Influence of sampling technique and bedding type on the milk microbiota: Results of a pilot study

Abstract

The objective of this pilot study was to evaluate the influence of sampling technique and exposure to different bedding types on the milk microbiome of healthy primiparous cows. Primiparous Holstein cows (n = 20) with no history of clinical mastitis or monthly somatic cell counts >150,000 cells/mL were selected for this study. From each enrolled cow, a composite milk sample was aseptically collected from all 4 mammary quarters (individual quarter somatic cell counts <100,000 cells/mL), 1 individual quarter milk sample was collected using conventional aseptic technique, and 2 individual quarter milk samples were collected directly from the gland cistern using a needle and vacuum tube. All milk samples were cultured using standard milk microbiological techniques and DNA was extracted. Extracted DNA was subjected to PCR and next-generation sequencing for microbiota determination. All samples yielded relatively little total DNA. Amplification of PCR was successful in 45, 40, and 83% of composite, conventional, and cisternal samples, respectively. Bacteria were successfully cultured from 35% of composite milk samples but from none of the quarter milk samples collected using conventional or cisternal sampling techniques. Bacterial DNA sequences were assigned to operational taxonomic units (OTU) based on 97% sequence similarity, and bacterial richness and diversity were determined. Most samples were dominated by low-prevalence OTU and of the 4,051 identified OTU, only 14 were prevalent at more than 1% each. These included bacteria typically recovered from environmental sources. Chao richness was greatest in composite samples and was 636, 347, and 356 for composite, conventional quarter, and cisternal milk samples, respectively. Shannon diversity was similar among sample types and ranged from 3.88 (quarter) to 4.17 (composite). Richness and diversity did not differ by bedding type among cisternal samples, but the power of this pilot study was limited due to small sample size. Despite the small sample size, for milk samples collected from the gland cistern, overall bacterial community composition differed among bedding types. These results demonstrate that sampling technique and bedding type may be associated with the microbiota detected in bovine milk, and we suggest that these variables should be considered in designing and reporting studies about the milk microbiota.

Milk Microbiota: What Are We Exactly Talking About?

“…Whether microbiota can be associated with milk inside the mammary gland remains to be determined. A recent study based on metataxonomics reported the existence of microbiota associated with bovine milk collected by direct sampling into the cistern using a needle and vacuum tube, and this was observed for healthy quarters of the mammary gland with low somatic cell counts (<100,000 cells/ml) (Metzger et al, 2018a). Its composition differed from the microbiota associated with milk collected by conventional septic techniques.…”

“…Considering this major bias of molecular approaches, methods have been developed to selectively amplify DNA of the intact fraction of the population, based on the use of propidium monoazide (Erkus et al, 2016). Moreover, even within these metataxonomics-based studies, several factors will introduce some variability: the milk fraction used, the lysis method (enzymatic, mechanical, or both), the purification kit, the target gene (or region) used to generate amplicons, the number of PCR cycles, PCR primers, and also the sequencing platform (Tremblay et al, 2015;Fouhy et al, 2016;Knudsen et al, 2016;Castelino et al, 2017;Rintala et al, 2017;Lima et al, 2018;Metzger et al, 2018a; Supplementary Tables S1-S4). This results in variation in overall coverage and in differential amplification efficiencies between taxa as illustrated by in silico PCR on a 16S rRNA gene database (Klindworth et al, 2013) and experimental validation on mock communities (Tremblay et al, 2015;Fouhy et al, 2016).…”

“…However, these geographical differences could be due to several factors, such as environment, diet, or even genetics. In bovines, while the influence of "geography" per se has not been studied, milk microbiota was found to be related to the farm environment and management practices (Doyle et al, 2017;Derakhshani et al, 2018a;Frétin et al, 2018;Metzger et al, 2018a). Metzger et al (2018a) reported a relation between the bedding material and the bovine milk microbiota profiles.…”

“…In bovines, while the influence of "geography" per se has not been studied, milk microbiota was found to be related to the farm environment and management practices (Doyle et al, 2017;Derakhshani et al, 2018a;Frétin et al, 2018;Metzger et al, 2018a). Metzger et al (2018a) reported a relation between the bedding material and the bovine milk microbiota profiles. Likewise, milking practices including premilking teat preparation treatment were reported to affect milk microbiota (Doyle et al, 2017).…”

Diversity and temporal dynamics of primate milk microbiomes

“…However, compelling evidence has shown that colostrum and breast milk contain a microbiome. For instance, milk has a distinct bacterial community from other maternal and infant body sites (Biagi et al, ; Pannaraj et al, ), diverse bacterial communities can still be recovered when using extreme sterile technique to collect samples (Metzger et al, ), and the bacterial community diminishes sharply after weaning (Fernández et al, ). Bacteria likely colonize breast milk through retrograde flow bringing infant saliva into the mammary gland (Cabrera‐Rubio et al, ; Hunt et al, ) and/or through entero‐mammary trafficking (EMT; Perez et al, ; Stagg, Hart, Knight, & Kamm, ).…”