Production Research Production Research Priorities and PRG Priorities and PRG Endophyte ToxicityEndophyte Toxicity

ByByR A LengR A Leng

Emeritus ProfessorEmeritus ProfessorUNEUNE

An animal nutritionist interest An animal nutritionist interest in endophyte toxicity?in endophyte toxicity?

Prior to 1996 –As a nutrition researcher never considered Prior to 1996 –As a nutrition researcher never considered possibility of endophyte ill thrift—Puzzled by low possibility of endophyte ill thrift—Puzzled by low productivity of experimental animals particularly in New productivity of experimental animals particularly in New Zealand grazing PRG. Zealand grazing PRG.

Since 1996 ---increasing awareness of ill thrift and sudden Since 1996 ---increasing awareness of ill thrift and sudden death of livestock in many different grazing areas I visited death of livestock in many different grazing areas I visited on lecture tourson lecture tours

Led me to research literature on ill thrift which ultimately Led me to research literature on ill thrift which ultimately convinced me of major insidious problems associated with convinced me of major insidious problems associated with endophytic fungus [Leng 2000]endophytic fungus [Leng 2000]

Few if any nutrition or production researchers consider Few if any nutrition or production researchers consider endophytes. e g team at Lincoln NZ working on effects of endophytes. e g team at Lincoln NZ working on effects of parasites/nutrition interaction. Blaxter’s energy parasites/nutrition interaction. Blaxter’s energy requirements team [Autumn PRG vs Spring PRG]requirements team [Autumn PRG vs Spring PRG]

•Producers who are not looking for a fescue toxicity problem [and PRG toxicity] are much less likely to detect one than someone who is - so endophyte effects may go unnoticed [Ball 1997].

•The effects of endophytes on annual gains and reproduction are much greater than is generally realized. [Ball 1997]

•Research fund providers in Australia have not recognised the economic losses from endophyte toxicosis [Leng 2000]

Endophyte Toxicosis Goes Endophyte Toxicosis Goes Largely UnrecognisedLargely Unrecognised

Australia’s research Australia’s research contribution has been contribution has been

miniscule and has not even miniscule and has not even attempted a survey of the attempted a survey of the

economic losses economic losses Most research has been done on endophyte Most research has been done on endophyte

toxicosis intoxicosis in Southern states of USA with Tall Fescue pastures Southern states of USA with Tall Fescue pastures

growing in hot conditionsgrowing in hot conditions New Zealand with perennial rye grass under cool New Zealand with perennial rye grass under cool

conditionsconditions Research in Australia has been minimal.Research in Australia has been minimal. Considerable production research has been Considerable production research has been

done on PRG without knowing alkaloid done on PRG without knowing alkaloid content.content.

Rye Grass Pastures Are Normally Rye Grass Pastures Are Normally Heavily Infected [60-80%] With Heavily Infected [60-80%] With

N.Lolli N.Lolli in Australiain Australia

Endophyte Endophyte [[Neotyphodium LolliNeotyphodium Lolli]] in the stem of PRG.in the stem of PRG.

Neotyphodium sppNeotyphodium spp are not are not restricted to PRGrestricted to PRG

A number of grasses A number of grasses contain Endophytes contain Endophytes Including Australian Including Australian native native Echinopogan Echinopogan Ovatus that is found Ovatus that is found in all Statesin all States and and contains a contains a Neotyphodium sp that Neotyphodium sp that caused staggers and caused staggers and death of sheep in the death of sheep in the Tenterfield area as far Tenterfield area as far back as 1890back as 1890

Anecdotal evidence of Anecdotal evidence of production losses in cattle production losses in cattle

and sheep in Australiaand sheep in Australia

PRG staggers is common in Victoria and Tasmania on the spring PRG shoot

Diarrhea in lambs on PRG, appears to be associated with Endophyte toxicosis.The amounts of dags and fly strike are highly correlated. Diarrhea and endogenous proteinloss are associated

Heat stress has been associated with consumption of endophyte PRG. Death rates from crowding into dams with subsequent drowning have been recorded on PRGpastures in hot weather

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Dry weigtht of dags[g/lamb]

% la

mbs

wit

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rike

Sheep and cattle even on relatively cool days will get into water or seek shade for no apparent reason

When you go looking for endophyte toxicity symptoms they are apparent through out the country

Evidence of likely production Evidence of likely production losses from NZ [cool climate] losses from NZ [cool climate]

and Southern USA [warm and Southern USA [warm climate] researchclimate] research

Growth rates of lambs on rye Growth rates of lambs on rye grass and cattle on fescue grass and cattle on fescue

pasturespastures

0102030405060708090

Live weight change of lambs[g/d]

0 25 50 75 100

% PRG pasture as endophyte infected

00.10.20.30.40.50.60.70.80.9

Live weight change [kg/d]

Ala. Geo. Miss. Tenn. Virg.

Plus endophyte No endophyte

Lambs on rye grass in NZ

Cattle on fescue in Southern USA

Heat stress associated with lowered Heat stress associated with lowered feed intake, is an important factor in feed intake, is an important factor in

ill-thrift on endophyte infected ill-thrift on endophyte infected

pasturespastures Cattle generally are poor thermo-regulators. Cattle generally are poor thermo-regulators.

[Their capacity to sweat is about 0.2 of [Their capacity to sweat is about 0.2 of humans]humans]

Breeds with higher capacity to regulate body Breeds with higher capacity to regulate body temperature [greater ability to sweat] are less temperature [greater ability to sweat] are less effected.effected.

The major measured effectsThe major measured effects Depressed cow and calf live weightDepressed cow and calf live weight Reduced fertility particularly in lactating dairy and Reduced fertility particularly in lactating dairy and

beef cowsbeef cows Reduced milk production in dairy and beef cattleReduced milk production in dairy and beef cattle

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-18 -12 -9 0 10 20 25 27.5 30 40

Environmental temperature [oC]

[kg

/da

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Thermal humidity index

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Sheep decrease feed intake a environmental temperature rises

Friesians reduce feed intakeand milk yield as environmental Heat stress increases

Grazing Friesian cows changetheir feeding behavior astemperature rises

Ergovaline and lolitrem are Ergovaline and lolitrem are both implicated in heat stress both implicated in heat stress

in ruminantsin ruminants Ergovaline decreases blood flow to skin reducing heat dissipation but Ergovaline decreases blood flow to skin reducing heat dissipation but

lolitrem has a heating effect. High body temperatures are associated lolitrem has a heating effect. High body temperatures are associated with low feed intake and often assumed to be the main cause of ill with low feed intake and often assumed to be the main cause of ill thrift.thrift.

Body temperature and respiration rate in sheep given a dose of Body temperature and respiration rate in sheep given a dose of lolitrem B[75ug/kg LW] under cool conditions [McLaey 1999lolitrem B[75ug/kg LW] under cool conditions [McLaey 1999]]

36.4

36.6

36.8

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37.2

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-1 0 1 2 3 4 5 6 7 8

Hours from injection of lolitrem

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Body temp. Respiration rate

Australia Is the Sun Burnt Australia Is the Sun Burnt Country and THI Is Often Very Country and THI Is Often Very

HighHigh

One January day in Australia

Research Priorities-1. Research Priorities-1. MonitoringMonitoring

Pasture monitoringPasture monitoring To provide information on variations in To provide information on variations in

endophyte alkaloids endophyte alkaloids levels of toxins in PRG and how they are effected by levels of toxins in PRG and how they are effected by

soil, soil fertility climate and grazing management soil, soil fertility climate and grazing management level of toxin effects intake or selection of pasture by level of toxin effects intake or selection of pasture by

cattle, sheep or other herbivorescattle, sheep or other herbivores To provide information on economic losses To provide information on economic losses

deaths, body weight, milk, wool, reproductive deaths, body weight, milk, wool, reproductive efficiencyefficiency

To provide information where research is being To provide information where research is being undertaken on other management factor e g undertaken on other management factor e g

Parasite control and productionParasite control and production Supplementary feeding strategies etcSupplementary feeding strategies etc

Research Priorities 2.Thermal Research Priorities 2.Thermal stressstress

Establish the production responses to Establish the production responses to endophyte alkaloids in relation to thermal endophyte alkaloids in relation to thermal stress [THI].stress [THI].

Potential for early warning of risk problems Potential for early warning of risk problems and for establishing heat ameliorating and for establishing heat ameliorating management practices.management practices. ShadeShade Yard workYard work Supplementary feeding [e g substitute feeding or Supplementary feeding [e g substitute feeding or

alternative pastures]alternative pastures]

Research Priorities 3 –other Research Priorities 3 –other economic effectseconomic effects

DiarrhoeaDiarrhoea IncidenceIncidence Effects in animals under thermo-neutral Effects in animals under thermo-neutral

and hot conditions and hot conditions Cost Cost

DaggingDagging Fly strikeFly strike Production lossesProduction losses

Implication of diarrhoeaImplication of diarrhoea

Ill-thrift has many causes either single or Ill-thrift has many causes either single or multiple and interactivemultiple and interactive

The symptoms are simply reduced production The symptoms are simply reduced production [which is often clear to the producer] [which is often clear to the producer] associated with reduced feed intake [which is associated with reduced feed intake [which is not apparent to the producer]not apparent to the producer]

Reduced feed intake is very often associated Reduced feed intake is very often associated with inflammatory cytokines and leptin with inflammatory cytokines and leptin secretionsecretion

The persistent diarrhoea may implicate The persistent diarrhoea may implicate inflammation of the gut wall and indirectly the inflammation of the gut wall and indirectly the immune systemimmune system

Research Priorities 4 Research Priorities 4 InteractionsInteractions

Seems safe to suggest that if there are Seems safe to suggest that if there are animals dying from heat stress or animals dying from heat stress or misadventure caused by heat stress then misadventure caused by heat stress then most animals will be subject to reduced feed most animals will be subject to reduced feed intake and some form of ill-thrift.intake and some form of ill-thrift.

Grazing animal production is complex and Grazing animal production is complex and there are potentially many interacting there are potentially many interacting factors as is clear from recent studies on the factors as is clear from recent studies on the interaction of protein requirements with interaction of protein requirements with parasites, immuno nutrition and parasites, immuno nutrition and physiological state physiological state

Research Priorities 5. Protein Research Priorities 5. Protein nutrition, parasite/disease nutrition, parasite/disease

challenge/climate challenge/climate

The highest levels of alkaloids in PRG The highest levels of alkaloids in PRG occur in mature pasture on fertilised occur in mature pasture on fertilised

land.land.

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Pasture availability Green Pasture Rainfall [mm]

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Concentration [ppm]

Winter Spring Summer Autumn

Peramine Lolitrem Ergovaline

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Ergovaline [ppm]

Oct. Nov. Dec. Jan. Feb.

Plus N fertilizer No fertizer0

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Cru

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in in

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ture

[%

]

Green pasture

Dry pasture

Deficientmedium

Digestibleforage

Fermentationintermediates

Microbialcells

Short chain organic acids

+methane

+heat

Digestibleforage

Fermentationintermediates

Microbialcells

Short chain organic acids

+methane

+heat

Well balance growth medium

10. 10.

017. 083034

066

Deficiencies of Growth Factors Alter the Flow of Fer mentation Intermediates Into Absorbed Nutrients

Priorities for Research—6 Priorities for Research—6 Implications of the immune Implications of the immune

systemsystem Environments where the sum of Environments where the sum of

environmental and immunological stressors is environmental and immunological stressors is high divert nutrients away from accretion in high divert nutrients away from accretion in muscle, milk and wool to liver anabolism and muscle, milk and wool to liver anabolism and host defence mechanisms [Husband 1995]host defence mechanisms [Husband 1995]

Physical and psychological stressors can Physical and psychological stressors can activate the acute phase response with activate the acute phase response with potential [Colditz 2002] [calves on slippery potential [Colditz 2002] [calves on slippery floors]floors]

Immune activation then is a primary factor in Immune activation then is a primary factor in reduced feed intake and reduced productionreduced feed intake and reduced production

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Feed

intake[g/

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ControlInfected

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Days prior to and after lambing

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Normal diet Plus bypass protein

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-36 -18 0 18 36 44 62 80 98 114 130 148

Days of infection

Cumulative Live Weight

[kg]

100g fish meal 100g FM infected 0g FM 0g FM infected

Parasites of the small intestine progressively reduce feed intakeSteele 1996

Essential amino acids, in late pregnancy and early lactation are prioritised for lamb and milk production. Lowering immune function Donaldson 1999

Both the immune competence and the feed intake effects are overcome by increasing in metabolisable proteinintakeVan Houtert et al 1996

Gut

Epithelia cellturnover tissue repair

Plasmaproteinleakage

Local inflammatory response

PeripheralInflammatory response

Albumin/globulinsfrom liver

Microbe &diet protein

VFA Energy

Muscle

Skeletal

Wool orHair

Milk

decreaseincrease

Effects of parasitism on the nutrient economy of small ruminants

Muscleprotein breakdownincrease

Growth of foetus

Endophyte toxicosis is likely to interact Endophyte toxicosis is likely to interact with climate, physiological state and with climate, physiological state and

demand for nutrients and disease demand for nutrients and disease incidenceincidence

Ruminants on imbalanced feeds[ dry forages] reduce feed Ruminants on imbalanced feeds[ dry forages] reduce feed intake and increase wasteful metabolism and therefore are intake and increase wasteful metabolism and therefore are more prone to heat stress[ the need for a balanced rumen more prone to heat stress[ the need for a balanced rumen vs imbalanced]vs imbalanced]

Metabolisable protein requirements are increased Metabolisable protein requirements are increased in late preg. early lactation and at weaningin late preg. early lactation and at weaning when the immune system is stimulated by foreign bodies, when the immune system is stimulated by foreign bodies,

stress and deficiencies of key nutrientsstress and deficiencies of key nutrients by the need to repair damaged areas of the gut by the need to repair damaged areas of the gut by increased secretion of mucous in the intestinesby increased secretion of mucous in the intestines by increased drain of endogenous and microbial protein to the by increased drain of endogenous and microbial protein to the

lower gutlower gut The metabolisable protein requirements to prevent The metabolisable protein requirements to prevent

the periparturient rise in parasite egg production in the periparturient rise in parasite egg production in ewes is about 350g protein or 30% greater then book ewes is about 350g protein or 30% greater then book requirements requirements

Research Priorities- 7 Discipline Research Priorities- 7 Discipline oriented oriented

Study metabolism in the rumen and the site of Study metabolism in the rumen and the site of absorption of the alkaloids--- using labelled absorption of the alkaloids--- using labelled alkaloids and cannulated animal [Hill 2005]alkaloids and cannulated animal [Hill 2005]

Study potential of a range of chemicals to adsorb Study potential of a range of chemicals to adsorb the alkaloidsthe alkaloids

Examine the effects of alkaloids on the immune Examine the effects of alkaloids on the immune systemsystem

Understand the effects on the gutUnderstand the effects on the gut Understand transfer to foetus and milk[?].Understand transfer to foetus and milk[?]. Measure their effects on the immune system, illeum Measure their effects on the immune system, illeum

wall and protein flow to the caecum large wall and protein flow to the caecum large intestine[ site of provitamin B12 absorption]intestine[ site of provitamin B12 absorption]

Radiolabelled Peptide Ergot Alkaloids. 83.Mitteilung über MutterkornalkaloideEmil SchreierBiopharmaceutical Department, Division of Pharmaceutical Research and Development, Sandoz Ltd., CH-4002 BaselHerrn Dr. Albert Hofmann zum 70. Geburtstag gewidmet

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Aflatoxin Reduction in

Milk [%]

Bentonite-Na Bentonite-Ca Mycosorb ActivatedCharcoal

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Relative

wool

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Bentonite given[g/day]

A number of binders including bentoniteprevent absorption of aflatoxins

Bentonite increases wool growth [Fenn and Leng 1985] in sheep by increasing microbial protein supply [Klieve 2002]

Bentonite clays strongly bind ergot alkaloids between ph 2-8but dissociate at Ph 8.5Site of absorption of lysergic acid is from the rumen [Hill 2005]

Grazier observation. Under field conditions wool growth is increased by feeding bentonite and scouring is eliminated or reduced in sheep .

Recombinant DNA ?Recombinant DNA ?

Is their a role for recombinant RNA to Is their a role for recombinant RNA to metabolise endophyte alkaloids.metabolise endophyte alkaloids.

Soil organisms will degrade alkaloidsSoil organisms will degrade alkaloids Recent work inducing Fluroacetate Recent work inducing Fluroacetate

detoxification by rumen organisms detoxification by rumen organisms suggests any thing is possible[ Greig suggests any thing is possible[ Greig et al 2004] et al 2004]

It is likely that the industry will have to live with endophyte infected pastures for a long time, therefore remedial strategies must be developed.

Wild card could be climate change in the long term The 3 hottest years of the past century have occurred since 2000

The Aussie Cow is just as susceptible to endophyte toxicosis as overseas cows

The End