Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells

Research (Published online: 21-05-2015)

14. Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells - A. Sasikalaveni, K. G. Tirumurugaan, S. Manoharan, G. Dhinakar Raj and K. Kumanan

Veterinary World, 8(5): 636-639

   doi: 10.14202/vetworld.2015.636-639

Abstract

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Background: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA) cells, which enable quicker isolation of street rabies virus with minimum passages.

Objective: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells.

Materials and Methods: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test.

Results: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided.

Conclusion: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

Keywords: co-culture, isolation, fluorescent-antibody test, murine neuroblastoma, rapid tissue culture infection test.

Critical sources of bacterial contamination and adoption of standard sanitary protocol during semen collection and processing in Semen Station

Research (Published online: 21-05-2015)

13. Critical sources of bacterial contamination and adoption of standard sanitary protocol during semen collection and processing in Semen Station - Chandrahas Sannat, Ajit Nair, S. B. Sahu, S. A. Sahasrabudhe, Ashish Kumar, Amit Kumar Gupta and R. K. Shende

Veterinary World, 8(5): 631-635

   doi: 10.14202/vetworld.2015.631-635

Abstract

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Aim: The present investigation was conducted to locate the critical sources of bacterial contamination and to evaluate the standard sanitation protocol so as to improve the hygienic conditions during collection, evaluation, and processing of bull semen in the Semen Station.

Materials and Methods: The study compared two different hygienic procedures during the collection, evaluation and processing of semen in Central Semen Station, Anjora, Durg. Routinely used materials including artificial vagina (AV) inner liner, cone, semen collection tube, buffer, extender/diluter, straws; and the laboratory environment like processing lab, pass box and laminar air flow (LAF) cabinet of extender preparation lab, processing lab, sealing filling machine, and bacteriological lab were subjected to bacteriological examination in two phases of study using two different sanitary protocols. Bacterial load in above items/environment was measured using standard plate count method and expressed as colony forming unit (CFU).

Results: Bacterial load in a laboratory environment and AV equipments during two different sanitary protocol in present investigation differed highly significantly (p<0.001). Potential sources of bacterial contamination during semen collection and processing included laboratory environment like processing lab, pass box, and LAF cabinets; AV equipments, including AV Liner and cone. Bacterial load was reduced highly significantly (p<0.001) in AV liner (from 2.33±0.67 to 0.50±0.52), cone (from 4.16±1.20 to 1.91±0.55), and extender (from 1.33±0.38 to 0) after application of improved practices of packaging, handling, and sterilization in Phase II of study. Glasswares, buffers, and straws showed nil bacterial contamination in both the phases of study. With slight modification in fumigation protocol (formalin @600 ml/1000 ft3), bacterial load was significantly decreased (p<0.001) up to 0-6 CFU in processing lab (from 6.43±1.34 to 2.86±0.59), pass box (from 12.13±2.53 to 3.78±0.79), and nil bacterial load was reported in LAFs.
Conclusion: Appropriate and careful management considering critical points step by step starting right from collection of semen to their processing can significantly minimize bacterial contamination.
Keywords: bacterial contamination, critical sources, environment, laboratory equipments, Semen Station.

Serum metabolic and minerals profile in norgestomet primed postpartum anestrous surti buffaloes

Research (Published online: 21-05-2015)

12. Serum metabolic and minerals profile in norgestomet primed postpartum anestrous surti buffaloes - Sanjay C. Parmar, C. T. Khasatiya, J. K. Chaudhary, R. V. Patel and H. B. Dhamsaniya

Veterinary World, 8(5): 625-630

   doi: 10.14202/vetworld.2015.625-630

Abstract

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Aim: The study was undertaken to find out the serum metabolic and minerals profile in postpartum anestrous surti buffaloes treated with norgestomet ear implants alone and in combination with pregnant mare serum gonadotropin (PMSG).

Materials and Methods: The study was conducted on 18 postpartum anestrous Surti buffaloes divided into three groups of six animals each at random to conduct the experiment. The buffaloes in Group-I and Group-II were implanted with Crestar ear implant for 9 days together with 2 ml injection of Crestar solution given i/m on the day of the implant insertion. In Group-II, additionally 500 IU PMSG was given i/m on the day of implant removal, whereas the buffaloes in Group-III served as anestrous control group and received 5 ml Normal Saline i/m on day 0 and 9 as a placebo treatment.

Results: The overall serum total protein values did not differ significantly (p > 0.05) between time (days) intervals in any of the groups. The mean serum total cholesterol levels at 10th day and on the day of estrus were found significantly lower (p < 0.05) in the control group as compared to treatment Groups I and II. However, there was no significant difference (p > 0.05) at 10th day and on the day of estrus between treatment groups (T1 and T2). The overall mean serum cobalt, zinc, iron, and manganese values did not differ significantly (p > 0.05) between different time intervals among any of the groups, except copper which was significantly lower (p < 0.05) at 10th day in control group as compared to treatment groups.

Conclusion: Microelements cannot be synthesized in the body. Hence, it is concluded that the mineral mixture should be supplied daily in the animals ration to suffice the requirement of the trace elements. The mean serum metabolic and micro-minerals profiles in treatment and control groups revealed that overall mean serum total protein, cholesterol, copper, and zinc levels were apparently higher in treatment groups whereas, mean serum cobalt, iron, and manganese concentration had no consistent trend between treatment and control groups of Surti buffaloes.

Keywords: anestrous, buffaloes, cholesterol, micro-minerals, norgestomet, pregnant mare serum gonadotropin, protein.

Isolation and purification of beta-lactoglobulin from cow milk

Research (Published online: 15-05-2015)

11. Isolation and purification of beta-lactoglobulin from cow milk - Ranjit Aich, Subhasis Batabyal and Siddhartha Narayan Joardar

Veterinary World, 8(5): 621-624

   doi: 10.14202/vetworld.2015.621-624

Abstract

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Aim: The present study was undertaken to standardize a convenient method for isolation and purification of β-lactoglobulin (β-lg) from cow milk keeping its antigenicity intact, so that the purified β-lg can be used for detection of cow milk protein intolerance (CMPI).

Materials and Methods: Raw milk was collected from Gir breed of cattle reared in Haringhata Farm, West Bengal. Milk was then converted to skimmed milk by removing fat globules and casein protein was removed by acidification to pH 4.6 by adding 3 M HCl. β-lg was isolated by gel filtration chromatography using Sephacryl S-200 from the supernatant whey protein fraction. Further, β-lg was purified by anion-exchange chromatography in diethylaminoethyl-sepharose. Molecular weight of the purified cattle β-lg was determined by 15 percent one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was analyzed by gel documentation system using standard molecular weight marker.

Results: The molecular weight of the purified cattle β-lg was detected as 17.44 kDa. The isolated β-lg was almost in pure form as the molecular weight of purified β-lg monomer is 18kDa.

Conclusion: The study revealed a simple and suitable method for isolation of β-lg from whey protein in pure form which may be used for detection of CMPI.

Keywords: beta-lactoglobulin, ion-exchange chromatography, milk protein intolerance, sodium dodecyl sulfate polyacrylamide gel electrophoresis, whey protein.

Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

Research (Published online: 15-05-2015)

10. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows - Pooja H. Gupta, Nirmal A. Patel, D. N. Rank and C. G. Joshi

Veterinary World, 8(5): 615-620

   doi: 10.14202/vetworld.2015.615-620

Abstract

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Aim: An attempt has been made to study the toll-like receptors 4 (TLR4) gene polymorphism from cattle DNA to correlate with mastitis cows.

Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR), respectively from Kankrej (22) and Triple cross (24) cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one.

Results: Results showed that both alleles (A and B) of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p˂0.05). Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS) indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05). Thus, the allele A might play an important role in mastitis resistance in cows.

Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance.

Keywords: cattle, mastitis, restriction fragment length polymorphisms, single-strand conformational polymorphism, somatic cell score, toll-like receptors 4 gene.

Cloning and sequencing of hfq (host factor required for synthesis of bacteriophage Q beta RNA) gene of Salmonella Typhimurium isolated from poultry

Research (Published online: 14-05-2015)

9. Cloning and sequencing of hfq (host factor required for synthesis of bacteriophage Q beta RNA) gene of Salmonella Typhimurium isolated from poultry - Parthasarathi Behera, Muhammed Kutty, Bhaskar Sharma, Ajay Kumar and Meeta Saxena

Veterinary World, 8(5): 610-614

   doi: 10.14202/vetworld.2015.610-614

Abstract

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Aim: The aim was to clone and sequence hfq gene of Salmonella Typhimurium strain PM-45 and compare its sequence with hfq gene of other serovar of Salmonella.

Materials and Methods: Salmonella Typhimurium strain PM-45 was procured from the G. B. Pant University of Agriculture and Technology, Pantnagar, India. The genomic DNA was isolated from Salmonella Typhimurium. Hfq gene was polymerase chain reaction (PCR) amplified from the DNA using specific primers, which was subsequently cloned into pET32a vector and transformed intoEscherichia coli BL21 pLys cells. The recombinant plasmid was isolated and subjected to restriction enzyme digestion as well as PCR. The clone was then sequenced. The sequence was analyzed and submitted in GenBank.

Results: PCR produced an amplicon of 309 bp. Restriction digestion of the recombinant plasmid released the desired insert. The hfqsequence shows 100% homology with similar sequences from other Salmonella Typhimurium isolates. Both nucleotide and amino acid sequences are highly conserved. The submitted sequence is having Genbank accession no KM998764.

Conclusion: Hfq, the hexameric RNA binding protein is one of the most important post-transcriptional regulator of bacteria. The sequence of hfq gene of Salmonella Typhimurium is highly conserved within and between Salmonella enterica serovars. This gene sequence is probably under heavy selection pressure to maintain the conformational integrity of its product in spite of its being not a survival gene.

Keywords: cloning, hfq, RNA binding protein, sequencing, Salmonella Typhimurium.

Evaluation of various feedstuffs of ruminants in terms of chemical composition and metabolisable energy content

Research (Published online: 14-05-2015)

8. Evaluation of various feedstuffs of ruminants in terms of chemical composition and metabolisable energy content - Dinesh Kumar, Chander Datt, L. K. Das and S. S. Kundu

Veterinary World, 8(5): 605-609

   doi: 10.14202/vetworld.2015.605-609

Abstract

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Aim: The aim was to determine the chemical composition and metabolisable energy (ME) content of feedstuffs used in ruminant animals using in vitro method.

Materials and Methods: A total of 18 feedstuffs used for ruminant feeding including cultivated non-leguminous fodders like maize, sorghum, pearl millet, and oat; leguminous fodders like cowpea and berseem; agro-industrial by-products such as wheat bran, deoiled rice bran, rice polish, wheat straw, and concentrates such as mustard oil cake, groundnut cake, soybean meal, cotton seed cake, grains like maize, oat, wheat, and barley were taken for this study. Chemical compositions and cell wall constituents of test feeds were determined in triplicate. The crude protein (CP) content was calculated as nitrogen (N) × 6.25. True dry matter digestibility (TDMD), true organic matter digestibility (TOMD), ME, and partitioning factor (PF) values were determined by in vitro gas production technique (IVGPT).

Results: The CP content of non-leguminous fodders varied from 7.29% (sorghum) to 9.51% (maize), but leguminous fodders had less variation in CP. Oilseed cakes/meals had high CP and ether extract (EE) content than other feedstuffs except rice polish, which had 12.80% EE. Wheat straw contained highest fiber fractions than the other ingredients. ME content was highest in grains (wheat-12.02 MJ/kg) and lowest in wheat straw (4.65 MJ/kg) and other roughages. TDMD of grains and oilseed cakes/meals were higher than the fodders and agro-industrial by-products. The same trend was observed for TOMD.

Conclusions: It was concluded that the energy feeds showed a great variation in chemical composition and ME content. The results of this study demonstrated that the kinetics of gas production of energy feed sources differed among themselves. Evaluation of various feedstuffs is helpful in balanced ration formulation for field animals and under farm conditions for better utilization of these commonly available feed resources.

Keywords: chemical compositions, feedstuffs, in vitro method, metabolisable energy, ruminants.