Polyethylenimine mediated recovery of SARS-CoV-2 and total viral RNA: Impact of aqueous conditions on behaviour and recovery.

Wastewater-based epidemiology (WBE) is an emerging, practical surveillance tool for monitoring community levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SC2). However, a paucity of data exists regarding SARS-CoV-2 and viral biomarker behaviour in aqueous and wastewater environments. Therefore, there is a pressing need to develop efficient and robust methods that both improve method sensitivity and reduce time and cost. We present a novel method for SARS-CoV-2, Human Coronavirus 229E (229E), and Pepper Mild Mottle Virus (PMMoV) recovery utilizing surface charge-based attraction via the branched cationic polymer, polyethylenimine (PEI). Initially, dose-optimization experiments demonstrated that low concentrations of PEI (0.001% w/v) proved most effective at flocculating suspended viruses and viral material, including additional unbound SC2 viral fragments and/or RNA from raw wastewater. A design-of-experiments (DOE) approach was used to optimize virus and/or viral material aggregation behaviour and recovery across varying aqueous conditions, revealing pH as a major influence on recoverability in this system, combinatorially due to both a reduction in viral material surface charge and increased protonation of PEI-bound amine groups. Overall, this method has shown great promise in significantly improving quantitative viral recovery, providing a straightforward and effective augmentation to standard centrifugation techniques.

First report of pathogenic Leptospira spp. isolated from urine and kidneys of naturally infected cats.

Leptospirosis is one of the most widespread zoonotic diseases and can infect both humans and animals worldwide. Healthy cat, as a potential source of exposure to humans, are likely underestimated owing to the lack of overt clinical signs associated with Leptospira spp. infection in this species. The aim of the study was to determine the exposure, shedding, and carrier status of leptospires in shelter cats in Malaysia by using serological, molecular, and bacteriological methods. For this study, 82 healthy cats from two shelters were sampled. The blood, urine, and kidneys were tested using the microscopic agglutination test (MAT), polymerase chain reaction (PCR), and bacterial culture. On the basis of serological, molecular, and/or culture techniques, the total detection of leptospiral infection was 29.3% (n = 24/82). Through culture techniques, 16.7% (n = 4/24) of the cats that tested positive were carriers with positive kidney cultures, and one cat was culture positive for both urine and kidney. The Leptospira spp. isolates were identified as pathogenic L. interrogans serovar Bataviae through serological and molecular methods. Through serological techniques, 87.5% (n = 21/24) had positive antibody titers (100-1600) and most of the Bataviae serogroup (n = 19/21). Using PCR, 16.7% (n = 4/24) of cats were shown to have pathogenic Leptospira spp. DNA in their urine. Furthermore, three out of four culture positive cats were serology negative. The present study reports the first retrieval of pathogenic leptospires from urine and kidneys obtained from naturally infected cats. The results provide evidence of the potential role of naturally infected cats in the transmission of leptospires. Additionally, leptospiral infection occurs sub-clinically in cats. The culture isolation provides evidence that healthy cats could be reservoirs of leptospiral infection, and this information may promote the development of disease prevention strategies for the cat population.

Serological Detection of Anti-Leptospira Antibodies in Shelter Cats in Malaysia.

Leptospirosis is one of the most widespread zoonotic diseases and despite extensive research, there is still a paucity of information regarding this disease in cats. The aim of this study was to investigate the prevalence of leptospirosis among the shelter cat population in Malaysia and to determine the most common infective Leptospira serogroups among them. Blood samples were collected from a total of 110 cats from 4 different shelters. The sampled cats appeared healthy, with minimal evidence of feline upper respiratory disease. The Microscopic Agglutination Test was used to detect anti-Leptospira antibodies against 20 pathogenic serovars. Based on a cut-off antibody titer of ≥1:100, 20 of 110 sheltered cats, showed presence of anti-Leptospira antibodies against at least 1 serovar. The serodetection of leptospirosis was 18.18% (95% confidence interval 12.09-26.42). The most commonly detected serogroups were Bataviae, Javanica, and Ballum, with antibody titers ranging from 1:100 to 1:1600. Knowledge of the predominant infective serovars in hosts worldwide and regionally is imperative for understanding the epidemiology of this zoonotic disease. Serosurveillance is the first step in this process. Further studies are warranted for investigation of urinary shedding in naturally infected cats with leptospirosis, using Polymerase chain reaction (PCR) and organism isolation followed by serovars identification.