Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus

Research (Published online: 10-04-2018)

7. Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus

I. Karthika Lakshmi, Kalyani Putty, Satya Samparna Raut, Sunil R. Patil, P. P. Rao, B. Bhagyalakshmi, Y. Krishna Jyothi, B. Susmitha, Y. Vishnuvardhan Reddy, Sowmya Kasulanati, J. Shiva Jyothi and Y. N. Reddy

Veterinary World, 11(4): 452-458

ABSTRACT

Aim: The present study was designed to standardize real-time polymerase chain reaction (PCR) for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India.

Materials and Methods: A 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV) NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 105 ml and RNA was isolated by the Trizol method. Both reverse transcription -PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD). The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect) and molecular confirmation (by BTV-NS1 group-specific PCR). The standardized technique was then applied to field samples (blood) for detecting BTV.

Results: The slope of the standard curve obtained was -3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269Ex103 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 103 TCID 50/ml and 104 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 102 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative.

Conclusion: Real-time PCR was found to be a very sensitive as well as reliable method to detect BTV present in different types of samples, including blood samples collected from BTV-infected sheep, compared to RT-PCR. The LoD of BTV is likely influenced by sample type, possibly by the interference by the other components present in the sample.

Keywords: bluetongue virus, limit of detection, real-time polymerase chain reaction.

Effects of lipid extraction on nutritive composition of winged bean (Psophocarpus tetragonolobus), rubber seed (Hevea brasiliensis), and tropical almond (Terminalia catappa)

Research (Published online: 10-04-2018)

6. Effects of lipid extraction on nutritive composition of winged bean (Psophocarpus tetragonolobus), rubber seed (Hevea brasiliensis), and tropical almond (Terminalia catappa)

Anuraga Jayanegara, Rakhmad P. Harahap, Richard F. Rozi and Nahrowi

Veterinary World, 11(4): 446-451

ABSTRACT

Aim: This experiment aimed to evaluate the nutritive composition and in vitro rumen fermentability and digestibility of intact and lipid-extracted winged bean, rubber seed, and tropical almond.

Materials and Methods: Soybean, winged bean, rubber seed, and tropical almond were subjected to lipid extraction and chemical composition determination. Lipid extraction was performed through solvent extraction by Soxhlet procedure. Non-extracted and extracted samples of these materials were evaluated for in vitro rumen fermentation and digestibility assay using rumen: Buffer mixture. Parameters measured were gas production kinetics, total volatile fatty acid (VFA) concentration, ammonia, in vitro dry matter (IVDMD) and in vitro organic matter digestibility (IVOMD). Data were analyzed by analysis of variance and Duncan's multiple range test.

Results: Soybean, winged bean, rubber seed, and tropical almond contained high amounts of ether extract, i.e., above 20% DM. Crude protein contents of soybean, winged bean, rubber seed, and tropical almond increased by 17.7, 4.7, 55.2, and 126.5% after lipid extraction, respectively. In vitro gas production of intact winged bean was the highest among other materials at various time point intervals (p<0.05), followed by soybean > rubber seed > tropical almond. Extraction of lipid increased in vitro gas production, total VFA concentration, IVDMD, and IVOMD of soybean, winged bean, rubber seed, and tropical almond (p<0.05). After lipid extraction, all feed materials had similar IVDMD and IVOMD values.

Conclusion: Lipid extraction improved the nutritional quality of winged bean, rubber seed, and tropical almond.

Keywords: Hevea brasiliensis, in vitro rumen, lipid extraction, Psophocarpus tetragonolobus, Terminalia catappa.

Validation of immunomodulatory effects of lipopolysaccharide through expression profiling of Th1 and Th2 biased genes in Newcastle disease virus vaccinated indigenous chicken

Research (Published online: 09-04-2018)

5. Validation of immunomodulatory effects of lipopolysaccharide through expression profiling of Th1 and Th2 biased genes in Newcastle disease virus vaccinated indigenous chicken

Rabia Bhardwaj, Ramneek Verma, Dipak Deka, P. P. Dubey, J. S. Arora, R. S. Sethi, T. C. Tolenkhomba and C. S. Mukhopadhyay

Veterinary World, 11(4): 437-445

ABSTRACT

Background and Aim: Newcastle disease (ND) is considered one of the most important poultry diseases with chicken morbidity and mortality rates up to 100%. Current vaccination programs allow the use of live attenuated vaccines in the field to protect against the disease, which alone is inefficient and requires repeat booster doses. Toll-like receptor agonists (e.g., lipopolysaccharide [LPS]) as adjuvants are the ones, most extensively studied and have shown to be very promising in delivering a robust balanced immune response. In the present study, we have evaluated the potential of LPS to elicit a strong immune response with respect to the elicitation of both Th1 (cell-mediated) and Th2 (humoral) immune arms.

Materials and Methods: A total of 72 apparently healthy 1-day-old indigenous unvaccinated chicks were randomly divided into six experimental Groups A to F (n=12). At 8-week of age chicks in Group A, C, and E were vaccinated with live attenuated La Sota strain ND vaccine along with LPS, bovine serum albumin, and normal saline solution, respectively, and those in Group B, D, and E were kept separately without vaccination. Sampling was done on days 0, 1, 3, 7, 14, 21, 35, and 60 after vaccination. After vaccination and respective adjuvant application, Th1 and Th2 cytokine expression were measured in mRNA of both blood and tissue samples.

Results: The results were validated by, hemagglutination inhibition and enzyme-linked immunosorbent assay tests, to check for the humoral as well as cell-mediated immune response in blood serum levels. The results showed an increase in mRNA expression of the Th1 biased cytokines in Group A (LPS+NDV) as compared to the control groups. Similar mRNA expression pattern was seen in blood as well as tissue samples. Validation of results also indicates an increase in Cell-mediated Immunity as well as a humoral immune response in Group A (LPS+NDV).

Conclusion: The results of the study provided enough evidence to consider LPS as a potential vaccine adjuvants candidate against ND in chicken.

Keywords: adjuvants, Aseel, lipopolysaccharide, Newcastle disease, vaccine.

Molecular detection of serotype groups of Listeria monocytogenes isolated from gallbladder of cattle and sheep in Iraq

Research (Published online: 07-04-2018)

4. Molecular detection of serotype groups of Listeria monocytogenes isolated from gallbladder of cattle and sheep in Iraq

Hamza Jawad Al-Ali, Mohsen Abd Al-Rodhan, Samer Abdulsahib Al-Hilali, Alaa Hani Al-Charrakh, Ali Muhsin Al-Mohana and Zainab Jaber Hadi

Veterinary World, 11(4): 431-436

ABSTRACT

Aim: This study was designed to investigate the occurrence of serotypes of Listeria monocytogenes, an important food-borne pathogen, in gallbladder samples from cattle and sheep.

Materials and Methods: Three hundred samples were collected and screened for the presence of L. monocytogenes. The identification of the isolates was confirmed by API-Listeria system and by the presence of hemolysin (hyl) gene. The isolates were subjected to polymerase chain reaction-based serotype identification with d1 (division 1), d2 (division 2), glt, mama (mismatch amplification mutation assay), and flaA (flagellin protein) genes.

Results: A total of 8 (2.7%) L. monocytogenes were recovered from 6 (4.0%) samples of sheep and 2 (1.3%) samples of cattle. All isolates showed positive results with Hly primers. Four isolates carried d1 gene, did not possess glt gene and harbored mama gene. The serotypes of these isolates were identified as 4a or 4c. The other 4 isolates carried d2 gene, 3 of them were positive with the FlaA primers, and hence, determined to be a 1/2a or 3a serotype, and 1 isolate was determined to be 1/2c or 3c serotype.

Conclusion: This study concluded that the presence of 1/2a serotype in gallbladder samples indicates public health risk through cross-contamination of meat at slaughterhouses.

Keywords: cattle, gallbladder, Listeria monocytogenes, molecular detection, sheep.

Molecular typing of Staphylococcus aureus based on coagulase gene

Research (Published online: 06-04-2018)

3. Molecular typing of Staphylococcus aureus based on coagulase gene

Faizan Javid, Anil Taku, Mohd Altaf Bhat, Gulzar Ahmad Badroo, Mir Mudasir and Tanveer Ahmad Sofi

Veterinary World, 11(4): 423-430

ABSTRACT

Aim: This study was conducted to study the coagulase gene-based genetic diversity of Staphylococcus aureus, isolated from different samples of cattle using restriction fragment length polymorphism (RFLP) and their sequence-based phylogenetic analysis.

Materials and Methods: A total of 192 different samples from mastitic milk, nasal cavity, and pus from skin wounds of cattle from Military Dairy Farm, Jammu, India, were screened for the presence of S. aureus. The presumptive isolates were confirmed by nuc gene-based polymerase chain reaction (PCR). The confirmed S. aureus isolates were subjected to coagulase (coa) gene PCR. Different coa genotypes observed were subjected to RFLP using restriction enzymes Hae111 and Alu1, to obtain the different restriction patterns. One isolate from each restriction pattern was sequenced. These sequences were aligned for maximum homology using the Bioedit software and similarity in the sequences was inferred with the help of sequence identity matrix.

Results: Of 192 different samples, 39 (20.31%) isolates of S. aureus were confirmed by targeting nuc gene using PCR. Of 39 S. aureus isolates, 25 (64.10%) isolates carried coa gene. Four different genotypes of coa gene, i.e., 514 bp, 595 bp, 757 bp, and 802 bp were obtained. Two coa genotypes, 595 bp (15 isolates) and 802 bp (4 isolates), were observed in mastitic milk. 514 bp (2 isolates) and 757 bp (4 isolates) coa genotypes were observed from nasal cavity and pus from skin wounds, respectively. On RFLP using both restriction enzymes, four different restriction patterns P1, P2, P3, and P4 were observed. On sequencing, four different sequences having unique restriction patterns were obtained. The most identical sequences with the value of 0.810 were found between isolate S. aureus 514 (nasal cavity) and S. aureus 595 (mastitic milk), and thus, they are most closely related. While as the most distant sequences with the value of 0.483 were found between S. aureus 514 and S. aureus 802 isolates.

Conclusion: The study, being localized to only one farm, yielded different RFLP patterns as observed from different sampling sites, which indicates that different S. aureus coagulase types have a site-specific predilection. Two coa patterns were observed in mastitic milk indicating multiple origins of infection, with 595 bp coa genotype being predominant in mastitic milk. The coa genotypes and their restriction patterns observed in the present study are novel, not published earlier. 514 and 595 coa variants of S. aureus are genetically most related.

Keywords: coagulase, restriction fragment length polymorphism, sequence-based phylogenetic analysis, Staphylococcus aureus.

Comparative occurrence of diabetes in canine, feline, and few wild animals and their association with pancreatic diseases and ketoacidosis with therapeutic approach

Review (Published online: 05-04-2018)

2. Comparative occurrence of diabetes in canine, feline, and few wild animals and their association with pancreatic diseases and ketoacidosis with therapeutic approach

Kamal Niaz, Faheem Maqbool, Fazlullah Khan, Fatima Ismail Hassan, Saeideh Momtaz and Mohammad Abdollahi

Veterinary World, 11(4): 410-422

ABSTRACT

Diabetes mellitus (DM) is a chronic metabolic disorder in which blood glucose level raises that can result in severe complications. However, the incidence increased mostly by obesity, pregnancy, persistent corpus luteum, and diestrus phase in humans and animals. This review has focused on addressing the possible understanding and pathogenesis of spontaneous DM in canine, feline, and few wild animals. Furthermore, pancreatic associated disorders, diabetic ketoacidosis, hormonal and drug interaction with diabetes, and herbal remedies associated with DM are elucidated. Bibliographic search for the present review was done using PubMed, Scopus, and Google Scholar for articles on concurrent DM in small and wild animals. Persistent corpus luteal and pseudopregnancy in female dogs generate gestational DM (GDM). GDM can also be caused by extensive use of drugs/hormones such as glucocorticosteroids. Although many similarities are present between diabetic cats and diabetic humans which present islet amyloidosis, there was a progressive loss of β- and α-cells and the normal number of δ-cells. The most prominent similarity is the occurrence of islet amyloidosis in all cases of diabetic cat and over 90% of human non-insulin dependent DM Type-2. Acute pancreatic necrosis (APN) occurs due to predisposing factors such as insulin antagonism, insulin resistance, alteration in glucose tolerance, obesity, hyperadrenocorticism, and persistent usage of glucocorticoids, as these play a vital role in the progression of APN. To manage such conditions, it is important to deal with the etiological agent, risk factors, diagnosis of diabetes, and hormonal and drug interaction along with its termination with suitable therapy (herbal) protocols. It should be noted that the protocols used for the diagnosis and treatment of human DM are not appropriate for animals. Further investigations regarding diabetic conditions of pets and wild animals are required, which will benefit the health status of all animals health worldwide.

Keywords: amyloidosis, diabetes mellitus, gestational diabetes mellitus, hypercortisolism, necrosis, pancreatitis.

Immunohistochemical, histopathological study and chemoprotective effect of Solanum nigrum in N-nitrosodiethylamine-induced hepatocellular carcinoma in Wistar rats

Research (Published online: 04-04-2018)

1. Immunohistochemical, histopathological study and chemoprotective effect of Solanum nigrum in N-nitrosodiethylamine-induced hepatocellular carcinoma in Wistar rats

G. M. Akshatha, S. K. Raval, G. M. Arpitha, S. H. Raval and D. J. Ghodasara

Veterinary World, 11(4): 402-409

ABSTRACT

Background and Aim: Cancer is a devastating disease with a severe impact on the physical and psychological well-being of patients. Hepatocellular carcinoma (HCC) has been reported in various species of animals including dogs, cats, sheep, and pigs. The present study aimed to study the immunohistochemical and histopathological changes and chemoprotective effect of aqueous and alcoholic extracts of Solanum nigrum on N-nitrosodiethylamine (NDEA)-induced HCC rat model.

Materials and Methods: Eighty-two male Wistar rats of 15 weeks of age weighing 200-250 g were selected for the experiment. They were randomly divided into ten groups. Group I served as normal control consisted of healthy rats. HCC was induced in Group II, IV, V, VI, VII, and X rats using NDEA as inducing agent followed by phenobarbitone as a promoter for 16 weeks. Group II rats were kept untreated as HCC control. Group III rats were kept as vehicle control (0.05% Sodium bicarbonate). Group IV and V rats were treated with aqueous extract of S. nigrum at 200 mg/kg and 400 mg/kg, respectively, and Group VI and VII rats were treated with an alcoholic extract of S. nigrum at 200 mg/kg and 400 mg/kg, respectively, daily orally for 28 days. Group X rats were treated with sorafenib as reference drug at a dose of 11.4 mg/kg daily orally for 28 days. Group VIII and IX rats were kept as aqueous and alcoholic extract control for studying the effect of the same on normal rats. Liver samples were collected to study the gross and histopathological lesions and the activity of cleaved caspase-3 and chemopreventive effect of aqueous and alcoholic extracts of S. nigrum on HCC.

Results: The liver sections of rats from HCC control (Group II) showed loss of lobular architecture, necrosis, fatty change, enlarged and darkened nuclei with variable size, dilatation of hepatic sinusoids with Kupffer cell hyperplasia, dilatation and proliferation of bile duct, and intranuclear vacuoles and also showed the presence of more than one nucleolus. Administration of alcoholic extract of S. nigrum and sorafenib to NDEA/phenobarbital-treated rats reduced the severity of lesions in the liver. Immunohistochemical analysis of liver sections for caspase-3-positive cells of hepatic cancer-induced group showed immunoreactivity to rarely few. The immunoreactivity of the hepatocytes treated with a higher dose of alcoholic extract of S. nigrum was limited and was comparable to a standard drug, sorafenib.

Conclusion: Oral administration of aqueous and alcoholic extracts of S. nigrum for 28 days showed significant rejuvenation in the structure of the liver in the histopathological section in a dose-dependent manner in rats.

Keywords: hepatocellular carcinoma, histopathology, immunohistochemistry, Solanum nigrum.