Tavola rotonda: Biodiversità e resistenza alla mastite nelle bovine da latte MICROBIOTA DEL LATTE E BIODIVERSITA Paola Cremonesi IBBA-CNR cremonesi@ibba.cnr.it
starting from. INTRODUCTION Microbiota: community of living microrganisms that inhabits a specific environment Microbiome: the collective genome of microbiota Dysbiosis: alteration of a healthy microbiota
What is metagenomics? INTRODUCTION Metagenomics is the study of genomic DNA obtained from microorganisms that cannot be cultured in the laboratory Metagenomic analysis is... A culture-independent technique Targeted on bacterial DNA A high-throughput sequencing technology A technological advance
Genomics versus Metagenomics INTRODUCTION Time consuming Laborious Expensive 1 colony 1 analysis 1 bacterial identification 20 colonies 20 analyses 20 bacterial identifications... Bacterial diversity profile Over 5.000 identifications amongst the most important populations
INTRODUCTION Addis et al., Mol. BioSyst., 2016, 12, 2359
Animal and microbiota INTRODUCTION Animals host on their skin gut oro-pharyngeal urinary, and genital tracts a wide diversity of microbial communities that have evolved with them because of complex and mutualistic interactions, playing crucial roles in their biology and health. Recently, the mammary gland, previously considered as a sterile organ, has also been included among these sites.
Mammary gland and microbiota INTRODUCTION There are commensal microbial communities within the mammary gland The milk microbiota composition depends on the composition of microbial ecosystems in direct contact with the milk (teat canal, milking machine, bedding material, faeces, feed, drinking and washing water, milker) Several studies also support the hypothesis that the presence of bacteria in milk is not only the result of external colonization Some authors described the ability of some microbes to move from intestinal lumen to the mammary gland throughout an enteromammary pathway Addis et al., Mol. BioSyst., 2016, 12, 2359
Most studies on the dairy ruminant milk microbiota have focused on INTRODUCTION how the microbial flora changes during food production and its impact on milk quality, product maturation, flavor, taste, texture development and product shelf life the impact of different dairy cattle diets on milk microbial communities how the milk microbiota changes during mastitis or following antimicrobial treatment, and the effects on milk microbiota of different therapy conditions during the dry period
Livestock animal Human milk INTRODUCTION Healthy and mastitic cow or buffalo
some problems INTRODUCTION Sample collection: quantity, first streams, teat end cleaning, healthy and/or mastitic quarters DNA extraction protocol: commercial kit, home-made procedure PCR amplification: one or more target on 16S rrna, number of cycle
INTRODUCTION UNDER REVIEW Milk microbiome diversity and bacterial group prevalence in a comparison between healthy Holstein Friesian and Rendena cows Paola Cremonesi 1, Camilla Ceccarani 2, Giulio Curone 3, Marco Severgnini 2, Claudia Pollera 3, Valerio Bronzo 3, Federica Riva 3, Maria Filippa Addis 3, Joel Filipe 3, Massimo Amadori 4, Erminio Trevisi 5, Daniele Vigo 3, Paolo Moroni 3,6, Bianca Castiglioni 1 1 Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, SS-Lodi, Italy 2 Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, Milano, Italy 3 Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, Milan, Italy 4 Laboratorio di Immunologia Cellulare, Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia-Romagna, Brescia, Italy 5 Istituto di Zootecnica, Facoltà di Scienze Agrarie, Alimentari ed Ambientali, Università Cattolica del Sacro Cuore, Piacenza, Italy 7 Cornell University, Animal Heath Diagnostic Center, Quality Milk Production Services, Ithaca, NY, USA
INTRODUCTION A.N.A.F.I - 2016 1,106,461 lactating cows 9,742 kg = average milk yield 3.75% = fat content 3.32% = protein content Considerable impact of these performances on animal welfare and health Higher risk of metabolic and infectious diseases A.N.A.R.E. - 2012 4,000 lactating cows 5,238 kg = average milk yield 3.47% = fat content 3.24% = protein content Local breed native of the Rendena Valley (NE of Italy) Rustic and double-purpose animal Reduced fertility
INTRODUCTION The three weeks before and the three weeks after calving is an important and vulnerable period for the dairy cow. In the calving period, high-yielding dairy cattle are probably more susceptible to common environmental stressors in terms of housing, hygiene and feeding conditions. > 40% of cows treated for subclinical mastitis > 4% (US, Moroni P., personal communication) to a lesser extent of ketosis, ovarian cysts and lameness
INTRODUCTION Our questions What underlines mastitis resistance in local breeds? How can we investigate it? Are there markers of innate immune response that can be defined?
AIM The objective of this study was to characterize and compare the milk microbiota of HF and REN cows reared in a single mixed-breed farm and under the same management conditions, to define bacterial group prevalence with a plausible effect on mammary gland health
Starting from a mixed herd in Pavia MATERIALS AND METHODS Mean parity: 4 1 No antibiotic treatments at dry-off Milk sampling after cleaning and teat disinfection Blood and quarter milk samples were collected from three Rendena cows and six pluriparous Holstein Friesian cows at different time points: Dryoff 1 DIM 7-10 DIM 340 60 Colostrum 30 DIM
MILK MICROBIOME MATERIALS AND METHODS 10 ml of milk from each quarter Sample pre-treatment Microbial DNA extraction (Cremonesi et al., 2006) Milk microbiome Library preparation MiSeq Sequencing Data analysis TARGET 16S rrna (V3-V4)
OUTLIERS RESULTS Paired-end 2 x 250 bp MEAN: 44370 reads per sample Holstein Friesian Phylum Rendena breed
Quarter samples distribution RESULTS Rendena breed Comparison between breeds Holstein Friesian p=0.001 HF REN
RESULTS COMPARISON BETWEEN BREEDS AND TIME POINTS p=0.001 T1 T2 T3 T4
CONCLUSIONS In spite of the same farming conditions: Differences in terms of general microbial diversity, taxonomy and predicted functional profiles REN showed a lower biodiversity in terms of bacterial phyla, genera, species those differences might also have an impact on their mammary gland health for what concerns disease and pathogen resistance, since they seem related with inflammo-metabolic changes presence of some species prevents pathogens colonization???...
Istituto di Biologia e Biotecnologia Agraria Bianca Castiglioni Dipartimento di Medicina Veterinaria UNIMI Federica Riva Claudia Pollera Joel Filipe Giulio Curone Lauretta Turin Alessandra Pagnoni Daniele Vigo Paolo Moroni Valerio Bronzo Istituto di Tecnologie Biomediche Camilla Ceccarani Marco Severgnini Porto Conte Ricerche Maria Filippa Addis Vittorio Tedde Istituto di Zootecnica, Facoltà di Scienze Agrarie Erminio Trevisi Andrea Minuti Istituto Zooprofilattico della Lombradia e dell Emilia Massimo Amadori
THANKS FOR YOUR ATTENTION