The critical need for surveillance of SARS-CoV-2 variants of concern has prompted the development of methods that can track variants in wastewater. Here, we develop and present an open-source method based on allele-specific RT-qPCR (AS RT-qPCR) that detects and quantifies the B.1.1.7 variant, targeting spike protein mutations at three independent genomic loci that are highly predictive of B.1.1.7 (HV69/70del, Y144del, and A570D). Our assays can reliably detect and quantify low levels of B.1.1.7 with low cross-reactivity, and at variant proportions down to 1% in a background of mixed SARS-CoV-2. Applying our method to wastewater samples from the United States, we track the occurrence of B.1.1.7 over time in 19 communities. AS RT-qPCR results align with clinical trends, and summation of B.1.1.7 and wild-type sequences quantified by our assays matches SARS-CoV-2 levels indicated by the U.S. CDC N1 and N2 assays. This work paves the way for AS RT-qPCR as a method for rapid inexpensive surveillance of SARS-CoV-2 variants in wastewater.

Mastitis in dairy ruminants is considered to be the most expensive disease to farmers worldwide. Recently, the intramammary infusion of lactic acid bacteria has emerged as a potential new alternative to antibiotics for preventing and treating bovine mastitis. In this study we have investigated in vitro the probiotic potential of Lactococcus lactis LMG 7930, a food-grade and nisin-producing strain, against mastitis-causing pathogens. We have characterized its carbohydrate fermentation and antibiotic susceptibility profiles, cell surface properties and antimicrobial activity, as well as its capabilities to adhere to and inhibit the invasion of pathogens into the bovine mammary epithelial cell line BME-UV1d. We found that L. lactis LMG 7930 was sensitive to tested drugs, according to the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), and showed an improved carbohydrate fermentation capacity compared to starter strains. Moreover, the strain exhibited antagonistic properties towards many of the pathogens tested. It presented medium surface hydrophobicity, a low basic property and no electron acceptor capability. It showed low auto-aggregation and no co-aggregation abilities towards any of the tested pathogens. The strain was one of the most adhesive to bovine mammary epithelial cells among tested bacteria, but its internalisation was low. The strain did not affect significantly pathogen invasion; however, a trend to decrease internalization of some pathogens tested was observed. In conclusion, our results suggest that this strain might be a promising candidate for the development of new strategies of mastitis control in ruminants. Future investigations are needed to evaluate its safety and efficacy under field conditions.

Arboviruses are viral pathogens transmitted by blood-borne vectors that impose a great social and economic burden globally. Most clinical surveillance of arbovirus outbreaks underestimates the true prevalence as a large proportion of cases exhibit no or only mild clinical symptoms (i.e., are subclinical). Reports of urinary shedding of several arboviruses such as Dengue (DENV), yellow fever (YFV), and Zika (ZIKV) viruses suggest the possibility of utilizing wastewater surveillance to assess the prevalence of arboviral outbreaks. To determine the feasibility of wastewater surveillance, we investigated the decay of representative arboviruses (including DENV-2, DENV-3, YFV, and ZIKV) along with murine hepatitis virus (MHV) as a surrogate for human coronavirus within a wastewater matrix at 6, 25, or 37 °C using RT-qPCR. DENV-2, DENV-3, YFV, ZIKV, and MHV experienced a one log₁₀ reduction within 3.95–6.21 days at 25 °C and within 2.60–5.12 days at 37 °C, while incubation at 6 °C did not indicate substantial decay within 21 days. Our work suggests that these arboviruses or their RNA could persist sufficiently in wastewater over a range of temperatures, supporting the potential for wastewater-based surveillance of arboviral outbreaks.