Evidence on the prevalence of emerging and re-emerging tick- and flea-borne rickettsial agents in acute encephalitis syndrome endemic areas of northeast Uttar Pradesh, India.

Outbreaks of acute encephalitis syndrome (AES) with unknown aetiology are reported every year in Gorakhpur district, Uttar Pradesh, India, and Orientia tsutsugamushi, the rickettsial pathogen, responsible for scrub typhus has been attributed as the primary cause of AES problem. However, information on the prevalence of other rickettsial infections is lacking. Hence, this study was carried out to assess any occurrence of tick- and flea-borne rickettsial agents in villages reporting AES cases in this district. In total, 825 peridomestic small mammals were trapped, by setting 9254 Sherman traps in four villages with a trap success rate of 8.9%. The Asian house shrew, Suncus murinus, constituted the predominant animal species (56.2%) and contributed to the maximum number (87.37%) of ectoparasites. In total, 1552 ectoparasites comprising two species of ticks and one species each of flea and louse were retrieved from the trapped rodents/shrews. Rhipicephalus sanguineus, the brown dog tick, was the predominant species retrieved from the trapped rodents/shrews, and the overall infestation rate was 1.75 per animal. In total, 4428 ectoparasites comprising five tick species, three louse species and one flea species were collected from 1798 domestic animals screened. Rhipicephalus microplus was the predominant tick species collected from the domestic animals. The cat flea, Ctenocephalides felis, constituted 1.5% of the total ectoparasites. Of all the ectoparasite samples (5980) from domestic animals and rodents, tested as 1211 pools through real-time PCR assays, 64 pools were positive for 23S rRNA gene of rickettsial agents. The PCR-positive samples were subjected to multi-locus sequence typing (MLST). In BLAST and phylogenetic analysis, the ectoparasites were found to harbour Rickettsia asembonensis (n = 9), Rickettsia conorii (n = 3), Rickettsia massiliae (n = 29) and Candidatus Rickettsia senegalensis (n = 1). A total of 22 pools were detected to have multiple rickettsial agents. The prevalence of fleas and high abundance of tick vectors with natural infections of rickettsial agents indicates the risk of transmission of tick- and flea-borne rickettsial diseases in rural villages of Gorakhpur. Further epidemiological studies are required to confirm the transmission of these agents to humans.

Molecular detection of spotted fever group of Rickettsiae in acute encephalitis syndrome cases from eastern Uttar Pradesh region of India.

The Eastern Uttar Pradesh region of India is known for its endemicity of acute encephalitis syndrome (AES). Decades of research have established that Orientia tsutsugamushi, a causative of scrub typhus, is a substantial contributor (>60%) for the AES cases besides other aetiology, but additional factors in the remaining proportion are still unidentified. Rickettsial infections are challenging to diagnose in clinical settings due to overlapping clinical symptoms, the absence of definitive indicators, a low index of suspicion, and the lack of low-cost, rapid diagnostic tools. Hence, the present study was designed to determine the load of rickettsial infections among AES cases. Furthermore, we aim to find out the prevalent rickettsial species in AES cases as well as in the vector population at this location. The study included the whole blood/cerebrospinal fluid of AES patients and arthropod specimens from rodents. The molecular identification was performed using the 23S-5S intergenic spacer region and ompB gene with genomic DNA obtained from studied specimens. We detected 5.34% (62/1160) of rickettsial infection in AES cases. Among these, phylogenetic analysis confirmed the presence of 54.8% Rickettsia conorii (n = 34) and 16.1% of Rickettsia felis (n = 10), while the rest proportion of the isolates was unidentified at the species level. Furthermore, R. felis was identified in one CSF sample from AES patients and three flea samples from Xenopsylla cheopis. Rickettsia spp. was also confirmed in one Ornithonyssus bacoti mite sample. The results of this investigation concluded the presence of spotted fever group Rickettsia spp. among AES identified cases as well as in the mite and flea vectors that infest rodents.

Molecular Detection and Genetic Characterization of Orientia tsutsugamushi from Hospitalized Acute Encephalitis Syndrome Cases During Two Consecutive Outbreaks in Eastern Uttar Pradesh, India.

Background: Seasonal outbreaks of acute encephalitis syndrome (AES) have been reported especially in the pediatric population with a high case fatality rate in Eastern Uttar Pradesh, India. Orientia tsutsugamushi (OT) is a causative agent of scrub typhus that has been recently identified as a major cause of AES. However, the specific genotypes of OT responsible for AES cases of this region are not known. Therefore, the present study was undertaken to understand the molecular epidemiology of OT prevailing in the AES endemic Eastern Uttar Pradesh region of India. Methods: The study was conducted on 2529 hospitalized AES cases from August 2016 to December 2017. The presence of antibodies against OT from cerebrospinal fluid (CSF) and serum samples were tested using OT IgM enzyme-linked immunosorbent assay (ELISA), whereas OT DNA was tested from whole blood and CSF specimens targeting the partial gene of 56 kDa using nested PCR. Phylogenetic analysis was conducted with sequences (n = 241) generated in this study. Findings: Among the studied AES cases, 50% were found positive for antibodies against OT, whereas 37% of cases were positive for OT DNA. The genetic analysis study revealed that Gilliam (93.8%) is the prevailing genotype of OT followed by Karp (6.16%) genotype in AES cases. Furthermore, the Gilliam strains of this study showed they were >99% identical to earlier reported Gilliam strains from AES cases. Conclusion: We observed the presence of two main OT genotypes in AES cases, among which the majority of OT genotypes fall under the Gilliam clade. The understanding of predominant genotype will be beneficial for its future implications in vaccine development strategies and the development of rapid diagnostic tests.

Acute encephalopathy in a 2-year-old pot-bellied pig following accidental intoxication with clonazepam.

OBJECTIVE: To describe a case of successful management of clonazepam toxicity causing encephalopathy in a pot-bellied pig. CASE SUMMARY: A 2-year-old female pot-bellied pig weighing 13.5 kg was presented for evaluation of clinical signs of acute encephalopathy. Based on the animal's history and clinical signs, a tentative diagnosis of benzodiazepine (BZP) intoxication was made. The results of a urinary drug screening test designed to detect illicit substances in human urine indicated benzodiazepine exposure. Gas chromatography and mass spectrometry analysis later confirmed clonazepam (urinary concentration 496 ng/mL) as the intoxicating substance. The pig responded favorably to treatment which included administration of flumazenil, decontamination with enteral activated charcoal, and intravenous isotonic crystalloid administration. The pig had a rapid improvement in mentation 10 minutes following IV flumazenil administration and was considered mentally appropriate following 24 hours of hospitalization. The pig was discharged from the hospital after 48 hours of care, and was reported to be doing well 6 months later. NEW INFORMATION PROVIDED: Intoxication with prescription benzodiazepines can occur in companion animals and result in clinical signs of acute encephalopathy. Urinary drug screening tests designed for human use may provide rapid results to indicate drug intoxication and guide therapeutic intervention in veterinary species. Administration of flumazenil resulted in a rapid improvement in mentation following clonazepam intoxication in a pot-bellied pig.