1. Impact of fungi on animal and food sector
Agha Waqar Yunus
(Senior Scientific Officer)
Animal Sciences Institute
National Agricultural Research Center, Islamabad

2. Fungal diseases in animals
Fugal infections:
Ringworm of cattle, sheep (Microsporon/ Trichophyton)
Mastitis (Cryptococcus)
Reproductive disorders (Aspergillus)
Aspergillosis / lung infection of chickens (Aspergillus)
Degnala disease of cattle (Fusarium)
Chronic nature, low occurrence, and neglected
Fungal toxic metabolites are common
Mycotoxin producing fungi affect feed and food
Ringworm/dermatomycosis = Microsporon, Trichophyton;
Mastitis = Cryptococcus (also causes lung or brain infection);
Aspergillosis of chickens (Aspergillus fumigatus)

3. Mycotoxin producing fungi
Myco-toxin is a cytotoxic secondary metabolite of fungi, produced
under special conditions of moisture and temperature.
> 350 known compounds
Ingestion exposure: grains, fruits, spices, milk
Inhalation exposure also possible
Wheat (Islamabad 2015) Dairy feed (Rawalpindi 2006) Walls (Lahore 2012)

4. Toxigenic Fungi : examples
Aspergillus : emodin, ochratoxin, citrinin, patulin
aflatoxins, cyclopiazonic acid
Penicillium : emodin, ochratoxin, citrinin, patulin,
rubratoxins
Fusarium : trichothecenes, zearalenone, fumonisns, enniatins, culmorins, moniliformin
Alternaria : Alternaria toxins
Claviceps : Ergotamins

5. Mycotoxins: why are we concerned ?
Mutagenic and carcinogenic (aflatoxins, ochratoxins, citrinin)
Specific blockers and inhibitors (trichothecenes)
Cell membrane dysfunction (patulin)
Unknown mechanisms (curvularin)
CNS
Liver, kidneys
Immune and hematopoietic system
Epithelia
(gastrointestinal/respiratory tract)
Food safety issues - deadly at small doses
Huge losses in plant/animal productivity

6. Mycotoxins: why are we concerned ?
Small molecular weight (~ )
No immunoprotection !
Very stable against environmental influences and detoxification
Dispose contaminated stuff !

7. Mycotoxins: points of contamination
Biological
factors
Susceptible crop
Toxigenic fungus
Environment
Moisture
Temperature
Insects/birds
Harvesting
Crop maturity
Detection/diversion
Storage
Distribution
and
Processing
All these points are checked and mechanisms for checking/monitoring is being improved to get early warning systems

8. Mycotoxins: why are we concerned ?
The popular slogan since early 1990’s
25% of cereals are affected globally
also in milk, eggs, meat

9. Mycotoxins: Real global situation
Real Situation in food and feed chain
Year
Numb of samples analyzed
% Positive
25,944
76%
2013
3,000
81%

10. Mycotoxins: Real global situation
Why is the trend of higher contamination?
Better methods of analyses (LC-MS/MS)
Climate change
New crops

11. Mycotoxins: Implications in trade
Notifications in 2014 by the EU Rapid Alert System for Food and Feed (RASFF)

12. Mycotoxins: Maximum tolerable limits
USA EU
China
Pakistan
Aflatoxins Y
Ochratoxin A -
Citrinin
Patulin
Fumonisins
Deoxynivalenol
T-2, HT-2
Zeralenone
Ergot alkaloids
Sterigmatocystin
to be

13. Mycotoxins: What we know in Pakistan?
Ochratoxins: (A, B)
Grains, dry fruits, animal feed, milk
Limit = 1 – 5 ppb
Breast, kidney, urinary tract cancer
30% poultry feeds contaminated – 12% exceed limit
72% export raisins contaminated – 5% exceed limit
26% medicinal plants positive
Over 92% blood samples positive in Karachi
Possible contribution to kidney cancer - risks not determined.
Mycotoxic Nephropathy in pigs (Stoev, 2008)
(1) Abidin etal. 2017; Sherazi etal (2) Asghar etal (4) Aslam etal., 2005

14. Mycotoxins: What we know in Pakistan?
Patulin: Aspergillus, Penicillium
Apple, fruits, vegetable, grains
Genotoxic, neurotoxicity, pulmonary congest.
57% fruit samples positive; 33% exceed limit
Trichothecenes: Fusarium
Grains, legumes, animal feeds
GIT, lymphoid, cardiovascular tissues
Cause of DegNala disease
Patulin is toxic primarily through affinity to sulfhydryl groups (SH), which results in inhibition of enzymes.

15. Mysterious diseases: Cottonseed cake toxicity
Occasional Cottonseed cake Toxicity in Pakistan
Mysterious illness of cattle, buffalo
thought to be due to cottonseed cake
Myths on Bt cotton toxicity to livestock and humans

18. Cottonseed cake toxicity: Fact vs Myth

19. Cottonseed cake toxicity: Expert opinion

20. Mysterious diseases: Cottonseed cake toxicity
Studies at NARC on Cottonseed cake Toxicity:
Survey, and analyses:
Complaints arising after 4 months post-production.
Screening for 220 toxins
Microbiological investigations etc
Results:
Co-contamination with 70 mycotoxins
Yunus, A. W., et al Mycotoxin cocktail in the samples of oilseed cake from early maturing cotton varieties associated with cattle feeding problems. Toxins. 7.

21. Mysterious cotton cake disease: NARC study
Mycotoxin
Fungi
Level (µg/kg)
Guide
µg/kg
Min
Max
Tenuazonic acid
Alternaria/Phoma
550
9,900
Aflatoxin B1 + B2
Aspergillus flavus
5,500 20
Ochratoxin A+B
Aspergillus/Penicillium
500
2,340
250
Citrinin
100
350
Aflatrem -
14,700
Cyclopiazonic acid
1,100
6,706
Paspalitrem A
Claviceps/Fusarium
11,870
107,600
Paspalin
3,700
34,500
Paspalinin
700
3-Nitropropionate
Arthrinium
1,000
Yunus, A. W., et al Mycotoxin cocktail in the samples of oilseed cake from early maturing cotton varieties associated with cattle feeding problems. Toxins. 7.

22. Mysterious cotton cake toxicity: NARC study
Mycotoxin
Primary effect
Carryover
Tenuazonic acid
Cancer (esophagus)
Aflatoxin B1 + B2
Cancer (liver, respiratory tract)
Yes
Ochratoxin A+B
Cancer (kidney), CNS
Citrinin
Cancer (kidney)
Aflatrem
Neurotoxin
Cyclopiazonic acid
Paspalitrem A
Paspalin
Paspalinin
3-Nitropropionate
Neurotoxin (Healthy animals)
Ochratoxins also associated with Alzheimer, Parkinson; The toxicity of andrastin A is not known but the parent fungi is known to results in fatal poisoning of cattle
Yunus, A. W., et al Mycotoxin cocktail in the samples of oilseed cake from early maturing cotton varieties associated with cattle feeding problems. Toxins. 7.

23. Mycotoxins: What we know in Pakistan?
Focus had been on aflatoxins

24. Aflatoxin: effects on health
Potent liver carcinogen.
25,000 to 155,000 cases/year globally (estimate)
Stunted growth and immunosuppression.
Acute exposure results in death
Many reports of contamination in South-East Asia

25. Normal thymus
Normal liver

26. Aflatoxin: Globally 5-30% cases of liver cancer
Liu and Wu, 2010

27. Scenario in Pakistan: Aflatoxins in foods
Aflatoxin levels in various foods – different studies
Commodity
> 4 ppb
> 20 ppb
Rice % 1%
Red Chili %
4 - 46%
Peanuts %
0 - 50%
Milk %
0 - 81%
Specific study areas – Small sample size

28. What did you drink last year?

29. What did you drink last year?
Milk collected from Rawalpindi-Islamabad:
Longitudinal study Jan to Dec 2016
Blind study
Analyses:
Aflatoxin in milk using ELISA

30. Study 1: UHT milk

31. Study 1: Pasteurized milk

32. Study 1: Baby milk powder

33. Study 1: Raw milk

34. Study 1: Overall comparison

35. Study 1: UHT 87% 258 770 Pasteurized 60% 939 4808 Raw 9% 1580 7461 Dry
Type
Samples under 500 ppt AFM1
AFM1 Mean
(ppt)
AFM1 Max
UHT
87%
258
770
Pasteurized
60%
939
4808
Raw 9%
1580
7461
Dry
42%
528
1935

36. Second Study All provincial capitals in high season

37. Methodology
450 dairy farms in all provincial capitals during Nov/Dec 2016.
Milk samples for AFM1 analysis
1800 feed samples for aflatoxin analysis

38. Study 2:

39. Study 2: Sampling

40. Study 2: Survey

41. Study 2: Results – AFM1 in milk

42. Study 2: Results – AFM1 in milk

43. Study 2: Results – AFM1 in milk

44. Study 2: Results – AFM1 in milk

45. From where is the milk being contaminated?
Dairy Feed !
Including oilseed cakes, mixed concentrates, brans, straws

46. Aflatoxin – From dairy feed to milk
Contaminated
dairy feed
B1, B2, G1, G2
M1, M2
M1, M2

47. Aflatoxin contribution of dairy feeds
High Contamination
Moderate Contamination
Low Contamination
Cottonseed cake
Vanda
Canola meal
Bakery waste
Pulses
Palm oilcake
Pulse bran
Bread waste
Coconut cake
Grains
Maize oilcake
Wheat bran
Rice polish
Wheat straw

48. Risky areas to encounter aflatoxin in feed
High Risk
Area
Moderate Risk
Low Risk
Lahore
Peshawar
Hunza
Islamabad
Gilgit
Muzaffarabad
Quetta
Karachi

49. Conclusions: Aflatoxin risks need to be reconsidered.
One Health - Urgent need to improve dairy feeds.
Growing evidence of presence of mycotoxins other than aflatoxin.
Long term studies and control laws on other mycotoxins
Need to educate food processors and health agencies.
Coupled with monitoring and control strategy.
No need to panic – please!

50. Thank you for the kind attention