Genetic and phylogenetic analysis of the outer capsid protein genes of Indian isolates of bluetongue virus serotype-16

Research (Published online: 01-08-2018)

1. Genetic and phylogenetic analysis of the outer capsid protein genes of Indian isolates of bluetongue virus serotype-16

Arpit Saxena, Sanchay K. Biswas, Karam Chand, Jishnu Naskar, Ankita Chauhan, Gulam Mohd, Neha Tewari, Kurat-ul-Ain, Muthannan A. Ramakrishnan and Awadh Bihari Pandey

Veterinary World, 11(8): 1025-1029

ABSTRACT

Aim: The aim of the study was to characterize bluetongue virus serotype 16 (BTV-16), recently isolated from different states of India. The evolutionary relationship of newly isolated BTV-16 and previously reported Indian and global BTV-16 isolates were compared using molecular analysis.

Materials and Methods: In the present study, five (n=5) BTV-16 isolates were used to amplify gene segment-2 and segment-6 encoding the outer capsid proteins VP2 and VP5, respectively. The amplified products were purified and sequenced by the Sanger sequencing method. The phylogenetic relationship and nucleotide identity of all five BTV-16 isolates were compared with previously reported Indian and global BTV-16 isolates. Nucleotide sequence data were aligned using the CLUSTAL W algorithm implemented in the MegAlign of DNASTAR program package (MegAlign 5.00, DNASTAR Inc., Madison, USA). Phylogenetic analyses were carried out using MEGA version 6.0 software with the best nucleotide substitution model.

Results: Phylogenetic analysis based on the VP2 and VP5 encoding genes, segregates Indian BTV-16 isolates in a distinct cluster with proximity to the Eastern topotype. Indian isolates make a monophyletic cluster with Eastern topotypes with Western topotype BTV-16 (BTV-16/NIG/AJ586694) occupying a separate cluster. Indian isolates were found to share 91.5%- 97.5% and 96.5%-98.9% identity at the nucleotide and deduced amino acid (aa) level, respectively, to the global BTV-16 isolates. There is a high degree of variation with the Nigerian isolate with 27.0-27.7% and 26.0-26.9% at the nucleotide and aa sequence level, respectively. These data suggest that Indian BTV-16 isolates might have evolved separately within the Eastern BTV topotype.

Conclusion: Phylogenetic analyses and nucleotide identity of BTV-16 isolates at the VP2 and VP5 gene encoded level indicate that isolates used in the present study might have evolved from a common Eastern topotype ancestor. The data presented in this study will be helpful for future selection of reference strains in a serological and molecular epidemiology study.

Keywords: bluetongue virus, phylogenetic analysis, VP2 gene, VP5 gene.

Blood biochemical profiles of Brahman crossbred cattle supplemented with different protein and energy sources

Research (Published online: 31-07-2018)

21. Blood biochemical profiles of Brahman crossbred cattle supplemented with different protein and energy sources

Nguyen Hong Xuan, Huynh Tan Loc and Nguyen Trong Ngu

Veterinary World, 11(7): 1021-1024

ABSTRACT

Aim: The experiment was carried out to evaluate the effects of supplementing different levels of protein and energy sources on blood biochemical profiles of Brahman crossbred cattle.

Materials and Methods: The study consisted of two experiments in Brahman crossbred cattle in An Giang Province. In trial 1, 28 cattle of 178±12.5 kg were arranged in a completely randomized block design. In the second trial, another 24 cattle of 182±14.3 kg were allocated in a 2 × 3 factorial design. The experiments lasted for 90 days. Blood samples were taken at the end of the experiments, and plasma concentrations of metabolites and enzymes were analyzed by an automated biochemical analyzer (Humalyzer 3000, USA).

Results: The glucose concentration was highest at 1.83 mmol/L when supplemented with urea (60 g/head/d). Urea and creatinine content was not significantly different between treatments when cattle were supplemented with different protein and energy sources. In the treatment with 360 g/head/d soybean meal supplementation, cholesterol concentration was lowest (2.50 mmol/L), compared with the highest concentration (3.86 mmol/L) in the treatment with soybean meal at 720 g/head/ day. The total protein concentration showed the highest values at 94.5 g/L and 96.3 g/L when supplemented with soybean meal (720 g/head/day) and fish oil, respectively.

Conclusion: There were slightly altered blood biochemical profiles among cattle at different protein and energy source supplements.

Keywords: cattle, concentrate, oil, soybean, supplementation.

The effects of Saccharomyces cerevisiae supplementation on intake, nutrient digestibility, and rumen fluid pH in Awassi female lambs

Research (Published online: 30-07-2018)

20. The effects of Saccharomyces cerevisiae supplementation on intake, nutrient digestibility, and rumen fluid pH in Awassi female lambs

Belal S. Obeidat, Kamel Z. Mahmoud, Mohammad D. Obeidat, Mysaa Ata, Rami T. Kridli, Serhan G. Haddad, Hosam H. Titi, Khaleel I. Jawasreh, Hosam J. Altamimi, Hadil S. Subih, Safaa M. Hatamleh, Majdi A. Abu Ishmais and Ruba Abu Affan

Veterinary World, 11(7): 1015-1020

ABSTRACT

Aim: The aim of this study was to evaluate the effect of feeding low (LO)- or high (HI)-fiber diets supplemented with Saccharomyces cerevisiae (SC) on nutrient intake, digestibility, nitrogen balance, rumen fluid pH, and serum concentrations of glucose and urea nitrogen in Awassi female lambs in a 2×2 factorial arrangement of treatments.

Materials and Methods: Experimental diets were as follows: (1) LO-fiber diet with no SC supplementation (-LO), (2) LO-fiber diet supplemented with SC (+LO), (3) HI-fiber diet with no SC supplementation (-HI), or (4) HI-fiber diet supplemented with SC (+HI). Eight female lambs were used in a replicated 4×4 Latin square design with 15-day experimental periods (10-day adaptation period and 5-day collection period).

Results: A fiber×SC interaction (p≤0.05) was detected for dry matter (DM) and crude protein (CP) intake among diets showing greater DM and CP intake for +LO diet compared to +HI group supplemented with SC, whereas -LO and -HI were intermediate. A fiber×SC interaction (p=0.05) was also detected for the neutral detergent fiber (NDF) intake among diets. Intake of NDF was greater for the -HI diet compared with +LO and -LO diets. Similarly, NDF intake was greater for +HI diet than -LO diet. A tendency (p=0.07) for a fiber×SC interaction was detected for acid detergent fiber (ADF) intake among diets as well. ADF intake tended to be greater for HI-fiber diets. No difference was observed in the rumen fluid pH for lambs fed with the different diets. No fiber×SC interactions were detected for the digestibility of DM, CP, NDF, and ADF among dietary treatments. Digestibility of DM was greater (72.9 g/100 g vs. 67.1 g/100 g; p=0.0002) for LO versus HI fiber. However, NDF and ADF digestibilities were greater (60.8 and 61.9 g/100 g vs. 55.8 and 52.7 g/100 g for NDF and ADF digestibility, respectively; p≤0.01) for the HI-fiber than the LO-fiber diets.

Conclusion: Results obtained in the current study indicate that SC supplementation has a minimal effect on the performance of Awassi female lambs fed with varying fiber levels.

Keywords: Awassi female lamb, intake, nutrients digestibility, yeast supplementation.

Use of molecular biology tools for rapid identification and characterization of Pasteurella spp.

Research (Published online: 29-07-2018)

19. Use of molecular biology tools for rapid identification and characterization of Pasteurella spp.

Ashraf M. Abbas, Dalia A. M. Abd El-Moaty, Eman S. A. Zaki, Elham F. El-Sergany, Nadine A. El-Sebay, Hala A. Fadl, and Ayman A. Samy

Veterinary World, 11(7): 1006-1014

ABSTRACT

Aim: This study aimed to create rapid characterization and genotyping of Pasteurella multocida (PM) protocol using modern molecular biology techniques.

Materials and Methods: Thirty bacterial isolates were characterized by capsular and somatic identification using conventional procedure followed by multiplex polymerase chain reaction (PCR), restriction endonucleases analysis (REA), and finally confirmed by sequence analysis. Two local vaccine strains and two field isolates were identified as PM Type A and B.

Results: A total of 30 isolates were found positive for PM either morphologically and biochemically; however, multiplex PCR technique identified only 22 isolates as Pasteurella species using universal primers while 8 isolates were found negative for PM. 12 of 22 isolates (54%) were characterized at the same reaction into PM Type A, five isolates (23%) were Type B and the rest five isolates (23%) of tested isolates were negative for Types A, B, and D. Hemorrhagic septicemia Type B: 2 or B: 5 could be identified somatically within PM capsular serogroup B using PCR technique. Somatic characterization of PM was done using REA that could identify all PM Type A into A:1 and all PM Type B into B: 2. These protocols were verified for its accuracy and reliability by sequence analysis of two vaccine strains of PM Type A and B that were characterized previously by biochemical and serological methods as well as two selected isolates from the 22 positive isolates representing PM Type A and B.

Conclusion: PCR and REA could confirm the identity of PM and provide a rapid and reliable characterization in comparison with biochemical analysis and conventional serotyping that may take up to 2 weeks. Hence, they can reduce the time needed for polyvalent vaccine production and when the reference antisera are unavailable. Moreover, the identity of Omp-H for vaccine and field strains may provide better data to control Pasteurellosis in Egypt.

Keywords: multiplex polymerase chain reaction, outer membrane protein H, Pasteurella multocida, restriction endonucleases analysis.

Aflatoxicosis in African greater cane rats (Thryonomys swinderianus)

Research (Published online: 27-07-2018)

18. Aflatoxicosis in African greater cane rats (Thryonomys swinderianus)

Henry O. Jegede, Ahmed O. Akeem, Oluwafemi B. Daodu and Afolabi A. Adegboye

Veterinary World, 11(7): 1001-1005

ABSTRACT

Aim: Aflatoxicosis is a widespread problem in captive animals fed on stored food and has been reported in various animals both domestic and wild. This report documents the clinicopathologic, microbial diagnostic findings and therapeutic regime for a study on the presentation, management, and outcome of aflatoxicosis in greater cane rats.

Materials and Methods: A total of 65 greater cane rats suspected to be exposed to the toxin were examined clinically along with their environment. Feed samples, recently deceased carcasses and some moribund carcasses were collected for the study. Carcasses were subjected to gross and histopathologic investigations while feed and organs were subjected to microbiological investigations.

Results: Gross lesions included hepatic lipidosis with ecchymotic hemorrhages, distended gallbladder, and renomegaly with ecchymosis among others. Histopathology revealed loss of hepatocellular architecture with massive centrilobular hepatocyte necrosis and diffuse steatotic damage characterized by macrovacuoles. Other histologic findings included pulmonary congestion, moderate renal tubular degeneration, and necrosis of epithelial tubular cells. Aspergillus flavus was isolated from the feed and ingesta. Total aflatoxin detected in feed sample was found to be over 400 ppm. Klebsiella species, Staphylococcus species, and Bacillus species were isolated from the liver and intestinal content. Management was attempted using Fungizal® (Avico, Jordan) (which contains Thymol, benzoic acid, sorbic acid, and kaolin) and Orego-Stim® (Saife, USA) (which contains carvacrol and thymol) which were instituted in feed and Superliv® (Ayurvet, India) (polyherbal) liquid was instituted in water for 5 days at manufacturers' dosage. All clinical signs disappeared, and no more deaths were recorded following management.

Conclusion: This report concludes that aflatoxicosis causes severe mortality in greater cane rats and can be prevented and managed successfully.

Keywords: aflatoxicosis, African greater cane rat, management, pathology, Thryonomys swinderianus.

Modifications and optimization of manual methods for polymerase chain reaction and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta

Research (Published online: 27-07-2018)

17. Modifications and optimization of manual methods for polymerase chain reaction and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta

Durgadevi Aphale and Aarohi Kulkarni

Veterinary World, 11(7): 990-1000

ABSTRACT

Background and Aim: A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen.

Materials and Methods: Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers of targeted rumen bacteria. Methods denoting the presence of targeted rumen bacteria were assessed further with 16S rRNA gene sequencing and identification studies. It led toward methods efficacy estimation for molecular biology applications. Effect of rumen sample preservation on community DNA extraction was also studied. Their mean standard deviation values were calculated to understand sampling criticality.

Results: Modified Chemical method (Cetrimonium bromide) and Enzymatic+Chemical+Physical (ECP) method (Lysozyme- Cetrimonium bromide-Sodium Dodecyl Sulfate-freeze-thaw) could extract 835 ng/μl and 161 ng/μl community DNA from 1.5 g solid and 2 ml squeezed rumen digesta with purity ratios of 1.8 (A260nm/A280nm) and 2.3 (A260nm/A230nm) respectively. Comparative analysis showed the better efficiency of ECP method and chemical method toward freshly squeezed rumen digesta and solid rumen digesta. However, sample preservation at -80°C for 1.5 months drastically affected the yield and purity ratios of community DNA. New protocol revealed targeted microbial community having Gram-positive as well as Gram-negative bacteria such as Prevotella ruminicola, Streptococcus lutetiensis, Ruminococcus flavefaciens, Fibrobacter succinogenes, and Selenomonas ruminantium.

Conclusion: To date, this is the first report of modified methods wherein least chemicals and steps lead toward PCR and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Detection of targeted rumen bacteria in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that in turn differs the performance of each different method employed and yield of nucleic acid obtained. It also leaves a possibility of the presence of complex microbial consortia in squeezed rumen digesta whose DNA extraction methods need more attention. Finally, manual protocols of community DNA extraction may vary in different ruminant which suggests undertaking rigorous research in their establishment.

Keywords: 16S rRNA gene sequencing, community DNA extraction, goat, polymerase chain reaction, rumen digesta.

An investigation on the predominant diseases, its diagnosis, and commonly used drugs in the poultry farms in the North-Eastern regions of Algeria

Research (Published online: 24-07-2018)

16. An investigation on the predominant diseases, its diagnosis, and commonly used drugs in the poultry farms in the North-Eastern regions of Algeria

Amine Berghiche, Tarek Khenenou, Ahmed Kouzi and Ibtissem Labiad

Veterinary World, 11(7): 986-989

ABSTRACT

Aim: An investigation was carried out to assess the occurrence of diseases, its method of diagnosis, and commonly used drugs in poultry farms in North-Eastern regions of Algeria.

Materials and Methods: A total of 265 veterinary doctors were surveyed to obtain information on the dominant diseases, its frequency of occurrence, method of diagnosis, and commonly used drugs in poultry farms.

Results: A study revealed that about 68% of bacterial diseases are due to colibacillosis, mycoplasmosis, and salmonellosis, 22% of viral diseases are due to Newcastle, Gumboro, and infectious bronchitis, and 10% others including coccidiosis and ascites syndrome. The study also showed that about 57% of cases were diagnosed by clinical signs, 36% by necropsy findings, and the remaining 7% through therapeutic and laboratory analysis. Antibiotics, a predominance of the anarchic veterinary drugs, were massively used to control the diseases. Hence, there is a need for strict regulations on the use of veterinary drugs to guarantee food safety.

Conclusion: These results remain non-exhaustive but contribute strongly to determine the status of health of the birds in the region.

Keywords: Algeria, diagnosis, disease, investigation, poultry.