Molecular and epidemiological characterization of Staphylococcus aureus causing bovine mastitis in China.

BACKGROUND: Staphylococcus aureus is one of the most prevalent pathogens in bovine mastitis, which leads to substantial financial losses for the dairy industry. RESULTS: In this study, S. aureus (n = 72) was isolated from 18 dairy farms in 15 provinces across China in 2021. The identification of these isolates at the species level was achieved by employing 16S rRNA sequencing. An isothermal amplification method for auxiliary detection of S. aureus was established, which can be employed not only for laboratory detection but also for point-of-care testing (POCT). Molecular characteristics of S. aureus mastitis in Chinese dairy cows were determined through MLST and spa typing. Finally, methicillin-resistant Staphylococcus aureus (MRSA) and MRSA resistance genes were detected using MIC and PCR amplification techniques. 72 isolates were identified as 12 sequence types (STs) and 7 clonal complexes (CC). ST1/CC1 was the dominant prevalent accounting for 33.3 % of the total, and exhibiting a wide distribution range. In terms of spa types, t114 was the dominant type, accounting for 31.9 % of the total, followed by t529 as the second major type. Four S. aureus strains were classified as MRSA according to their levels of oxacillin resistance (MIC ≥4 µg/mL). Among these four MRSA strains, one of them was found to be mecA positive. However, the presence of drug-resistance genes mecA and mecC was not detected in the remaining three MRSA strains, indicating the possible existence of new resistance genes. CONCLUSIONS: Our study investigated the prevalence of S. aureus mastitis in dairy cows in China, while also examined the molecular characteristics and MRSA strains. This information will help with the clinical monitoring, prevention, and control of S. aureus mastitis in dairy cattle.

Circular replication-associated protein-encoding single-stranded DNA virus with risk of spillover is widely prevalent in domestic animals in China.

Circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses are highly diverse and have a broad range of hosts. In this study, we report the detection of Bo-Circo-like virus AH20-1 in the feces of diarrheal cattle. The virus has a circular genome of 3,912 nucleotides, three major putative open reading frames, and encodes a Rep gene of 310 amino acids. We found that the virus is closely related to the Bo-Circo-like virus CH strain, which belongs to the novel Kirkoviridae family. Furthermore, we conducted a nationwide surveillance program and found that the virus is prevalent in China (23.6%, 205/868), with the BCLa subtype being the predominant strain. Our findings suggest that the virus can infect sheep, highlighting the potential for cross-species transmission. Our pressure analysis indicates that the CRESS-DNA Kirkoviridae family has broad host adaptation, and that selection pressure played an important role in the evolution of its Rep genes. Our study underscores the need for continued epidemiological surveillance of this virus due to its widespread prevalence in our ruminant population and potential for cross-species transmission.

Integrating filter paper extraction, isothermal amplification, and lateral flow dipstick methods to detect Streptococcus agalactiae in milk within 15 min.

Introduction: Mastitis is one of the most serious diseases affecting dairy farming, causing huge economic losses worldwide. Streptococcus agalactiae is the main pathogenic bacterium of contagious mastitis and can deliver a devastating blow to a farm's economy. Rapid detection is the key to disease control. Methods: In this study, a rapid detection method for S. agalactiae was established. This method combines filter paper extraction, multienzyme isothermal rapid amplification (MIRA), and lateral flow dipsticks (LFD). To simplify the extraction procedure, we designed a disposable extraction device (DED). First, DED performance was evaluated by polymerase chain reaction (PCR) and then the lysis formula and extraction time were optimized. Second, this study compared the extraction performance of a filter paper and an automatic nucleic acid extraction instrument. After screening primers, MIRA for S. agalactiae was established and combined with LFD. Specificity and sensitivity were evaluated after optimizing the reaction conditions. Results: The results showed that the lowest extraction line for DED was 0.01-0.001 ng/µl. In the specificity study, 12 different bacteria were tested, and only S. agalactiae was found to be positive. In the sensitivity study, seven dilution gradients were established, and the lowest detection line was 3.52 × 102 CFU/ml. Discussion: In summary, the method established in this study does not require laboratory equipment and is suitable for on-site detection. The entire method takes only 15 min, is low in cost, has high precision and low technical requirements for operators, which is in contrast with the high cost and cumbersome operation of traditional methods, and is suitable for on-site testing in areas with limited facilities.

Thioredoxin reductase from Toxoplasma gondii: an essential virulence effector with antioxidant function.

Thioredoxin reductase (TR) can help pathogens resist oxidative-burst injury from host immune cells by maintaining a thioredoxin-reduction state during NADPH consumption. TR is a necessary virulence factor that enables the persistent infection of some parasites. We performed bioinformatics analyses and biochemical assays to characterize the activity, subcellular localization, and genetic ablation of Toxoplasma gondii TR (TgTR), to shed light on its biologic function. We expressed the TgTR protein with an Escherichia coli expression system and analyzed its enzyme activity, reporting a Km for the recombinant TgTR of 11.47-15.57 µM, using NADPH as a substrate, and 130.48-151.09 µM with dithio-bis-nitrobenzoic acid as a substrate. The TgTR sequence shared homology with that of TR, but lacked a selenocysteine residue in the C-terminal region and was thought to contain 2 flavin adenine dinucleotide (FAD) domains and 1 NADPH domain. In addition, immunoelectron microscopy results showed that TgTR was widely dispersed in the cytoplasm, and we observed that parasite antioxidant capacity, invasion efficiency, and proliferation were decreased in TR-knockout (TR-KO) strains in vitro, although this strain still stimulated the release of reactive oxygen species release in mouse macrophages while being more sensitive to H2O2 toxicity in vitro Furthermore, our in vivo results revealed that the survival time of mice infected with the TR-KO strain was significantly prolonged relative to that of mice infected with the wild-type strain. These results suggest that TgTR plays an important role in resistance to oxidative damage and can be considered a virulence factor associated with T. gondii infection.-Xue, J., Jiang, W., Chen, Y., Gong, F., Wang, M., Zeng, P., Xia, C., Wang, Q., Huang, K. Thioredoxin reductase from Toxoplasma gondii: an essential virulence effector with antioxidant function.