Isolation and identification of avirulent strains of Bacillus anthracis from environmental samples in Central Java, Indonesia

Research (Published online: 13-10-2021)

8. Isolation and identification of avirulent strains of Bacillus anthracis from environmental samples in Central Java, Indonesia

Ully Apriliana, Hendra Wibawa, Endang Ruhiat, Tri Untari and Soedarmanto Indarjulianto

International Journal of One Health, 7(2): 204-211

ABSTRACT

Background and Aim: Anthrax is a non-contagious infectious disease caused by Bacillus anthracis. The bacteria form spores that are resistant to extreme conditions and can contaminate the environment for decades. This study aimed to detect and characterize B. anthracis found in endemic areas of anthrax in Yogyakarta and Central Java province, Indonesia.

Materials and Methods: Soil samples were collected from Gunungkidul regency, Yogyakarta province (n=315) and Boyolali regency, Central Java province (n=100). Additional soil samples (n=10) and straw samples (n=5) were obtained from Pati regency, Central Java province. The isolation and identification of B. anthracis were performed using conventional methods: Morphology of bacteria colony in solid media, Gram staining, capsule staining, spores staining, and motility test. Isolates were further identified using polymerase chain reaction (PCR) against Ba813, lef (pXO1), and capC (pXO2) gene. An avirulent vaccine strain of B. anthracis (strain 34F2) was used as a control.

Results: Only four samples grew on blood agar with a ground-glass appearance, white-gray colony (Gunungkidul and avirulent strain) or yellowish (Boyolali and Pati). All were Gram-positive, presented chains, square-ended rods, spores, and were then identified as B. anthracis. Boyolali, Pati, and avirulent strain isolates had slightly different characteristics, including the growth of non-mucoid in the bicarbonate agar medium, and their uncapsulated form. The PCR showed two Gunungkidul isolates which amplified three genes, including Ba813, lef, and capC. Contrarily, the other isolates did not amplify the capC gene.

Conclusion: Gunungkidul isolates were identified as virulent strains of B. anthracis while Boyolali and Pati isolates were proposed as avirulent strains. This is the first report of isolation and identification of avirulent strains of B. anthracis in Central Java, Indonesia.

Keywords: avirulent Bacillus anthracis, Boyolali, identification, pati, polymerase chain reaction multiplex.

Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

Research (Published online: 13-10-2021)

7. Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

Fitrine Ekawasti, Umi Cahyaningsih, N. L. P. Indi Dharmayanti, Siti Sa'diah, Didik Tulus Subekti, Zul Azmi and Muhammad Ibrahim Desem

International Journal of One Health, 7(2): 196-203

ABSTRACT

Background and Aim: Toxoplasma gondii is a unicellular coccidian parasite distributed globally and is an important zoonotic pathogen. Approximately 30% of the human population worldwide is chronically infected with T. gondii. The pathogenicity of this species depends on the type originating from the clonal population. Techniques for more accurately determining the type of T. gondii have recently been developed using genetic markers. Specifically, T. gondii has been typed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This study aimed to identify sets of PCR-RFLP markers that have high power to discriminate genotyping of T. gondii and are easy to use and are easy to use. The objective of this study was to characterize virulent strain isolates of T. gondii by PCR-RFLP using 10 markers with DdeI.

Materials and Methods: T. gondii tachyzoites (RH virulent strain) were derived from culture cells at the Indonesian Research Center for Veterinary Sciences. Genotyping was performed on T. gondii DNA extracted from cell cultured tachyzoites using 10 genetic markers of PCR-RFLP, namely, B1#1, B1#2, B1#3, SAG1#1, SAG1#2, P30, BAG1, ROP1, GRA1, and GRA7, with digestion using the restriction enzyme DdeI.

Results: The 10 genes were amplified by PCR. Among them, three genetic markers, B1#3, ROP1, and GRA1, were genotyped by the PCR-RFLP using restriction enzyme DdeI. Overall, the findings showed that the specific RFLP profile of digestion of gene regions by DdeI could be used as a specific marker for the virulent biotype causative of toxoplasmosis. In addition, virulent strains of T. gondii can be easily detected by these markers.

Conclusion: Three pairs of primers (B1#3, ROP1, and GRA1) with DdeI have proven useful for the diagnosis of acute toxoplasmosis (virulent strain biotype I). This proposed method is relatively simple, rapid, cheap, and can be performed in most laboratories, providing a practical approach for the routine analysis of T. gondii strains.

Keywords: DdeI, genotype, restriction enzyme, Toxoplasma gondii.

Animal rabies epidemiology in Nepal from 2005 to 2017

Research (Published online: 13-10-2021)

6. Animal rabies epidemiology in Nepal from 2005 to 2017

Pushkar Pal, Adisorn Yawongsa, Rajesh Bhatta, Hiroshi Shimoda and Theera Rukkwamsuk

International Journal of One Health, 7(2): 190-195

ABSTRACT

Background and Aim: Animal rabies is endemic in Nepal, and it occurs in two forms. Although governmental and non-governmental agencies are working toward the control of rabies by mass dog vaccination and stray dog population management, there is still massive number of rabies incidence reported to the reference veterinary laboratory, Nepal. Therefore, this study aimed to assess animal species, temporal, regional, and agro-ecological distribution patterns of animal rabies in Nepal from 2005 to 2017.

Materials and Methods: The epidemiological data on animal rabies from the period of 2005 to 2017 were obtained from the Central Veterinary Laboratory, Tripureshwor, Kathmandu, Nepal. The laboratory-confirmed rabies cases were analyzed according to animal species, temporal, regional (developmental zones), and agro-ecological distributions. In addition, descriptive statistics were used to evaluate the distribution patterns of rabies.

Results: From 2005 to 2017, a total of 2771 suspected rabies cases in animals were reported to The Central Veterinary Hospital, Kathmandu. Of which, 1302 were found laboratory-confirmed cases. The rabies cases were most commonly reported and confirmed in dogs followed by other domestic animals. The high occurrences were recorded between 2005 and 2007. However, the incidence was increased during 2016 and 2017. The highest number of rabies cases was recorded in the eastern development zone, and the least number in the central zone at regional level. Likewise, it was highest in the Terai (plain) region and lowest in mountainous areas at agro-ecological zones. The findings also revealed that the occurrences of rabies significantly differed among seasons.

Conclusion: Rabies is present in Nepal throughout the year and all seasons with seasonal variation. Among the animal species, dogs are the primary animals affected with rabies followed by cattle and other domestic animals. At the regional level, eastern development zone had the highest incidence and Central development zone recorded the least. Similarly, the Terai region had the highest incidence rates, and the least overall prevalence rate was observed in mountainous regions among agro-ecological zones. Therefore, the government should implement the strict enforcement of mass dog vaccination and dog population management through one health approach to control rabies incidence in the country.

Keywords: agro-ecological zones, animals, epidemiology, Nepal, rabies.

Adoption of the One Health approach to improve zoonosis control in low-income countries: Insights from the case of rabies management in Burkina Faso

Research (Published online: 12-10-2021)

5. Adoption of the One Health approach to improve zoonosis control in low-income countries: Insights from the case of rabies management in Burkina Faso

Madi Savadogo, Hamidou Zangré, Sougrenoma Désiré Nana, Abdoul Kader Ilboudo, Laibané Dieudonné Dahourou, Sidwatta Guy Ilboudo, Virginie Simonis, Kongnimissom Apoline Sondo, Ayayi Justin Akakpo, Zékiba Tarnagda and Rianatou Bada Alambedji

International Journal of One Health, 7(2): 182-189

ABSTRACT

Background and Aim: Rabies is a neglected zoonotic disease transmissible to humans and domestic and wild animals through biting, scratching, or licking. This study aimed to analyze the adoption of the One Health approach by the stakeholders involved in rabies control in Burkina Faso.

Materials and Methods: The stakeholders involved in rabies control were from the Ministry of Livestock, Ministry of Health, Ministry of the Territory Administration, Ministry of Environment and Wildlife, and Ministry of Higher Education and Research. A structured questionnaire was used in face-to-face interviews to collect data from the stakeholders. The collected data included stakeholders' knowledge of rabies and the One Health approach and their levels of involvement in the multisectoral collaboration.

Results: Most participants could not describe rabies correctly (80%), and only 52.9% had heard of the One Health approach. In addition, there was no significant association between knowledge of rabies and participants' characteristics, and the knowledge of the One Health approach was significantly influenced by a participant's affiliation (place of work).

Conclusion: The results call for an increase in One Health education for its effective adoption by all the rabies control stakeholders. Additional efforts should focus on continual training of the One Health workforce, from policy-makers to frontline personnel.

Keywords: Burkina Faso, zoonosis, One Health, rabies control system, stakeholders, multisectoral collaboration, public health.

In ovo inhibition of avian pox virus replication by mangosteen rind and red ginger ethanolic extracts

Research (Published online: 11-10-2021)

9. In ovo inhibition of avian pox virus replication by mangosteen rind and red ginger ethanolic extracts

Annisaa' Lu'lu Nur'aini, Sri Hartati and Tri Untari

Veterinary World, 14(10): 2640-2645

ABSTRACT

Background and Aim: Avian pox is a contagious disease caused by the avian pox virus (APV). Mangostin and γ-mangostin in mangosteen rind (MR) and gingerol in red ginger (RG) exhibit antiviral activity. In this study, we evaluated the effect of MR and RG ethanolic extracts on APV based on pock lesions on the chorioallantoic membrane (CAM) of specific pathogen-free (SPF) embryonated chicken eggs (ECEs).

Materials and Methods: Three APVs from chicken isolates (C1, C2, and C3), one APV from a pigeon isolate (P), 1.5% and 3% MR ethanolic extract, 5% and 10% RG ethanolic extract, and a combination of 1.5% MR and 5% RG at 0.1 mL/ egg were inoculated in ovo (7th day incubation, chorioallantoic route) in SPF ECEs. A control group inoculated in ovo with APV alone was also established. Each treatment consisted of three replicates. Parameters including embryo survival, CAM lesions, and average number of pock lesions were determined.

Results: In ovo inoculation of MR and RG ethanolic extracts was not harmful to the ECEs and did not induce CAM lesions. The average number of pock lesions in the control group (C1, C2, C3, and P) was 35, 14, 10, and 17, respectively, whereas in all treatment groups, the number was 0, except in the 5% RG group of C1, which had a value of 10.

Conclusion: In ovo inoculation of 1.5% and 3% MR, 5% and 10% RG, and the combination of 1.5% MR plus 5% RG ethanolic extract s at 0.1 mL/egg inhibit APV by reducing the number of pock lesions on the CAM of the ECE.

Keywords: avian pox virus, in ovo, mangosteen rind, pock lesions, red ginger.

Pathology and molecular characterization of Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand

Research (Published online: 09-10-2021)

8. Pathology and molecular characterization of Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand

Tawatchai Pohuang, Suphattra Jittimanee and Sucheeva Junnu

Veterinary World, 14(10): 2634-2639

ABSTRACT

Aim: The aim of this study was to characterize Leucocytozoon caulleryi from backyard chickens in Khon Kaen Province, Thailand.

Materials and Methods: Tissue samples were collected from backyard chickens suspected to have leucocytozoonosis and subjected to histopathology examination. The BLAST Tool at NCBI GenBank (Basic Local Alignment Research Tools) (http://www.ncbi.nlm.nih.gov/BLAST) was used to identify the nucleotide sequence of the partial cytochrome c oxidase subunit I (cox I) gene. A Phylogenetic tree for analysis of L. caulleryi was constructed by using MEGA 7.0 software (https:// www.megasoftware.net/).

Results: The necropsy results revealed the subcutaneous hemorrhages of pectoral muscles, multifocal hemorrhages of the thymus and pectoral muscles, hemorrhage of the proventriculus and peritoneal cavity, and megaloschizonts of the pancreas and intestine, including subcapsular hemorrhages of the liver. Microscopic examination revealed numerous megaloschizonts of Leucocytozoon spp. in the pectoral muscles, intestine, pancreas, and thymus. Molecular analysis of the partial cox I gene showed that the causal agent was closely related to L. caulleryi reported in Japan.

Conclusion: From these results, L. caulleryi was determined to be the causal agent of leucocytozoonosis and was closely associated with L. caulleryi reported in Japan.

Keywords: chickens, cytochrome c oxidase subunit I gene, leucocytozoonosis, megaloschizonts.

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.