Therapeutic administration of Luteolin protects against Escherichia coli-derived Lipopolysaccharide-triggered inflammatory response and oxidative injury.

Endometritis reduces reproductive effectiveness and leads to significant financial losses in the dairy sector. Luteolin is a natural phyto-flavonoid compound with many biological activities. However, the therapeutic effect of Luteolin against lipopolysaccharides (LPS)-induced endometritis has not yet been explored. A total of eighty female Kunming mice were randomly assigned into four treatment groups (n = 20). Following a successful initiation of the endometritis model by LPS, Luteolin was intraperitoneally administered three times, at six-hour intervals between each injection in the Luteolin groups. The histopathological findings revealed that Luteolin significantly alleviated uterine injury induced by LPS. Moreover, Luteolin suppressed the synthesis of pro-inflammatory mediators [interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α] while promoting the synthesis of an anti-inflammatory mediator (IL-10) altered by LPS. Furthermore, Luteolin significantly mitigated the LPS-induced oxidative stress by scavenging malondialdehyde (MDA) and reactive oxygen species (ROS), accumulation and boosting the capacity of antioxidant enzyme activities such as superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase 1 (Gpx1) in the uterine tissue of mice. Additionally, injection of Luteolin markedly increased the expression of Toll-like receptors (TLR) 4 both at mRNA and protein levels under LPS stimulation. Western blotting and ELISA findings demonstrated that Luteolin suppressed the activation of the NF-κB pathway in response to LPS exposure in the uterine tissue of mice. Notably, Luteolin enhanced the anti-oxidant defense system by activating the Nrf2 signaling pathway under LPS exposure in the uterine tissue of mice. Conclusively, our findings demonstrated that Luteolin effectively alleviated LPS-induced endometritis via modulation of TLR4-associated Nrf2 and NF-κB signaling pathways.

Autologous Platelet Lysate Is an Alternative to Fetal Bovine Serum for Canine Adipose-Derived Mesenchymal Stem Cell Culture and Differentiation.

The use of fetal bovine serum (FBS) in regenerative medicine raises serious ethical and scientific concerns. We have cultured and differentiated the canine mesenchymal stem cells (cMSCs) in five different media combinations of autologous platelet lysate (A-PL) and FBS; consisting of 0% A-PL and 10% FBS (M-1), 2.5% A-PL and 7.5% FBS (M-2), 5% A-PL and 5% FBS (M-3), 7.5% A-PL and 2.5% FBS (M-4), and 10% A-PL and 0% FBS (M-5). The cMSCs were evaluated for their doubling time, differentiation efficiency, and expression of CD73, CD90, CD105, and PDGFRα. The mRNA expression of NT5E, THY1, ENG, PPARγ, FABP4, FAS, SP7, BGLAP, and SPP1 was also assessed. The results indicated non-significant differences in cellular proliferation/viability; positive expression of surface markers, and PDGFRα with substantial adipo/osteogenic differentiation. The expression of adipogenic (PPARγ, FABP4, FAS), and osteogenic (SP7, BGLAP, SPP1) genes were higher (p < 0.05) in the M5 group. In conclusion, A-PL in cMSCs culture did not negatively affect cellular proliferation and viability but also enhanced their genetic potential for multilineage differentiation. Our results indicate that A-PL can be used as an alternative for FBS to develop potent cMSCs under good manufacturing practice protocol for regenerative medicine.

Epidemiological survey of bovine gammaherpesvirus 4 (BoHV-4) infection in cattle and buffalo from Pakistan.

This study was conducted to evaluate the prevalence of bovine gammaherpesvirus 4 (BoHV-4) among healthy cattle and buffaloes as well as those associated with different diseases (respiratory tract infection, mastitis and reproductive tract infection) in District Chakwal, Pakistan. Blood, swab and milk samples of cattle and buffaloes were randomly collected from different areas of Chakwal. DNA was isolated from the samples and subjected to nested PCR using thymidine kinase gene primers. Out of 300 samples (200 blood, 50 swab and 50 milk samples) from both species (cattle and buffalo), an overall prevalence of BoHV-4 of 3.33% was obtained. Samples from cattle showed a higher species-specific prevalence (4.16%) than samples from buffalo (2.78%). One sample out of 50 swab samples and 1 out of 50 milk samples were also positive for BoHV-4. DNA sequencing of a positive PCR product from cattle confirmed that the sequence was from the thymidine kinase gene of BoHV-4. Phylogenetic analysis also revealed close similarities with other BOHV-4 thymidine kinase sequences. To detect BoHV-4 antibodies, an indirect ELISA was also performed. Two hundred blood samples were also collected from the same animals in nonanticoagulant-containing tubes for the isolation of serum and were subjected to indirect ELISA. Sixteen samples (8%) were positive for BoHV-4 antibodies. This study will be useful in further diagnoses of BoHV-4 in Pakistan and in devising measures to control the spread of BoHV-4.

Characterization and differentiation potential of mesenchymal stem cells isolated from multiple canine adipose tissue sources.

BACKGROUND: Mesenchymal stem cells (MSCs) are undifferentiated cells that can give rise to a mesoderm lineage. Adipose-derived MSCs are an easy and accessible source for MSCs isolation, although each source of MSC has its own advantages and disadvantages. Our study identifies a promising source for the isolation and differentiation of canines MSCs. For this purpose, adipose tissue from inguinal subcutaneous (SC), perirenal (PR), omental (OM), and infrapatellar fat pad (IPFP) was isolated and processed for MSCs isolation. In the third passage, MSCs proliferation/metabolism, surface markers expression, in vitro differentiation potential and quantitative reverse transcription PCR (CD73, CD90, CD105, PPARγ, FabP4, FAS, SP7, Osteopontin, and Osteocalcin) were evaluated. RESULTS: Our results showed that MSCs derived from IPFP have a higher proliferation rate, while OM-derived MSCs have higher cell metabolism. In addition, MSCs from all adipose tissue sources showed positive expression of CD73 (NT5E), CD90 (THY1), CD105 (ENDOGLIN), and very low expression of CD45. The isolated canine MSCs were successfully differentiated into adipogenic and osteogenic lineages. The oil-red-O quantification and adipogenic gene expression (FAS, FabP4, and PPARγ) were higher in OM-derived cells, followed by IPFP-MSCs. Similarly, in osteogenic differentiation, alkaline phosphatase activity and osteogenic gene (SP7 and Osteocalcin) expression were higher in OM-derived MSCs, while osteopontin expression was higher in PR-derived MSCs. CONCLUSION: In summary, among all four adipose tissue sources, OM-derived MSCs have better differentiation potential toward adipo- and osteogenic lineages, followed by IPFP-MSCs. Interestingly, among all adipose tissue sources, MSCs derived from IPFP have the maximum proliferation potential. The characterization and differentiation potential of canine MSCs isolated from four different adipose tissue sources are useful to assess their potential for application in regenerative medicine.

Molecular detection and characterization of ovine herpesvirus-2 using heminested PCR in Pakistan.

BACKGROUND: Malignant catarrhal fever (MCF) is a highly fatal lymphoproliferative disease of cattle, deer, bison, water buffalo, and pigs caused by the gamma-herpesviruses alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2). OBJECTIVES: This study aimed to determine the prevalence of OvHV-2 in sheep, goats, cattle, and buffalo in Rawalpindi and Islamabad, Pakistan, by applying molecular and phylogenetic methods. METHODS: Blood samples were aspirated from sheep (n = 54), goat (n = 50), cattle (n = 46) and buffalo (n= 50) at a slaughterhouse and several farms. The samples were subjected to heminested polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the OvHV-2 POL gene and the OvHV-2 ORF75 tegument protein gene. RESULTS: The highest percentage of MCF positive samples was in sheep (13%), whereas goat, cattle, and buffalo had lower positive percentages, 11%, 9%, and 6.5%, respectively. Four OvHV-2-positive PCR products obtained from sheep samples were sequenced. The sequences obtained were submitted to the NCBI GenBank database (MK852173 for the POL gene; MK840962, MK852171, and MK852172 for the ORF75 tegument protein gene). Phylogenetic analysis revealed a close similarity of study sequences with those of worldwide samples. CONCLUSIONS: This study is the first cross-sectional study on the prevalence and molecular detection of OvHV-2 in apparently healthy cattle and buffalo that could be carrying OvHV-2 acquired from OvHV-2-positive sheep and goats. The results indicate that OvHV-2 is circulating in Pakistan. Further studies are needed to characterize OvHV-2 and elucidate further its prevalence.

First detection and molecular characterization of avian polyomavirus in young parrots in Pakistan.

Avian polyomavirus (APV) infection, also called as budgerigar fledgling disease (BFD) causes various health problems in many psittacine species which may cause untimely death. The aims of this study were to investigate, for the first time, the detection, molecular characterization and phylogenetic analysis of avian polyomavirus (APV) in Pakistani psittacine birds. In an aviary a disease similar to APV was found and 90% of the nestlings died within a few weeks. Seven to ten-day-old parrot nestlings (n = 3) from the aviary were presented with feather abnormalities, plumage defect and were clinically depressed. Birds died at 11th, 14th and 16th day of age. Samples of hearts, livers, spleen, feathers and kidneys were collected from the dead birds. Samples were analyzed for the presence of APV DNA by using PCR. APV VP1 gene was partially sequenced, and phylogenetic analysis was performed. The APV strain was similar to those previously reported in other areas of the world. The results of this investigation indicate presence of a high frequency of APV infections in psittacine birds in Pakistan.

Occurrence and identification of Emeria species in broiler rearing under traditional system.

BACKGROUND: The present study was intended to determine the prevalence and identification of species involved causing coccidiosis in broilers rearing under traditional farming system in Mirpur, Azad Kashmir, Pakistan. For the current study, a convenient sampling technique was carried out. A total of 7814 broilers (aged 1 to 6 weeks) were submitted to the Disease Diagnostic Laboratory of Livestock and Animal husbandry Department Mirpur, Azad Kashmir. RESULTS: From the total screened, 750 were found positive for coccidiosis representing an overall prevalence of 9.59 %. Age-wise highest prevalence (10.88 %) recorded in the middle age birds (0 to 3 week old) were found more susceptible to infection than those aged above 3 weeks. Higher prevalence (12.49 %) of coccidiosis in broilers was observed in spring as compared with 6.60 % in summer season. In this study two main coccidiosis causing species, Emeria tenella and Emeria maxima were identified on the basis of their morphological feature and habitat (caeca and intestine), However, E. tenella was dominant compared to E. maxima. CONCLUSION: The study provides an insight to the occurrence of Emeria species which must be taken into consideration when rearing the broilers.

Anticoccidial activity of hydrochloric acid (HCl) against Eimeria tenella in broiler chickens

The present study was planned to evaluate the anticoccidial activity of the different concentrations of the HCl against Eimeria tenella infection in broiler chickens in comparison with the amprolium anticoccidial. For this purpose, a total of 198 chicks were placed 11 per pen with three pens per treatment. The different concentrations of HCl (1000ppm, 2000ppm and 3000ppm) and amproilum (at the dose rate of 125ppm) were given to the experimental groups in drinking water from 10 to 19th days of age. One group was kept as infected non medicated control and one as non infected non medicated control. At the 12th day of age, all the groups were inoculated orally with 75,000 sporulated oocysts except non infected non medicated control. Anticoccidial activity was evaluated on the basis of performance (weight gain, feed conversion ratio) and pathogenic (oocyst score, lesion score and mortality percentage) parameters. Among HCl medicated groups, the maximum anticoccidial effect was seen in the group medicated with 1000ppm HCl followed by 2000ppm and 3000ppm HCl medicated groups. Amprolium and 1000ppm HCl were almost equivalent in suppressing the negative performance and pathogenic effects associated with coccidiosis (Eimeria tenella) challenge. In summary, the lower doses of HCl have the potential to be used as alternative to chemotherapeutic drugs for Eimeria tenella control. It is therefore suggested that further studies should be carried out to determine the possible minimum safe levels of HCl with least toxic effects to be used as anticoccidial.

Anticoccidial effects of acetic acid on performance and pathogenic parameters in broiler chickens challenged with Eimeria tenella

The objective of the present study was to evaluate the anticoccidial effect of the different concentrations of the acetic acid in the broiler chickens in comparison with the amprolium anticoccidial. A total of 198 chicks were placed 11 per pen with three pens per treatment. The different concentrations (1 percent, 2 percent and 3 percent) of acetic acid and amproilum (at the dose rate of 125ppm) were given to the experimental groups in drinking water from 10-19th days of age. One group was kept as infected non medicated control and one as non infected non medicated control. All the groups were inoculated orally with 75,000 sporulated oocysts at the 12th day of age except non infected non medicated control. Anticoccidial effect was evaluated on the basis of performance (weight gain, feed conversion ratio) and pathogenic (oocyst score, lesion score and mortality percentage) parameters. Among acetic acid medicated groups, the maximum anticoccidial effect was seen in the group medicated with 3 percent acetic acid followed by 2 percent and 1 percent acetic acid medicated groups. Amprolium and 3 percent acetic acid were almost equivalent in suppressing the negative performance and pathogenic effects associated with coccidiosis (Eimeria tenella) challenge. In summary, acetic acid has the potential to be used as alternative to chemotherapeutic drugs for Eimeria tenella control. Concentration-dependent anticoccidial effect of acetic acid suggests that further studies should be carried out to determine the possible maximum safe levels of acetic acid with least toxic effects to be used as anticoccidial.

Toxicopathological effects of lambda-cyhalothrin in female rabbits (Oryctolagus cuniculus).

The aim of this study was to investigate effects of lambda-cyhalothrin (LCT) on clinical, hematological, biochemical and pathological alterations in rabbits (Oryctolagus cuniculus). New Zealand white female rabbits (n = 24) of 4-5 months age having 997.92 ± 32.83 g weight were divided into four equal groups. Group A (control) received normal saline intraperitoneally (ip). Animals in groups B, C and D were treated with LCT 1.0, 4.0 and 8.0 mg/kg bw ip. Each group received seven consecutive doses at an interval of 48 hours. Blood and serum samples were collected at an interval of 96 hours. Blood analysis revealed a significant (p < 0.05) decrease in red blood cell and white blood cell counts, hemoglobin concentration and lymphocytes, while mean corpuscular hemoglobin concentration, mean corpuscular volume, neutrophils, monocytes and eosinophils were increased. Serum biochemical analysis revealed significant (p < 0.05) decrease in serum total proteins and serum albumin, while an increase was seen in serum alanine aminotransferase and aspartate aminotransferase activities compared with the control group. Serum globulin values varied non-significantly in all treatment groups as compared to control group. A dose-dependent increase in the incidence of micronucleated polychromatic erythrocyte was observed. All gross and histopathological lesions observed in LCT-treated rabbits were dose-dependent. Liver of the treated rabbits exhibited extensive perihepatitis, hyperplasia of bile duct, necrosis, hemorrhages and congestion. In lungs, there were hemorrhages, thickened alveolar walls, congestion, emphysema, collapsed alveoli and accumulation of extensive inflammatory cells. Kidneys were congested and hemorrhagic whereas renal parenchyma and stroma were normal. Microscopically, heart showed congestion of blood vessels and nuclear pyknosis, myodegeneration. It was concluded from the study that LCT produced toxicopathological alterations in rabbits in a dose-dependent manner. On the basis of the results, it can be suggested that overdosing of LCT be avoided while treating animals for ectoparasites.

Cutaneous form of pox infection among captive peafowl (Pavo cristatus) chicks.

The present study was carried out to investigate the epidemiology and lesions of avian pox in captive peafowl chicks. Overall values of morbidity, mortality and case fatality were 45.2%, 27.1% and 60.0%, respectively. The chicks of 9 to 12 weeks of age showed a significantly (P<0.001) higher prevalence rate than other age groups. The morbidity and mortality due to avian pox in peafowl chicks was significantly (P<0.001) reduced when kept in mosquito-proof cages and hatched under broody chicken hens. Morbidity due to poxvirus infection on the peafowl farm was 82%, 26% and 12% in successive years. This reduction might have been the result of the introduction of mosquito-proof nets after year 1, although this was not the subject of a controlled experiment. All of the peafowl chicks suffering from dry pox showed pustular and nodular lesions on eye lids, beak, legs and toes. Distribution of lesions in different body parts varied significantly (P<0.023). Lesion diameters were less than 1 cm (59.73%), 1 to 2 cm (23.75%) and more than 2 cm (16.87%). Histopathological studies revealed extensive proliferation of subdermal connective tissue and infiltration of heterophils and macrophages. The keratinocytes showed degenerative changes in the form of cytoplasmic vacuolation, ballooning and hyper-chromatic nuclei. Eosinophilic intracytoplasmic inclusions (Bollinger bodies) in keratinocytes were consistently present. It was concluded that avian pox rendered high morbidity, mortality and case fatality in peafowl chicks.