Significance: There is an urgent need for new antibiotics active against resistant bacterial pathogens like Pseudomonas aeruginosa . Target-directed drug development provides a potential path to such drugs, and essential gene products represent potential targets. Accordingly, the work reported here defines a highly verified set of such functions for P. aeruginosa required for growth under a variety of different conditions.

Burkholderia pseudomallei is the causative agent of melioidosis, an overwhelming, rapidly fatal septic infection, and B. thailandensis is a closely related, less virulent species. Both organisms are naturally competent for DNA transformation, and this report describes a procedure exploiting this property for the rapid generation of marked deletion mutations by using PCR products. The method was employed to create 61 mutant strains. Several selectable elements were employed, including elements carrying loxP and FRT recombinase recognition sites to facilitate resistance marker excision. Chromosomal mutations could also be transferred readily between strains by transformation. The availability of simple procedures for creating defined chromosomal mutations and moving them between strains should facilitate genetic analysis of virulence and other traits of these two Burkholderia species.

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.

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.

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.

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.