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.

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.