Novel CRISPR/Cas13-based assay for detection of bovine coronavirus associated with severe diarrhea in calves.

Bovine coronavirus (BCoV) is one of the important causes of diarrhoea in cattle. The virus is responsible for the high fatality rate associated with acute diarrhoea in calves. Rapid and accurate tests need to be conducted to detect the virus and minimise economic losses associated with the disease. Nucleic acid-based detection assays including PCR is an accurate test for detecting pathogens. However, these tests need skilled personnel, time and expensive devices. In this study, we developed a novel assay for the detection of BCoV in clinical cases. This novel assay combined reverse transcription-recombinase polymerase amplification with CRISPR/Cas13 and conducted a rapid visualisation of cleavage activity using a Lateral Flow Device. A conserved sequence of the BCV M gene was used as a target gene and the assays were tested in terms of specificity, sensitivity and time consumption. The result showed the specificity of the assay as 100% with no false positives being detected. Ten copies of the input RNA were enough to detect the virus and perform the assay. It took up to forty minutes for reading the results. Conducted together, the assay should be used as a rapid test to clinically diagnose infectious pathogens including bovine coronavirus. However, the assay needed the RNA to be extracted from the clinical sample in order to detect the virus. Therefore, more studies are needed to optimise the assay to be able to detect the virus in the clinical sample without extracting the RNA.

Staphylococcus aureus FadB is a dehydrogenase that mediates cholate resistance and survival under human colonic conditions.

Staphylococcus aureus is a common colonizer of the human gut and in doing so it must be able to resist the actions of the host's innate defences. Bile salts are a class of molecules that possess potent antibacterial activity that control growth. Bacteria that colonize and survive in that niche must be able to resist the action of bile salts, but the mechanisms by which S. aureus does so are poorly understood. Here we show that FadB is a bile-induced oxidoreductase which mediates bile salt resistance and when heterologously expressed in Escherichia coli renders them resistant. Deletion of fadB attenuated survival of S. aureus in a model of the human distal colon.

Designing a multiepitope mRNA-based vaccine against enterotoxigenic Escherichia coli infection in calves: Immunoinformatics and molecular modeling approach.

Background: Escherichia coli is one of the serious pathogens causing various infections in the animal field, such as neonatal calf diarrhea, which is responsible for mortality associated with diarrhea during the first days of life. Aim: Current work is aimed at designing an effective and safe multiepitope vaccine candidate against E. coli infection in calves based on the fimbrial protein K99 of Enterotoxigenic E. coli (ETEC) and Immuno-informatics. Methods: A conserved sequence of K99 protein was generated, and then highly antigenic, nonallergic, and overlapped epitopes were used to construct a multiepitope vaccine. Five THL, six MHC II, and four beta cell epitopes were targeted to create the candidate. The candidate vaccine was produced utilizing 15 epitopes and three types of linkers, two types of untranslated region (UTR) human hemoglobin subunit beta (HBB), UTR beta-globin (Rabb), and RpfE protein as an immunomodulation adjuvant. Results: Immuno-informatics analysis of the constructed protein showed that the protein was antigenic (antigenic score of 0.8841), stable, nonallergen, and soluble. Furthermore, the Immuno-informatics and physiochemical analysis of the constructed protein showed a stable, nonallergic, soluble, hydrophilic, and acidic PI (isoelectric point). of 9.34. Docking of the candidate vaccine with the toll-like receptor TLR3 was performed, and results showed a strong interaction between the immune receptor and the vaccine. Finally, the expression efficiency of the construct in E. coli was estimated via computational cloning of the vaccine sequence into Pet28a. Conclusion: Results of immunoinformatics and in silico approaches reveal that the designed vaccine is antigenic, stable, and able to bind to the immune cell receptors. Our results interpret the proposed multiepitope mRNA vaccine as a good preventive option against E. coli infection in calves.

Molecular identification of Anaplasma platys in cattle by nested PCR.

Background and Objectives: Anaplasmosis is a zoonotic disease caused by Gram-negative bacterium from Anaplasmataceae family. Anaplasma causes high economic losses worldwide. 16S rRNA analysis was used to diagnose Anaplasma platys in Cattle. Phylogenetic tree and estimation of evolutionary divergence between A. platys isolates were performed. Materials and Methods: A total of 60 blood samples were collected from a cattle farm in AL-Diwaniyah province. 16S rRNA gene was identified using nested PCR. Overall, 40% of cattle that were chosen to collect the blood were identified to be infected with A. platys. Results: The results have shown presence of targeting partial region of 16S rRNA gene in 24 samples out of 60. Sequencing results of 10 samples have revealed that the phylogenetic tree was divided in to two separate clades. Five isolates of A. platys-Iraq (accession no. OP646782, OP646783, OP646784, OP646790, and OP646791) were located in one clade with the A. platys-China (accession no. MN193068.1). While, five isolates (accession no. OP646785, OP646786, OP646787, OP646788, OP646789) were in different clade with two isolates of A. platys-Africa and A. platys-Zambia in distinct branches, close to the Rickettsiales. Conclusion: The phylogenetic study of A. platys sequences indicated that the isolates were collected from a cattle farm in Al-Dewaniyah were similar and close related to A. platys-China, A. platys-Zambia and A. platys-Africa). This study suggests that cattle can be considered a reservoir of A. platys.

Evaluation of efflux pump inhibitory activity of some plant extracts and using them as adjuvants to potentiate the inhibitory activity of some antibiotics against Staphylococcus aureus.

Background: Antibiotic-resistant pathogens became a real global threat to human and animal health. This needs to concentrate the efforts to minimize and control these organisms. Efflux pumps are considered one of the important strategies used by bacteria to exclude harmful materials from the cell. Inhibition of these pumps can be an active strategy against multidrug resistance pathogens. There are two sources of efflux pump inhibitors that can be used, chemical and natural inhibitors. The chemical origin efflux pump inhibitors have many toxic side effects while the natural origin is characterized by a wide margin of safety for the host cell. Aim: In this study, the ability of some plant extracts like (propolis show rosemary, clove, capsaicin, and cumin) to potentiate the inhibitory activity of some antibiotics such as (ciprofloxacin, erythromycin, gentamycin, tetracycline, and ampicillin) against Staphylococcus aureus pathogen were tested. Methods: Efflux pump inhibitory activity of the selected plant extracts was tested using an ethidium bromide (EtBr) accumulation assay. Results: The results have shown that Propolis has a significant synergistic effect in combination with ciprofloxacin, erythromycin, and gentamycin. While it has no effect with tetracycline or ampicillin. Also, no synergic effect was noticed in a combination of the minimum inhibitory concentration for the selected plant extracts (rosemary, clove, capsaicin, and cumin) with any of the tested antibiotics. Interestingly, according to the results of the EtBr accumulation assay, Propolis has potent inhibitory activity against the S. aureus (MRS usa300) pump system. Conclusion: This study suggests that Propolis might act as a resistance breaker that is able to restore the activity of ciprofloxacin, erythromycin, and gentamycin against S. aureus strains, in case of the efflux-mediated antimicrobial resistance mechanisms.