Evaluation of a novel multiplex qPCR method for rapid detection and quantification of pathogens associated with calf diarrhoea.

AIMS: Diarrhoea is a common health problem in calves and a main reason for use of antimicrobials. It is associated with several bacterial, viral and parasitic pathogens, most of which are commonly present in healthy animals. Methods, which quantify the causative agents, may therefore improve confidence in associating a pathogen to the disease. This study evaluated a novel commercially available, multiplex quantitative polymerase chain reaction (qPCR) assay (Enterit4Calves) for detection and quantification of pathogens associated with calf-diarrhoea. METHODS AND RESULTS: Performance of the method was first evaluated under laboratory conditions. Then it was compared with current routine methods for detection of pathogens in faecal samples from 65 calves with diarrhoea and in 30 spiked faecal samples. The qPCR efficiencies were between 84%-103% and detection limits of 100-1000 copies of nucleic acids per sample were observed. Correct identification was obtained on 42 strains of cultured target bacteria, with only one false positive reaction from 135 nontarget bacteria. Kappa values for agreement between the novel assay and current routine methods varied between 0.38 and 0.83. CONCLUSION: The novel qPCR method showed good performance under laboratory conditions and a fair to good agreement with current routine methods when used for testing of field samples. SIGNIFICANCE AND IMPACT OF STUDY: In addition to having fair to good detection abilities, the novel qPCR method allowed quantification of pathogens. In the future, use of quantification may improve diagnosis and hence treatment of calf diarrhoea.

A bioinformatic approach to identify core genome difference between Salmonella Pullorum and Salmonella Enteritidis In silico screening of natural compounds against COVID-19 by targeting Mpro and ACE2 using molecular docking

S. Pullorum and S. Enteritidis are closely related in genetic terms, but they show very different pathogenicity and host range. S. Enteritidis infects many different hosts, usually causing acute gastroenteritis, while S. Pullorum is restricted to avian, where it causes systemic disease in young animals. The reason why they differ in host range and pathogenicity is unknown. The core-genome denotes those genes that are present in all strains within a clade, and in the present work, an automated bioinformatics workflow was developed and applied to identify core-genome differences between these two serovars with the aim to identify genome features associated with host specificity of S. Pullorum. Results showed that S. Pullorum unique coding sequences (CDS) were mainly concentrated in three regions not present in S. Enteritidis, suggesting that such CDS were taken up probably during the separation of the two types from their common ancestor. One of the unique regions encoded Pathogenicity Islands 19 (SPI-19), which encodes a type VI secretion system (T6SS). Single-nucleotide polymorphism (SNP) analysis identified 1791 conserved SNPs in coding sequences between the two serovars, including several SNPs located in a type IV secretion system (T4SS). Analyzing of 100 bp regions upstream of coding sequences identified 443 conserved SNPs between the two serovars, including SNP variations in type III secretion system effector (T3SE). In conclusion, this analysis has identified genetic features encoding putative factors controlling host-specificity in S. Pullorum. The novel bioinformatic workflow and associated scripts can directly be applied to other bacteria to uncover the genome difference between clades. OBJECTIVE: Currently, Coronavirus COVID-19 is spreading worldwide very rapidly and its control is very difficult because there is no effective vaccine or drugs available in markets. This virus can infect both animals and people and cause illnesses of the respiratory tract. WHO has declared Coronavirus as pandemic and the whole world is fighting against Coronavirus. Globally, more than 199,478 people have been diagnosed with COVID-19. As of March 18, 2020, more than 167 countries have been affected and more than 8000 deaths have been reported. The main country being affected is China followed by Italy, Iran, Spain, France, and the USA. MATERIALS AND METHODS: Since there are no effective drugs available against Coronavirus, we conducted virtual screening of phytochemicals to find novel compounds against this virus. Hence, we created a phytochemical library of 318 phytochemicals from 11 plants which have been reported as antiviral, antibacterial and antifungal activity. The phytochemical library was subjected to virtual screening against molecular targets;Main protease (Mpro) and Angiotensin-Converting Enzyme 2 (ACE2). RESULTS: Top 10 compounds were selected from each target which had better and significantly low binding energy as compared to the reference molecule. CONCLUSIONS: Based on the binding energy score, we suggest that these compounds can be tested against Coronavirus and used to develop effective antiviral drugs.