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.

Enhanced pathogenicity of low-pathogenic H9N2 avian influenza virus after vaccination with infectious bronchitis live attenuated vaccine

Research (Published online: 24-07-2018)

15. Enhanced pathogenicity of low-pathogenic H9N2 avian influenza virus after vaccination with infectious bronchitis live attenuated vaccine

Zainab Mohamed Ismail, Ayman Hanea EL-Deeb, Mounir Mohamed EL-Safty and Hussein Aly Hussein

Veterinary World, 11(7): 977-985

ABSTRACT

Aim: In the present study, two experiments were carried out for studying the pathogenicity of H9N2 avian influenza virus (AIV) in broiler chickens after vaccination with different live respiratory viral vaccines.

Materials and Methods: One-day-old specific pathogen-free (SPF) chicks were divided into four groups in each experiment. In experiment 1, Groups 1 and 2 were inoculated with H9N2 AIV through nasal route in 1 day old, Groups 1 and 3 were vaccinated with live infectious bronchitis coronavirus (IBV) vaccine in 5 days old, and Group 4 was left as a negative control. In experiment 2, Groups 5 and 6 were inoculated with AIV subtype H9N2 through nasal route in 1 day old, Group 5 was vaccinated with live IBV vaccine and live Newcastle disease virus (NDV) vaccine in 5 and 18 days old, respectively, Groups 6 and 7 were vaccinated with live NDV vaccine in 18 days old, and Group 8 was left as a negative control. Chicks were kept in isolators for 18 days in the first experiment and 35 days in the second experiment. Tracheal and cloacal swabs were collected from 3, 5, 7, 10, 12, and 15 day's old chicks from all groups in experiment 1 and 21, 23, 25, and 28 days old from all groups in experiment 2. Quantitative real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) was applied on the collected tracheal swabs for detecting RNA copies of H9N2 AIV. Cloacal swabs and the positive rRT-PCR tracheal swabs were inoculated in 10-day-old SPF embryonated chicken eggs (ECE) to confirm rRT-PCR results. Internal organs (kidney, trachea, and spleen) from all chicken groups were collected weekly for histopathological examination to determine severity of the lesions. Serum samples were collected on a weekly basis for the detection of humoral immune response against H9N2, NDV, and IBV from all chicken groups.

Results: rRT-PCR results with virus titration in ECEs revealed a significant increase in H9N2 AIV titer with extension in the period of viral shedding in Groups 1 and 5. Severe lesion score was observed for Groups 1 and 5. The Humoral immune response against H9N2 AIV, NDV, and IBV revealed a significant increase in H9N2 AIV titer in Groups 1 and 5, NDV titer showed a significant increase in Group 7, and IBV titer increased in Groups 1, 3, and 5.

Conclusion: Results demonstrated the increase in pathogenicity of H9N2 AIV, especially when H9N2-infected chicks vaccinated with live IBV vaccine.

Keywords: coinfection, infectious bronchitis virus, low pathogenic H9N2.

Concurrent occurrence of acute bovine pulmonary edema and emphysema and endocardial fibroelastosis in cattle: A case history and literature review

Research (Published online: 21-07-2018)

14. Concurrent occurrence of acute bovine pulmonary edema and emphysema and endocardial fibroelastosis in cattle: A case history and literature review

W. M. Hananeh and Z. B. Ismail

Veterinary World, 11(7): 971-976

ABSTRACT

Aim: The aim of this study was to describe the clinical and pathological findings of acute bovine pulmonary edema and emphysema (ABPEE) and left endocardial fibroelastosis in an adult dairy cow. In addition, a review of recent literature of these two conditions is provided.

Materials and Methods: Necropsy and histopathological examination were performed using conventional techniques. A review of the literature was carried out using internet search engines such as PubMed and Google Scholar. Only published papers in scientific and refereed journals were reviewed.

Results: Concurrent pathologies of ABPEE and left endocardial fibroelastosis were described in an adult Holstein Friesian cow. A review of recent literature concerning ABPEE and endocardial fibroelastosis revealed seven and two scientific reports of these conditions in cattle, respectively.

Conclusion: Although rare, combined pathologies involving multiple organs such as the lungs and heart can be diagnosed in animals on careful clinical and histopathological examinations.

Keywords: bovine, cardiac anomaly, edema, emphysema, lungs.

Hypoxic preconditioning effect on stromal cells derived factor-1 and C-X-C chemokine receptor type 4 expression in Wistar rat's (Rattus norvegicus) bone marrow mesenchymal stem cells (in vitro study)

Research (Published online: 19-07-2018)

13. Hypoxic preconditioning effect on stromal cells derived factor-1 and C-X-C chemokine receptor type 4 expression in Wistar rat's (Rattus norvegicus) bone marrow mesenchymal stem cells (in vitro study)

Sri Wigati Mardi Mulyani, Diah Savitri Ernawati, Eha Renwi Astuti and Fedik Abdul Rantam

Veterinary World, 11(7): 965-970

ABSTRACT

Aim: To examine the effect of hypoxic preconditions on the ability of bone marrow stem cells culture mediated expression C-X-C chemokine receptor type 4 (CXCR4) and stromal cells derived factor-1 (SDF-1) in vitro.

Materials and Methods: Bone marrow mesenchymal stem cells (BMSCs) were derived from 12 femurs of 200 g Wistar male rats. The animals were euthanized before BMSCs isolation. BMSCs were divided into two groups, control group: Normoxic condition 21% O2 and treatment group: Hypoxic condition 1% O2. The characterization of BMSCs was analyzed using flow cytometry by cluster differentiation 34 and cluster differentiation 105. The expression of CXCR4 and SDF-1 measured using immunocytochemistry immunofluorescence label after 48-h incubation in a low-tension oxygen chamber with an internal atmosphere consisting of 95% N2, 5% CO2, and 1% O2. All data were subjected to a normality test and then analyzed using t-test statistic (p<0.05).

Results: The characterization of bone marrow stem cells showed positive cluster differentiation 34 and cluster differentiation 105. A hypoxic precondition (1% O2) in culture increases CXCR4 (p=0.000) and SDF-1 expression than normoxic conditions (p=0.000) (p<0.05).

Conclusion: Hypoxic preconditioning with 1% O2 increase CXCR4 and SDF1 expression.

Keywords: bone marrow stem cells, C-X-C chemokine receptor type 4, hypoxic preconditioning, mesenchymal stem cells, stromal cells derived factor-1.

Effect of dietary supplementation of cinnamon oil and sodium butyrate on carcass characteristics and meat quality of broiler chicken

Research (Published online: 18-07-2018)

12. Effect of dietary supplementation of cinnamon oil and sodium butyrate on carcass characteristics and meat quality of broiler chicken

Govindarajan Gomathi, Subramaniam Senthilkumar, Amirthalingam Natarajan, Ramasamy Amutha and Manika Ragavan Purushothaman

Veterinary World, 11(7): 959-964

ABSTRACT

Aim: An in vivo experiment was conducted to investigate the effect of supplementation of cinnamon oil (CO) and sodium butyrate on carcass characteristics and meat quality of broiler chicken compared with the antibiotic supplementation.

Materials and Methods: A biological experiment was carried out with 216-day-old Vencobb-400 broiler chicks randomly distributed to six experimental treatments with six replicates, each replicate containing six chicks with equal numbers of male and female chicks. The experimental diets were prepared with isocaloric and isonitrogenous basis. The experimental groups, namely control (T1), control with antibiotic (T2), control with CO at 250 mg/kg and coated sodium butyrate (CSB) either at 0.09 (T3) or 0.18% (T4), and control with CO at 500 mg/kg and CSB either at 0.09 (T5) or 0.18% (T6). The trial was carried out in deep litter pen for 35 days. The carcass characteristics such as ready to cooked yield, eviscerated weight, heart, liver, gizzard, giblet, and abdominal fat percent in slaughtered birds and meat quality properties such as pH, water-holding capacity (WHC), tyrosine, shear force, cooking loss, thiobarbituric acid, sensory characteristics, and muscle cholesterol in breast muscle samples were evaluated.

Results: The carcass characteristics such as ready-to-cook yield, eviscerated weight, and weight of heart, liver, gizzard, giblet, and abdominal fat as a percent of live body weight were not influenced by supplementation of CO and CSB at the levels attempted or by antibiotic supplementation in broilers. The pH, cooking loss, shear force and WHC of meat, appearance, flavor, texture, mouth coating, juiciness and overall acceptability of meat were not influenced by the supplementation of different levels of CO and CSB or by antibiotic supplementation but decreased meat cholesterol level in broilers.

Conclusion: The results indicated that the supplementation of CO and CSB in broiler diet did not alter the carcass characteristics and meat quality parameters except meat cholesterol content in broilers.

Keywords: antibiotic, broilers, carcass characteristics, cinnamon oil, coated sodium butyrate, meat quality.

Efficacy of acetamiprid and fipronil fly baits against the housefly (Musca domestica L.) under laboratory conditions

Research (Published online: 16-07-2018)

11. Efficacy of acetamiprid and fipronil fly baits against the housefly (Musca domestica L.) under laboratory conditions

Mikhail Alekseevich Levchenko, Elena Anatol'evna Silivanova, Ruzilya Khusanovna Bikinyaeva and Galina Fedorovna Balabanova

Veterinary World, 11(7): 953-958

ABSTRACT

Background: The housefly Musca domestica L. (Diptera: Muscidae) is permanent pests in livestock facilities. High fly density in livestock and poultry farms can increase the risks of economic loss and public health. Treatment with toxic baits is one of the methods for housefly control. However, development of resistance to insecticides makes it difficult to manage of flies. Anti-resistance strategies include the use of multiple pesticides with different modes of action.

Aim: This study was conducted to estimate the efficacy of neonicotinoid acetamiprid and phenylpyrazole fipronil, applied alone or in the mixture, against adults of M. domestica and to evaluate the efficacy of fly bait formulations containing acetamiprid and fipronil under laboratory conditions.

Materials and Methods: The adult flies, M. domestica of laboratory strain, were used in laboratory bioassays. The efficacy of acetamiprid and fipronil as technical substances, when applied alone and in the mixture, against adult flies was tested by no-choice feeding bioassays. The insecticidal efficacy of bait formulations (wet powder) with acetamiprid or fipronil or their mixture was tested against flies by choice feeding bioassays. The probit analysis was used to calculate lethal concentrations of insecticides, and the χ2 test was used to analyze the interaction between fipronil and acetamiprid in the mixture.

Results: Fipronil was more toxic to adults of M. domestica than acetamiprid in laboratory tests. Lethal concentrations for 50% mortality (95% confidence interval) of flies were 0.0159% (0.0124-0.0205) of acetamiprid and 0.000119% (0.000039- 0.0002640) of fipronil. The mixture containing fipronil at concentration 0.005% and acetamiprid at concentration 0.05% had the additive effect on fly mortality.

Conclusion: The results of laboratory feeding bioassays indicate that the mixture of fipronil and acetamiprid might have a potential to use in toxic bait formulations against houseflies.

Keywords: fly bait, housefly management, insecticide interaction, insecticide mixture.