Genetic detection and analysis of porcine norovirus in pigs farmed in north Vietnam.

Norovirus (NoV) causing gastroenteritis symptoms, which has been reported in several hosts, including humans, pigs, and rats. This study was conducted to identify porcine viral infection and to characterize NoV strains from pigs in some provinces in north Vietnam. Totally, 102 fecal samples from diarrheal pigs on farms in six cities and provinces in northern Vietnam during July 2022 to March 2023 were collected. Polymerase chain reaction was used to identify the viral genome. Positive samples were used for nucleotide sequencing of the partial RNA-dependent RNA polymerase gene sequence. Five (4.9 %) positive stool samples were detected from animals farmed in five different farms, with one positive animal identified in each farm. Genetic analysis indicated that nucleotide identity was in the range 97.77-99.62 % among the 5 NoVs in this study. Phylogenetic analysis pointed out that the five NoVs were Genotype II.19 viruses. Genetically, these strains were closely related to porcine NoV strains that were reported in China in 2009.

Molecular epidemiology and strain diversity of circulating feline Calicivirus in Thai cats.

Feline calicivirus (FCV) is a significant viral pathogen causing upper respiratory tract and oral diseases in cats. The emergence of the virulent systemic FCV variant (VS-FCV) has raised global concern in the past decade. This study aims to explore the epidemiology, genetic characterization, and diversity of FCV strains circulating among Thai cats. Various sample types, including nasal, oral, and oropharyngeal swabs and fresh tissues, were collected from 184 cats across different regions of Thailand from 2016 to 2021. Using reverse transcription real-time polymerase chain reaction (RT-qPCR), FCV infection was investigated, with additional screening for feline herpesvirus-1 (FHV-1) by qPCR. The detection rates for FCV, FHV-1, and co-infection were 46.7, 65.8, and 31.5%, respectively. Significantly, the odds ratio (OR) revealed a strong association between the detection of a single FCV and the presence of gingivostomatitis lesions (OR: 7.15, 95% CI: 1.89-26.99, p = 0.004). In addition, FCV detection is notably less likely in vaccinated cats (OR: 0.22, 95% CI: 0.07-0.75, p = 0.015). Amino acid sequence analysis based on the VP1 major capsid protein gene of the 14 FCV-Thai (FCV-TH) strains revealed genetic diversity compared to the other 43 global strains (0 to 86.6%). Intriguingly, a vaccine-like FCV variant was detected in one cat. In summary, this study provides insights into the epidemiology and molecular characteristics of FCV diversity within the Thai cat population for the first time. The identification of unique physicochemical characteristics in the capsid hypervariable region of some FCV-TH strains challenges previous hypotheses. Therefore, further exploration of vaccine-like FCV variants is crucial for a comprehensive understanding and to improve viral prevention and control strategies.

Duck Tembusu virus in North Vietnam: epidemiological and genetic analysis reveals novel virus strains.

Tembusu virus (TMUV) is an important infectious disease, causing economic losses in duck production. Since the first report of TMUV infection in Vietnam in 2020, the disease has persisted and affected poultry production in the country. This study conducted epidemiological and genetic characterization of the viral strains circulating in north Vietnam based on 130 pooled tissue samples collected in six provinces/cities during 2021. The TMUV genome was examined using conventional PCR. The results indicated that 21 (16.15%) samples and 9 (23.68%) farms were positive for the viral genome. The positive rate was 59.26% for ducks at ages 2-4 weeks, which was significantly higher than for ducks at ages >4 weeks and < 2 weeks. Genetic analysis of the partial envelope gene (891 bp) sequences indicated that the five Vietnamese TMUVs shared 99.55-100% nucleotide identity, while the rates were in the range 99.59-100% based on the pre-membrane gene sequences (498 bp). The five Vietnamese TMUV strains obtained formed a novel single subcluster. These strains were closely related to Chinese strains and differed from the vaccine strain, suggesting that Vietnamese TMUV strains were field viruses. It needs to be further studied on vaccine development to prevent effects of TMUV infection on poultry production across Vietnam.

Feline infectious peritonitis: A comprehensive evaluation of clinical manifestations, laboratory diagnosis, and therapeutic approaches.

Objective: This study aimed to investigate the clinical and laboratory characteristics of naturally occurring feline infectious peritonitis (FIP) and estimate the median survival time of FIP cats treated with prednisolone to guide further therapeutic planning. Materials and Methods: In this retrospective study, data from a total of 116 cats with effusion were fully recorded. Forty-five FIP-diagnosed cats were enrolled for analysis. Results: The study findings indicate that FIP was a disease affecting cats aged 1-2 years and was highly prevalent among male cats. Clinical manifestations of FIP affected the digestive (60%), hematological (53.3%), respiratory (33.3%), neurological (6.7%), and ocular (4.4%) systems. Blood profiles revealed mild anemia, lymphopenia, thrombocytopenia, hypoalbuminemia, hyperglobulinemia, and an albumin to globulin ratio of 0.4. Fluid analysis and cytology of FIP cats demonstrated a transparent yellow fluid with a protein content of 6 gm/dl and a total nucleated cell count of approximately 5,000-10,000 cells. During the observation period, FIP cats treated with prednisolone exhibited a median survival time of 31 days. Conclusion: Confirming FIP cases can be challenging; therefore, a tentative diagnosis of FIP must be made with care. This study provided practical diagnostic tools to diagnose FIP based on clinical signs and multiple abnormalities, which allowed for more efficient and rapid detection.

Detection of feline immunodeficiency virus by neutral red-based loop-mediated isothermal amplification assay.

Background and Aim: Feline immunodeficiency virus (FIV) is a retroviral pathogen globally responsible for immunodeficiency disease in cats. However, the current diagnosis based on antibody detection has limitations and can also produce false-positive results. This study aimed to develop a one-pot loop-mediated isothermal amplification (LAMP) process integrated with neutral red (NR-LAMP) assay for detection of FIV proviral DNA. Materials and Methods: We developed a one-pot, gag gene-based NR-LAMP for convenient, rapid, specific, and sensitive colorimetric inspection of FIV proviral DNA. Results: The developed NR-LAMP was capable of amplifying at an optimum temperature of 65°C for 40 min. No cross-amplification was detected between FIV and other feline viruses tested, indicating the high specificity (98.44%) of the novel FIV-LAMP primer. Our NR-LAMP assay has a detection limit of 4.2 × 101 copies/µL. A total of 80 clinical samples with a background of FIV infection were collected and tested using the proposed method. The NR-LAMP assay showed a high sensitivity of 100% compared to conventional polymerase chain reaction assay. Conclusion: These results support the suitability of NR-LAMP as a potential future alternative clinical molecular approach for further use in the diagnosis of FIV-infected cats.

Biochemical and structural comparisons of non-nucleoside reverse transcriptase inhibitors against feline and human immunodeficiency viruses.

BACKGROUND: Feline immunodeficiency virus (FIV) causes an acquired immunodeficiency-like syndrome in cats. FIV is latent. No effective treatment has been developed for treatment the infected cats. The first and second generations non-nucleoside reverse transcriptase inhibitors (NNRTIs) for HIV treatment, nevirapine (NVP) and efavirenz (EFV), and rilpivirine (RPV), were used to investigate the potential of NNRTIs for treatment of FIV infection. OBJECTIVE: This study aims to use experimental and in silico approaches to investigate the potential of NNRTIs, NVP, EFV, and RPV, for inhibition of FIV reverse transcriptase (FIV-RT). METHODS: The FIV-RT and human immunodeficiency virus reverse transcriptase (HIV-RT) were expressed and purified using chromatography approaches. The purified proteins were used to determine the IC50 values with NVP, EFV, and RPV. Surface plasmon resonance (SPR) analysis was used to calculate the binding affinities of NNRTIs to HIV-RT and FIV-RT. The molecular docking and molecular dynamic simulations were used to demonstrate the mechanism of FIV-RT and HIV-RT with first and second generation NNRTI complexes. RESULTS: The IC50 values of NNRTIs NVP, EFV, and RPV against FIV-RT were in comparable ranges to HIV-RT. The SPR analysis showed that NVP, EFV, and RPV could bind to both enzymes. Computational calculation also supports that these NNRTIs can bind with both FIV-RT and HIV-RT. CONCLUSIONS: Our results suggest the first and second generation NNRTIs (NVP, EFV, and RPV) could inhibit both FIV-RT and HIV-RT.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus.

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Generation of a Single-Chain Variable Fragment Antibody against Feline Immunoglobulin G for Biosensor Applications.

For many decades, feline infectious disease has been among the most common health problems and a leading cause of death in cats. These diseases include toxoplasmosis, feline leukemia virus (FeLV), and particularly feline immunodeficiency virus (FIV) disease. Early diagnosis is essential to increase the chance of successful treatment. Generally, measurement of the IgG level is considered to be indicative of an individual's immune status for a particular pathogen. The antibodies specific to feline IgG are crucial components for the development of a detection kit. In this study, feline IgG-bound scFv was selected using phage display technology. Three rounds of biopanning were conducted against purified feline IgG. Through an indirect enzyme-linked immunosorbent assay (ELISA), two scFv clones demonstrating the best binding ability to feline IgG were chosen for biochemical characterization. In addition, the selected scFv (N14) was expressed and purified in a bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of the purified N14 was 29 kDa. A sandwich ELISA was used to evaluate the binding capacity of the purified scFv to feline IgG. As expected, the purified N14 had the capacity to bind feline IgG. Furthermore, N14 was modified to create a scFv-alkaline phosphatase (scFv-AP) fusion platform. The surface plasmon resonance (SPR) results revealed that N14-AP bound to feline IgG with an affinity binding value of 0.3 ± 0.496 µM. Additionally, the direct ELISA demonstrated the binding capacity of N14-AP to feline IgG in both cell lysate and purified protein. Moreover, N14-AP could be applied to detect feline IgG based on electrosensing with a detection limit of 10.42 nM. Overall, this study successfully selected a feline IgG-bound scFv and developed a scFv-AP platform that could be further engineered and applied in a feline infectious disease detection kit.

Porcine Sapovirus in Northern Vietnam: Genetic Detection and Characterization Reveals Co-Circulation of Multiple Genotypes.

Porcine sapovirus (PoSaV) has been reported in many countries over the world, which may cause gastroenteritis symptoms in pigs with all ages. There has been no report on PoSaV infection in Vietnam up to now. In this study, a total of 102 samples were collected from piglets, fattening pigs, and sows with diarrhea in several cities and provinces in northern Vietnam. The PoSaV genome was examined using polymerase chain reaction (PCR). Sequencing of the partial RNA-dependent RNA polymerase (RdRp) gene sequences (324 bp) was performed. Of the 102 tested samples, 10 (9.8%) and 7/20 (35%) were detected as positive for the PoSaV RdRp gene using the PCR method at the individual and farm levels, respectively. Genetic analysis of the partial RdRp gene region of about 324 bp indicated that the nucleotide identity of the current 10 Vietnamese viral strains ranged from 61.39% to 100%. Among the 10 strains obtained, 8 belonged to genotype III and the remaining 2 strains were clustered in genotype VIII. The Vietnamese genotype III viruses formed two sub-clusters. The Vietnamese PoSaV strains were closely related to PoSaVs reported in South Korea, Venezuela, and the Netherlands. This research was the first to describe PoSaV infection in northern Vietnam during 2022-2023.

The first study on clinicopathological changes in cats with feline infectious peritonitis with and without retrovirus coinfection.

Background and Aim: Feline infectious peritonitis (FIP) is an infectious, immune-mediated, and fatal disease in cats caused by a mutant feline coronavirus (FCoV) infection. Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are two common retroviruses that play a role in reducing feline immune function with opportunistic retrovirus infection being a predisposing factor for the development of FIP. This study aimed to evaluate the clinicopathological parameters of FIP in cats with and without retrovirus coinfection. Materials and Methods: In total, 62 cats presenting with pleural and/or peritoneal effusion at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand, were selected for the study. Effusion samples were collected and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay was performed on all samples using the 3' untranslated region primer. All FCoV-positive cats were tested for retrovirus infection using a commercial kit (Witness FeLV-FIV [Zoetis]; United States). Clinical signs, hematological, and biochemical parameters of these cats were investigated and grouped. Results: Of the 62 cats with pleural and/or peritoneal effusion, FCoV was detected in 32, of which 21 were highly suspicious for FIP. The cats suspected of FIP were divided into three subgroups following viral detection. A total of 14 had only FCoV infection (Group A), four had FCoV and FeLV infection (Group B), and three had FCoV, FeLV, and FIV infection (Group C). Of the rest, 11 had definitive diagnoses, which included three being FCoV and FeLV-positive (Group D), and eight were retrovirus-negative (Group E). Mild anemia and lymphopenia were found in cats infected with these three viruses. An albumin-to-globulin ratio lower than 0.5 was found in FIP cats with only FCoV infection. Conclusion: Typically, cats with clinical effusion and FIP, with and without retrovirus coinfection, had similar hematological findings. Clinical signs, blood parameters, fluid analysis with cytological assessment, and RT-PCR assays could identify better criteria to diagnose FIP with and without retrovirus coinfection.

Molecularly Imprinted Nanoparticle Ensembles for Rapidly Identifying S. epidermidis.

Staphylococcus epidermidis (S. epidermidis) belongs to methicillin-resistant bacteria strains that cause severe disease in humans. Herein, molecularly imprinted polymer (MIP) nanoparticles resulting from solid-phase synthesis on entire cells were employed as a sensing material to identify the species. MIP nanoparticles revealed spherical shapes with diameters of approximately 70 nm to 200 nm in scanning electron microscopy (SEM), which atomic force microscopy (AFM) confirmed. The interaction between nanoparticles and bacteria was assessed using height image analysis in AFM. Selective binding between MIP nanoparticles and S. epidermidis leads to uneven surfaces on bacteria. The surface roughness of S. epidermidis cells was increased to approximately 6.3 ± 1.2 nm after binding to MIP nanoparticles from around 1 nm in the case of native cells. This binding behavior is selective: when exposing Escherichia coli and Bacillus subtilis to the same MIP nanoparticle solutions, one cannot observe binding. Fluorescence microscopy confirms both sensitivity and selectivity. Hence, the developed MIP nanoparticles are a promising approach to identify (pathogenic) bacteria species.

Risk factors and clinical and laboratory findings associated with feline immunodeficiency virus and feline leukemia virus infections in Bangkok, Thailand.

Background and Aim: Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are retroviruses associated with chronic and neoplastic diseases in domestic and non-domestic cats. There has been increasing interest in the clinical importance of feline retroviruses in Thailand and the identification of associated risk factors in domestic cats. To prevent the spread of retroviral diseases and improve the management of retrovirus-infected cats, risk factors and associated clinical laboratory data must be clearly understood. This study aimed to identify the influence of household, lifestyle, health status, sterilization, clinical presentations, and laboratory findings on FIV- and FeLV-infected cats in Bangkok, Thailand. Materials and Methods: A total of 480 cats were evaluated for FeLV p27 antigen and FIV antibodies using Witness FeLV-FIV Rapid Test and SNAP FIV/FeLV Combo Test at a veterinary hospital service. Results: Of the 480 cats tested, 113 were positive for virus infection, including 60 for FeLV (12.5%), 40 for FIV (8.3%), and 13 for both FeLV and FIV (2.7%). The findings revealed that the risk factors for cats infected with FeLV, FIV, or both FeLV and FIV were significantly different compared with those for non-infected cats (p < 0.05). Multivariate analysis showed that multi-cat ownership is a risk factor for the high prevalence of feline retrovirus infection, as multi-cat households exhibited a higher prevalence of infection than single-cat households. Anemic and sick cats were also at a greater risk of testing positive for specific retrovirus infections. FeLV-infected cats had a higher risk of anemia and low erythrocyte and thrombocyte counts (p ≤ 0.0001), whereas FIV-infected cats were more likely to have anemia and leukocytopenia than controls. Conclusion: Knowledge of the risk factors for retroviral diseases and associated clinical and laboratory findings can be used to develop strategies to reduce FIV and FeLV infections in cats.

Epidemiological Analysis and Genetic Characterization of Parvovirus in Ducks in Northern Vietnam Reveal Evidence of Recombination.

In total, 130 tissue-pooled samples collected from ducks in some provinces/cities in north Vietnam were examined for waterfowl parvovirus genome identification. Twenty-six (20%) samples were positive for the parvovirus infection, based on polymerase chain reaction analysis. Of the 38 farms tested, 14 (36.84%) were positive for the waterfowl parvovirus genome. The rate of the parvovirus genome detection in ducks aged 2−4 weeks (37.04%) was significantly (p < 0.05) higher than that at ages <2 weeks (9.09%) and >4 weeks (16.30%). The positive rate on medium-scale farms (9.36%) was significantly (p < 0.05) lower than for small-scale (31.03%) and large-scale (29.73%) farms. The lengths of the four Vietnamese waterfowl parvovirus genomes identified were 4750 nucleotides. Among the four Vietnamese parvovirus genomes, nucleotide identities were from 99.29% to 99.87%. Phylogenetic analysis of the near-complete genomes indicated that the waterfowl circulating in northern Vietnam belonged to the novel goose parvovirus (NGPV) group. The Vietnamese NGPV group was closely related to the Chinese group. Recombination analysis suggested that the Vietnam/VNUA-26/2021 strain was generated by a recombination event. One positive selection site of the capsid protein was detected.

Development of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Detecting Feline Coronavirus.

Feline infectious peritonitis (FIP) is a worldwide fatal disease caused by a mutant feline coronavirus (FCoV). Simple and efficient molecular detection methods are needed. Here, sensitive, specific, rapid, and reliable colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the ORF1a/1b gene of FCoV from cats with suspected FIP using neutral red as an indicator. Novel LAMP primers were specifically designed based on the gene of interest. The isothermal assay could visually detect FCoV at 58 °C for 50 min. The RT-LAMP assay was highly specific and had no cross-reactivity with other related feline viruses. The detection limit of FCoV detection by RT-LAMP was 20 fg/µL. A blind clinical test (n = 81) of the developed RT-LAMP procedure was in good agreement with the conventional PCR method. In the light of its performance specificity, sensitivity, and easy visualization, this neutral-red-based RT-LAMP approach would be a fruitful alternative molecular diagnostic tool for veterinary inspection of FCoV when combined with nucleotide sequencing or specific PCR to affirm the highly virulent FIP-associated FCoV.

Investigation of Bacterial Isolations and Antimicrobial Susceptibility of Chronic Rhinitis in Cats.

Chronic rhinitis is a quite common upper respiratory tract (URT) disease in cats. As a result of unclear etiology, frequently, multidrug-resistant bacteria are identified. This study investigated bacterial isolations and an antimicrobial susceptibility test (AST) in chronic rhinitis in cats. The medical records of 395 cats with chronic URT signs were reviewed at the Kasetsart University Veterinary Teaching Hospital (KUVTH) between 2016 and 2021 to survey the underlying causes of URT. Then, apart from rhinitis, other causes were excluded to identify the bacterial species and antimicrobial susceptibility. The results indicated that the most frequent finding was neoplasia, followed by rhinitis and anatomical defects. Furthermore, the only significant association was between the age range and disease group, with gender, FIV, or FeLV infection not being significant. Rhinitis was 4.7 times more likely to occur than neoplasia in younger and young adult cats in the age range < 1−3 years compared to the group > 10 years. The main bacterial species was the Pseudomonas species. Antimicrobials with a susceptibility rate of more than 90% were amikacin, gentamicin, ciprofloxacin, norfloxacin, marbofloxacin, imipenem, and meropenem. In conclusion, rhinitis was the second most common chronic URT disease in cats and was more common in younger and young adult cats. The predominant bacteria with AST in this study reflect the antimicrobial resistance situation. Thus, antimicrobial usage should follow antimicrobial use guidelines first.

Pro-inflammatory cytokine release from chicken peripheral blood mononuclear cells stimulated with lipopolysaccharide.

Background and Aim: The principal cytokines released by the host on infection include pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF). These cytokines were regarded as regulators of the host's response to infection. This study aimed to determine the release of pro-inflammatory cytokines from chicken peripheral blood mononuclear cells (PBMCs) following lipopolysaccharide (LPS) stimulation. Materials and Methods: Blood samples were collected from six Betong chickens. To isolate PBMCs, density gradient centrifugation was utilized. PBMC culture in RPMI1640 with 10% fetal bovine serum was stimulated with various concentrations of LPS (0, 0.01, 0.1, and 1.0 µg/mL). The production of TNF-α, IL-1ß, and IL-6 was determined using an enzyme-linked immunosorbent assay. Results: When the PBMCs were cultured for 24 h with varying doses of LPS, there was no significant variation in cell viability. TNF-α, IL-1ß, and IL-6 levels were measured in Betong chicken PBMC. The release of these cytokines increased considerably as LPS concentration (0.01-1 µg/mL) increased (p<0.05). Conclusion: In vitro studies of the chicken immune response, notably the release of pro-inflammatory cytokines, can be conducted using PBMCs obtained from chicken blood.

Effectiveness and Adverse Events of Cyclophosphamide, Vincristine, and Prednisolone Chemotherapy in Feline Mediastinal Lymphoma Naturally Infected with Feline Leukemia Virus.

Feline leukemia virus (FeLV) infection is considered a poor prognostic factor for feline lymphoma. This study investigated the prevalence of cats suffering from feline lymphoma with natural infection of the feline leukemia virus, as well as clinical signs, adverse events, and survival time after cyclophosphamide, vincristine and prednisolone (COP) chemotherapy. This retrospective study involved 92 cats diagnosed with mediastinal or mediastinal plus other anatomical sites of lymphoma and treated with COP chemotherapy. FeLV-antigen-positive was observed in all cats. Clinical signs and adverse events were observed after the 1st, 2nd, and 3rd inductions. Clinical signs improved after the 3rd induction of COP chemotherapy. The response rate was 96.74% (81.52% complete response, 15.22% partial response, and 3.26% no response). The overall median survival time was 338 days (range 62−1057 days). The overall response rate and median survival time of cats with feline lymphoma that were FeLV-antigen-positive and treated with COP chemotherapy were higher than from other studies. This study found that cats aged <4 years survived longer than those aged at least 4 years. Anemia (before COP), azotemia (after 2nd induction), and elevated alanine aminotransferase (after 1st induction) were associated with an increased chance of mortality.

Assessment of chicken peripheral blood mononuclear cells isolated from freshly drawn blood versus 24 h refrigerated blood.

BACKGROUND AND AIM: The peripheral blood mononuclear cell (PBMC) is an excellent cell source for in vitro studies, particularly those involving immunology. The aim of this study was to determine the quality and quantity of chicken PBMCs isolated from freshly drawn blood as well as blood that had been chilled for 24 h. In addition, the survival of PBMCs cultured in medium was investigated. MATERIALS AND METHODS: Blood samples were collected from 12 Betong and 12 Leghorn chickens. Hemograms were analyzed. Density gradient centrifugation was used to isolate PBMCs. PBMCs (2×106 cells/mL) were cultured in a culture medium and incubated in a CO2 incubator for 5 consecutive days. The number of viable cells was determined using the trypan blue dye exclusion method. RESULTS: Blood samples were obtained from healthy chickens. There was no statistically significant difference in the total amount of PBMC between fresh and refrigerated blood samples from both chicken breeds. The viability of PBMCs isolated from fresh blood (95%) was significantly greater than blood refrigerated for 24 h (90-92%) in both breeds. Furthermore, the viability of PBMCs isolated from both blood samples decreased significantly over time, from 90-95% to 60-65%. CONCLUSION: The total number of PBMC in fresh and refrigerated blood was not significantly different. Fresh blood-derived PBMCs had significantly higher viability than 24 h refrigerated blood PBMCs. Furthermore, the viability of PBMCs decreased significantly over time.

Efficacy of cyclic lipopeptides obtained from Bacillus subtilis to inhibit the growth of Microsporum canis isolated from cats.

BACKGROUND AND AIM: Microsporum canis (M. canis) is a dermatophyte fungal pathogen that causes ringworms. Cats are considered to be a dominant reservoir host enabling M. canis transmission to humans. The concerns of dermatophyte resistance were raised among the usage of antifungal drugs to treat the ringworm. This study aimed to evaluate the fungal activity of cyclic lipopeptides (CLPs) obtained from Bacillus subtilis (B. subtilis) as an alternative method for the inhibition of M. canis growth. MATERIALS AND METHODS: The culture plate of M. canis from confirmed cats with ringworm infection was provided. The purification of CLP extract, fengycin, iturin A, and surfactin was carried out from B. subtilis by preparative thin-layer chromatography (PTLC) coupled with solid-phase extraction (SPE) methods. Half-maximal effective concentration (EC50) and agar well diffusion assays were performed to determine the efficacy of Bacillus CLP extract, fengycin, iturin A, and surfactin to inhibit the growth of M. canis isolated from cats. RESULTS: All purified Bacillus substances displayed antifungal activity to control the growth of M. canis when compared with 80% ethanol (control). EC50 values for CLP extract, fengycin, iturin A, and surfactin were 0.23, 0.05, 0.17, and 0.08 mg/mL, respectively. In agar well diffusion assay, the ability of CLP extract, fengycin, iturin A, and surfactin on fungal inhibition had no statistically significant difference at 24 and 48 h after treatment (p < 0.05). However, CLP extract showed a statistically significant difference on M. canis inhibition at 62.21% followed by surfactin with 59.04% at 72 h after treatment. CONCLUSION: In vitro, Bacillus CLPs revealed an inhibitory effect on M. canis growth which is a zoonotic pathogen that causes ringworms. This study suggests an alternative approach to control the growth of M. canis using substances obtained from B. subtilis as a biomedicine agent with antifungal activity.

Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosa.

BACKGROUND AND AIM: Helicobacter pylori is an important pathogen in humans and animals involved in chronic gastritis, leading to the development of gastric cancer. Urease produced by H. pylori is an enzyme that promotes bacterial colonization and can be used clinically as a biomarker of H. pylori infection as part of a rapid urease test (RUT). A test with high specificity (95-100%) would be more convenient and faster than histopathology, bacterial culture, and polymerase chain reaction (PCR). The aim of this study was to develop a simple, cheap, and fast kit for detecting H. pylori infection in the gastric mucosa of canines, which can be used in clinical practice for diagnosing infection with this bacterium. MATERIALS AND METHODS: The RUT assays developed were prepared using 1% agar, 1% sodium phosphate monobasic, and 1% urea followed by the addition of 3% methyl red indicator. The cutoff value of sensitivity of the RUT assay was established using the urease of H. pylori ATCC 43504 and color change was monitored for 24 h. Comparisons of the sensitivity to H. pylori ATCC 43504 were made between the developed RUT assays and the Hp Fast™ commercial kit. Then, the limit of detection for H. pylori ATCC 43504 number was analyzed by the SYBR Green real-time PCR assay to measure the copy number of the ureC gene. Gastric biopsy samples from the antrum, body, and fundus of the stomach were collected from eight canines presenting with vomiting and gastroenteritis. Analyses were performed on fresh samples using the developed RUT assays and the Hp Fast™ commercial kit, which were read within 24 h; then, the results were confirmed with SYBR Green real-time PCR. The specificity of the RUT assays was tested with a number of different bacteria, including Staphylococcus pseudintermedius, Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus spp., Escherichia coli, and Salmonella spp.; H. pylori ATCC 43504 was used as a positive control. RESULTS: The results showed that the developed assays were sensitive to the urease enzyme at 0.1 mg/mL. The lowest detection limit of this assay for H. pylori ATCC 43504 was found to be 102 copies at 30 min. The sensitivity of detection of H. pylori in gastric biopsies of canines occurred in a minimum of 30 min. The RUT showed similar results to the Hp Fast™ commercial kit. In the developed RUT, the color change of the test from red to yellow could be clearly distinguished between the color of the positive test and the negative one; however, in the commercial Hp Fast™, it was difficult to observe the gel color changein the negative pH range of 5.8 and the positive pH of 6.5. The developed RUT was specific for H. pylori and did not detect any of the other tested bacteria. The test kit can also be stored for 6 months at 4°C. CONCLUSION: The sensitivity of the developed assays allowed the detection of urease enzyme at a minimum concentration of 0.1 mg/mL. Our RUT could also detect H. pylori from one in eight canine specimens at a minimum of 102 copies within 30 min. This RUT is specific to H. pylori as it did not detect any of the other tested bacteria.