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Background: Chikungunya virus (CHIKV) has reemerged as a major public health concern, causing chikungunya fever with increasing cases and neurological complications. Methods: In the present study, we investigated a low-passage human isolate of the East/ Central/South African (ECSA) lineage of CHIKV strain LK(EH)CH6708, which exhibited a mix of small and large viral plaques. The small and large plaque variants were isolated and designated as CHIKV-SP and CHIKV-BP, respectively. CHIKV-SP and CHIKV-BP were characterized in vitro and in vivo to compare their virus production and virulence. Additionally, whole viral genome analysis and reverse genetics were employed to identify genomic virulence factors. Results: CHIKV-SP demonstrated lower virus production in mammalian cells and attenuated virulence in a murine model. On the other hand, CHIKV-BP induced higher pro-inflammatory cytokine levels, compromised the integrity of the blood–brain barrier, and led to astrocyte infection in mouse brains. Furthermore, the CHIKV-SP variant had limited transmission potential in Aedesalbopictus mosquitoes, likely due to restricted dissemination. Whole viral genome analysis revealed multiple genetic mutations in the CHIKV-SP variant, including a Glycine (G) to Arginine (R) mutation at position 55 in the viral E2 glycoprotein. Reverse genetics experiments confirmed that the E2-G55R mutation alone was sufficient to reduce virus production in vitro and virulence in mice. Conclusions: These findings highlight the attenuating effects of the E2-G55R mutation on CHIKV pathogenicity and neurovirulence and emphasize the importance of monitoring this mutation in natural infections. [ABSTRACT FROM AUTHOR]

Background and Aims: Methicillin-resistant Staphylococci (MRS) seriously threatens animal and human health. Repeated antibiotic use allows the bacteria to develop resistance to several antibiotic classes and become multidrug-resistant (MDR). Canine pyoderma, a common skin condition in dogs, is mainly caused by Staphylococci, including MRS. Detecting this infection in all canine populations is crucial to develop a proper preventive plan. This study estimated the prevalence, antibiogram, and risk factors of MRS in canine patients at a referral animal hospital in Khon Kaen, Thailand. Materials and Methods: Skin swabs and relevant information were collected from 56 client-owned dogs that visited the hospital from September 2019 to September 2020. Staphylococci colonies were subjected to molecular identification and antibiotic susceptibility tests using an automated system (VITEK®2). These colonies were also genetically identified using multiplex-polymerase chain reaction (PCR) and sequencing. The mecA gene, encoding methicillin resistance, was detected using simplex-PCR. The risk factors of MRS infection and their association with MRS infection were analyzed using logistic regression and the Chi-square test, respectively. Results: The prevalence of MRS was found to be 35.7% (20/56 dogs). By species, methicillin-resistant Staphylococcus pseudintermedius was found in 24 of 104 isolates (23.1%), and all samples were MDR. Receiving systemic antibiotics in the past 6 months was a major risk factor associated with MRS infection (p < 0.05; odds ratio (OR) > 1). In addition to the MRS isolates, the mecA gene was also detected in methicillin-susceptible Staphylococci isolates. This might be because of the high expression of blaI, and mutations in c-di-AMP cyclase DacA, RelA, and Fem proteins. Conclusion: A high prevalence of MRS and MDR was observed in the studied population, which might be potentially due to improper antibiotic use by the owners and horizontal transfer of drug-resistance genes. [ABSTRACT FROM AUTHOR]

Mycotoxins are toxic, fungal secondary metabolites that contaminate agricultural commodities, food, and feed. Among them, T-2, HT-2, and diacetoxyscirpenol (DAS; the major type A trichothecene) are primarily produced from Fusarium species. These mycotoxins exert numerous toxicological effects in animals and humans, such as dermatotoxicity, haematotoxicity, hepatotoxicity, nephrotoxicity, neurotoxicity, and immunotoxicity. In the present study, human Jurkat T cells were used as a model to investigate apoptotic cell death induced by T-2, HT-2, and DAS. The results showed that T-2, HT-2, and DAS decreased cell viability and increased production of Reactive Oxygen Species in a time- and dose-dependency. Based on their IC 50 values, they could be ranked in decreasing order of cytotoxicity as T-2 > HT-2 > DAS. All tested mycotoxins caused DNA fragmentation, up-regulated cytochrome C, caspase 3, and caspase 9 mRNA levels, and down-regulated the relative expression of Bcl-2 and caspase 8. The effects of these trichothecenes on apoptosis were determined based on flow cytometry. At the IC 50 concentrations, the percentages of apoptotic cells were significantly higher than for the controls. Taken together, these data suggested that T-2, HT-2, and DAS could induce apoptosis through the mitochondrial apoptotic pathway. [Display omitted] • Trichothecene type A induce apoptotic cell death in human Jurkat T cells. • Cell viability, gene expression and protein expression were effected in human Jurkat cells exposed to T-2, HT-2 and DAS. • Trichothecene type A induce apoptosis through the mitochondrial apoptotic pathway. [ABSTRACT FROM AUTHOR]

Aim: This study aims to determine the prevalence of Cryptosporidium spp. infection and to identify the species of Cryptosporidium spp. in newborn dairy calves between December 2016 and March 2017 in Muang District, Khon Kaen Province, Thailand. Materials and Methods: A total of 200 fecal samples from newborn dairy calves of the ages 1 day up to 28 days were collected and the presence of Cryptosporidium oocysts was examined microscopically using the modified Kinyoun's acidfast staining technique. Then, Cryptosporidium species were identified using nested polymerase chain reaction amplification of 18S rRNA gene and sequencing. Results: The modified Kinyoun's acid-fast staining revealed the presence of Cryptosporidium oocysts in 51% (102/200). Sequence analysis of the 18S rRNA gene identified two species, namely, Cryptosporidium bovis (n=11) and Cryptosporidium ryanae (n=11) and one isolated strain could not be identified. Conclusion: This study indicated that newborn dairy calves aging up to 4 weeks were highly infected with Cryptosporidium spp., and the infection mostly occurred in diarrheic dairy calves. This is the first report of Cryptosporidium in dairy calves in Khon Kaen Province and the results provide baseline information for further studies and control of Cryptosporidium infection in dairy calves in the study area. [ABSTRACT FROM AUTHOR]

Abstract Food and feed commodities are often contaminated by more than one mycotoxin. Among the several combinations that frequently occur, fusariotoxins are often mentioned. The multi-mycotoxins contamination is a serious threat for health. In the present study we investigated the toxic interactions between deoxynivalenol (DON), nivalenol (NIV), and fusarenon-X (FX) in Jurkat T cells by using the MTT assay. The tested mycotoxins alone or in combination had a dose dependent effect on proliferating lymphocytes. According to IC 50 , it could be classified in decreasing order of toxicity: FX > NIV > DON > DON + FX > NIV + FX > DON + NIV > DON + NIV + FX. The type of mycotoxin interactions was assessed by calculating the combination index (CI) and the dose reduction index (DRI). Our data indicate that an antagonistic effect was strongly observed in binary combination between DON and NIV at higher concentrations and ternary mixtures. Meanwhile, the DON and FX mixtures generated moderate antagonism, while NIV and FX provoked different interactions. The effects of tested mycotoxins on apoptosis were also determined using a FACScan flow cytometer. At IC 75 , the percentages of apoptotic cells in all treatment groups were significantly different when compared to control group but no significant differences among treatment groups was observed. Taken together, our data suggest that immunotoxicity among multi-mycotoxins contamination cannot be predicted based on individual effects but depends on the mixtures, ratio and/or concentrations in the product, as well as type of target cells. Highlights • The natural co-occurrence of mycotoxins in cereal grains and animal feeds are frequently noted around the world. • DON, NIV, and FX alone or in combination induce cytotoxicity and apoptosis on Jurkat human T cells. • The toxic interaction depends on the mixed of mycotoxin, their ratio and concentrations, as well as type of target cells. [ABSTRACT FROM AUTHOR]

Fusarenon X, a member of the type B trichothecene mycotoxin group, has been frequently observed, along with deoxynivalenol (DON) and nivalenol (NIV) as a contaminant in cereals. Our previous study demonstrated that a 14-day FX exposure caused apoptosis in the lymphoid tissues of mice, especially at 0.5 mg/kg bodyweight. However, the relationship between low concentrations of FX and apoptotic molecular machinery remains unclear. In the present study, we investigated the genetic regulatory mechanisms in the thymus and Peyer's patches of mice after 14 days oral administration of FX at 0.5 mg/kg bodyweight. FX caused the up-regulation of Bax, Bid, Trp53 , and Caspase-9 mRNA but the relative expression of Fas, TNF , and Caspase-8 remained unchanged. Furthermore, we also determined the toxicity of FX in Jurkat T-cells. FX exhibited a concentration- and time-dependent inhibition of cell viability. Thus, incubation time and FX concentration influence the percentage of apoptotic cells. These data suggested that treatment with low dosage of FX can induce apoptosis in lymphocytes through an effect on Bax, Bid, Trp53, and Caspase-9 and therefore the mitochondrial apoptotic pathway. [ABSTRACT FROM AUTHOR]

One hundred and twenty chicken samples from feces (n = 80), the carcass surface at slaughter at 2 meat chicken farms (n = 20), and retail chicken meat from 5 markets (n = 20) collected during 2018 and 2019 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) in Escherichia coli. We detected qnrS -positive E. coli in a total of 74 samples from feces (n = 59), the carcass surface (n = 7), and retail meat (n = 8). These 74 qnrS -positive isolates were tested for antimicrobial susceptibility to determine the minimum inhibitory concentrations (MICs) of certain antimicrobials and genetically characterized. Ampicillin-resistance accounted for 71 of the 74 isolates (96%), followed by resistance to oxytetracycline (57/74; 77%), enrofloxacin (ERFX) (56/74; 76%), sulfisoxazole (SUL) (56/74; 76%), trimethoprim (TMP) (49/74; 66%), and dihydrostreptomycin (48/74; 65%). All farm-borne SUL- and TMP-resistant isolates except one were obtained from samples from farm A where a combination of sulfadiazine and TMP was administered to the chickens. Concentrations of ERFX at which 50 and 90% of isolates were inhibited were 2 μg/mL and 32 μg/mL, respectively. Diverse pulsed-field gel electrophoresis (PFGE) patterns of Xba I-digested genomic DNA were observed in the qnrS -positive isolates from fecal samples. Several isolates from feces and the carcass surface had identical Xba I-digested PFGE patterns. S1-nuclease PFGE and Southern blot analysis demonstrated that 7 of 11 dfrA13 -positive fecal isolates carried both the qnrS and dfrA13 genes on the same plasmid, and 2 of 3 dfrA1 -positive isolates similarly carried both qnrS and dfrA1 on the same plasmid, although the PFGE patterns of Xba I-digested genomic DNA of the isolates were different. These results suggest that the qnrS gene is prevalent in chicken farms via horizontal transfer of plasmids and may partly be co-selected under the use of TMP. [ABSTRACT FROM AUTHOR]

Abstract: Most human enterovirus 71 (HEV71) strains infect only primates and are unable to cause clinically apparent infection in mice. Here we describe a mouse-adapted HEV71 strain that belongs to sub-genogroup B5 with increased virulence in newborn BALB/c mice. The mouse-virulent strain was initially selected by serial passage of a HEV71 clinical isolate (HEV71-B5) in Chinese hamster ovary (CHO) cells (CHO-B5), followed by serial passage in newborn mice. Virus from the fifth mouse passage was cultured twice on Vero cells and designated as MP-B5. MP-B5 induces severe disease of high mortality in newborn mice in a dose-dependent manner. Skeletal muscle is the primary site of virus replication and results in severe myositis. CHO-B5 harbours a single amino acid substitution (K149 →I) in the VP2 capsid protein. Five additional nucleotide sequence changes were identified in MP-B5, two of which are located in the 5′ UTR and the three within the open reading frame (ORF). Two of the ORF mutations resulted in deduced amino acid changes in the capsid protein VP1: S241 →L and K244 →E; the third ORF mutation was a synonymous C→T change at nucleotide position 6072 within the 3D polymerase gene. Infectious cDNA clone-derived mutant virus populations of HEV71 belonging to sub-genogroup B3 (CHO-26M) that contain the VP1 mutations identified in MP-B5 were generated in order to determine the mutation(s) responsible for mouse virulence. Only viruses expressing the VP1 (K244 →E) mutation were virulent in 5-day-old BALB/c mice, indicating that the VP1 (K244 →E) change is the critical genetic determinant of mouse adaptation and virulence in this model. [Copyright &y& Elsevier]

Abstract: We selected Chinese hamster ovary (CHO) cell-adapted strains of human enterovirus 71 (HEV71) belonging to sub-genogroups B5 (HEV71-B5) and C2 (HEV71-C2) by serial passage in CHO cells at a high multiplicity of infection. During the course of CHO cell passage, virus growth improved significantly, with increasing virus titres and the presence of cytopathic effect observed. A study of virus growth kinetics revealed that the CHO cell-adapted strains of HEV71-B5 (CHO-B5) and HEV71-C2 (CHO-C2) grew efficiently in CHO cells with maximum titres >100-fold higher than unadapted parental virus. Both CHO-B5 and CHO-C2 harboured single amino acid mutations within the VP2 capsid protein gene. CHO-B5 has an amino acid substitution of K149 →I in VP2 and CHO-C2 has an amino acid substitution of K149 →M in VP2. An isolate of sub-genogroup C4 (HEV71-C4) failed to adapt to CHO cells during serial passage. Infectious cDNA clone-derived populations of HEV71-C4 containing the mutations K149 →I or K149 →M in VP2 were generated by site-directed mutagenesis. Both mutations resulted in the ability of the virus to replicate efficiently in CHO cells, indicating that amino acid position 149 in VP2 is critical for the adaptation of HEV71 to growth in CHO cells. [Copyright &y& Elsevier]

Human enterovirus 71 (HEV71) is the causative agent of hand, foot, and mouth disease and associated acute neurological disease. At present, little is known about the genetic determinants of HEV71 neurovirulence. Studies of related enteroviruses have indicated that the untranslated regions (UTRs), which control virus-directed translation and replication, also exert significant influence on neurovirulence. We used an infectious cDNA clone of a subgenogroup B3 strain to construct and characterize chimeras with 5′- and 3′-UTR modifications. Replacement of the entire HEV71 5′ UTR with that of human rhinovirus 2 (HRV2) resulted in a small reduction in growth efficiency in cells of both nonneuronal (rhabdomyosarcoma) and neuronal (SH-SY5Y) origin due to reduced translational efficiency. However, the introduction of a 17-nucleotide deletion into the proximal region of the 3′ UTR significantly decreased the growth of HEV71-HRV2 in SH-SY5Y cells. This observation is similar to that made with stem-loop domain Z (SLD Z)-deleted coxsackievirus B3-HRV2 5′-UTR chimeras reported previously and provides the first evidence of a potentially functional SLD Z in the 3′ UTR in human enterovirus A species viruses. We further showed that the cell-specific growth impairment was caused by the synergistic effects of cis-acting UTR control elements on different stages of the virus life cycle. These chimeras will further improve our understanding of the control of HEV71 replication and its relationship to neurovirulence. [ABSTRACT FROM AUTHOR]

Eighty isolates of Riemerella anatipestifer representing 71 outbreaks of riemerellosis in Thailand between 1994 and 1999 were serotyped using the gel diffusion precipitin test. Based on the precipitation patterns, 25 serological profiles containing one to three antigenic determinants were recognized. Heat-stable antigens of the organism reacted with antisera raised against 16 known serotypes and an untypable strain 698/95. The most prevalent serotype appeared to be serotype 7, followed by serotypes 5, 10, 21 and 1. Further study demonstrated that the untypable strain probably represents a new serotype. Analysis of the polymerase chain reaction-amplified rrs genes for restriction fragment length polymorphisms verified the inclusion of strain 698/95 within the species R. anatipestifer and supported earlier work excluding strain 670/89, which had originally been designated the reference strain of serotype 20. Therefore, it is suggested that the strain 698/95 could be adopted as a replacement for the reference strain of serotype 20. Attention should be paid to strains with multiple antigenic factors as they may be useful for the preparation of vaccines. [ABSTRACT FROM AUTHOR]

Lymphocytes are involved in the adaptive immune response and are highly sensitive to type B trichothecenes. In grains and their products, deoxynivalenol (DON) is the most widely distributed trichothecene. It usually co-occurs with other type B members, such as nivalenol (NIV) and fusarenon-X (FX), because they are all produced by the same Fusarium fungi. However, the combined effects of mycotoxins are complex and cannot be predicted based on individual toxicity. Thus, the adverse effects of combined toxins are of increasing concern. The aim of this study was to compare the toxicity to lymphoid tissues of mice of DON alone or mixed with NIV or FX. Forty, 3-week-old male ICR mice were given a single oral administration of a vehicle control, one toxin, binary, or ternary mixtures and then sacrificed at 12 h after exposure. Mice treated with FX alone showed marked nuclear condensation and fragmentation of lymphocytes in the cortical thymus and germinal center of Peyer's patches and spleen. Similarly, these animals clearly displayed TUNEL- and Caspase-3-positive cells in the regions. In contrast, minimal changes were noticed in the lymphoid tissues of mice receiving combined toxins when compared to this toxin alone. In addition, oral exposure to FX alone significantly up-regulated the relative expression of Bax , Caspase-3 , Caspase-9 , and Trp53. These data increase our understanding of the toxic actions of DON, NIV, and FX alone or in combination to lymphocytes and can be used to assess the possible risk associated with their co-occurrences in foodstuffs to human and animal health. • FX alone showed marked apoptotic cell death in the lymphoid tissues of mice after oral administration. • Mycotoxin mixtures are less toxicity to lymphocytes apoptosis than that of individual exposure. • In natural co-occurrence, the combined mycotoxins might be less impact on lymphoid mortality. [ABSTRACT FROM AUTHOR]

Background and Aim: Mastitis is an important disease that can reduce milk production and farmer income as well as negatively affect human health. This study aimed to summarize dairy mastitis in Indonesia, both subclinical mastitis (SCM) and clinical mastitis (CM), and its prevalence in different provinces, the diagnostic methods, and the animal species. Materials and Methods: Relevant studies on mastitis in dairy animals in Indonesia were obtained from PubMed, Scopus, ProQuest, Google Scholar, and Garuda. The title and abstract were screened for the eligibility of the studies. The full text of the selected studies was assessed and the data were extracted for analysis. To determine the pooled estimate of the prevalence of mastitis, a random-effects model was performed using the "Meta" and "Metaphor" packages in the R software version 4.2.2. The heterogeneity of several characteristics (mastitis type, provinces, animal species, and diagnostic methods) was evaluated through subgroup meta-analysis. Meta-regression analysis was conducted to assess the trend of mastitis prevalence reports over time. Publication bias was evaluated using Egger's test and a funnel plot. Results: A total of 735 studies were retrieved for the title and abstract screening, which resulted in the final selection of 37 studies with a total of 6050 samples for meta-analysis. The pooled estimate of mastitis prevalence in dairy animals in Indonesia was 59.44% (95% confidence interval [CI], 52.39%-66.49%). Based on mastitis type, SCM had a significantly higher prevalence than CM (58.24% [95% CI, 51.26%-65.23%] vs. 3.31% [95% CI, 1.42%-5.19%]). No significant difference was observed in the analysis of other subgroups. Among provinces, Central Java had the highest prevalence (66.62% [95% CI, 49.37%-83.87%]), whereas Yogyakarta had the lowest (41.77% [95% CI, 14.96%-68.58%]). Based on animal species, cow and goat had a prevalence of 63.42% (95% CI, 55.97%-70.86%) and 44.96% (95% CI, 28.26%-61.66%), respectively. Based on the diagnostic method, the California mastitis test resulted in 60.08% (95% CI, 52.11%- 68.06%) and the Institut Pertanian Bogor test, 56.00% (95% CI, 41.20%-70.81%). No significant change in the prevalence of mastitis in Indonesia was observed from 2003 to 2022. Conclusion: This study demonstrates that the pooled estimate of mastitis prevalence in dairy animals in Indonesia is >50%. Based on subgroup analysis, SCM had a higher prevalence than CM; however, the prevalence between provinces, detection methods, and animal species in the 2003-2022 periods was not significantly different. A mastitis control strategy needs to be developed to reduce the prevalence of mastitis and further loss in milk production. [ABSTRACT FROM AUTHOR]

Abstract: Human enterovirus 71 (HEV71) has emerged as a major cause of epidemics of hand, foot and mouth disease associated with severe neurological sequelae in the Asia-Pacific region. In this study, a passive protection mouse model was used to evaluate the protective efficacy of formalin-inactivated HEV71 vaccines derived from a Chinese C4 genotype strain. Pregnant mice were immunised using a prime/boost strategy and ≥50U of vaccine protected five-day-old pups from lethal challenge with a mouse-adapted (B3 genotype) strain of HEV71. Immunised mice developed a neutralising antibody response to both the immunising C4 strain and to the mouse-adapted strain. Mice born to immunised dams showed significantly less myositis and reduced viral loads in tissues. [Copyright &y& Elsevier]

Abstract: Human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are two major aetiological agents of hand, foot and mouth disease (HFMD) in children. Recently there have been several large outbreaks of HFMD in Vietnam and the Asia-Pacific region. In this study, a multiplex RT-PCR assay was developed in order to detect simultaneously HEV71, CVA16 and other human enteroviruses. Enterovirus detection was performed with a mixture of three pairs of oligonucleotide primers: one pair of published primers for amplifying all known enterovirus genomes and two new primer pairs specific for detection of the VP1 genes of HEV71 and CVA16. Enterovirus isolates, CVA16 and HEV71 strains identified previously from patients with HFMD were examined to evaluate the sensitivity and specificity of the multiplex RT-PCR assay. The assay was then applied to the direct detection of these viruses in clinical specimens obtained from HFMD cases identified at Children''s Hospital Number 2, Ho Chi Minh City, Vietnam. The multiplex RT-PCR assay showed 100% specificity in screening for enteroviruses and in identifying HEV71 and CVA16. Similar results were obtained when using the multiplex RT-PCR assay to screen for enteroviruses and to identify HEV71 and CVA16 in clinical specimens obtained from HFMD cases identified at the hospital. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of HEV71 or CVA16 infection in cases of HFMD and is also potentially useful for molecular epidemiological investigations. [Copyright &y& Elsevier]