPPS-10. RESULTS: On 200 cases chorionic villus sampling was done as an outdoor procedure. Most common indication was thalassaemia trait 75 (37.5%). Most procedures were done between 12-13 weeks. All placental positions including 104 (52%) posterior and 71 (35.5%) anterior were approachable. Most aspirations were easy, however, in 30 (15%) the aspiration was difficult. Overall success rate was 100%. In 158 (79%) of the cases sample yield was good. One (0.5%) patient had vaginal bleeding and three (1.5%) had placental haematoma formation. Most patients (84%) experienced mild pain during the procedure. The procedure related miscarriage occurred in 2 (1%) patients while another patient developed this complication after 6 weeks. CONCLUSION: First trimester TA-CVS is an accurate and safe invasive prenatal diagnostic procedure. Placentas in almost any position can be approached without any significant risk to mother and the foetus.