1.RUMEN MICROBES AND THEIR MANIPULATION A Symbiotic Journey Inside The Cow By Shakira Ghazanfar SO, ASI, NARC

2. In the next 20 minutes, well Define Ruminant Examine digestive system of cattle Discuss the rumen microbes Describe methods of manipulation Explain how Infection occurs 3. What is a Ruminant? Ruminant: (n.) any of the various cud-chewing mammals having a stomach divided into four compartments. Cows, sheep, moose, goats, antelope, and camels 4. Ruminants.. Has a stomach with 4 chambers: 1. rumen 2. reticulum 3. omasum 4. abomasum 5. What is RUMEN? Digestive part in ruminants 150 L liquid Natural fermentation tank and feed reservoir Microorganisms adhere to feed particles and mucosal of rumen A Fermentation Vat Volume: 180-240L Temp: 38- 42 C pH: 6.4 to 7.2 6. The Rumen A Fermentation Vat Volume: 180-240L Temp: 38- 42 C pH: 6.4 to 7.2 7. Quantities of Microorganisms in Rumen 1. Bacteria/gm = 1010 (99.99 anaerobes) Feed= mainly fibrous material (cellulose) Cellulose is a lignified material (beta 1-4 bond) in feed Animals cannot digest it without high quantities (60%) of cellulolytic bacteria Cellulolytic bacteria= Break that beta-1,4- linkages of cellulose and release energy for milk formation 2. Protozoa/gm =109 3. Fungi/gm =105 8. Microbial Population The rumen is home to billions and billions of microbes, including bacteria, protists, fungi, and viruses. These many different rumen microbes form a complex community of organisms that interact with one another, helping the animal digest its food. 9. Establishment of rumen microorganisms 0 5 15 20 25 10 Days after birth Facultative anaerobes Microaerophilic Cellulolytic population Fungi Caecomyces Piromyces Neocallimastix Protozoa, Mycoplasma Entodinia Diplodinia Holotrichs 50 10. Ruminal microflora 1. BACTERIA 107 1012/ ml rumen fluid Cellulolytic bacteria Bacteria producing VFA and lactic acid Methane producing bacteria Proteolytic bacteria Lipolytic bacteria 11. Ruminal microflora 2. PROTOZOA 105/ml rumen fluid Utilize soluble sugars and polysaccharides Utilize starch prevent a decrease in pH 12. Ruminal microflora 3. FUNGI High celullolytic and hemicellulolytic activity Improve rumen fermentation Increased body weight Improved milk production Improve overall animal production 13. Bacterial forms cocci rods Spirochete filamentous budding and appendaged 14. Cocci found in different arrangements single pairs groups chains 15. Groups of Bacteria in the Rumen Free-living in the liquid phase Loosely associated with feed particles Attached to surface of protozoa and fungi 16. Adherence of mixed rumen bacteria to plant material. Protuberances from cells probably are binding factors. 17. Fibrobacter succinogenes Gram - none motile rods but can become coccoid or oval on culture 18. Fibrobacter succinogenes Reportedly most widespread cellulolytic bacteria; about 5 % of all rumen isolates Most extensive lignocellulosic degradation in vitro; ferment cellobiose and glucose Do not hydrolyse xylan and make limited use of pentoses liberated from fiber digestion 19. Enhance beneficial processes Minimize, alter or delete inefficient processes Minimize, alter or delete processes harmful to the host Aim of manipulating ruminal fermentation 20. Dietary : Chemical & Physical treatment, Feeding frequency, Order, Additives, Plant genetics Animal : Intake, Saliva flow, Digestive system Microbial : Population, Activity, GMO Methods for Manipulation 21. Effect of diet on the number & size of papillae A B C D Poor Quality Feed High Quality Feed 22. A well-fed ruminant A starving ruminant A young ruminant 23. Isolation of rumen bacteria Usually total and cellulolytic bacteria amylolytic, xylanolytic and saccharolytic e.t.c. Sampling Cannulated animals Multi-site sampling Minimize headspace Homogenise under CO2; filter through cheese cloth into pre-warmed (ca 39oC) thermos 24. Viable cell count 1.dilute sample 1 ml 9 ml 107 106 105 104 1000 100 10 cell no/ml 2. plate out 0.1 ml 25. Isolation of bacteria Solid associated microorganisms may be detached using methyl cellulose (or tween-80 followed by chilling for 6 - 8 h) Dilute sample in a 10 fold dilution series using an anaerobic diluent Role tube method employed for all but cellulolytic Anaerobic and sterile procedures 26. Enumeration of bacteria For cellulolytics: MPN procedure. 1 ml from 10-6 to 10-8 dilutions inoculated in triplicate into 4 ml anaerobic growth media (39oC) in Hungate tubes, containing a strip of filter paper as sole C source. Incubate at 39oC for up to 2 weeks Numbers determined statistically based on numbers of tubes containing digested filter paper at each dilution For other groups, Same procedure but inoculate (10-6 to 10-9) into media containing 1.8 % agar(47oC) and either a mixture of energy sources for total counts, or just the specific substrate for each sub group. Spin tubes in ice slurry to solidify agar Incubate at 39oC for 2 to 3 days and count colonies (/ml rumen fluid) 27. Water + nutrients energy source;organic, inorganic or light carbon, nitrogen source Agar can be added to make the medium semi-solid and poured into Petri dishes Culture media 1. Biochemical requirements for growth 28. 2. Physical and environmental requirements for growth oxygen concentration pH (hydrogen ion concentration) temperature 29. Growth media Rumen fluid containing Clarified autoclaved rumen fluid (10 to 20 %) Mineral solution 1: KH2PO4 Mineral solution 2: NaCl, (NH4)2SO4, CaCl2, MgSO4 and microelements VFA and Vitamin solutions Resazurin Energy substrate(s) as required Casein hydrolysate (amino acids and peptides) pH adjusted to 6.5 Boil; replace headspace with stream of CO2 Reducing agent and bicarbonate Dispense in to tubes, seal and autoclave 30. Growth media Chemically defined media No rumen fluid; nutrient requirements usually met by rumen fluid must be supplied e.g phenylpropanoic acid for R. albus and 1, 4- napthoquinone for S. dextrinosolvens. 31. Anaerobic glove box Permits growth of anaerobes on Petri dishes, hence the use of techniques such as replica plating rapid identification systems such as strip tests avoids exposure of bacteria to molten agar (47oC) Sufficient CO2 in atmosphere to maintain medium pH for nutrition of microbes requiring CO2 95 % CO2/ 5% H2 ok for most rumen bacteria; for methanogens, 80% H2/20% CO2 32. An anaerobic glove box 33. Maintenance of cultures Storage at 4oC can retain viability for up to 1 month (storage usually for up to 1 week) of 12 to 24 h cultures in 20 % glycerol for up to 1 year under liquid nitrogen (-196oC); followed by rapid thawing in water at 32 to 35oC. Very successful. Freeze drying. Reports of viability for up to 5 years 34. Conversion of carbohydrates to pyruvate 35. Methanogenesis Three major catabolic pathways CO2 reduction* CO2 and H2 to form CH4; electrons mainly from free H2 but also formate Methyltrophic pathway reduce CH3 groups from compounds methanol, trimethylamine Aceticlastic pathway Split acetic acid and reduce CH3 to CH4 36. Cell Wall Structure of Bacteria Gram + Gram 37. Microbiology of the Rumen 1: Role of Bacteria Brings enzyme and substrate together in a poorly mixed system Allows bacteria to colonize the digestible surface of feed particles Reduces predatory activity of protozoa and help fungi function 38. Microbiology of the Rumen 2: Role of Protozoa Digestion and fermentation carbohydrates and proteins Ingest bacteria and feed particles Engulf feed particles and digest CHOH, proteins and fats. 39. Microbiology of the Rumen 3: Role of fungi Improved nutrient utilization More feed intake Increased body weight Improved milk production 40. Rumen Microorganisms (Nutritional Requirements) CO2 Energy: End products from digestion of CHOH Nitrogen: Ammonia (Majority of N needs) Amino acids (nonstructural CHOH digesters) Minerals: Co, S, P, Na, K, Ca, Mg, Mn, Fe, Zn, Mo, Se Vitamins: None required in mixed cultures 41. Rumen Digestion and Fermentation CO2 VFA Degradable Rumen Microbial cells Feed Microbes NH3, CH4 Heat Long-chain fatty acids H2S 42. ADP ATP NADP+ NADPH Sugars Catabolism Growth Maintenance Transport Microbial Metabolism VFA CO2 CH4 Heat 43. Types of microorganisms in the rumen Bacteria Archea Protozoa Fungi Mycoplasma Bacteriophages Considerable diversity in the population Traditional culturing techniques Bacteria: 22 Genera and 68 species Protozoa: 6 Genera and 15 species Fungi: 3 Genera and species Molecular techniques ????? 44. http://www.bacterialphylogeny.info/Reprints/Lecture-2%20Archaea-Eukaryote.pdf Domains of organisms ARCHEA 45. Reasons for the diverse population Wide range of substrates Rapid environmental changes Types and concentrations of nutrients Frequency of feeding pH Presence of O2 Range of environments and microenvironments Digesta particles Liquid Rumen wall Laminae of omasum Surfaces or inside of other organisms In terms of microbial growth, a group of microorganisms is more efficient than any single microorganism Maximum biochemical work 46. Properties of a true rumen microorganism Anerobic or facultative anerobic Produce endproducts found in the rumen or that are utilized by other microorganisms Numbers needed Bacterial species >106/ml 47. Quantities of microorganisms Viable organisms 1010 1011 bacteria/gm 105 protozoa/gm 105 fungi/gm 109 bacteriophages/gm Variability in counts Total counts are 2 to 3 x greater than viable counts Total counts decrease after feeding Causes for reduction in bacteria Lysis from O2 Movement of bacteria from fluid to solid digesta Washout with digesta flow Dilution with water and saliva Cause for reduction in protozoa Chemotaxis Problems with traditional techniques Bacteria (Culture techniques) Difficult to separate particulate-bound bacteria Inability to count viable, but non-dividing cells Colonies may be formed by clumps of cells Inability to grow some species on lab media Protozoa Chemotaxis Dilution of minor species 48. Quantity of protozoa Protozoa numbers < Bacteria numbers Protozoa size are 500 to 1,000,000 x > Bacteria Therefore normally, Protozoal volume = Bacterial volume 49. Methods of classifying rumen bacteria Traditional Morphology Shape Size Gram or + Groups Energy source Fermentation endproducts Special nutritional requirements Immunological Molecular RFLP (Restriction Fragment Length Polymorphisms) 16s RNA sequencing PCR (Polymerase Chain Reactions) 50. Classifying rumen bacteria by energy source Relationships Few species specialize in metabolizing a single substrate, but many prefer certain substrates Substrate concentration is important in controlling growth of specific species General relationship u = umax / (1+ Ks/[S]) where u = growth rate umax = theoretical maximum growth rate Ks = Affinity coefficient for a substrate (Lower = more affinity) [S] = Substrate concentration Therefore, if the substrate concentration is very high relative to the affinity, the closer the growth rate will be to the maximum. 51. Relationships If species A has a higher affinity (ie. lower affinity coefficient) and equal umax to species B, then species A will always predominate except at very high concentrations a If species A has a higher affinity (lower Ks) and lower umax than species B, then species A will predominate at low concentrations and species B will predominate at high concentrations A B u [Substrate] u [Substrate] A B 52. Classifying rumen bacteria by energy source Cellulolytic bacteria Cellulose Primary constituent of plant cell walls A chain of glucose units bound by beta-1,4-linkages Can only be digested by microorganisms Digestibility controlled by lignification Common cellulolytic bacteria Ruminococcus flavefaciens Ruminococcus albus Fibrobacter succinogenes Butyrvibrio fibrisolvens Clostridium lochheadii 53. Growth requirements of cellulolytic bacteria pH 6.0-7.0 Will not grow at pH < 6.0 Reasons: Depletion of HCO3 VFAs are inhibitory Destruction of membrane potential NH3* Branched chain VFA* Leucine > Isovaleric acid Isoleucine > 2 methyl-butyric acid Valine > Isobutyric acid Phenolic acids* Phenylalanine > Phenylacetic acid Phenylalanine or Cinnamic acids > 3-Phenylpropionic acid CO2 as HCO3* S- as Cysteine or Sulfate 54. Fermentation endproducts of cellulolytic bacteria Cellobiose Acetic acid* Butyric acid CO2* H2* Ethanol* Succinic acid* Formic acid Lactic acid *Major endproducts Not normally found; Used by other bacteria 55. Cellulose digestion In reticulorumen Approximately 90% of cellulose digestion Requires two steps Microbial attachment Hydrolysis Miron et al. JDS 84:1294 56. Attachment of cellulolytic bacteria on fiber Results in a lag period in digestion Phases Transport of bacteria to fiber Slow Dependent on number of bacteria Nonspecific adhesion of bacteria to sites on substrate Binds with Glycocalyx Mixture of polysaccharide, glycoprotein and protein on outside of cell membrane of gram- bacteria Peptidoglycan of gram+ bacteria Occurs mainly at cut or macerated sites of the plant Specific adhesions of bacteria with digestible cellulose Structures Cellulosome: Large, multienzyme complexes specialized for adhesion and hydrolysis of cellulose Fimbriae or Pili: Small (5-7 nm in width and 100-200 nm in length) structures in both gram + and bacteria Proliferation and colonization of bacteria 57. Structure of the cellulosome Cellulose hydrolysis Cellulases are extracellular Enzymes Endo-B-1,4-glucanase > Cleaves cellulose chains Exo-B-1,4-glucanase > Cleaves cellobiose units Cellobiase > Cleaves cellobiose Cellulose hydrolysis Cellulases are extracellular Enzymes Endo-B-1,4-glucanase > Cleaves cellulose chains Exo-B-1,4-glucanase > Cleaves cellobiose units Cellobiase > Cleaves cellobiose 58. Hemicellulolytic bacteria Hemicellulose A major component of plant cell walls A chain of hexoses, pentoses, and uronic acids bound by beta-1,4-linkages Digestibility controlled by lignification Common hemicellulolytic bacteria Most cellulolytic bacteria Prevotella ruminicola Growth requirements Similar to cellulolytic bacteria Fermentation endproducts Similar to cellulolytic bacteria 59. Pectinolytic bacteria Pectin Chains of uronic acids bound by alpha-1,4-linkages with pentose branch points 2-6% forage DM Highly digestible Pectinolytic bacteria Lachnospira multiparus Succinovibrio dextrinosolvens Fibrobacter succinogenes Butyrvibrio fibrisolvens Prevotella ruminicola Streptococcus bovis Fermentation endproducts Acetic acid Propionic acid Butyric acid Lactic acid Succinic acid Formic acid* Intermediates 60. Amylolytic bacteria Starch Polymer of glucose units bound by alpha-1,4-linkages with varying numbers of alpha-1,6-branch points Primary carbohydrate in grains Amylolytic bacteria Streptococcus bovis Normally present in low numbers in cattle either fed forages or adapted to grain diets Very high numbers in unadapted cattle that engorge on grain Reasons for increase High concentrations of glucose in rumen Low division time Loss of protozoa Fermentation 85% of starch is fermented to lactic acid Causes lactic acidosis 61. Lactic acidosis Grain engorgement Increased [VFA] in rumen Decreased rumen pH and free glucose Increased S. bovis Increased rumen D,l-lactic acid pH 5.0 Increased lactobacilli species More D,L-lactic acid production Lactic acid absorbed through rumen wall D-lactic acid is not metabolized by the animal Increases blood [D-Lactic acid] Reduces blood pH Decreases the [CO3] in blood Hemoconcentration Coma 62. More amylolytic bacterial species Ruminobacter amylophilus Prevotella ruminicola Succinomonas amylolytica Succinovibrio dextrinosolvens Growth requirements pH 5.0-6.0 CO2 NH3 Peptides Fermentation endproducts Oligosaccharides (Intermediate) Acetic acid* Propionic acid* Butyric acid CO2 Lactic acid Succinic acid Formic acid Intermediates 63. Sugar fermenters Free sugars rarely found in rumen Few sugar utilizers Streptococcus bovis Lactobacillus species Cellulodextrin utilizers Treponema bryantii Grows in co-culture with F. succinogenes 64. Acid-utilizing bacteria Acids that are usually intermediate metabolites Lactic acid Succinic acid Formic acid Acid-utilizing bacteria Lactate utilizers Megasphaera elsdenii Veillonella alcalescens Prevotella ruminicola Fermentation endproducts Acetic acid Propionic acid* Valeric acid Caproic acid Succinate utilizers Selenomonas ruminantium Veillonella alcalescens Anerovibrio lipolytica Fermentation endproducts Propionic acid* CO2 Formate utilizer Methanobrevibacter ruminantium 65. Proteolytic bacteria Few bacteria only use protein as their sole energy source 38% of isolates are proteolytic Most active proteolytic bacteria Prevotella ruminicola Ruminobacter amylophilus Butyrvibrio fibrisolvens Lipolytic bacteria Hydrolyze triglycerides and phospholipids Anerovibrio lipolytica Hydrolyze galactolipids, phospholipids, and sulfolipids Butyrvibrio fibrisolvens Biohydrogenation 66. Sulfur-reducing bacteria Species Desulfovibrio sapovorans Metabolism Low levels of sulfate Butyrate + EtOH - Acetate + H2 H2 cross-fed to Wolinella succinogenes in reaction H2 + Fumarate - succinate propionate High levels of sulfate (depletion of fumarate) Butyrate + sulfate - Acetate + H2S Sulfur reduction is preferred use of H2 over CH4 H2_threshold Km Vmax Sulfur reduction .0013 .1 .3 Methane production .067 6.6 6.0 67. Toxin-degrading bacteria Degradation of mimosine A goitrogen found in the tropical legume, Leucaena leucocephala Caused toxicity in Australian ruminants, but not Hawaiian goats Degraded by bacteria Synergistes jonesii Now used as an inoculant for ruminants across the world Degradation of oxalic acid A toxin found in Halogeton plants A noxious weed in the west Toxic at intakes of .3 to .5% of intake for short periods Mechanism Primarily binds Ca Degraded by bacteria Oxalobacter formigenes Oxalate Formic acid + CO2 68. Methanogenic archea Classes Free-living Methanomicrobiales sp. Methanosarcinales sp. Associated with protozoa Methanobrevibacter sp. Methanococcales sp. Methane production mechanisms Acetate or methanol > CH4 + CO2 CO2 + 4H2 > CH4 + H2O Formic acid + 3H2 > CH4 + 2H2O Effects Energy waste (5-6% of GE) Greenhouse gas Requirement to increase ATP and microbial growth 69. Rumen protozoa Most are ciliated Families Isotrichidae (Holotrichs) Cilia over entire body Genuses Isotricha Dasytricha Orphryscolidae (Oligotrichs) Cilia in mouth region Genuses Entodinium Eudiplodinium Epidinium Ophryoscolex Photos courtesy M. Rasmussen and S. Franklin, USDA-ARS http://www.rowett.ac.uk/ercule/html/rumen_protozoa.html 70. Protozoa motility Photos courtesy M. Rasmussen and S. Franklin, USDA-ARS 71. Additional properties of protozoa Much larger than bacteria Count is normally 105/gm Slow generation time Closely associated with feed particles Holotrichs exhibit chemotaxis moving to the back of the rumen when animals are eating before settling in ventral and cranial sacs Do not readily pass from the rumen Holotrichs near the rumen wall scavenge O2 All protozoa store soluble carbohydrates as an amylopectin-like storage polysaccharide Carbohydrate specificity Holotrichs store sugars Oligotrichs store starch Benefits To protozoa, it maintains a constant energy source To animal, it stabilizes fermentation Protozoa engulf and lyse bacteria Contributes to rumen protein turnover reducing efficiency of protein use Bacteria that resist lysing in the protozoa may have genes activated that result in resistant, more virulent pathogens Protozoa have close relationships with methanogens 72. Fermentation endproducts of protozoa Holotrichs Acetic acid Butyric acid Lactic acid H2 Oligotrichs CO2 H2 Acetic acid Butryric acid N requirements of protozoa Do not use NH3 Actively proteolytic 73. Factors affecting protozoa Diet Feed CHO pH Protozoa Pasture Sugars, Cellulose 6-7 Total high Mod. Grain Sugars, Cellulose, 6.0-6.5 Total higher, Starch Inc % Oligotrichs High Grains Starch Increases protozoa High liquid dilution rate > Decreases protozoa Defaunation Early attempts CuSO4 Aeration Detergents Recent attempts Lecithin or linoleic acid Tannins (Quebracho or Yucca plants) Saponins (Quillaja plants) Coconut oil (Lauric acid) Difficult to accomplish without affecting bacteria or host animal 74. The need for protozoa in the rumen Protozoa are not necessary for the animal (Commensalism) Advantages of protozoa Increased cellulose digestion 25 33% of total cellulose digestion Mechanisms More active than bacteria? Provide NH3 to bacteria Remove O2 Slower fermentation of starch and sugars Greater VFA production Increased transport on conjugated linoleic acid (CLA) and trans-11 (18:1) fatty acid to duodenum and meat and milk Disadvantages of protozoa Increased rumen protein turnover Reduced efficiency of protein use Increased rumen [NH3] Increased CH4 production Development of more virulent strains of pathogenic bacteria 75. Net effects of defaunation Increased daily gains Improved feed efficiency Decreased OM and cellulose digestion Increased total and microbial protein flow to the duodenum Decreased pH on high concentrate diets, but increased pH on high forage diets pH response to defaunation = 0.31 0.006 x % concentrate in diet Increased production of propionic acid and decreased production of butyric acid Increased rumen volume and liquid outflow rate 76. Eugene et al. (2004) 77. Eugene et al. (2004) 78. Rumen fungi Species Neocallismatix frontalis Sphaeromonas communis Piromonas communis Orpinomyces joyonii Occurrence Appear 8 10 days after birth More prevalent on grasses than legumes May be related to sulfur supplementation Function Fiber digestion Enzymes identified Cellulases Xylanases Lichenase Mannanase Feruloyl esterase* http://www.goatbiology.com/animations/funguslc.html 79. Establishment of the rumen microbial population At birth, rumen has no bacteria Normal pattern of establishment Appear Peak Microorganisms 5-8 hours 4 days E. coli, Clostridium welchii, Streptococcus bovis week 3 weeks Lactobacilli week 5 weeks Lactic acid-utilizing bacteria week 6 weeks Amylolytic bacteria Prevotella-wk 6 1 week 6-10 weeks Cellulolytic and Methanogenic bacteria Butyrvibrio-wk 1 Ruminococcus-wk 3 Fibrobacter-wk 1 1 week 12 weeks Proteolytic bacteria 3 weeks 5-9 weeks Protozoa - 9-13 weeks Normal population 80. Factors affecting establishment of population Presence of organisms Normally population is established through animal-to-animal contact Bacteria may establish without contact with mature ruminants Establishment of protozoa requires contact with mature ruminants Favorable environment Substrates and intermediates Increased rumen pH Digesta turnover 81. Altering the rumen population Diet High forage > High pH, cellulose, hemicellulose, sugars > High cellulolytic and hemicellulolytic bacteria > High methanogens > High protozoa High concentrate> Low pH, high starch > Low cellulolytic and hemicellulolytic bacteria > High amylolytic bacteria > Low methanogens > Low protozoa, primarily oligotrichs Buffers Same as high forage Antibiotics Ionophores Microbial inoculants 82. Ionophore effects on the rumen microbial population Ionophores Monensin Lasalocid Laidlomycin Actions Create pores in membranes of gram + bacteria Allows potassium to exit and hydrogen to enter cells Bacteria affected Inhibits Effects Ruminococcus albus Decreased acetate, formate and Ruminococcus flavefaciens methane Butyrvibrio fibrisolvens Streptococci Decreased lactate Lactobacilli Increases Fibrobacter succinogenes Increased propionate Prevotella ruminicola Selenomonas ruminantium 83. Net results of feeding ionophores Increased propionate Reduced protein degradation Reduced deamination Reduced methane production Reduced lactate production 84. Use of microbial inoculants Dosing with lactate-utilizing bacteria can reduce lactic acid build up in rumen Difficult to do long-term Antagonistic environment Difficult to get enough organisms Considerable gene exchange Mechanisms Transformation Conjugation Transduction Favorable conditions for gene transfer High population Intimate cell-to-cell contact Supply of phages Extrachromasomal plasmid DNA Transient non-rumen bacteria