DNA extraction from hydatid cyst protoscolices: Comparison of five different methods

Research (Published online: 22-02-2018)

23. DNA extraction from hydatid cyst protoscolices: Comparison of five different methods

Afshin Barazesh, Bahador Sarkari, Sepideh Ebrahimi and Mehdi Hami

Veterinary World, 11(2): 231-234

ABSTRACT

Aim: The current study aimed to find out a simple, practical and high throughput DNA isolation method for extraction of DNA from hydatid cyst samples.

Materials and Methods: Cattle and sheep isolate of hydatid cysts were obtained from the slaughterhouse, and hydatid fluid and protoscolices were collected in a sterile condition. Protoscolices were washed, 3 times with phosphate buffered saline, and DNA was extracted by different methods including manual extraction with freeze/thawing and phenol-chloroform, Triton X-100 extraction, and by a commercial kit (YTA, Yekta Tajhiz Azma, Iran) with three different modifications in the kit's manufacturer instructions. The obtained DNA from the different methods was evaluated by Nanodrop in terms of the yield of DNA and carbohydrates or protein contaminations. To compare the quality of the extracted DNA, two pieces of the mitochondrial genome of Echinococcus granulosus, cox1, and nad1, were polymerase chain reaction (PCR)-amplified, using each of the DNA prepared by different methods. Electrophoresis of PCR products was carried out on the agarose gel.

Results: The DNA extracted by manual method, using phenol/chloroform, had the highest yield, yet with the highest level of protein and carbohydrate contamination. The DNA extracted using two-step incubations, initially at 60°C for 2 h and then overnight at 37°C, was the most purified DNA with the lowest rate of contamination.

Conclusion: Findings of the study demonstrated that modification in the currently available commercially DNA extraction kit resulted in the development of a high throughput DNA isolation method. This method can be recommended for the extraction of DNA from hydatid cysts, especially the cattle isolate where the extraction of DNA in these samples are usually problematic.

Keywords: DNA extraction, hydatid cyst, protoscolices.

Interspecies comparison of probiotics isolated from different animals

Research (Published online: 21-02-2018)

22. Interspecies comparison of probiotics isolated from different animals

Amr M. Abdou, Riham H. Hedia, Shimaa T. Omara, Mohamed Abd El-Fatah Mahmoud, Mai M. Kandil, and M. A. Bakry

Veterinary World, 11(2): 227-230

ABSTRACT

Aim: The aim of the current study was to isolate and identify naturally occurring probiotic Lactobacillus species in different animals with the different environmental background including fish, and farm animals to investigate interspecies differences in probiotics on the species level.

Materials and Methods: A total of 44 fecal and milk samples were collected under aseptic conditions from cattle, buffalo, camel, sheep, goats, and fish. The samples were cultured, and the isolated strains were confirmed biochemically and molecularly using 16S rRNA multiplex polymerase chain reaction (PCR) analysis following DNA extraction from the bacterial isolates.

Results: A total of 31 isolates identified as lactobacilli were isolated from cattle milk, goat feces, sheep feces, fish feces, buffalo milk, camel milk, and goats' milk. Lactobacillus species were identified based on the size of the PCR product. The results showed that different species were different in their lactobacilli content. At the same time, there were some differences between individuals of the same species.

Conclusion: The diversity of probiotic strains isolated from different animal species implies different types of benefits to the host. Although it would be both money - and time-consuming research, discovering the benefit of each of these strains may provide very important information for the health of both human and animal. Furthermore, transferring these beneficial effects either to individuals within the same species or between different species would be of great importance.

Keywords: Lactobacillus, multiplex polymerase chain reaction, probiotics.

Intranasal administration of inactivated avian influenza virus of H5N1 subtype vaccine-induced systemic immune response in chicken and mice

Research (Published online: 20-02-2018)

21. Intranasal administration of inactivated avian influenza virus of H5N1 subtype vaccine-induced systemic immune response in chicken and mice

I N. Suartha, G. A. A. Suartini, I W. Wirata, N. M. A. R. K. Dewi, G. N. N. Putra, G. A. Y. Kencana and G. N. Mahardika

Veterinary World, 11(2): 221-226

ABSTRACT

Aim: The need for non-parenteral administration of inactivated avian influenza virus of H5N1 subtype (AIV-H5N1) vaccine is paramount. Here, we provide preliminary data on the immune response of chicken and mice after intranasal administration of AIV-H5N1-inactivated vaccine with ISCOMS, Inmunair (INM), and combined ISCOMS and INM as an adjuvant.

Materials and Methods: The AIV isolate of A/Chicken/Denpasar/01/2004 (H5N1) was cultivated in specific pathogen-free chicken eggs and inactivated with formaldehyde. The vaccine preparation was added with those adjuvants for intranasal administration and aluminum hydroxide for subcutaneous injection. The chicken and mouse were vaccinated at the age of 3 weeks or 1 month and repeated 2 weeks thereafter. In one experiment, chicken was injected with Newcastle disease virus (NDV) at the same time with AIV vaccine. The sera were collected at one (serum 1) and 2 w (serum 2) after booster vaccination. The anti-AIV-H5 and NDV antibodies in chicken sera were detected using hemagglutination inhibition (HI) assay. Mouse IgG anti-AIV-H5N1 antibody was detected using ELISA.

Results: The result shows that the geometric mean titers (GMTs) of chicken sera of intranasal vaccinated with inactivated AIV-H5N1 vaccine with mixed ISCOM- INM as adjuvant were <20.0 and 22.7 unit HI-unit (HIU) in serum 1 and serum 2, respectively. The GMTs of the positive control group were 23.7 and 25.7 HIU in serum 1 and serum 2, respectively. The result of the second experiment shows that IgG anti-AIV-H5N1 was detected in mouse sera. In the third experiment, the GMTs of anti-NDV in chicken vaccinated subsequently with inactivated NDV vaccine and AIV-H5N1 with mixed ISCOMS-INM administrated intranasally and aluminum hydroxide adjuvant administrated through subcutaneous injection as well as positive control group receiving NDV vaccine only were 28.0, 28.0, and 27.4 HIU in serum 1 while were 29.6, 29.2, and 28.2 HIU in serum 2, respectively.

Conclusion: Intranasal administration of inactivated AIV-H5N1 vaccine-induced a systemic immune response in chicken and mice after adding ISCOMS and/or INM as adjuvants. The adjuvant and the intranasal administration caused no immunosuppressive effect on the chicken immune response to NDV vaccine.

Keywords: inactivated vaccine, influenza-H5N1, ISCOMS, Inmunair, intranasal.

Comparative ovicidal activity of Moringa oleifera leaf extracts on Fasciola gigantica eggs

Research (Published online: 19-02-2018)

20. Comparative ovicidal activity of Moringa oleifera leaf extracts on Fasciola gigantica eggs

Ahmed G. Hegazi, Kadria N. Abdel Megeed, Soad E. Hassan, M. M. Abdelaziz, Nagwa I. Toaleb, Eman E. El Shanawany and Dina Aboelsoued

Veterinary World, 11(2): 215-220

ABSTRACT

Background: Fasciolosis is an important zoonotic disease affecting the productive performance of farm animals in Egypt.

Aim: The aim of the present study was comparing the ovicidal effect of different extracts as an alcoholic (Methanolic and Ethanolic) and aqueous Moringa oleifera leaf extracts on Fasciola gigantica non-embryonated and developed eggs.

Materials and Methods: Tested concentrations of extracts ranged from 12.5 to 800 mg/ml. Nitroxynil was used as reference drug with a dose of 100 mg/ml.

Results: M. oleifera alcoholic and aqueous extracts showed a concentration-dependent ovicidal effect on F. gigantica non-embryonated and developed eggs. Based on LC50 values, water extract showed the highest ovicidal activity since it registered the lowest values of 2.6 mg/ml on non-embryonated eggs. Non-embryonated eggs were more susceptible to aqueous extract than developed eggs. On the other hand, the developed eggs were more susceptible to ethanolic extract than non-embryonated eggs even the lowest LC50 (12.38 mg/ml).

Conclusion: M. oleifera leaf extracts especially aqueous extract could be a promising step in the field of controlling fascioliasis. Further, in vivo studies are needed to enlighten the therapeutic potential of M. oleiferaextracts in treating F. gigantica infection.

Keywords: Fasciola gigantica, leaf extract, Moringa oleifera, nitroxynil, ovicidal activity.

Immunomodulatory effects of probiotics and prilled fat supplementation on immune genes expression and lymphocyte proliferation of transition stage Karan Fries cows

Research (Published online: 18-02-2018)

19. Immunomodulatory effects of probiotics and prilled fat supplementation on immune genes expression and lymphocyte proliferation of transition stage Karan Fries cows

Meeti Punetha, A. K. Roy, H. M. Ajithakumar, Irshad Ahmed Para, Deepanshu Gupta, Mahendra Singh and Jaya Bharati

Veterinary World, 11(2): 209-214

ABSTRACT

Background and Aim: Probiotics are the living microorganism which when administered improves the digestion and health of the animal. Saccharomyces cerevisiae (SC) improves the humoral and innate immunity of the animal. Prilled fat is a hydrogenated palm oil triglyceride which has been reported to promote the release of cytokines from macrophages. The aim of the study was to evaluate the immunomodulatory effect of probiotic and prilled fat during transition stage in Karan Fries (KF) cows.

Materials and Methods: A total of 12 KF cows at 21 days prepartum were selected and divided into two groups of six animals each. The control group was fed as per the standard feeding practices and the supplemented group cows were supplemented daily with prilled fat at 100 g/cow, SC at 25 g/cow, and sweetener at 1 g/cow in addition to the standard feeding practices from -30 days of prepartum to 21 days of lactation. The sweetener was added to improve the palatability of the feed. The natural sweetener of an African plant leave had 105 times more sweetness than glucose with good aroma. The dry matter intake of the animal was recorded. Plasma samples were collected weekly from all cows for the analysis of blood metabolite beta-hydroxybutyric acid (BHBA). Lymphocytes were isolated from the blood for studying the expression of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) and for estimating lymphocyte proliferation index (LPI).

Results: The upregulated IL-1β and TNF-α around calving might be possibly associated to the metabolic changes occurring during the transition period and suggest a higher degree of inflammation around parturition. High concentrations of BHBA caused increased expression and synthesis of the pro-inflammatory factors such as TNF-α and IL-1β in supplemented group in primary calf hepatocytes. The LPI was higher in supplemented group as compared to control which suggests a stimulatory effect of unsaturated fatty acids on mitogen-stimulated T-cell proliferation.

Conclusion: Dietary supplementation of probiotics, prilled fat, and sweetener alleviated negative energy balance by stimulating feed intake and modulating hepatic lipid metabolism; and both of these additives improved the postpartum health (antioxidant status and immune function) of transition dairy cows.

Keywords: beta-hydroxybutyric acid, crossbred cows, dry matter intake, interleukin-1β, lymphocyte proliferation index, prilled fat, Saccharomyces cerevisiae, tumor necrosis factor alpha.

Comparison of four polymerase chain reaction assays for the detection of Brucella spp. in clinical samples from dogs

Research (Published online: 16-02-2018)

18. Comparison of four polymerase chain reaction assays for the detection of Brucella spp. in clinical samples from dogs

Eduardo J. Boeri, Maria M. Wanke, Maria J. Madariaga, Maria L. Teijeiro, Sebastian A. Elena and Marcos D. Trangoni

Veterinary World, 11(2): 201-208

ABSTRACT

Aim: This study aimed to compare the sensitivity (S), specificity (Sp), and positive likelihood ratios (LR+) of four polymerase chain reaction (PCR) assays for the detection of Brucella spp. in dog's clinical samples.

Materials and Methods: A total of 595 samples of whole blood, urine, and genital fluids were evaluated between October 2014 and November 2016. To compare PCR assays, the gold standard was defined using a combination of different serological and microbiological test. Bacterial isolation from urine and blood cultures was carried out. Serological methods such as rapid slide agglutination test, indirect enzyme-linked immunosorbent assay, agar gel immunodiffusion test, and buffered plate antigen test were performed. Four genes were evaluated: (i) The gene coding for the BCSP31 protein, (ii) the ribosomal gene coding for the 16S-23S intergenic spacer region, (iii) the gene coding for porins omp2a/omp2b, and (iv) the gene coding for the insertion sequence IS711.

Results: The results obtained were as follows: (1) For the primers that amplify the gene coding for the BCSP31 protein: S: 45.64% (confidence interval [CI] 39.81-51.46), Sp: 95.62% (CI 93.13-98.12), and LR+: 10.43 (CI 6.04-18); (2) for the primers that amplify the ribosomal gene of the 16S-23S rDNA intergenic spacer region: S: 69.80% (CI 64.42-75.18), Sp: 95.62 % (CI 93.13-98.12), and LR+: 11.52 (CI 7.31-18.13); (3) for the primers that amplify the omp2a and omp2b genes: S: 39.26% (CI 33.55-44.97), Sp: 97.31% (CI 95.30-99.32), and LR+ 14.58 (CI 7.25-29.29); and (4) for the primers that amplify the insertion sequence IS711: S: 22.82% (CI 17.89 - 27.75), Sp: 99.66% (CI 98.84-100), and LR+ 67.77 (CI 9.47-484.89).

Conclusion: We concluded that the gene coding for the 16S-23S rDNA intergenic spacer region was the one that best detected Brucella spp. in canine clinical samples.

Keywords: Brucella, Brucella canis, canine brucellosis, clinical samples, comparison, molecular, polymerase chain reaction.

Antimicrobial peptides of buffalo and their role in host defenses

Review (Published online: 15-02-2018)

17. Antimicrobial peptides of buffalo and their role in host defenses

Khangembam Victoria Chanu, Dimpal Thakuria and Satish Kumar

Veterinary World, 11(2): 192-200

ABSTRACT

Antimicrobial peptides (AMPs) are highly conserved components of the innate immune system found among all classes of life. Buffalo (Bubalus bubalis), an important livestock for milk and meat production, is known to have a better resistance to many diseases as compared to cattle. They are found to express many AMPs such as defensins, cathelicidins, and hepcidin which play an important role in neutralizing the invading pathogens. Buffalo AMPs exhibit broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Similar to its natural form, synthetic analogs of buffalo AMPs are also antimicrobial against bacteria and even fungus making them a good target for the development of therapeutic antimicrobials. In addition to its antimicrobial effect, AMPs have been demonstrated to have a number of immunomodulatory functions, and their genes are responsive to infections. Further, induction of their gene expression by external factors may help in preventing infectious diseases. This review briefly discusses the AMPs of buffalo identified to date and their possible role in innate immunity.

Keywords: antimicrobial peptides, Bubalus bubalis, cathelicidins, defensin, hepcidin.

Identification and antibiogram pattern of Bacillus cereus from the milk and milk products in and around Jammu region

Research (Published online: 14-02-2018)

16. Identification and antibiogram pattern of Bacillus cereus from the milk and milk products in and around Jammu region

Umar Yusuf, S. K. Kotwal, Sanjolly Gupta and Touqeer Ahmed

Veterinary World, 11(2): 186-191

ABSTRACT

Aim: The aims of the present study were to assess the prevalence, identification, and antibiogram pattern of Bacillus cereus from 215 samples of different milk and milk products in and around Jammu region.

Materials and Methods: In the present study, 215 samples of milk, rasgulla, burfi, rasmalai, kalaari, paneer, ice cream, and pastry were collected and analyzed for the isolation of the B. cereus using PEMBA, and antibiogram pattern was observed for all the milk and milk products.

Results: B. cereus was detected in 61/215 samples with an overall prevalence of 28.37%. Biotyping revealed predominantly 5, 7, and 2 biotypes in raw milk. Burfi and ice cream revealed 2, 3, 5, and 7 biotypes. Rasgulla had 2, 3, and 5 biotypes; paneer and rasmalai had biotypes 2 and 5, while kalaari revealed biotype 5. Antibiogram pattern revealed that isolates were highly sensitive to gentamicin (100%), intermediate to ampicillin (40.98%), tetracycline (31.14%), erythromycin (29.50%), and amoxicillin (26.22%), and high resistance against penicillin G (100%). Adulteration of starch was detected in 16.66 % raw milk samples. All starch positive samples were positive for B. cereus. However, 12 starch negative samples also yielded B. cereus.

Conclusion: From this study, it was concluded that highest prevalence of B. cereus was found in ice cream. Several isolates of B. cereus showed toxigenic activity, so the presence of B. cereus in milk and milk products may be of public health hazard. The antibiogram pattern of B. cereus isolates showed sensitivity to gentamicin, ciprofloxacin, chloramphenicol, streptomycin, and resistance to penicillin-G and cephalexin. The presence of B. cereus in milk and milk products showed a strong association besides establishing the fact that starch adulteration can be indicative of the presence of B. cereus.

Keywords: Antibiogram, Bacillus cereus, milk, milk products, prevalence.

End-threaded intramedullary positive profile screw ended self-tapping pin (Admit pin) - A cost-effective novel implant for fixing canine long bone fractures

Research (Published online: 13-02-2018)

15. End-threaded intramedullary positive profile screw ended self-tapping pin (Admit pin) - A cost-effective novel implant for fixing canine long bone fractures

Mitin Chanana, Adarsh Kumar, Som Prakash Tyagi, Amit Kumar Singla, Arvind Sharma and Uiase Bin Farooq

Veterinary World, 11(2): 181-185

ABSTRACT

Aim: The current study was undertaken to evaluate the clinical efficacy of end-threaded intramedullary pinning for management of various long bone fractures in canines.

Materials and Methods: This study was conducted in two phases, managing 25 client-owned dogs presented with different fractures. The technique of application of end-threaded intramedullary pinning in long bone fractures was initially standardized in 6 clinical patients presented with long bone fractures. In this phase, end-threaded pins of different profiles, i.e., positive and negative, were used as the internal fixation technique. On the basis of results obtained from standardization phase, 19 client-owned dogs clinically presented with different fractures were implanted with end-threaded intramedullary positive profile screw ended self-tapping pin in the clinical application phase.

Results: The patients, allocated randomly in two groups, when evaluated postoperatively revealed slight pin migration in Group-I (negative profile), which resulted in disruption of callus site causing delayed union in one case and large callus formation in other two cases whereas no pin migration was observed in Group-II (positive profile). Other observations in Group-I was reduced muscle girth and delayed healing time as compared to Group-II. In clinical application, phase 21st and 42nd day post-operative radiographic follow-up revealed no pin migration in any of the cases, and there was no bone shortening or fragment collapse in end-threaded intramedullary positive profile screw ended self-tapping pin.

Conclusion: The end-threaded intramedullary positive profile screw ended self-tapping pin used for fixation of long bone fractures in canines can resist pin migration, pin breakage, and all loads acting on the bone, i.e., compression, tension, bending, rotation, and shearing to an extent with no post-operative complications.

Keywords: Admit pin, canine, end-threaded, fracture, intramedullary, orthopedics, pinning, positive profile.

The effect of moderate exercise on the elevation of Bax/Bcl-2 ratio in oral squamous epithelial cells induced by benzopyrene

Research (Published online: 13-02-2018)

14. The effect of moderate exercise on the elevation of Bax/Bcl-2 ratio in oral squamous epithelial cells induced by benzopyrene

Anis Irmawati, Nadira Jasmin and Sidarningsih

Veterinary World, 11(2): 177-180

ABSTRACT

Aim: The aim of this study was to analyze the effect of moderate exercise on the elevation of Bax/Bcl-2 ratio.

Materials and Methods: Eighteen Mus musculus strain Swiss Webster (Balb/c) were divided into three groups (n=6). K1 and K2 had contact with water 3 times/week for 12 weeks, while the members of the K3 group swam 3 times/week for 12 weeks while carrying load weighed 3% of their body weight. After 5 weeks, they were induced with 0.04 ml oleum olivarum (K1), 0.08 mg benzopyrene/0.04 ml oleum olivarum (K2, K3) 3 times/week for 4 weeks. Immunohistochemistry assays were used to determine the ratio of Bax/Bcl-2 expression. The results were analyzed using an independent t-test.

Results: The Bax/Bcl-2 ratio increased significantly in K3 compared to K2 (p=0.00).

Conclusion: Moderate exercise could increase the Bax/Bcl-2 ratio in oral squamous epithelial cells induced by benzopyrene.

Keywords: Bax/Bcl-2 ratio, benzopyrene, moderate exercise, oral squamous carcinoma.