Development and evaluation of indirect enzyme-linked immunosorbent assay using recombinant dense granule antigen 7 protein for the detection of Toxoplasma gondii infection in cats in Thailand

Research (Published online: 12-03-2022)

8. Development and evaluation of indirect enzyme-linked immunosorbent assay using recombinant dense granule antigen 7 protein for the detection of Toxoplasma gondii infection in cats in Thailand

Eukote Suwan, Piangjai Chalermwong, Rucksak Rucksaken, Metita Sussadee, Sarawan Kaewmongkol, Ruenruetai Udonsom, Sathaporn Jittapalapong and Bandid Mangkit

Veterinary World, 15(3): 602-610

ABSTRACT

Background and Aim: Toxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite- dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats.

Materials and Methods: T. gondii-GRA7 was cloned and expressed in the Expresso® small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test.

Results: A 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 μg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite- GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94).

Conclusion: In this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats' sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

Keywords: cats, GRA7, indirect enzyme-linked immunosorbent assay, recombinant protein, serodiagnosis, Toxoplasma gondii.

Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene

Research (Published online: 12-03-2022)

7. Development of loop-mediated isothermal amplification-lateral flow dipstick as a rapid screening test for detecting Listeria monocytogenes in frozen food products using a specific region on the ferrous iron transport protein B gene

Wimvipa Srisawat, Chalermkiat Saengthongpinit and Wirawan Nuchchanart

Veterinary World, 15(3): 590-601

ABSTRACT

Background and Aim: Listeria monocytogenes is a critical foodborne pathogen that infects pregnant females and their newborns and older adults and individuals with comorbidities. It contaminates fresh vegetables, fruits, ready-to-eat foods, and frozen food products consumed by individuals. The culture conventional detection methods for L. monocytogenes are time-consuming, taking 4 days. This study aimed to describe the development and comparison of loop-mediated isothermal amplification (LAMP)- lateral flow dipstick (LFD), LAMP assay to PCR, and conventional culture for detecting L. monocytogenes in frozen food products.

Materials and Methods: Five LAMP primer sets, including F3, B3, forward inner primer, and backward inner primer, were designed from a specific region on ferrous iron transport protein B gene (feoB gene) to amplify LAMP products. The DNA probe was created, and the detection limit was determined in pure culture and purified DNA, as well as the detection in 20 frozen food product samples.

Results: The LMfeoB4 LAMP primer sets and DNA probe were LAMP products amplified at 60°C for 50 min. The specificity of the assay revealed no cross-reactivity with other pathogenic bacteria. The limit of detection (LOD) of the LAMP-LFD and LAMP assays using purified genomic DNA was 219 fg/μL both in LAMP and LAMP-LFD assays. The LOD of LAMP and LAMP-LFD assays in pure culture was 4.3×102 colony-forming unit (CFU)/mL and 43 CFU/mL, respectively. The LOD of the LAMP-LFD assay using artificially inoculated chicken in frozen food samples with pre-enrichment was 3.2×102 CFU/mL. The LAMP-LFD was also more sensitive than the LAMP assay and polymerase chain reaction. Finally, LAMP-LFD revealed no false positives in any of the 20 frozen food product samples.

Conclusion: LAMP-LFD assay using a specific region on the feoB gene to detect L. monocytogenes was highly specific, sensitive, faster, and convenient, making it a valuable tool for the monitoring and rapid screening of L. monocytogenes in frozen food products. This technique is applicable to the development of detection technologies for other pathogens in food products.

Keywords: ferrous iron transport protein B gene, frozen food product, Listeria monocytogenes, loop-mediated isothermal amplification, loop-mediated isothermal amplification-lateral flow dipstick.

Experimental and natural infections of severe acute respiratory syndrome-related coronavirus 2 in pets and wild and farm animals

Review (Published online: 10-03-2022)

6. Experimental and natural infections of severe acute respiratory syndrome-related coronavirus 2 in pets and wild and farm animals

Gondo Mastutik, Ali Rohman, Reny I'tishom, Ignacio Ruiz-Arrondo and Ignacio de Blas

Veterinary World, 15(3): 565-589

ABSTRACT

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has spread globally and has led to extremely high mortality rates. In addition to infecting humans, this virus also has infected animals. Experimental studies and natural infections showed that dogs have a low susceptibility to SARS-CoV-2 infection, whereas domesticated cats and other animals in the family Felidae, such as lions, tigers, snow leopards, and cougars, have a high susceptibility to viral infections. In addition, wild white-tailed deer, gorillas, and otters have been found to be infected by SARS-CoV-2. Furry farm animals, such as minks, have a high susceptibility to SARS-CoV-2 infection. The virus appears to spread among minks and generate several new mutations, resulting in increased viral virulence. Furthermore, livestock animals, such as cattle, sheep, and pigs, were found to have low susceptibility to the virus, whereas chicken, ducks, turkeys, quail, and geese did not show susceptibility to SARS-CoV-2 infection. This knowledge can provide insights for the development of SARS-CoV-2 mitigation strategies in animals and humans. Therefore, this review focuses on experimental (both replication and transmission) in vitro, ex vivo, and in vivo studies of SARS-CoV-2 infections in pets and in wild and farm animals, and to provide details on the mechanism associated with natural infection.

Keywords: animal disease, coronavirus disease 2019, infectious disease, pandemic, severe acute respiratory syndrome-related coronavirus 2.

Genetic relationship of Staphylococcus aureus isolated from humans, animals, environment, and Dangke products in dairy farms of South Sulawesi Province, Indonesia

Research (Published online: 10-03-2022)

5. Genetic relationship of Staphylococcus aureus isolated from humans, animals, environment, and Dangke products in dairy farms of South Sulawesi Province, Indonesia

Sartika Juwita, Agustin Indrawati, Retno Damajanti, Safika Safika and Ni Luh Putu Ika Mayasari

Veterinary World, 15(3): 558-564

ABSTRACT

Background and Aim: Staphylococcus aureus is a bacterium that causes several infectious diseases, including mastitis, endocarditis, and osteomyelitis, and poses a threat to human and animal health. This study aims to phenotypically and genetically identify S. aureus from the isolates collected from humans, animals, environment, and Dangke products in the dairy farms of South Sulawesi Province, Indonesia, as well as to establish a genetic relationship among the isolated S. aureus strains.

Materials and Methods: The total number of samples was 142, comprising 30 humans (skin swab), 58 animals (raw milk), 14 dairy products (Dangke), and 40 environmental samples (water). S. aureus was phenotypically identified using the culture method, followed by Gram staining, catalase test, and coagulase test. Simultaneously, genotypic identification of S. aureus was performed using the conventional polymerase chain reaction and sequencing methods. Sequencing data were analyzed using the MEGA X software by comparing BLAST National Center for Biotechnology Information databases.

Results: The phenotypic methods revealed that 56/142 (39.4%) animal, human, and Dangke samples grew on culture, and 56/56 (100%) were Gram stain positive, 56/56 (100%) catalase-positive, and 23/56 (41.1%) coagulase positive. The genotypic method revealed that 32/56 (57.1%) samples amplified the nuc gene. The phylogenetic analysis of 12 isolates revealed that they are all closely related and do not belong to distinct clades.

Conclusion: It indicates that S. aureus isolates from animals (S30) are probably the same strain as human isolates (H2, H3, H4, and H5). The findings of this study can be used as information regarding the importance of preventing and controlling diseases caused by S. aureus using a health approach involving the human, animal, and environmental sectors. This study was limited to the sequencing analysis of the nuc gene.

Keywords: dairy farm, environment, human, nuc gene, Staphylococcus aureus.

Dehydrated husks and cake of prickly pear (Opuntia ficus-indica) processing for broiler feed: Effects on growth performance, carcass characteristics, and meat quality

Research (Published online: 09-03-2022)

4. Dehydrated husks and cake of prickly pear (Opuntia ficus-indica) processing for broiler feed: Effects on growth performance, carcass characteristics, and meat quality

Imene Cherif, Rafik Arbouche, Yasmine Arbouche, Achour Mennani and Fodil Arbouche

Veterinary World, 15(3): 551-557

ABSTRACT

Background and Aim: The potential solution is to use agro-industrial by-products as an unconventional source of raw materials for broiler feed. This study aims to determine the effects of substituting prickly pear (FB; Opuntia ficus-indica) husks for corn and FB seed cake for soybean meal on the production performance, slaughter characteristics, and chemical composition of broiler meat.

Materials and Methods: Two hundred day-old chicks of equal sex ratio (1:1) of Big Fast strain, weighing on average 37±2g, were randomly divided into four homogeneous groups of 50 subjects each. Each group was subdivided into 10 packs of five animals, which were banded and numbered. Rations with substitution rates of 0%, 10%, 20%, and 30% of corn and soybean meal by dehydrated husks and FB cake were randomly distributed among the groups.

Results: Average daily gains and body weights on 48 days were improved (p<0.05) in 10% and 20% groups, while the 30% group performed identically to the control. Cold carcass yield was optimal in 10% and 20% groups. The liver weight of the experimental groups decreased significantly (p<0.05), while their gizzard weight increased significantly (+24 points). The meat protein rate evolved proportionally to the substitution rate, whereas the fat rate depreciated by up to –1.08 points for the 30% group compared to the control.

Conclusion: Incorporating FB processing by-products into broiler feed at rates of 10% and 20% improves zootechnical performance, carcass yields, and the chemical composition of the meat.

Keywords: broiler feed, prickly pear processing, production cost.

Risk factors associated with Salmonella prevalence, its antibiotic resistance, and egg antibiotic residues in the layer farming environment

Research (Published online: 09-03-2022)

3. Risk factors associated with Salmonella prevalence, its antibiotic resistance, and egg antibiotic residues in the layer farming environment

Pairat Sornplang, Jareerat Aieamsaard, Chuleeporn Saksangawong and Naritsara Suayroop

Veterinary World, 15(3): 543-550

ABSTRACT

Background and Aim: Human salmonellosis with non-typhoidal Salmonella remains a global public health concern related to the consumption of contaminated eggs and egg-based products. This study aimed to examine the prevalence of Salmonella, antimicrobial-resistant Salmonella, and egg antibiotic residues concerning risk factors associated with Salmonella contamination in eggs, the layer farming environment, and laying hens kept in battery-cage closed-housing systems.

Materials and Methods: This study used a repeated cross-sectional design to collect 488 samples from eggs, laying hens, and the farm environment on one laying farm for Salmonella detection according to ISO 6579:2002/AMD 1:2007. Salmonella-positive samples were further tested for serotype and antimicrobial susceptibility using the disk diffusion test. The layer farm contact person was interviewed at the sampling time to evaluate the risk factors associated with Salmonella contamination using logistic regression analysis. For each month, 24 eggs (144 eggs in total) were also randomly sampled from the collection egg area at the farm for antibiotic residue detection using the European Four Plate Test.

Results: The highest Salmonella prevalence rates were in the samples from the layer pen floors, followed by the egg sizing machine (ESM) and eggshells at 65.5%, 52.5%, and 15%, respectively. Salmonella enterica serovar Corvallis was the dominant serovar (48.38%), followed by Mbandaka (37.76%), Braenderup (14.29%), and Typhimurium (4.08%). Rodent presence at the farm and the frequency of changing the disinfectant foot dip were significant factors related to Salmonella contamination on the pen floors (odds ratio [OR]=22.5, 95% confidence interval [CI]=2.11-240.48, p=0.01; OR=24, 95% CI=2.78-206.96, p=0.004, respectively). Hand-washing before sorting and cleaning the ESM were the significant factors (OR=13, 95% CI=1.2-140.73, p=0.04). The most resistant Salmonella isolates were resistant to oxytetracycline. One isolate of S. enterica Typhimurium was resistant to cefotaxime, enrofloxacin, and oxytetracycline. The antibiotic residues in the egg yolks were streptomycin, enrofloxacin, and tetracycline at prevalence rates of 36.11%, 11.81%, and 7.64%, respectively. Streptomycin was the most abundant residue in the albumen and yolk, followed by tetracycline.

Conclusion: Salmonella prevalence in layer farming with a closed-housing system is related to effective biosecurity and hygiene issues, such as rodent control, clean farm equipment, and good worker hygiene. In addition, eggs' antibiotic residues may be related to treating antimicrobial-resistant Salmonella isolates and medicated feed with inappropriate antibiotic withdrawal time.

Keywords: antibiotic residue, antibiotic resistance, eggs, laying hens, risk factors, Salmonella prevalence.

Evaluation of the anesthetic depth and bispectral index during propofol sequential target-controlled infusion in dogs

Research (Published online: 08-03-2022)

2. Evaluation of the anesthetic depth and bispectral index during propofol sequential target-controlled infusion in dogs

Matheus Luis Cunha Ubiali, Guilherme Paes Meirelles, Julia Milczewski Vilani, Henrique Erick da Luz, Sabrine Marangoni, Raisa Braul Rodrigues and Ricardo Guilherme D'OCtaviano de Castro Vilani

Veterinary World, 15(3): 537-542

ABSTRACT

Background and Aim: The use of anesthetic infusions based on pharmacokinetic values associated with anesthetic plan and bispectral index in dogs have not been well-documented in the literature. This study aimed to evaluate the bispectral index (BIS) change based on pre-propofol and establish clinical anesthetic depth changes during propofol sequential target-controlled infusion (STCI) in dogs with a plasma target of 5 μg/mL.

Materials and Methods: Twenty healthy male dogs aged 1-3 years and weighing 9.8-44 kg were recruited. These dogs were pre-medicated intramuscularly with methadone (0.2 mg/kg) and acepromazine (0.03 mg/kg). After 30 min, propofol anesthetic induction and maintenance were initiated using STCI according to dog pharmacokinetic (PK) parameters. Subsequently, the target plasma concentration of propofol was set at 5 μg/mL for both anesthetic induction and the 120 min maintenance. Then, TivaTrainer v.9.1 software was used to calculate anesthetic infusion rates in a TCI plasmatic concentration mode using the PKs model optimized by covariates for propofol TCI in dogs. The BIS value was recorded every 5 min from the beginning of induction until the end of anesthesia. Finally, analysis of variance was performed on numerical data using the Friedman test, followed by the Bonferroni adjustment (p<0.05).

Results: A statistical difference was observed between the baseline BIS value (T0), with a median value of 84.5 (81-97), and BIS after every 15 min (T15) of inducing anesthesia. Surgical anesthetic depth was also reached in 18 of 20 dogs after 10 min of infusion and in all dogs after 20 min, with a median BIS value of 72 (53-89) at the time of surgical anesthesia depth. Results also showed no BIS variation (p<0.05) between anesthetic moments after anesthetic induction with a substantial amplitude of BIS in the surgical anesthetic depth. Moreover, the maximum depth of anesthesia in all dogs by clinical evaluation was reached after 20 min of anesthesia and then remained stable throughout the anesthetic period.

Conclusion: This study suggested that most dogs (90%) attained a surgical depth of anesthesia within 15 min of STCI onset, with a plasma target of 5 μg/mL and no change in anesthetic depth throughout the period anesthesia lasted. Furthermore, median BIS values remained high even after dogs reached the surgical depth of anesthesia, indicating that the comparison of BIS values of dogs and humans should not be considered for classifying anesthetic and hypnotic depths in dogs.

Keywords: bispectral index, target-controlled infusion, total intravenous infusion.