Antibacterial Efficacy of Neorautanenia brachypus (Harms) CA Sm (Zhombwe) in Management of Mastitis in Dairy Cows

Abstract

The control of bacterial mastitis in dairy farming has raised attention due to the growing threat of antibiotic resistance. Some herbal plants have been documented as having medicinal properties making them potential alternatives. However, their antimicrobial potential against bovine mammary pathogens is not well studied. The present study aimed at investigating efficacy of Neorautanenia brachypus, a medicinal herbal plant, in the management of mastitis in dairy cows. To achieve this, different plant parts; seeds, leaves, brown tuber and white tuber were extracted using 10 different solvents. The percentage yield and qualitatively screening of the extracts for presence of phytochemicals was done to inform further tests. Further analysis was done to quantify total phenolic content (TPC) and total flavonoid content using the Folin-Ciocalteu reagent and the aluminium chloride colorimetric method respectively. The screened extracts were evaluated for their antimicrobial activity against Staphylococcus aureus and Escherichia coli (ATCC and mastitis clinical strains) using agar well diffusion method (AWDM). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the plant extracts were determined using microbroth dilution and planting method respectively. The findings made room for further exploration of extracts from the fresh and dried plant extracted as crude methanolic and three phytochemicals; alkaloids, saponins and flavonoids obtained from selective extraction. These were evaluated for yield, phytochemical confirmatory tests and antibacterial activity of mastitis field strains. The field strains were obtained from 4 different farms (n=73) and isolated using microbiological techniques followed by profiling for antimicrobial resistance (AMR) using 7 different antibiotics. The strains were also evaluated for their susceptibility to the tuber methanolic extract using AWDM, MIC and MBC using methods described earlier. The determined MIC was used to evaluate the in vitro mechanism of action (MOA) of the tuber methanolic extract based on the kill kinetics, membrane permeability, nucleic acid and protein leakage assay. The tuber methanolic extract was evaluated for cytotoxicity and compounds present using gas chromatography-mass spectrometry (GC-MS). The well evaluated methanolic extract was used to formulate an oil based intramammary infusion. The infusion was evaluated for stability using the accelerated and real time condition. The organoleptic and physicochemical parameters, antimicrobial efficacy and microbial quality were evaluated at set timelines. The infusion was also evaluated for efficacy in a clinical trial using 15 lactating dairy cows screened and found to have mastitis. The treatments for the trial were the infusion at 3 doses, infusion at 10 doses and a known commercial infusion. The efficacy was evaluated based on the reduction in somatic cell counts (SCC), change in milk vii quality and re-isolation of mastitis bacteria post treatment over 35 days. The findings showed that the yield using different solvent was in the order seeds>leaves>brown tuber> white tuber based on the mean rank scores with water giving the highest yield. However, there was no significant difference between the brown and the white tubers. The TPC and TFC quantification showed the tubers were the least in content. The results however showed that yield does not translate to high antibacterial potency. The combination of the tuber and methanol had higher antibacterial activity showing significant differences (P < 0.05) with extracts from the seeds and leaves. This could be attributed to the tuber extracts having more of the bioavailable active compounds to suppress microbial growth. The crude methanolic extracts exhibited to be low in yield when compared to the selectively isolated saponins and alkaloids but had the highest antibacterial potency against the multi-drug-resistant field strains; S. aureus and E. coli. The strains were regarded as multidrug resistant as they were only susceptible to 2 out of 7 antibiotics tested. When evaluated for MOA, the methanol extract showed to have bactericidal properties, membrane disruption activity causing leakage of electrolytes, nucleic acids and proteins in a dose dependant manner where higher doses caused the highest effect. The high activity in methanol is likely because of the high solvating power allowing extraction of diverse bioactive compounds in a bioavailable form. The cytotoxicity test showed that the extract was safe because of very insignificant haemolysis to red blood cells. The GC-MS results showed presence of compounds with antibacterial properties but a greater number of the compounds have not been recently characterised as isolated compounds. The extract was therefore a good candidate and was used in formulating oil based intramammary infusion which was very stable throughout the 24 months period.The infusion also performed well as it showed reduction in SCC over the 35 days with no difference (P > 0.05) between treatments. The milk was also evaluated to be good quality. The quality of the infusion is likely to have been maintained by the choice of unreactive reagents and aided by the preservative. The antibacterial activity displayed shows the presence of stable bioactive compounds in the tuber extracts. In conclusion, N. brachypus is a good candidate for combating mastitis in dairy cows. It is important to increase the clinical trial population with varied cases of mastitis to strengthen efficacy data. Also, research on methods of scaling production is valuable.