Effects of glycolytic and cytoskeletal inhibitors on phagocytic and nitroblue tetrazolium reductive activities of bovine neutrophils

Phagocytic and oxidative metabolic activities of bovine blood neutrophils were determined in the presence of glycolytic (NaF) and cytoskeletal (colchicine, cytochalasin B, and prostaglandin E1) inhibitors. Phagocytosis and post-phagocytic oxidative metabolic activity, measured by nitroblue tetrazolium reduction, were determined using zymosan, Escherichia coli, Staphylococcus aureus, or Streptococcus agalactiae. Sodium fluoride (1.25 microM to 1.25 mM concentrations) did not significantly (P greater than 0.05) inhibit phagocytosis of S aureus and Str agalactiae, whereas phagocytosis of zymosan and E coli was significantly (P less than 0.05) inhibited only at 1.25 mM concentration. Colchicine at 1.25 nM to 1.25 microM concentrations significantly inhibited phagocytosis of zymosan and E coli, but not of S aureus and Str agalactiae. Cytochalasin B at 1.25 nM to 1.25 microM concentrations significantly inhibited phagocytosis of zymosan and all 3 bacteria, whereas prostaglandin E1 was noninhibitory at similar concentrations. Nitroblue tetrazolium reduction, in general, was not significantly affected by NaF and cytoskeletal inhibitors.     

Assessment of Genetic Variability in the Coding Sequence of Melatonin Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane‐bound G‐coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out‐of‐season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen‐2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out‐of‐season breeding. GG and CC genotypes were having a higher prevalence in the studied population.     

Effects of various treatments on induced chronic aflatoxicosis in rabbits

Male New Zealand White rabbits were orally given 0.05 mg of aflatoxin B1 (AFB1)/kg of body weight daily for 10 days and were treated with glutathione-precursors and depletor, antibacterial agents, or sodium thiosulfate. The drug administered, the mortality, and the mean survival time were as follows: corn-oil controls (0), euthanatized at 25 days; AFB1-controls (2), 21 days; AFB1 and saline controls (2), 22 days; cysteine and AFB1 (5), 13 days; methionine and AFB1 (5), 12 days; sodium thiosulfate and AFB1 (2), 21 days; sulfadimethoxine and AFB1 (1), 24 days; oxytetracycline and AFB1 (0), euthanatized at 25 days; and ethyl maleate and AFB1 (3), 21 days. Clinical signs of toxicosis included decreased feed consumption during AFB1 administration, loss of body weight or failure to gain, and death. Clinicopathologic changes included increases in serum bilirubin concentration and alanine aminotransferase and aspartate aminotransferase activities. Prothrombin and activated partial thromboplastin times were lengthened. Plasma fibrinogen concentration was decreased. Changes in PCV, hemoglobin concentration, and serum alkaline phosphatase were unremarkable. Oxytetracycline had protective effects against chronic aflatoxicosis in rabbits. Cysteine and methionine enhanced chronic aflatoxicosis.     

In vitro platelet release by rat megakaryocytes: effect of metabolic inhibitors and cytoskeletal disrupting agents

Development of an in vitro visual assay facilitated the study of large numbers of megakaryocytes undergoing proplatelet formation in short-term cultures. Approximately 9% of megakaryocytes formed platelets during a 24-hour period. In the presence of an inhibitor of anaerobic glycolysis (NaF), proplatelet formation was inhibited, whereas inhibitors of respiration (NaCN) did not significantly (P greater than 0.05) decrease proplatelet formation. Presence of the microtubule-disrupting agents colchicine and vincristine sulfate in culture medium inhibited proplatelet formation, whereas the microfilament-disrupting agent cytochalasin B had a less pronounced inhibition.     

Methods for detection of immune-mediated neutropenia in horses, using antineutrophil serum of rabbit origin

Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity greater than 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils. Specific bright membranous fluorescence was seen in neutrophils treated with the antiserum and exposed to fluorescein-conjugated goat anti-rabbit immunoglobulin, and staphylococcal protein A and streptococcal protein G. Whereas the indirect immunofluorescence and protein G-binding tests were equally sensitive and resulted in titer of 1:256, the protein A-binding test was less sensitive and resulted in titer of only 1:32. Nonspecific binding of protein A and protein G was noticed as uniform or patchy cellular fluorescence in a small number of neutrophils. Treatment of neutrophils with antiserum up to dilution of 1:8 resulted in a significant (P less than 0.05) suppression of phagocytosis of opsonized zymosan particles. Thus, protein G-binding and indirect immunofluorescence tests are highly sensitive to detect neutrophil antibody and may be used to diagnose immune-mediated neutropenias in horses and, possibly, in other animal species.     

Pharmacokinetics and urinary excretion of sulphadimidine in buffalo calves

Pharmacokinetics and urinary excretion of sulphadimidine (SDI) were determined in buffalo calves following single oral administration (150 mg/kg). The plasma levels of free sulphadimidine were above minimum effective therapeutic concentration (> 40 micrograms/ml) between 4 and 12 h and the N4-acetylated form of the drug was in the range of 7.2-19.3%. Kinetic evaluation of plasma levels was performed using a two-compartment open model. The absorption and elimination half-lives of SDI were 3.01 and 11.94 h, respectively. Based on this study, an optimal dosage regimen of sulphadimidine in buffalo calves would be 100 mg/kg, followed by 50 mg/kg at 12 h intervals. Sulphadimidine was mainly excreted in the urine as free amine. The percentage of N4-acetyl sulphadimidine in urine was comparatively higher than in plasma.     

Preliminary observations on the pharmacokinetics of CDRI compound 81/470 in calves

Veterinary Research Communications -