Impact of intramammary inoculation of inactivated Lactobacillus rhamnosus and antibiotics on the milk microbiota of water buffalo with subclinical mastitis

Abstract

Water buffalo mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies to avoid or reduce antibiotics’ based therapies, in particular during subclinical mastitis. The use of Generally Regarded As Safe bacteria (GRAS) such as Lactobacillus rhamnosus to restore the unbalance in mammary gland microbiota could provide potential corrective measures. The aim of this study was to investigate the changes in milk microbiota after the intramammary treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters naturally affected by subclinical mastitis as compared to antibiotic therapy.A number of 43 quarters affected by subclinical mastitis with no signs of clinical inflammation and aerobic culture positive for pathogens were included in the study. The experimental design was as follows: 11 quarters were treated with antibiotics, 15 with inactivated cultures of Lactobacillus rhmnosus and 17 with PBS as negative control, by means of intrammary injection. Samples were collected at eight time points, pre- (T-29, T-21, T-15, T-7, T0 days) and post- treatment (T1, T2, and T6 days). Microbiological culture and Somatic Cell Count (SCC) were perfomed on all the samples, and microbiota was determined on milk samples collected at T0 and T6 by amplifying the V4 region of 16S rRNA gene by PCR and sequencing using next generation sequencing technique. Treatment with Lactobacillus rhamnosus elicited a strong chemotactic response, as determined by a significant increase of leukocytes in milk, but did not change the microbiological culture results of the treated quarters. For what concerns the analysis of the microbiota, the treatment with Lactobacillus rhamnosus induced the modification in relative abundance of some genera such as Pseudomonas and 5-7N15. As expected, antibiotic treatment caused major changes in microbiota structure with an increase of Methylobacterium relative abundance. No changes were detected after PBS treatment. In conclusion, the present findings demonstrated that the in vivo intrammmary treatment with Lactobacillus rhamnosus has a transient pro-inflammatory activity by increasing SCC and is capable to modify the microbiota of milk after six days from inoculation, albeit slightly, even when the bacterial cultures were heat inactivated. Further studies are necessary to assess the potential use of this GRAS as supportive therapy against mastitis.

Heat inactivation partially preserved barrier and immunomodulatory effects of Lactobacillus gasseri LA806 in an in vitro model of bovine mastitis

“…Inactivated strains have already been assessed in mastitis. One study showed a transient pro-inflammatory effect of intramammary infusion of a heat-inactivated Lactobacillus rhamnosus strain, revealed by an increase in somatic cell count and a slight modification of milk microbiota after six days (Catozzi et al, 2019). However, despite stimulation of the innate immune system, administration of the heatinactivated L. rhamnosus strain did not enhance pathogen clearance in this study.…”

“…However, whereas some studies have reported the need for bacteria to be alive to exert their effects (Aguilar-Toalá et al, 2018;Brodmann et al, 2017;Douillard and Vos, 2019;Terpou et al, 2019), others have demonstrated that viability is not mandatory for all probiotic effects, as not all mechanisms are directly linked to viability, particularly when the effect depends on surface-exposed components (Aguilar-Toalá et al, 2018;Brodmann et al, 2017;Douillard and Vos, 2019;Terpou et al, 2019). In addition, it cannot be denied that non-viable (inactivated) bacteria may have advantages over live bacteria in terms of safety, by reducing the risk of microbial translocation, infection and inflammatory responses (Aguilar-Toalá et al, 2018;Catozzi et al, 2019;Piqué et al, 2019). The use of inactivated bacteria may therefore be safer -particularly in humans or animals with a weak immune system or under inflammatory conditions -and still valuable, provided that the beneficial effects of the bacteria are at least partially preserved.…”

Impact of yeast and lactic acid bacteria on mastitis and milk microbiota composition of dairy cows

“…Higher milk percentage of fat and lactose caused by dietary supplementation of yeast has been reported in dairy goats (Ma et al 2019) and dairy cows (Olagaray et al 2019), while similar study with LAB are rare. Catozzi et al (2019) reported that intramammary incubation of inactivated Lactobacillus rhamnosus caused significant increase of SCC on cows with subclinical at the beginning but decreased afterwards, as well as modulation of milk microbiota. Combined with our study, oral intake of LAB also decreased milk SCC and improved the milk quality in mastitis cows, indicating the relieving effects of LAB on mastitis.…”

Short communication: Milk microbiota profiling on water buffalo with full-length 16S rRNA using nanopore sequencing

“…The milk microbiota in ruminants has already been investigated through massive sequencing methods in cows and water buffaloes (Oikonomou et al, 2012;Quigley et al, 2013;McInnis et al, 2015;Catozzi et al, 2017). The association between milk microbiota and health status is particularly relevant for understanding mastitis, by unravelling previously unreported microorganisms potentially related to mammary gland pathogenesis (Hoque et al, 2019) and assessing the milk microbiota recovery after treatment (Catozzi et al, 2019). Microbiological culture technique is considered the gold standard for identifying pathogens in mastitis; however, bacterial DNA is present in culture-negative samples collected from animals with clinical mastitis (Kuehn et al, 2013).…”

“…DNA extraction was carried out and quality assessed as previously reported, starting from 1 mL of milk (Catozzi et al, 2017). First, V4 16S amplification (about 250 bp) was performed using 515F and R806 primers, as already described (Catozzi et al, 2019). Briefly, the forward and reverse primers were…”