Sero-epidemiological investigation and cross-neutralization activity against SARS-CoV-2 variants in cats and dogs, Thailand.

Epidemiological data on SARS-CoV-2 infection in companion animals have been thoroughly investigated in many countries. However, information on the neutralizing cross-reactivity against SARS-CoV-2 variants in companion animals is still limited. Here, we explored the neutralizing antibodies against SARS-CoV-2 in cats and dogs between May 2020 and December 2021 during the first wave (a Wuhan-Hu-1-dominant period) and the fourth wave (a Delta-dominant period) of the Thailand COVID-19 outbreak. Archival plasma samples of 1,304 cats and 1,795 dogs (total = 3,099) submitted for diagnosis and health checks were collected at the Prasu-Arthorn Veterinary Teaching Hospital, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom. A microneutralization test was used to detect neutralizing antibodies against the ancestral Wuhan-Hu-1 and the Delta variants. A plasma sample with neutralizing titers ≥10 was considered positive. Our results showed relatively low seroprevalence with seropositive samples detected in 8 out of 3,099 individuals (0.26, 95% CI 0.11-0.51%). Among these cases, SARS-CoV-2 neutralizing antibodies from both the ancestral Wuhan-Hu-1 and the Delta variants were found in three out of eight cases in two cats (n = 2) and one dog (n = 1). Furthermore, neutralizing antibodies specific to only the ancestral Wuhan-Hu-1 variant were exclusively found in one cat (n = 1), while antibodies against only the Delta variant were detected in four dogs (n = 4). Additionally, the neutralizing cross-activities against SARS-CoV-2 variants (Alpha, Beta, and Omicron BA.2) were observed in the seropositive cats with limited capacity to neutralize the Omicron BA.2 variant. In summary, the seropositivity among cats and dogs in households with an unknown COVID-19 status was relatively low in Thailand. Moreover, the neutralizing antibodies against SARS-CoV-2 found in the seropositive cats and dogs had limited or no ability to neutralize the Omicron BA.2 variant. Thus, monitoring SARS-CoV-2 infection and sero-surveillance, particularly in cats, is imperative for tracking virus susceptibility to the emergence of new SARS-CoV-2 variants.

Isolation and characterisation of a novel Silviavirus bacteriophage promising antimicrobial agent against methicillin-resistant Staphylococcus aureus infections.

The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) emphasises the urgent need for novel antimicrobial agents as alternatives to antibiotics. Bacteriophage therapy is one of the most promising antimicrobial strategies. Here, we isolated and comprehensively characterized a novel Staphylococcus phage, vB_SauM_VL10 (VL10), from urban sewage. The VL10 genome displays 141,746 bp of linear double-stranded DNA, containing 193 open reading frames and lacking tRNA, virulence, or antibiotic resistance genes. Phylogenetic analysis categorizes VL10 as a novel species within the Silviavirus genus, Twortvirinae subfamily. VL10 exhibits lytic behaviour characterized by efficient adsorption, a short latent period, and substantial burst size, with environmental stability. It demonstrates lytic activity against 79.06% of tested S. aureus strains, highlighting its species specificity. Additionally, VL10 effectively targets MRSA biofilms, reducing biomass and viable cells. In MRSA-infected G. mellonella larvae, VL10 enhances survival rates, supporting its potential for phage therapy applications. Moreover, the emergence of VL10-resistant S. aureus strains associated with fitness trade-offs, including reduced growth, biofilm formation, and virulence. Altogether, these findings emphasize VL10 as a promising candidate for developing therapeutic agents against MRSA infections, providing insights into phage biology and resistance dynamics.

Diversity and genetic characterization of Chlamydia isolated from Siamese crocodiles (Crocodylus siamensis).

Chlamydiosis, an infection caused by several Chlamydia species, has been reported in Nile, saltwater, and Siamese crocodiles. Despite its widespread reports in various countries, including Thailand, genetic information on Chlamydia species remains limited. This study presents a whole-genome-based characterization of Siamese crocodile-isolated Chlamydia. The results showed that Siamese crocodile Chlamydia contained a single circular chromosome with a size of 1.22-1.23 Mbp and a plasmid with a size of 7.7-8.0 kbp. A plasmid containing eight coding sequences (CDSs) was grouped in a ß lineage. A chromosome sequence had approximately 1,018-1,031 CDSs. Chlamydial factors involving virulence were documented in terms of the presence of cytotoxins and several virulence factors in the chromosomes of Siamese crocodile Chlamydia. The analysis of antimicrobial resistance genes in the Chlamydia genome revealed that the most common resistance genes were associated with aminoglycosides, fluoroquinolones, macrolides, tetracyclines, and cephalosporins, with loose matching (identities between 21.12 % and 74.65 %). Phylogenetic analyses, encompassing the assessments of both whole proteome and nine taxonomic markers, revealed that Siamese crocodile Chlamydia was separated into three lineages (lineages I-III) with high bootstrapping statistic support. Interestingly, isolate 12-01 differed genetically from the others, suggesting that it is a new member of Chlamydia. The study findings indicate that Siamese crocodiles are susceptible hosts to Chlamydia, involving more than one species. This study is the first employing the highest number of whole-genome data on Siamese crocodile Chlamydia and provides better insights into pathogen genetics.

Epidemiology of Chlamydia sp. infection in farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

BACKGROUND: Although Chlamydia sp. causes widespread disease outbreaks in juvenile crocodiles in Thailand, data regarding the epidemiology, and risk factors of such infections are limited. The aim of this study was to investigate the prevalence and possible risk factors associated with Chlamydia sp. infections on Siamese crocodile (Crocodylus siamensis) farms in Thailand. A cross-sectional study was conducted from July to December 2019. Samples were collected from 40 farms across six regions in Thailand. Conjunctival, pharyngeal, and cloacal swab samples were analyzed for Chlamydiaceae nucleic acids using semi-nested PCR followed by phylogenetic analysis based on the ompA gene fragment. Risk factors of infection were analyzed using chi-square and univariate regression to calculate odds ratios. RESULTS: The prevalence of Chlamydia sp. infection across all regions was 65%. The ompA phylogenetic analysis showed that Chlamydia sp. detected in this study was genetically closely related to Chlamydia crocodili and Chlamydia caviae. The risk factors for infection were water source, reusing treated wastewater from the treatment pond, not disposing of leftover food, low frequency of water replacement in the enclosure of juvenile crocodiles, and lack of water replacement after the death of a crocodile. CONCLUSION: The prevalence of Chlamydia sp. infection in farmed crocodiles in Thailand was 65% during the study period. Cloacal swabs were superior to conjunctival and pharyngeal swabs due to their higher sensitivity in detecting Chlamydia sp., as well as their lower invasiveness. Good management and biosecurity in crocodile farming can reduce the risk of Chlamydia sp.

A novel virulent Litunavirus phage possesses therapeutic value against multidrug resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a notable nosocomial pathogen that can cause severe infections in humans and animals. The emergence of multidrug resistant (MDR) P. aeruginosa has motivated the development of phages to treat the infections. In this study, a novel Pseudomonas phage, vB_PaeS_VL1 (VL1), was isolated from urban sewage. Phylogenetic analyses revealed that VL1 is a novel species in the genus Litunavirus of subfamily Migulavirinae. The VL1 is a virulent phage as no genes encoding lysogeny, toxins or antibiotic resistance were identified. The therapeutic potential of phage VL1 was investigated and revealed that approximately 56% (34/60 strains) of MDR P. aeruginosa strains, isolated from companion animal diseases, could be lysed by VL1. In contrast, VL1 did not lyse other Gram-negative and Gram-positive bacteria suggesting its specificity of infection. Phage VL1 demonstrated high efficiency to reduce bacterial load (~ 6 log cell number reduction) and ~ 75% reduction of biofilm in pre-formed biofilms of MDR P. aeruginosa. The result of two of the three MDR P. aeruginosa infected Galleria mellonella larvae showed that VL1 could significantly increase the survival rate of infected larvae. Taken together, phage VL1 has genetic and biological properties that make it a potential candidate for phage therapy against P. aeruginosa infections.

Serological and Molecular Surveillance for SARS-CoV-2 Infection in Captive Tigers (Panthera tigris), Thailand.

Coronavirus disease (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Given the emergence of SARS-CoV-2 variants, continuous surveillance of SARS-CoV-2 in animals is important. To monitor SARS-CoV-2 infection in wildlife in Thailand, we collected 62 blood samples and nine nasal- and rectal-swab samples from captive tigers (Panthera tigris) in Ratchaburi province in Thailand during 2020-2021. A plaque reduction neutralization test (PRNT) was employed to detect SARS-CoV-2 neutralizing antibodies. A real-time RT-PCR assay was performed to detect SARS-CoV-2 RNA. Our findings demonstrated that four captive tigers (6.5%, 4/62) had SARS-CoV-2 neutralizing antibodies against Wuhan Hu-1 and the Delta variant, while no SARS-CoV-2 RNA genome could be detected in all swab samples. Moreover, a low-level titer of neutralizing antibodies against the Omicron BA.2 subvariant could be found in only one seropositive tiger. The source of SARS-CoV-2 infection in these tigers most likely came from close contact with the infected animals' caretakers who engaged in activities such as tiger petting and feeding. In summary, we described the first case of natural SARS-CoV-2 infection in captive tigers during the COVID-19 outbreak in Thailand and provided seroepidemiological-based evidence of human-to-animal transmission. Our findings highlight the need for continuous surveillance of COVID-19 among the captive tiger population and emphasize the need to adopt a One Health approach for preventing and controlling outbreaks of COVID-19 zoonotic disease.

Coding-Complete Genome Sequence of a Lumpy Skin Disease Virus Isolated during the 2021 Thailand Outbreak.

Lumpy skin disease (LSD) is an economically devastating and transboundary disease in cattle. Here, we report the coding-complete genome sequence of the LSDV72/PrachuapKhiriKhan/Thailand/2021 strain, which was isolated from an affected cow during the first LSD outbreak in Thailand in 2021. The sequence will be beneficial for future genomic studies of the virus.

Molecular detection and characterization of lumpy skin disease viruses from outbreaks in Thailand in 2021.

Lumpy skin disease (LSD) is one of the most important transboundary and emerging diseases in cattle. The disease causes significant economic losses in animal production and trade worldwide. The first LSD outbreak was recorded in March 2021, at Roi-Et province in the northeastern region of Thailand. Thereafter, the disease had rapidly spread into neighbouring provinces and throughout the country. The aim of the present study was to provide information regarding to the molecular detection and characterization of LSD viruses from outbreaks in Thailand in 2021. There were 1,748,112 susceptible and 604,404 affected animals (n = 588,512 [36.30%], beef cattle; n = 12,367 [15.74%], dairy cattle and n = 3524 [7.35%], buffaloes). The morbidity and mortality rates were 34.57% and 3.47%, respectively, and the case fatality rate was 10.05% (60,713 deaths). Based on real-time polymerase chain reaction results, the p32 gene of LSD virus (LSDV) was detected more frequently in skin nodule samples (54/77, 70.13%) than in nasal swabs (26/55, 42.57%) and EDTA blood (16/77, 20.78%) samples. Moreover, the copy number of the p32 gene was higher in skin nodule samples than in nasal swab and EDTA blood samples (cycle threshold value = 21.94 ± 0.62 vs. 31.52 ± 0.66 and 34.27 ± 0.32, respectively). Furthermore, 29 (53.70%) of 54 capripoxvirus-positive skin nodule samples were successfully isolated from Madin-Darby bovine kidney cells, and the cytopathic effect was observed 72 h after inoculation. Based on the phylogenetic trees of the GPCR, ANK and RPO30 gene sequences, the LSDV isolates from Thailand were distinct from both the LSDV-field and LSDV-vaccine groups and were closely correlated with the LSDV strains isolated from mainland China, Hong Kong territory and Vietnam in 2020. Additionally, they could be a potential virulent vaccine-recombinant LSDV strain.

Evidence of Influenza A Virus Infection in Cynomolgus Macaques, Thailand.

Little is known about the ecology of influenza A virus (IAV) in nonhuman primates (NHPs). We conducted active surveillance of IAV among 672 cynomolgus macaques (Macaca fascicularis) living in 27 free-ranging colonies in Thailand between March and November 2019. A hemagglutination inhibition (HI) assay was employed as the screening test against 16 subtypes of avian influenza virus (AIV) and two strains of the H1 subtype of human influenza virus. The serum samples with HI titers ≥20 were further confirmed by microneutralization (MN) assay. Real-time RT-PCR assay was performed to detect the conserved region of the influenza matrix (M) gene. The seropositive rate for subtypes of IAV, including AIV H1 (1.6%, 11/672), AIV H2 (15.2%, 102/672), AIV H3 (0.3%, 2/672), AIV H9 (3.4%, 23/672), and human H1 (NP-045) (0.9%, 6/672), was demonstrated. We also found antibody against more than one subtype of IAV in 15 out of 128 positive tested sera (11.7%). Moreover, influenza genome could be detected in 1 out of 245 pool swab samples (0.41%). Evidence of IAV infection presented here emphasizes the role of NHPs in the ecology of the virus. Our findings highlight the need to further conduct a continuous active surveillance program in NHP populations.

Serologic evidence of pandemic (H1N1) 2009 virus infection in camel and Eld's deer, Thailand.

BACKGROUND AND AIM: The pandemic (H1N1) 2009 influenza (H1N1pdm09) virus has affected both human and animal populations worldwide. The transmission of the H1N1pdm09 virus from humans to animals is increasingly more evident. Captive animals, particularly zoo animals, are at risk of H1N1pdm09 virus infection through close contact with humans. Evidence of exposure to the H1N1pdm09 virus has been reported in several species of animals in captivity. However, there is limited information on the H1N1pdm09 virus infection and circulation in captive animals. To extend the body of knowledge on exposure to the H1N1pdm09 virus among captive animals in Thailand, our study investigated the presence of antibodies against the H1N1pdm09 virus in two captive animals: Camelids and Eld's deer. MATERIALS AND METHODS: We investigated H1N1pdm09 virus infection among four domestic camelid species and wild Eld's deer that were kept in different zoos in Thailand. In total, 72 archival serum samples from camelid species and Eld's deer collected between 2013 and 2014 in seven provinces in Thailand were analyzed for influenza antibodies using hemagglutination inhibition (HI), microneutralization, and western blotting (WB) assays. RESULTS: The presence of antibodies against the H1N1pdm09 virus was detected in 2.4% (1/42) of dromedary camel serum samples and 15.4% (2/13) of Eld's deer serum samples. No antibodies were detected in the rest of the serum samples derived from other investigated camelids, including Bactrian camels (0/3), alpacas (0/5), and llamas (0/9). The three positive serum samples showed HI antibody titers of 80, whereas the neutralization titers were in the range of 320-640. Antibodies specific to HA and NP proteins in the H1N1pdm09 virus were detected in positive camel serum samples using WB. Conversely, the presence of the specific antibodies in the positive Eld's deer serum samples could not be determined using WB due to the lack of commercially labeled secondary antibodies. CONCLUSION: The present study provided evidence of H1N1pdm09 virus infection in the captive dromedary camel and Eld's deer in Thailand. Our findings highlight the need for continuous surveillance for influenza A virus in the population of dromedary camels and Eld's deer. The susceptible animal populations in close contact with humans should be closely monitored. Further study is warranted to determine whether Eld's deer are indeed a competent reservoir for human influenza virus.

Coinfection of Chlamydia spp. and herpesvirus in juvenile farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

BACKGROUND AND AIM: For a decade, chlamydial and herpesvirus infections have caused significant morbidity and mortality in farmed crocodiles. In September 2017, a total of 160 juvenile freshwater Siamese crocodiles (Crocodylus siamensis) with conjunctivitis/pharyngitis lesions were admitted at the Veterinary Aquatic Animal Research Health Care Unit, Faculty of Veterinary Science, Mahidol University. All crocodiles did not respond well to antibiotics or supportive treatments and died. This study aimed to detect and identify the causative agents associated with conjunctivitis/pharyngitis and fatal outcomes in juvenile farmed Siamese crocodiles. MATERIALS AND METHODS: A total of 138 pharyngeal and conjunctival swabs and conjunctival scrapes were collected from live crocodiles. All swab and scrape samples were DNA-extracted and amplified by polymerase chain reaction (PCR) using Chlamydiaceae- and herpesvirus-specific primers. Tissue samples (brain, lung, liver, heart, spleen, and intestine) were collected from two representative postmortem animals. All tissue samples were processed for molecular and pathological analyses. RESULTS: PCR examinations identified chlamydial and herpesvirus DNA in 92% (126/138) and 100% (138/138), respectively, of the tested swab and scrape samples. Of those positive samples, 79% (26/33), 67% (4/6), and 98% (97/99) of the pharyngeal swabs, conjunctival swabs, and conjunctival scrapes, respectively, were positive for both chlamydial and herpesvirus DNA. Histopathological examination indicated necrosis and mononuclear cell infiltration in the liver, kidney, and intestine of the affected animals. The intracytoplasmic accumulation of Chlamydia was randomly observed in the examined tissue sample. Moreover, the presence of chlamydial and herpesvirus DNA was also detected in the tissue samples, including the heart, intestine, brain, lung, liver, and spleen, of the affected animals by PCR. Phylogenetic analyses revealed that Chlamydia spp. detected in the juvenile Siamese crocodiles was notably different from other known species in the Chlamydia genus, while the herpesvirus detected in the crocodiles was closely related to crocodyline herpesvirus 1. CONCLUSION: Based on histopathological and molecular examinations, this report provided the first evidence of coinfection of Chlamydia spp. and crocodyline herpesvirus 1 in juvenile Siamese crocodiles in Thailand.

A new species of Chlamydia isolated from Siamese crocodiles (Crocodylus siamensis).

Chlamydia is a known pathogen in both saltwater and freshwater crocodiles. However, the exact species/strain has not been clearly identified. In this study, we successfully cultivated Siamese crocodile Chlamydia in McCoy cells at a temperature of 30°C. Electron microscopy; phylogeny based on nine conserved taxonomically informative markers, on ompA, or on seven housekeeping genes; and whole-genome sequencing and analysis of the isolate confirmed the identity of the isolate as a new member of the genus Chlamydia, a new species that we name Chlamydia crocodili.

Human, animal, water source interactions and leptospirosis in Thailand.

In Thailand, leptospirosis is primarily associated with those who work in agricultural occupations. Leptospirosis control is hampered by a poor understanding of the complex interactions between humans, animal reservoirs, Leptospira, and the variable spatial environment in which these factors coexist. We aimed to address key knowledge gaps concerning leptospirosis disease dynamics and the human-animal-water-source interface in two high-risk areas in Thailand. We conducted a cross-sectional survey among 746 study participants in two high-risk areas for leptospirosis in Thailand: Sisaket (SSK) and Nakhon Si Thammarat (NST). Interactions among humans, animals and water sources were quantified and analyzed. The presence of different animal species and thus contact patterns were different in NST and SSK. The consumption of water from the shared sources between the two areas was different. Those whose occupations were related to animals or environmental water and those who consumed water from more than two sources were more likely to have been infected with leptospirosis, with adjusted odds ratios 4.31 (95% CI 1.17-15.83) and 10.74 (95% CI 2.28-50.53), respectively. Understanding specific water-source sharing networks and human-animal contact patterns is useful when designing national and area-specific control programmes to prevent and control leptospirosis outbreaks.

Potentially Pathogenic Leptospira in the Environment of an Elephant Camp in Thailand.

Leptospira is the causative agent of leptospirosis, a globally emerging zoonotic disease. The infection is commonly acquired through contact with the contaminated environment. To extend the knowledge on environmental source of leptospirosis, we investigated the presence of Leptospira in an elephant camp setting where the interaction between humans, animals, and the shared environment occur particularly when engaging in recreational activities. In this study, a total of 24 environmental samples were collected from an elephant camp area in western Thailand. All samples were processed for Leptospira isolation using the EMJH medium. The identification of Leptospira species was carried out by partial 16S rRNA and secY gene sequencing. Of those 24 samples, 18 samples (75%) were culture-positive for Leptospira. The recovered leptospires were mostly derived from water and soil sampled from a river and a mud pond, the main areas for recreational activities. The majority of the isolates were classified into "Pathogens" clade (89%, 16/18) and more than half of the isolates (61%, 11/18) contained species of the "Saprophytes" clade. Notably, two soil isolates from the river beach sampling area were found to contain leptospiral DNA with high similarity to the pathogenic L. interrogans and L. santarosai. The evidence of diverse Leptospira species, particularly those belonging to the "Pathogens" clade, suggest that the shared environments of an elephant camp can serve as potential infection source and may pose a risk to the elephant camp tourists and workers.

Functional redundancy of Burkholderia pseudomallei phospholipase C enzymes and their role in virulence.

Phospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Burkholderia pseudomallei, the causative agent of melioidosis, possesses at least three plc genes (plc1, plc2 and plc3). We found that in culture medium plc1 gene expression increased with increasing pH, whilst expression of the plc3 gene was pH (4.5 to 9.0) independent. Expression of the plc2 gene was not detected in culture medium. All three plc genes were expressed during macrophage infection by B. pseudomallei K96243. Comparing B. pseudomallei wild-type with plc mutants revealed that plc2, plc12 or plc123 mutants showed reduced intracellular survival in macrophages and reduced plaque formation in HeLa cells. However, plc1 or plc3 mutants showed no significant differences in plaque formation compared to wild-type bacteria. These findings suggest that Plc2, but not Plc1 or Plc3 are required for infection of host cells. In Galleria mellonella, plc1, plc2 or plc3 mutants were not attenuated compared to the wild-type strain, but multiple plc mutants showed reduced virulence. These findings indicate functional redundancy of the B. pseudomallei phospholipases in virulence.

Optimization of Culture Protocols to Isolate Leptospira spp. from Environmental Water, Field Investigation, and Identification of Factors Associated with the Presence of Leptospira spp. in the Environment.

The successful culture of Leptospira spp. from the environment is challenging. Here, we optimized the isolation of Leptospira spp. from water samples spiked with different species and initial concentrations of this organism. The time periods between water sampling and the isolation process were varied (0, 2, and 4 weeks). Bacterial cultures were observed under a microscope, and cultures were graded for cell density, weekly, for 12 weeks. Most pathogenic Leptospira spp. were difficult to culture under all conditions. All conditions of water samples spiked with novel species of Leptospira subclade P1 were culture positive within 2 weeks. For Leptospira subclade P2, storing samples for 2 weeks prior to isolation resulted in more successful isolation compared with isolation after other storage conditions. For subclade S1, all samples with initial bacterial concentrations of more than 103 colonies/mL, under all storage conditions, were successfully cultured. These results suggest that storing contaminated water samples for 2 to 4 weeks in the dark at an ambient temperature prior to culturing can improve the isolation of Leptospira spp. from the samples. We implemented this protocol and collected water samples from natural sources accessed by both humans and animals. Leptospira spp. was identified in 32% (35/109) of water samples. The animal species using a water source influenced the likelihood of water samples being contaminated with Leptospira spp. Cultures of Leptospira spp. from environmental samples can provide useful information for understanding the complex interactions between humans, animals and the environment in the transmission of leptospirosis.

Serological evidence of influenza virus infection in captive wild felids, Thailand.

Influenza virus is known to affect wild felids. To explore the prevalence of influenza viruses in these animal species, 196 archival sera from 5 felid species including Panthera tigris (N=147), Prionailurus viverrinus (N=35), Panthera leo (N=5), Pardofelis temminckii (N=8) and Neofelis nebulosa (N=1) collected between 2011 and 2015 in 10 provinces of Thailand were determined for the presence of antibody to avian and human influenza viruses. Blocking enzyme-linked immunosorbent (ELISA) assay and hemagglutination inhibition (HI) assay were employed as the screening tests, which the serum samples with HI antibody titers ≥20 were further confirmed by cytopathic effect/hemagglutination based-microneutralization (CPE/HA-based microNT) test. Based on HI and microNT assays, the seropositive rates of low pathogenic avian influenza (LPAI) H5 virus, highly pathogenic avian influenza (HPAI) H5 virus and human H1 virus were 1.53% (3/196), 2.04% (4/196) and 6.63% (13/196), respectively. In addition, we also found antibody against both LPAI H5 virus and HPAI H5 virus in 2 out of 196 tested sera (1.02%). Evidences of influenza virus infection were found in captive P. tigris in Kanchanaburi, Nakhon Sawan and Ratchaburi provinces of Thailand. The findings of our study highlights the need of a continuous active surveillance program of influenza viruses in wild felid species.

A Novel Genotype of Leptospira interrogans Recovered from Leptospirosis Outbreak Samples from Southern Thailand.

We performed Leptospira culture analysis of 76 clinical samples collected from animals and of six soil samples for the investigation of a leptospirosis outbreak in southern Thailand in 2017. Leptospires were recovered from a kidney sample (a fatal canine leptospirosis case) and from all the soil samples. Next, 16S rRNA sequence analysis demonstrated that the clinical isolate was closely related to the pathogenic L. interrogans, whereas the soil isolates represented different species, including pathogenic L. ellisii, intermediate L. wolffii, and nonpathogenic L. yanagawae. Multilocus sequence typing identified an isolate of L. interrogans as a novel sequence type (ST263), suggesting that the causative agent of the canine leptospirosis in the southern Thailand outbreak has a unique genetic profile.

Potentially Pathogenic Leptospira Species Isolated from a Waterfall in Thailand.

We collected water and soil samples from a waterfall in Thailand to investigate the presence of potentially pathogenic Leptospira. Isolation of Leptospira from all the 17 environmental samples was successful. Based on 16S rRNA gene sequence analysis, a diverse group of Leptospira species was recovered from waterfall samples including 2 pathogenic species (Leptospira alstonii [5/17, 29%] and Leptospira kmetyi [1/17, 6%]); 1 intermediate species (Leptospira wolffii [9/17, 53%]); and 2 non-pathogenic species (Leptospira meyeri [1/17, 6%] and Leptospira idonii [1/17, 6%]). The high prevalence of pathogenic and intermediate Leptospira indicates that a waterfall may serve as a natural reservoir of possible pathogens of leptospirosis.

General and condition-specific essential functions of Pseudomonas aeruginosa.

The essential functions of a bacterial pathogen reflect the most basic processes required for its viability and growth, and represent potential therapeutic targets. Most screens for essential genes have assayed a single condition--growth in a rich undefined medium--and thus have not distinguished genes that are generally essential from those that are specific to this particular condition. To help define these classes for Pseudomonas aeruginosa, we identified genes required for growth on six different media, including a medium made from cystic fibrosis patient sputum. The analysis used the Tn-seq circle method to achieve high genome coverage and analyzed more than 1,000,000 unique insertion positions (an average of one insertion every 6.0 bp). We identified 352 general and 199 condition-specific essential genes. A subset of assignments was verified in individual strains with regulated expression alleles. The profile of essential genes revealed that, compared with Escherichia coli, P. aeruginosa is highly vulnerable to mutations disrupting central carbon-energy metabolism and reactive oxygen defenses. These vulnerabilities may arise from the stripped-down architecture of the organism's carbohydrate utilization pathways and its reliance on respiration for energy generation. The essential function profile thus provides fundamental insights into P. aeruginosa physiology as well as identifying candidate targets for new antibacterial agents.