Background: ICMR-Vector Control Research Centre, Puducherry, India, developed two colonies ofAedes aegyptiinfected withwMel andwAlbBWolbaciastrains calledAe. aegypti(Pud) lines for dengue control. The sensitivity ofwMel andwAlbB strains inAe. aegypti(Pud) lines to heat stress was studied.Methods: wMel andwAlbB infected and uninfectedAe. aegyptilarvae (first to fourth instars) were reared in the laboratory to adults at 26 °C, 30 °C, 36 °C and 40 °C constant temperatures and also 26–30 °C, 26–36 °C and 26–40 °C diurnal cyclic temperatures. The adults were tested forWolbachiainfection. Experiments were also carried out rearing the larvae under simulated field conditions in summer (April and June) under sunlight using fully open and half open bowls and also under sunlight and natural shade.Results: At 36 °C and 40 °C constant temperatures, complete larval mortality was observed. At 30 °C and 26 °C, no larval mortality occurred, butWolbachiadensity was relatively low inwMel infected males compared to control (maintained at 26 ± 1 °C). At diurnal cyclic temperature of 26–40 °C,Wolbachiadensity was reduced in males of both the (Pud) lines, but not in females. At 26–36 °C, reduction inWolbachiadensity was observed inwMel males but not inwAlbB males. At 26–30 °C, no significant reduction inWolbachiadensity was observed withwMel andwAlbB strains. In simulated field conditions (April), under sunlight, the daytime water temperature reached a maximum of 35.7 °C in both full and half open bowls. No larval mortality occurred.Wolbachiafrequency and density was reduced inwMel-infectedAe. aegypti(Pud) males from both type of bowls and in females from full open bowls, and inwAlbB males from half open bowls. In June, rearing of larvae under sunlight, the first-instar larvae experienced a maximum daytime water temperature of > 38 °C that caused complete mortality. No larval mortality was observed in bowls kept under shade (< 32 °C).Conclusions: Exposure of larvae to higher rearing temperatures in the laboratory and simulated-field conditions reduced the densities ofwMel andwAlbB strains particularly in males, but the impact was more pronounced forwMel strain. The actual effect of heat stress on the stability of these twoWolbachiastrains needs to be tested under natural field conditions.Graphical

Antimicrobial resistance (AMR) is a global public health concern and needs to be monitored for control. In this study, synanthropic rodents trapped from humans and animal habitats in Puducherry, India, were screened as sentinels for bacterial pathogens of public health importance and antimicrobial resistance spillover. From the trapped rodents and shrews (n = 100) pathogens viz., Staphylococcus sp, E. coli and Salmonella sp were isolated from oropharyngeal and rectal swabs on Mannitol salt, Mac Conkey and Xylose lysine deoxycholate media respectively. The AMR genes in these isolates were screened by PCR. A total of 76, S. aureus and 19, Staphylococcus non aureus were isolated. E. coli was isolated in 89 samples and among the Salmonella sp (n = 59), 16, were S. enteritidis and 29, were S. typhimurium. A total of 46 MRSA isolates with mec A (n = 40) and mec C (n = 6) were detected. Also, 36.84% and 5.3% Staphylococcus non aureus isolates were tested to have mec A and mec C genes. AMR genes encoding ESBL [bla_TEM in 21, bla_SHV in 45 and bla_CTX-M in 11] was tested positive in 77 E. coli isolates. Among, Salmonella isolates 44/45 were screened to have AMR genes [tet in 13, sul3 & sul4 in 20 and qnrA in 11]. Antibiotic sensitivity test confirmed the antimicrobial resistance. Isolation of pathogens of public health importance and demonstration of genetic elements conferring antimicrobial resistance in the synanthropic rodents confirms that they act as reservoirs and appropriate sentinels to monitor AMR spillover in the environment.

Globally, India has a high zoonotic disease burden and lacks surveillance data in humans and animals. Rodents are known reservoirs for many zoonotic diseases and their synanthropic behavior poses a great public health threat. In this study, trapped rodents/shrews from randomly selected villages within Puducherry, India, and their ectoparasites were screened for zoonotic pathogens, namely, Orientia tsutsugamushi, other pathogenic rickettsiae, Leptospira spp., Cryptosporidium spp., Coxiella burnetii and methicillin-resistant Staphylococcus aureus (MRSA) using conventional PCR. A total of 58 rodents/shrews were trapped from 11 villages. The species trapped were Suncus murinus (49/58, 84.48%), Rattus rattus (8/58, 13.79%) and Rattus norvegicus (1/58, 1.72%). All ectoparasites collected were identified as mites and its infestation rate was 46.55% (27/58). Real-time PCR targeting the 47 kDa gene of O. tsutsugamushi revealed positivity in one rodent and one shrew (3.45%) and two mite pools (7.41%). Conventional PCR targeting the 56 kDa gene revealed positivity in one shrew and two mite pools and the phylogenetic analysis of all three amplicons indicated the circulation of the Gilliam-related serotype. MRSA was detected in the alimentary tract of a shrew (1/32, 3.13%). Leptospira spp., Rickettsia, Cryptosporidium spp. and Co. burnetii tested negative. The detection of zoonotic pathogens within reservoir hosts and vectors poses a risk of transmission to humans. This study signifies the need for zoonotic pathogen surveillance in synanthropic rodents/shrews.

Background: Scrub typhus (ST), is a vector borne zoonotic disease, transmitted by the larva of the trombiculid mites. The enzootic cycle of the pathogen involves rodents/shrews as the animal reservoirs and humans are the accidental dead-end host. A transposon-like activity in its major antigen 56 kDa, has led to the evolution of several serotypes/strains, and more than 40 serotypes are reported globally. Puducherry, India, is endemic to scrub, but limited data exist on local serotype distribution across hosts and vectors.Methodology: A longitudinal molecular surveillance was conducted in Puducherry to investigate the genetic diversity of Orientia tsutsugamushi among humans, animal reservoirs, and vectors. Samples from febrile patients, trapped rodents/shrews, and their infesting mites were screened using real-time and nested PCR. Serotype analysis was performed by partial amplification and sequencing of the 56 kDa gene, followed by phylogenetic, pairwise genetic distance and amino acid analysis.Result: ST infection was detected in 4.37% (95% CI: 3.05–5.71%) of human, 11.52% (95% CI: 8.6–14.4%) of rodent/shrew, and 2.36% (95% CI: 0.95–4.87%) of mite samples. Karp-like (51.72%) and Gilliam-like (41.38%) strains were predominant in both humans and animal hosts, with pairwise genetic distance (85%) analysis revealing a close relationship between the strains identified across the region. Notably, the only mite pool that tested positive for the 56 kDa gene, along with a shrew, was identified to belong to the TA678-like serotype (6.90%), which has not been previously reported from Puducherry.Conclusion: This study provides molecular evidence of the enzootic maintenance and active human transmission of O. tsutsugamushi in Puducherry, with multiple co-circulating serotypes. The first detection of the TA678-like strain in the region suggests the possible introduction of new strains and underscores the need to monitor for strain-specific clinical manifestations in future studies.

Scrub typhus is a re-emerging disease caused by Orientia tsutsugamushi, transmitted by mites belonging to the family Trombiculidae. Humans and rodents acquire the infection by the bite of larval mites/chiggers. Suncus murinus, the Asian house shrew, has been reported to harbor the vector mites and has been naturally infected with O. tsutsugamushi. The present study aimed to localize and record O. tsutsugamushi in the tissues and the host response in shrews naturally infected with O. tsutsugamushi. Sheehan’s modified May–Grunwald Giemsa staining was carried out in 365 tissues from 87 animals, and rickettsiae were documented in 87 tissues from 20 animals. Immunohistochemical (IHC) staining, using polyclonal antibodies raised against selected epitopes of the 56-kDa antigen, was carried out, and 81/87 tissue sections were tested positive for O. tsutsugamushi. By IHC, in addition to the endothelium, the pathogen was also demonstrated by IHC in cardiomyocytes, the bronchiolar epithelium, stroma of the lungs, hepatocytes, the bile duct epithelium, the epithelium and goblet cells of intestine, the tubular epithelium of the kidney, and splenic macrophages. Furthermore, the pathogen was confirmed by real-time PCR using blood (n = 20) and tissues (n = 81) of the IHC-positive animals. None of the blood samples and only 22 out of 81 IHC-positive tissues were tested positive by PCR. By nucleotide sequencing of the 56-kDa gene, Gilliam and Karp strains were found circulating among these animals. Although these bacterial strains are highly virulent and cause a wide range of pathological alterations, hence exploring their adaptive mechanisms of survival in shrews will be of significance. Given that the pathogen localizes in various organs following a transient bacteremia, we recommend the inclusion of tissues from the heart, lung, intestine, and kidney of reservoir animals, in addition to blood samples, for future molecular surveillance of scrub typhus.