Development of antibodies against recombinant staphylococcal enterotoxin B from food poisoning cases.

Background and Aim: Staphylococcal enterotoxin B (SEB) is the most common serotype involved in food poisoning. The aim of this study was to develop immunoassay detection methods using a recombinant enterotoxin B antigen protein to produce recombinant polyclonal antibodies in vivo. Materials and Methods: Staphylococcus aureus isolated from a food poisoning case (strain JH5800) was analyzed by polymerase chain reaction (PCR) and confirmed to contain a seb gene of 477 bp. A SEB segment was amplified, cloned, sequenced, and aligned. The PCR product corresponding to the predicted mature SEB peptide was inserted into Escherichia coli BL21 (DE-3) expression vector and expressed as a hexahistidine-SEB fusion protein. Antiserum against recombinant SEB protein was produced by immunization of Balb/c mice. Results: In the indirect enzyme-linked immunosorbent assay (ELISA), the polyclonal antibodies produced had a titer of 1:3200. The seb gene of Staphylococcus aureus isolated from a poisoning case (JH5800) had a molecular size of about 477 bp and a band of recombinant SEB toxin was observed at approximately 30 kDa on SDS-PAGE gel. The polyclonal anti-SEB antibody titer, as revealed by indirect ELISA, was 1:3200 at 59 days. Conclusion: SEB recombinant protein could be used to produce polyclonal antibodies. ELISA and Western blotting were used to analyze the specificity and sensitivity of the recombinant polyclonal antibodies. Polyclonal antibodies produced could be used to detect SEB on a large-scale.

Methicillin-resistant Staphylococcus aureus isolates derived from humans and animals in Yogyakarta, Indonesia.

Background and Aim: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) as a highly pathogenic strain in veterinary and human medicine is a growing global problem. This study aimed to evaluate MRSA isolates of human and animal origin against various antibiotics in Yogyakarta, Indonesia. Materials and Methods: The susceptibility test was carried out by the disk diffusion method using Mueller-Hinton agar against nine antibiotic disks. Methicillin-resistant S. aureus strains were genetically confirmed through mecA gene detection encoding for methicillin resistance by polymerase chain reaction. Results: All 240 S. aureus strains isolated from animals and humans were resistant to penicillin G (P) (100% and 99%, respectively), followed by ampicillin (AMP), amoxicillin (AML), oxacillin (OX), erythromycin (E), clindamycin (DA), tetracycline (TE), gentamicin (GEN), and ciprofloxacin (CIP). Eighty-three MRSA strains were resistant to OX (100%), P (100%), AMP (99.27%), AML (95.52%), E (87.77%), TE (71.33%), DA (63.24%), GEN (38.81%), and CIP (26.87%). Conclusion: The antimicrobial resistance pattern of S. aureus human isolates was similar to their animal counterpart, with 77.20% of MRSA strains classified as multidrug-resistant (MDR) bacteria. These findings indicate an increase in MDR S. aureus strains of animal origin in Yogyakarta, thus raising public health concerns about MRSA zoonotic spread.

A G3P[9] rotavirus strain with an unusual genome constellation in a diarrheic cat in Thailand.

Rotavirus infection can cause diarrhea in many animal species. A 2-year-old indoor female Siamese cat with bloody mucoid diarrhea tested positive for rotavirus (RV) group A by real-time reverse transcription polymerase chain reaction (RT-PCR). Subsequent conventional RT-PCR amplification of the 11 RV segments and sequencing revealed a G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3 genome constellation. Phylogenetic analysis showed that the VP4, VP7, NSP1, NSP3, NSP4, and NSP5 genes were closely related to those of human feline-like rotaviruses, while the VP1, VP2, VP3, VP6, and NSP2 genes were genetically closest to those of human bovine-like rotaviruses, suggesting that genetic reassortment had occurred. The uniqueness of this G3P[9] feline rotavirus strain expands our knowledge about feline rotaviruses.

Cellular immune response of Staphylococcus aureus enterotoxin B in Balb/c mice through intranasal infection.

Background and Aim: Staphylococcus aureus produces various superantigen exotoxins, including staphylococcal enterotoxin B (SEB). It causes fatal anaphylactic reactions and toxic shock. This study aimed to evaluate the reaction of leukocytes and histopathological changes in the respiratory organs of Balb/c mice after intranasal infection with enterotoxigenic S. aureus (SEB). Materials and Methods: The presence of the seb gene in S. aureus was established in this study using polymerase chain reaction-specific primer. Two groups of 8-week-old male Balb-c mice consist of six mice in each group. The treated group was infected with 50 µL and 100 µL of SEB intranasal on days 1 and 14, respectively. NaCl was administered in the second group and was considered as a control group. Blood samples were collected through the retro-orbital plexus on days 1, 4, 7, 14, and 22 after infections. Total cell counts were analyzed with an independent sample t-test and compared using the statistical package for the social sciences (SPSS) version 16.0 (IBM Corp., NY, USA). The infected tissues of the respiratory organ were observed descriptively and compared to the control group. Results: The seb gene with a molecular size of 478 bp, indicating the SEB strain, is present in S. aureus used in this study. Intranasal administration of SEB showed increased leukocytes, lymphocytes, monocytes, and eosinophils on day 22 post-infection. Significant leukocytosis was seen on days 6 and 14; lymphocytosis on days 1, 4, 6, and 16; and eosinophilia on days 6, 14, and 22 compared with the control group (p > 0.05). In contrast, the neutrophil decreased after an increase of immature band cells compared to the control group, indicating a severe acute infection with SEB. The lungs and trachea of the test group had an inflammatory cell accumulation in the respiratory organ. Conclusion: Intranasal route infection of S. aureus containing seb gene significantly induced the cellular immune response and caused pathological changes in the respiratory tissues of the Balb/c mice model. The hematological changes were aligned with marked pathological changes in the respiratory tract. Balb/c mice could be an excellent experimental model to study toxic and anaphylactic shock against SEB to define the future therapeutic agents.

Diverse human and bat-like rotavirus G3 strains circulating in suburban Bangkok.

Although rotavirus vaccines are available in many parts of the world and are effective in reducing the overall incidence of rotavirus infection, it remains a major cause of diarrhea in less-developed countries. Among various rotavirus group A (RVA) strains, the increasingly common genotype G3 (defined by the VP7 gene) has been identified in both humans and animals. Our previous epidemiological surveillance in Bangkok found several unusual non-vaccine-like G3 strains in patients with diarrhea. In this study, we sequenced and characterized the genomes of seven of these G3 strains, which formed combinations with genotypes P[4], P[6], P[9], and P[10] (defined by the VP4 gene). Interestingly, we identified a bat-like RVA strain with the genome constellation G3-P[10]-I3-R3-C3-M3-A9-N3-T3-E3-H6, which has not been previously reported in the literature. The amino acid residues deduced from the nucleotide sequences of our G3 strains differed at the antigenic epitopes to those of the VP7 capsid protein of the G3 strain in RotaTeq vaccine. Although it is not unusual for the segmented genomes of RVA to reassort and give rise to emerging novel strains, the atypical G3 strains identified in this study suggest possible animal-to-human RVA zoonotic spillover even in urban areas.

The impact of COVID-19 and control measures on public health in Thailand, 2020.

BACKGROUND: The COVID-19 virus has been an emerging disease causing global outbreaks for over a year. In Thailand, transmission may be controlled by strict measures that could positively and negatively impact physical health and suicidal behavior. METHODS: The incidence of COVID-19 was retrieved from the Department of Disease Control (DDC). The impact of viral diseases was retrieved from the open-source of the DDC and King Chulalongkorn Memorial Hospital. The road accidents data were from the Thai Ministry of Transport. The suicidal behavior data were obtained from the Department of Mental Health. We compared data from the year 2019 with the pandemic COVID-19 outbreak period in 2020, before lockdown, during lockdown, easing, and new wave period using unpaired t-test and least-squares linear regression. We compared the impact of the outbreak on various data records in 2020 with corresponding non-outbreak from 2019. RESULTS: There was a significant decline in cases of influenza (p < 0.001) and norovirus (p = 0.01). However, there was no significant difference in RSV cases (p = 0.17). There was a dramatic increase in attempt to suicides and suicides (p < 0.001). There was no impact on roadside accidents and outpatient department visits. DISCUSSION: The extensive intervention measures during lockdown during the first wave positively impacted total cases for each period for acute respiratory and gastrointestinal tract diseases, car accidents, and injuries and negatively impacted indicators of suicidal behavior. The data support government policies that would be effective against the next outbreak by promoting the "new normal" lifestyle.

Investigation of chlamydophilosis from naturally infected cats.

BACKGROUND: Chlamydophila felis, formerly known as Chlamydia psittaci var. felis, is frequently associated with ocular, respiratory, and occasionally reproduction tract infections. Even though the infection is sometimes asymptomatic, it potentially results in a latent immunosuppressive infection. OBJECTIVE: This study aimed to identify occurrences of feline chlamydophilosis, rarely reported in cats in Indonesia. METHODS: The observation was conducted in three cats with clinical signs of Cp. felis infection, particularly relapsing conjunctivitis. The cats' histories were recorded based on owners' information. Conjunctival swabs were sampled for cytology examination and molecular assay detection. A phylogenetic tree was generated using MEGA-X software to reveal group clustering. A post-mortem examination was performed on the cat that died during an examination. RESULTS: Cp. felis was detected in both cytological examination and polymerase chain reaction assay. The phylogenetic tree demonstrated that the Cp. felis isolated in this study clustered with several other isolates from the other countries. Cp. felis can be isolated from cats with different clinical manifestations and levels of severity. The chronic fatal infection demonstrated interstitial broncho-pneumonia under histopathological examination. CONCLUSIONS: Molecular assay of Cp. felis is always recommended to obtain a definitive diagnosis of feline chlamydophilosis since the disease can have various clinical manifestations. Even though it may be subclinical and is often not fatal, an infected cat may be a carrier that could spread the pathogen in the surrounding environment. Serious disease management is suggested to avoid high costs associated with regularly relapsing disease.

Streptococcus equi subsp. zooepidemicus finding in confirmed feline infectious peritonitis cat patient.

BACKGROUND: Feline infectious peritonitis (FIP) is a fatal immune-mediated disease in cat, caused by mutated feline coronavirus (FCoV). Due to its difficulties in diagnosis, FIP is sometimes underdiagnosed. Therefore, several laboratory procedures were performed to gain high index suspicion of FIP. However, through several laboratory findings, not only FIP but also SEZ infection was confirmed in this case. CASE DESCRIPTION: A-year-old male, domestic cat was admitted to Veterinary Medicine Clinical Pathology Laboratory, Universitas Gadjah Mada, for further effusion examination due to its high suspicion of feline infectious peritonitis (FIP). Further examination using molecular and post-mortem analysis resulted on confirmed SEZ infection and FIP. This study informed the manifestation and pathological changes in patient with SEZ and FIP in the same time. CONCLUSIONS: This study showed that viral infection followed by bacterial infection could be fatal and untreatable. After these findings, clinicians may consider SEZ infection in cat with respiratory disorder followed by thoracic effusion besides FIP. Companion animal, especially outdoor-kept animal, possibly become infected from its contact to another human or animal in the environment.

High prevalence of circulating DS-1-like human rotavirus A and genotype diversity in children with acute gastroenteritis in Thailand from 2016 to 2019.

BACKGROUND: Human rotavirus A (RVA) infection is the primary cause of acute gastroenteritis (AGE) in infants and young children worldwide, especially in children under 5 years of age and is a major public health problem causing severe diarrhea in children in Thailand. This study aimed to investigate the prevalence, genotype diversity, and molecular characterization of rotavirus infection circulating in children under 15 years of age diagnosed with AGE in Thailand from January 2016 to December 2019. METHODS: A total of 2,001 stool samples were collected from children with gastroenteritis (neonates to children <15 years of age) and tested for RVA by real-time polymerase chain reaction (RT-PCR). Amplified products were sequenced and submitted to an online genotyping tool for analysis. RESULTS: Overall, 301 (15.0%) stool samples were positive for RVA. RVA occurred most frequently among children aged 0-24 months. The seasonal incidence of rotavirus infection occurred typically in Thailand during the winter months (December-March). The G3P[8] genotype was identified as the most prevalent genotype (33.2%, 100/301), followed by G8P[8] (10.6%, 32/301), G9P[8] (6.3%, 19/301), G2P[4] (6.0%, 18/301), and G1P[6] (5.3%, 16/301). Uncommon G and P combinations such as G9P[4], G2P[8], G3P[4] and G3P[9] were also detected at low frequencies. In terms of genetic backbone, the unusual DS-1-like G3P[8] was the most frequently detected (28.2%, 85/301), and the phylogenetic analysis demonstrated high nucleotide identity with unusual DS-1-like G3P[8] detected in Thailand and several countries. CONCLUSIONS: A genetic association between RVA isolates from Thailand and other countries ought to be investigated given the local and global dissemination of rotavirus as it is crucial for controlling viral gastroenteritis, and implications for the national vaccination programs.

High prevalence of DS-1-like rotavirus infection in Thai adults between 2016 and 2019.

Rotavirus infection is the most common cause of viral diarrhea in infants and young children but uncommon and usually asymptomatic in adults. In the winter of 2017-2018, a large-scale outbreak of rotavirus in both children and adults was reported in Thailand. The current study focused on the prevalence, genotyping, and molecular characterization of rotavirus infections in Thai adults from July 2016 to December 2019. In 2,598 stool samples collected from adult residents of Bangkok (aged #x2265; 15 years) with acute gastroenteritis, rotavirus was detected via real-time RT-PCR analysis of the VP6 gene. G, P and I genotypes were determined by direct sequencing of VP7, VP4, and VP6 genes, respectively. Our results showed 8.7% (226/2,598) of stool samples were positive for rotavirus. The incidence of rotavirus was high during the winter season of 2017-2018 (17.7%) compared to another studied periods (4.5% between July 2016- October 2017 and 2.8% between March 2018- December 2019). Nucleotide sequencing of VP7 and VP4 revealed G3P[8] as the predominant strain (33.2%,75/226), followed by G9P[8] (17.3%,39/226), and G2P[4] (15.0%,34/226). Uncommon G and P combinations were additionally detected at low frequencies. VP6 sequencing was conducted to discriminate I genotype between the Wa and DS-1 genogroup. The unusual DS-1-like G3P[8] strain was most prevalent amomg rotavirus strains detected in this study (29.6%, 67/226), and the corresponding VP7 sequences showed high nucleotide identity with unusual DS-1-like globally circulating strains. Our study demonstrates that rotavirus outbreaks in adults are attributable not only to high prevalence of RV infection but also the unusual DS-like genogroup. The collective findings reinforce the importance of investigating rotavirus diagnosis in adults suffering from acute gastroenteritis and taking appropriate preventive measures.

Rotavirus infection in children in Southeast Asia 2008-2018: disease burden, genotype distribution, seasonality, and vaccination.

BACKGROUND: Rotaviruses (RVs) are recognized as a major cause of acute gastroenteritis (AGE) in infants and young children worldwide. Here we summarize the virology, disease burden, prevalence, distribution of genotypes and seasonality of RVs, and the current status of RV vaccination in Southeast Asia (Cambodia, Indonesia, Lao People's Democratic Republic, Malaysia, Myanmar, Philippines, Singapore, Thailand, and Vietnam) from 2008 to 2018. METHODS: Rotavirus infection in Children in Southeast Asia countries was assessed using data from Pubmed and Google Scholars. Most countries in Southeast Asia have not yet introduced national RV vaccination programs. We exclude Brunei Darussalam, and Timor Leste because there were no eligible studies identified during that time. RESULTS: According to the 2008-2018 RV surveillance data for Southeast Asia, 40.78% of all diarrheal disease in children were caused by RV infection, which is still a major cause of morbidity and mortality in children under 5 years old in Southeast Asia. Mortality was inversely related to socioeconomic status. The most predominant genotype distribution of RV changed from G1P[8] and G2P[4] into the rare and unusual genotypes G3P[8], G8P[8], and G9P[8]. Although the predominat strain has changed, but the seasonality of RV infection remains unchanged. One of the best strategies for decreasing the global burden of the disease is the development and implementation of effective vaccines. CONCLUSIONS: The most predominant genotype distribution of RV was changed time by time. Rotavirus vaccine is highly cost effective in Southeast Asian countries because the ratio between cost per disability-adjusted life years (DALY) averted and gross domestic product (GDP) per capita is less than one. These data are important for healthcare practitioners and officials to make appropriate policies and recommendations about RV vaccination.