Robusta coffee extracts inhibit quorum sensing activity in Chromobacterium violaceum and reduce biofilms against Bacillus cereus and Staphylococcus aureus

Research (Published online: 12-10-2022)

4. Robusta coffee extracts inhibit quorum sensing activity in Chromobacterium violaceum and reduce biofilms against Bacillus cereus and Staphylococcus aureus

Porwornwisit Tritripmongkol, Suthinee Sangkanu, Ratchadaporn Boripun, Juthatip Jeenkeawpieam, Julalak Chuprom, Veeranoot Nissapatorn, Maria de Lourdes Pereira, Alok K. Paul, and Watcharapong Mitsuwan

Veterinary World, 15(10): 2391-2398

ABSTRACT

Background and Aim: Bacillus cereus and Staphylococcus aureus cause foodborne intoxication in humans and animals. Pathogens can produce biofilms controlled by the quorum sensing system. The study aimed to investigate the antibacterial, antibiofilm, and anti-quorum sensing activities of Coffea canephora P. ex Fr. (Robusta coffee) extracts against B. cereus and S. aureus.

Materials and Methods: Ethanol extracts of fruit peels and seeds of Robusta coffee were tested for antibacterial activity against B. cereus and S. aureus using a broth microdilution assay. Reduction of the biofilm formation and elimination of the viability of mature biofilm-grown cells of B. cereus and S. aureus were determined. Inhibition of quorum sensing activity in Chromobacterium violaceum by the extracts was investigated using the disk diffusion method and flask incubation assay.

Results: Fresh fruit peel extract showed the strongest antibacterial activity against B. cereus and S. aureus with minimum inhibitory concentration (MIC) values of 2 and 4 mg/mL, respectively. However, the extracts did not inhibit Escherichia coli, avian pathogenic E. coli, and Pseudomonas aeruginosa at 8 mg/mL. Significant inhibition of biofilm formation at 1/2 × MIC of the fresh peel extract was detected in B. cereus (56.37%) and S. aureus (39.69 %), respectively. At 8 × MIC of the fresh peel extract, a significant elimination of the mature biofilm viability was detected in B. cereus (92.48%) and S. aureus (74.49%), respectively. The results showed that fresh and dried peel fruit extracts at 1/2 × MIC significantly reduced violacein production with the highest percentage inhibition ranging from 44.53 to 47.48% at 24 h (p ≤ 0.05).

Conclusion: The results of the present study suggest the potential therapeutic benefits of Robusta coffee extracts in inhibiting the growth, biofilm, and quorum sensing of both B. cereus and S. aureus. The results put forward an alternative strategy to control the foodborne intoxications caused by both pathogens.

Keywords: Bacillus cereus, biofilms, quorum sensing, Robusta coffee extract, Staphylococcus aureus.

Effect of thioredoxin on the immunogenicity of the recombinant P32 protein of lumpy skin disease virus

Research (Published online: 11-10-2022)

3. Effect of thioredoxin on the immunogenicity of the recombinant P32 protein of lumpy skin disease virus

Kanat Tursunov, Laura Tokhtarova, Darkhan Kanayev, Raikhan Mustafina, and Kanatbek Mukantayev

Veterinary World, 15(10): 2384-2390

ABSTRACT

Background and Aim: The rapid spread of lumpy skin disease (LSD) globally poses a serious threat to the agricultural sector. The timely and accurate diagnosis of the disease is crucial to control LSD. This study aimed to determine the effect of thioredoxin on the immunogenicity of the recombinant P32 (rP32) protein of LSD virus (LSDV). Since the P32 protein is poorly soluble, it is often expressed by adding an auxiliary sequence of a highly soluble partner protein such as thioredoxin.

Materials and Methods: The P32 gene fragment was amplified using a polymerase chain reaction from genomic DNA used as a template. The resulting DNA fragments were cloned into the pET32a vector, and transformed into Escherichia coli BL21 (DE3) cells through electroporation. Purification of the rP32 protein was performed using a HisTrap column. Purified rP32 protein fused with thioredoxin (rP32Trx) was characterized by western blotting, liquid chromatography with tandem mass spectrometry and indirect enzyme-linked immunosorbent assay (ELISA).

Results: Indirect ELISA revealed that, despite the lower molecular weight, the main part of the antibodies in the serum of immunized mice was directed against thioredoxin and not the target P32 protein. Thus, the antibody titers against rP32Trx were 1:102400, whereas antibody titers against heterologous recombinant 3BTrx and PD1Trx proteins were 1:25600 and 1:51200, respectively. Concurrently, the antibodies did not bind to the heterologous recombinant PD1 protein, which did not contain thioredoxin.

Conclusion: The results showed that the rP32 protein fused with the partner protein thioredoxin could not be used to obtain polyclonal and monoclonal antibodies. However, the recombinant fusion protein rP32Trx can be used to develop a serological test to detect antibodies, since antibodies against thioredoxin were not detected in the animal sera.

Keywords: immunogenicity, lumpy skin disease virus, P32 protein, recombinant antigen, thioredoxin.

Expression and epitope prediction of MPT64 recombinant proteins from clinical isolates of Mycobacterium tuberculosis as immunoserodiagnostic candidates

Research (Published online: 08-10-2022)

2. Expression and epitope prediction of MPT64 recombinant proteins from clinical isolates of Mycobacterium tuberculosis as immunoserodiagnostic candidates

Fihiruddin Fihiruddin, Nurul Inayati, Raudatul Jannah, Lalu Unsunnidhal, and Asmarani Kusumawati

Veterinary World, 15(10): 2376-2383

ABSTRACT

Background and Aim: The success in the handling and prevention of tuberculosis (TB) cases is highly dependent on their rapid detection, monitoring, and treatment. The efficacy of the Bacille Calmette–Guerin (BCG) vaccine is inconclusive in eastern Indonesia. The RV1980c gene of Mycobacterium tuberculosis encodes an antigenic protein that is considered to be a virulence factor, as it can stimulate the immune response in patients with TB. This study aimed to study the expression and epitope indicator of MPT64 recombinant proteins from clinical isolates of M. tuberculosis as immunoserodiagnostic candidates for pET SUMO plasmids from clinical isolates as candidates for serodiagnostic tests and recombinant vaccines.

Materials and Methods: The polymerase chain reaction (PCR) product of the RV1980c gene was inserted into the SUMO pET plasmid, which was then transformed into Escherichia coli BL21 (DE3) cells and expressed in Luria Bertani media induced by 1.0 M IPTG. Subsequently, sequencing was performed and the results were analyzed using the ClustalW and National Center for Biotechnology Information BLAST software. The T-cell epitope prognosis was then explained by GENETYX version 8.0., for the prediction of B-cell epitope, as assessed using an Immune Epitope Database analysis.

Results: The PCR product of the RV1980c gene had a length of 619 bp. Moreover, SDS–polyacrylamide gel electrophoresis and Western blotting revealed that the protein encoded by the Rv1980c gene weighed 36 kDa. We gained nine specific T-cell epitopes according to Iad Pattern position and eight epitopes according to Rothbard/Taylor Pattern Position; furthermore, we detected five B-cell epitopes in the RV1980c gene.

Conclusion: The MPT64 protein encoded by the RV1980c gene carries epitopes that are realized by lymphocytes and represent potential immunoserodiagnostic candidates in diagnostic immunology.

Keywords: Escherichia coli, immune response, lymphocytes, tuberculosis.

Comparative immune responses after vaccination with the formulated inactivated African horse sickness vaccine serotype 1 between naïve horses and pretreated horses with the live-attenuated African horse sickness vaccine

Research (Published online: 07-10-2022)

1. Comparative immune responses after vaccination with the formulated inactivated African horse sickness vaccine serotype 1 between naïve horses and pretreated horses with the live-attenuated African horse sickness vaccine

Narongsak Chaiyabutr, Suphot Wattanaphansak, Rachod Tantilerdcharoen, Surasak Akesowan, Suraseha Ouisuwan, and Darm Naraporn

Veterinary World, 15(10): 2365-2375

ABSTRACT

Background and Aim: African horse sickness (AHS) is a non-contagious, high mortality, and insect-borne disease caused by a double-stranded RNA virus from the genus Orbivirus. The study aimed to develop inactivated vaccines serotype 1 inactivated AHS vaccine (IAV) and to compare the effect of IAV on antibody responses in young naïve horses and adult horses pre-immunized with live-attenuated AHS virus (AHSV) serotypes 1, 3, and 4 live-attenuated vaccine (LAV).

Materials and Methods: A total of 27 horses were vaccinated in two trials. Twelve AHS naïve young horses and 15 adult horses were divided into three groups of 4 and 5 horses each, respectively. Horses in control Group 1 were treated with phosphate-buffered saline. Horses in Group 2 were subcutaneously vaccinated with 2 mL of formulated IAV with 10% Gel 01™ (Seppic, France) on day 0 and horses in Group 3 were subcutaneously vaccinated with 2 mL of IAV on day 0 and a booster on day 28. The IAV vaccine was prepared by isolating the AHSV serotype 1 growing on Vero cells, 10× virus titer was concentrated by ultrafiltration and chemically killed by formalin, using 10% Gel 01™ as an adjuvant. Ethylenediaminetetraacetic acid blood samples were taken for hematology, blood biochemistry, and antibody titers using an immunoperoxidase monolayer assay on 158th day post-vaccination.

Results: Vaccination with IAV serotype 1 in adult horses pretreated with LAV increased antibody titers more than in young naïve vaccinated horses. The total leukocyte count and %neutrophils significantly increased, while %lymphocytes and %eosinophils significantly decreased on day 1 after vaccination; no local reactions were observed at the site of injection in any group. All biochemical and electrolyte analyte values were within the normal range after vaccination.

Conclusion: The formulation of IAV serotype 1 using Gel 01™ as an adjuvant is safe and induces high antibody titers. This IAV formulation induced a high antibody response in horses without causing local reactions and mild systemic effects. However, AHS naïve horses still required ≥2 vaccinations and an annual booster vaccination to achieve high antibody titers.

Keywords: African horse sickness, inactivated vaccine, naïve young horse.

Effect of honey bee venom on the histological changes of testes and hormonal disturbance in diabetic mice

Research (Published online: 30-09-2022)

30. Effect of honey bee venom on the histological changes of testes and hormonal disturbance in diabetic mice

Sattar J. J. AL-Shaeli, Talal Jabal Hussen, and Ali M. Ethaeb

Veterinary World, 15(9): 2357-2364

ABSTRACT

Background and Aim: Hyperglycemia associated with hyper- or hypo-insulinemia is a hallmark of type 2 diabetes mellitus, which is firmly linked to decreased male infertility. Recently, bee venom (BV) has shown potential health prosperities, including antidiabetic; however, no study focuses on the effect of BV on male fertility in diabetic conditions. This study aimed to detect the effect of BV on histological and hormonal alteration of the testis in diabetic mice.

Materials and Methods: Twenty adult male mice were selected and assigned to four groups: Control, diabetic (150 mg/kg alloxan), BV1 (diabetic + 0.5 mg/kg BV), and BV2 (diabetic + 1 mg/kg BV). After 35 days, the serum levels of glucose, insulin, testosterone, follicular-stimulating hormone, luteinizing hormone, and prolactin were estimated. The histological structure of the testes was also evaluated.

Results: Alloxan-induced hyperglycemia and decreased insulin concentrations were reversed significantly by BV. Furthermore, diabetic mice exhibited various alterations in fertility hormones, while these disturbances were improved considerably to normal concentrations by BV. Similarly, alloxan-induced changes in sperm and testis histological parameters such as motility, viability, abnormality, sperm count, the number and diameter of seminiferous tubules, and the number of Leydig and Sertoli cells were significantly ameliorated to the normal condition by BV. Changes in the number, size, and shape of seminiferous tubules, the number of Leydig and Sertoli cells, and initial degeneration and vacuolization in interstitial cells and spermatogonia and spermatocyte were seen in diabetic mice. All these changes were shifted almost to normal structure by BV.

Conclusion: The BV could be used as an alternative therapeutic agent that manages the markers related to diabetic conditions concomitant with the improved histological structure of the testes and hormone production to accelerate male fertility.

Keywords: diabetic mice, hyperglycemia, Sertoli cells, spermatogonia.

Impact of hygiene and sanitation in ruminant slaughterhouses on the bacterial contamination of meat in Central Java Province, Indonesia

Research (Published online: 30-09-2022)

29. Impact of hygiene and sanitation in ruminant slaughterhouses on the bacterial contamination of meat in Central Java Province, Indonesia

Edy Dharma, Haryono Haryono, Aldi Salman, Pangesti Rahayu, and Widagdo Sri Nugroho

Veterinary World, 15(9): 2348-2356

ABSTRACT

Background and Aim: Ruminant slaughterhouse is one of the food-producing units to meet the protein demand of the people in Central Java. This study aimed to evaluate the implementation of sanitation and hygiene in ruminant slaughterhouses in Central Java based on their veterinary control number (NKV) certification and the microbiological quality of the meat produced.

Materials and Methods: This study was conducted from September 2021 to December 2021. Thirty-three priority slaughterhouses, representing 33 districts/cities in Central Java Province, were assessed for their hygiene and sanitation practices according to the NKV criteria mandated by The Minister of Agriculture Regulation No. 11/2020 on NKV Certification for Animal Production Unit. Sixty-six meat samples from these slaughterhouses were obtained for microbiological analysis. The total plate count (TPC), counts of Escherichia coli and Staphylococcus aureus, and the presence of Salmonella spp. were determined. The microbiological tests followed the standard national testing procedure according to the Indonesian National Standard 2897:2008 on Method of Analysis for Microbiological Contaminants in Meat, Eggs, Milk, and its derived products.

Results: The sanitation hygiene assessment of the 33 slaughterhouses showed that seven (21.2%) met the NKV criteria level 3, while the others did not. The average TPC of the meat samples was 1.57 × 105 CFU/g (4.93 log10), the S. aureus count was 7.6 CFU/g, and the E. coli count was 9.2 most probable number/g. Only one sample (1.50%) tested positive for Salmonella spp. A comprehensive assessment comparing the NKV criteria with the level of meat contamination showed that the ruminant slaughterhouses that satisfied the NKV criteria had more meat samples (85.71%), on average, that complied with the Indonesian National Standard for microbial contamination compared with those that did not satisfy the NKV criteria (69.23%). The odds ratio was 2.67.

Conclusion: Most of the priority ruminant slaughterhouses in Central Java did not meet the NKV standards. The research only looks at the level of hygiene sanitation according to NKV standards in slaughterhouses, the level of contamination produced does not reflect the level of the consumer; therefore, the level of contamination should continue to be investigated at the post-production stage.

Keywords: bacterial contamination, hygiene sanitation, meat, slaughterhouse.

Quality control procedure for Coccidial vaccines versus different routes of immunization

Research (Published online: 29-09-2022)

28. Quality control procedure for Coccidial vaccines versus different routes of immunization

Arwa Elnaggar, Hala Mahmoud, and Sahar Saber

Veterinary World, 15(9): 2342-2347

ABSTRACT

Background and Aim: Coccidiosis is an enteric infection caused by a protozoon (Eimeria tenella). Coccidiosis is known to have a negative impact on the economy. Coccidiosis is controlled using anticoccidial drugs, antibiotics, and vaccines. Various coccidial vaccines differ in application technique, attenuation method, and the species used. Coccidial vaccines can be spray or gel-based (Form). This study aimed to compare the effect of application and approaches between spray and gel vaccines for coccidiosis.

Materials and Methods: Specific pathogen-free chicks were vaccinated with different vaccines. Fecal samples were taken on 21 days post-vaccination for vaccine take, and then a challenge test was done on day 21.

Results: Post-vaccination oocyst counts in gel vaccinated groups were more than the spray vaccinated ones as it recorded (1400 and 2200) oocyst/g, but the gel vaccines resulted in lower post vaccinal titer which was (10000 and 12500) oocyst/g. Results of quantitative real-time polymerase chain reaction test post-vaccination were (23.72, 20.29) cycle threshold (CT) for spray vaccines and (18.75, 17.62) CT for gel vaccinated group. By challenging all the experimental groups, the microscopic and macroscopic lesion of gel vaccines resulted in score 1, while spray vaccines groups recorded score 2 and the control non-vaccinated challenged chickens showed score 4. The non-vaccinated/non-challenged group recorded a score of zero.

Conclusion: These results can help poultry producers to decide which delivery system will provide the best results for their production system. The gel vaccines showed a better protection rate and lower shedding, which means more protection of birds and public health.

Keywords: coccidia, Eimeria tenella, anticoccidial drugs and vaccinations, spray.