In silico analysis of highly conserved cytotoxic T-cell epitopes in the structural proteins of African swine fever virus

Research (Published online: 09-10-2021)

7. In silico analysis of highly conserved cytotoxic T-cell epitopes in the structural proteins of African swine fever virus

Leana Rich De Mesa Herrera and Elizabeth Paulino Bisa

Veterinary World, 14(10): 2625-2633

ABSTRACT

Background and Aim: African swine fever (ASF) is a viral disease of pigs caused by ASF virus (ASFV). High mortality and the lack of available treatments have severely impacted the swine industry resulting in huge global economic losses. In response to the dire necessity for vaccines, this study aims to identify highly conserved cytotoxic T-cell epitopes in ASFV structural proteins pp220, pp62, p72, p30, and CD2v through immunoinformatics approach.

Materials and Methods: The amino acid sequences of the structural proteins were retrieved from the National Center for Biotechnology Information protein database. The sequences were evaluated in CD-HIT Suite wherein resulting representative sequences were aligned in Clustal Omega. Highly conserved sequences were identified in the Protein Variability Server which were used as reference sequences for the cytotoxic T-cell epitope mapping. Epitopes were predicted using the tools in Immune Epitope Database. Peptides which bind to the swine major histocompatibility complex with IC50 binding scores >500 nM were filtered out. Epitopes which are classified to be potentially toxic and cross-reactive with the swine proteome sequences were all excluded from the study. The epitopes were docked with the swine leukocyte antigen-1*0401 (SLA-1*0401) wherein the binding affinity, the binding energy, and the root-mean-square deviation (RMSD) per residue of epitope-SLA complexes formed were determined and compared with the influenza epitope as positive control.

Results: A total of 112 highly conserved fragments with Shannon variability index ≤0.1 were identified. These include 66, 12, 26, 6, and 2 highly conserved fragments from ASFV proteins pp220, pp62, p72, p30, and CD2v, respectively. From these reference sequences, 35 nonameric peptides were selected for the list of candidate cytotoxic T-cell epitopes. These include 26 epitopes for pp220, 7 for pp62, 6 for p72, and one each for p30 and CD2v. Bioinformatics analysis classified the peptides as non-toxic. Further evaluations of epitopes showed that these are less likely to cross-react with the domestic swine proteome sequences. This study identified candidate epitopes from pp220 (IADAINQEF, FLNKSTQAY, QIYKTLLEY, and SLYPTQFDY), and pp62 (GTDLYQSAM, FINSTDFLY, and STDFLYTAI) which can bind to at least two widely distributed SLAs in pig populations. The immunogenicity of candidate peptides RSNPGSFYW, DFDPLVTFY, AIPSVSIPF, and VVFHAGSLY was validated by the acceptable binding affinities, binding energies, and RMSD of the peptide-SLA complexes formed. Results were also comparable with the crystal structure of an SLA-epitope complex in the database.

Conclusion: This is the first study to identify highly conserved cytotoxic T-cell epitopes in the structural proteins of ASFV. Overall, the results of in silico evaluations showed that the identified highly conserved cytotoxic T-cell epitopes may be used as part of future vaccine formulations against ASFV infection in domesticated pigs. Nonetheless, these findings require in vitro and in vivo validation before application.

Keywords: African swine fever virus, cytotoxic T-cell epitopes, immunoinformatics, in silico.

Actinomycetes of secondary metabolite producers from mangrove sediments, Central Java, Indonesia

Research (Published online: 07-10-2021)

6. Actinomycetes of secondary metabolite producers from mangrove sediments, Central Java, Indonesia

Wilis Ari Setyati, Delianis Pringgenies, Nirwani Soenardjo and Rini Pramesti

Veterinary World, 14(10): 2620-2624

ABSTRACT

Background and Aim: Actinomycetes are a group of Gram-positive bacteria with a fungus-like morphology. Their natural habitat encompasses terrestrial and water areas, including mangrove ecosystems. This study aimed to assess the PKS and NRPS genes as the producers of secondary metabolites and to determine the target bacterial species using molecular DNA tests.

Materials and Methods: In this study, we isolated bacteria from sediment samples from mangrove forests located on Karimunjawa Islands and in Semarang city, purified bacteria, screened for antibacterial activity, extracted bacterial DNA, amplified the NRPS gene, detected and amplified the PKS-I and PKS-II genes, amplified and sequenced the 16S rRNA, processed molecular data, and simulated a map of secondary metabolite producing genes.

Results: Samples from the Karimunjawa Islands yielded 19 bacterial isolates, whereas samples from Semarang yielded 11 bacterial isolates after culture in different media. Further experiments identified three active isolates, which were termed PN.SB.6.2, S.SK.6.3, and S.SK.7.1, against pathogenic species of Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. Isolate PN.SB.6.2 was determined to possess three biosynthetic gene clusters (BGCs), whereas the remaining two isolates, S.SK.6.3 and S.SK.7.1, only possessed two BGCs, namely, NRPS and PKS II.

Conclusion: Products were estimated to be in the NRPS, thiopeptide, RiPP-like, siderophore, betalactone, terpene, Type III PKS, CDPS, and lassopeptide groups. DNA identification of the isolates found three species of actinomycetes with antibacterial potential, namely, Virgibacillus salaries, Bacillus licheniformis, and Priestia flexa.

Keywords: actinomycetes, biosynthetic gene cluster, Karimunjawa Island, NRPS, PKS.

First report on molecular prevalence and identification of Anaplasma platys in dogs in Khon Kaen, Thailand

Research (Published online: 06-10-2021)

5. First report on molecular prevalence and identification of Anaplasma platys in dogs in Khon Kaen, Thailand

Biethee Rani Sarker, Thongphet Mitpasa, Arayaporn Macotpet, Pattara-Anong Bupata, Somboon Sangmaneedet and Weerapol Taweenan

Veterinary World, 14(10): 2613-2619

ABSTRACT

Background and Aim: Anaplasma platys is a blood parasite that infects platelets, causing thrombocytopenia. Rhipicephalus sanguineus ticks are believed to transmit A. platys. To identify A. platys, nested polymerase chain reaction (PCR) has proven to be an effective diagnostic tool. In this study, the molecular prevalence of A. platys infection in dogs was investigated for the 1st time in the Khon Kaen region of Thailand. The association between risk factors and A. platys infection was also evaluated.

Materials and Methods: A total of 130 blood samples were collected from dogs in Khon Kaen, Thailand. DNA from the samples was extracted and nested PCR was applied for molecular analysis. Platelet count and packed cell volume (PCV) levels were measured. Platelet counts were categorized into four grades: Non-thrombocytopenia (platelets >200,000 cells/μL), mild thrombocytopenia (platelets 150,000-200,000 cells/μL), moderate thrombocytopenia (platelets 100,000-150,000 cells/μL), and severe thrombocytopenia (platelets <100,000 cells/μL). Four categories for PCV levels of >37%, 30-37%, 20-29%, and <20% were defined as no anemia, mild anemia, moderate anemia, and severe anemia, respectively. DNA sequencing was analyzed using BTSeq™ (Barcode-Tagged Sequencing; CELEMICS, Seoul, South Korea) for similarity index.

Results: Among the 130 samples, 9 (6.9%) were positive for A. platys infection. There was an association between low platelet count and infection (p<0.05). PCV level was also associated with A. platys infection (p<0.05). DNA sequencing results of the nine positive samples showed similarity to known sequences of A. platys with 99.36-100% nucleotide identity. These results suggested low genetic diversity in A. platys infecting dogs in the Khon Kaen area.

Conclusion: By amplifying 16S rRNA, A. platys infection was detected in the blood of Thai dogs. Further work should be performed to identify risk factors potentially associated with A. platys infection in dogs in Khon Kaen. Other related factors should also be considered, such as location and breeding, as well as the environmental characteristics of each locality. In addition, sampling a larger number of animals may reveal predictors for the positivity of A. platys in dogs in this region.

Keywords: Anaplasma platys, molecular prevalence, nested polymerase chain reaction, thrombocytotropic anaplasmosis.

Efficacy of bubaline blood derived fibrin glue in silk ligature-induced acute periodontitis in Wistar rats

Research (Published online: 06-10-2021)

4. Efficacy of bubaline blood derived fibrin glue in silk ligature-induced acute periodontitis in Wistar rats

Poranee Banyatworakul, Nopadon Pirarat, Sujin Sirisawadi, Thanaphum Osathanon and Chanin Kalpravidh

Veterinary World, 14(10): 2602-2612

ABSTRACT

Background and Aim: Fibrin forms in the coagulation process, enhancing local hemostatic properties and promoting wound healing. The study aimed to evaluate the efficacy of bubaline-derived fibrin glue in silk ligature-induced periodontitis rats.

Materials and Methods: Bubaline blood–derived fibrin glue was prepared using cryoprecipitation and cryocentrifugation. Periodontitis was induced in rats by placing 5-0 silk ligatures around the mandibular first molars. The animals were divided into two groups: (1) Non-treatment and (2) bubaline fibrin glue–treated groups. Plaque, gingival inflammation, and mobility index were scored on days 1, 7, and 14 after intervention. Histological examinations were performed. The mRNA expression of inflammatory cytokines and growth factors was evaluated using a real-time polymerase chain reaction. Ligature-induced periodontitis was confirmed by the increase in inflammatory cell infiltration as well as histological bone and attachment loss.

Results: Compared to the non-treatment group, bubaline fibrin glue application reduced mononuclear cell infiltration into periodontal tissues corresponding to the reduction of collagen destruction. On days 7 and 14 after intervention, the inflammatory score and histological attachment loss were significantly lower in the bubaline fibrin glue–treated group than in the non-treatment group. A significant reduction in histological bone loss was observed in the treated group on day 7. Bubaline fibrin glue application led to a significant reduction of Tnfa and Il1b mRNA levels, while an increased expression of Pdgfa, Tgfb1, and Il10 was observed compared with the control.

Conclusion: Bubaline fibrin glue could be beneficial in periodontitis treatment aiming to reduce inflammation and delay the progression of periodontal disease.

Keywords: bubaline, fibrin, inflammation, ligature-induced, periodontitis.

Serologic evidence of pandemic (H1N1) 2009 virus infection in camel and Eld's deer, Thailand

Research (Published online: 05-10-2021)

3. Serologic evidence of pandemic (H1N1) 2009 virus infection in camel and Eld's deer, Thailand

Somjit Chaiwattanarungruengpaisan, Natthaphat Ketchim, Wanvisa Surarith, Metawee Thongdee, Phirom Prompiram, Kanittha Tonchiangsai, Wanlaya Tipkantha, Witthawat Wiriyarat and Weena Paungpin

Veterinary World, 14(10): 2596-2601

ABSTRACT

Background and Aim: The pandemic (H1N1) 2009 influenza (H1N1pdm09) virus has affected both human and animal populations worldwide. The transmission of the H1N1pdm09 virus from humans to animals is increasingly more evident. Captive animals, particularly zoo animals, are at risk of H1N1pdm09 virus infection through close contact with humans. Evidence of exposure to the H1N1pdm09 virus has been reported in several species of animals in captivity. However, there is limited information on the H1N1pdm09 virus infection and circulation in captive animals. To extend the body of knowledge on exposure to the H1N1pdm09 virus among captive animals in Thailand, our study investigated the presence of antibodies against the H1N1pdm09 virus in two captive animals: Camelids and Eld's deer.

Materials and Methods: We investigated H1N1pdm09 virus infection among four domestic camelid species and wild Eld's deer that were kept in different zoos in Thailand. In total, 72 archival serum samples from camelid species and Eld's deer collected between 2013 and 2014 in seven provinces in Thailand were analyzed for influenza antibodies using hemagglutination inhibition (HI), microneutralization, and western blotting (WB) assays.

Results: The presence of antibodies against the H1N1pdm09 virus was detected in 2.4% (1/42) of dromedary camel serum samples and 15.4% (2/13) of Eld's deer serum samples. No antibodies were detected in the rest of the serum samples derived from other investigated camelids, including Bactrian camels (0/3), alpacas (0/5), and llamas (0/9). The three positive serum samples showed HI antibody titers of 80, whereas the neutralization titers were in the range of 320-640. Antibodies specific to HA and NP proteins in the H1N1pdm09 virus were detected in positive camel serum samples using WB. Conversely, the presence of the specific antibodies in the positive Eld's deer serum samples could not be determined using WB due to the lack of commercially labeled secondary antibodies.

Conclusion: The present study provided evidence of H1N1pdm09 virus infection in the captive dromedary camel and Eld's deer in Thailand. Our findings highlight the need for continuous surveillance for influenza A virus in the population of dromedary camels and Eld's deer. The susceptible animal populations in close contact with humans should be closely monitored. Further study is warranted to determine whether Eld's deer are indeed a competent reservoir for human influenza virus.

Keywords: camel, Eld's deer, pandemic (H1N1) 2009 virus, serosurveillance, Thailand.

Conditioned medium derived from bovine umbilical mesenchymal stem cells as an alternative source of cell-free therapy

Review (Published online: 05-10-2021)

2. Conditioned medium derived from bovine umbilical mesenchymal stem cells as an alternative source of cell-free therapy

Dwi Liliek Kusindarta and Hevi Wihadmadyatami

Veterinary World, 14(10): 2588-2595

ABSTRACT

Umbilical cord blood (UCB) cells are an important source of mesenchymal stem cells (MSCs). It is known that the umbilical cord is rich in hematopoietic stem cells, which influenced research on ontogeny and transplantation (allogeneic transplantation). In recent years, stem cell research has emerged as an area of major interest due to its prospective applications in various aspects of both human and veterinary medicine. Moreover, it is known that the application of MSCs has several weaknesses. The use of these cells has limitations in terms of tumorigenesis effect, delivery, safety, and variability of therapeutic response, which led to the use of secretomes as an alternative to cell-free therapy. The main obstacle in its use is the availability of human UCB as an origin of MSCs and MSCs' secretomes, which are often difficult to obtain. Ethical issues regarding the use of stem cells based on human origin are another challenge, so an alternative is needed. Several studies have demonstrated that MSCs obtained from bovine umbilical cords have the same properties and express the same surface markers as MSCs obtained from human umbilical cords. Therefore, secretomes from MSCs derived from domestic animals (bovine) can possibly be used in human and veterinary medicine. This finding would contribute significantly to improve cell-free therapy. At present, the use of UCB MSCs derived from domestic animals, especially bovines, is very restricted, and only limited data about bovine UCB are available. Therefore, the aim of this review was to provide an updated overview of cell-free therapy and discuss the new possibilities introduced by the generation of this therapy derived from bovine umbilical MSCs as a promising tool in developing modern and efficient treatment strategies.

Keywords: bovine umbilical cord blood, cell-free therapy, mesenchymal stem cells, secretome.

Effects of dietary fiber sources on bacterial diversity in separate segments of the gastrointestinal tract of native and exotic pig breeds raised in Vietnam

Research (Published online: 02-10-2021)

1. Effects of dietary fiber sources on bacterial diversity in separate segments of the gastrointestinal tract of native and exotic pig breeds raised in Vietnam

Tran Thi Bich Ngoc, Nguyen Cong Oanh, Tran Thi Thu Hong and Pham Kim Dang

Veterinary World, 14(10): 2579-2587

ABSTRACT

Background and Aim: Dietary fiber has distinctive effects on the environment and microbiota of the pig's intestinal tract. This study was conducted at the naturally ventilated facility of the experimental station, National Institute of Animal Sciences, Vietnam, to examine the effects of fiber sources in diets on the intestinal microbiota of two different pig breeds raised in Vietnam.

Materials and Methods: A total of 18 native and 18 exotic pigs with average initial body weights of 9.5±0.4 and 16.5±0.4 kg, respectively, were each divided into three dietary treatments, including a low-fiber diet containing approximately 200 g NDF per kg dry matter (DM) and two high-fiber diets containing cassava by-products or brewer's grains containing approximately 300 g NDF per kg DM. At the end of the experiment (28 days), the bacterial diversity of digesta samples collected from the stomach, ileum, and colon segments was analyzed through DGGE analysis of the V3 variable regions of 16S-rDNA and by cloning and sequencing.

Results: Among the diets, significant differences were observed in the number of DNA bands in the stomach between the native and exotic pigs (p<0.05), but not in the ileum and colon. The dietary fiber affected the number of DNA bands in the ileum (p<0.05), but not in the stomach and colon. A significant interaction effect was found between diet and breed on the number of DNA bands in the ileum (p<0.05). Dietary fiber and breed had a greater effect on microbiota in the ileum and colon than that in the stomach.

Conclusion: The fiber sources affected the number of DNA bands in the ileum, and breed affected the number of DNA bands in the stomach. The microbial compositions in the ileum and colon segments were significantly affected by the dietary fiber and breed.

Keywords: bacterial diversity, brewer's grain, cassava by-products, exotic pig, fiber, native pig.