1

WASTEWATER WASTEWATER –– FOOD FOOD ––HEALTH CHAINHEALTH CHAIN

Avrelija Cencič

Infectious Diseases

• Infectious diseases can be defined as those diseases, which are caused by microorganism or their toxic secretionsthat penetrate the natural barriers in the body and multiply within to cause disease.

• Infectious diseases continue to be the third leading cause of death in the United States annually and the second leading cause of death worldwide (Fauci, Touchette, & Folkers, 2005).

• There are approximately 57 milliondeaths occurring throughout the world each year.

2

Emerging pathogensEmerging pathogens

• A newly emerging disease is a disease that has never been recognized before.

• HIV/AIDS, • Severe acute respiratory syndrome

(SARS), • Nipah virus encephalitis, and • variant Creutzfeld-Jakob disease (vCJD)

Re-emerging pathogensRe-emerging pathogens• Re-emerging, or resurging, diseases are those

that have been around for decades or centuries, but have come back in a different form or a different location.

• HEV• West Nile virus in the Western hemisphere, • Monkeypox in the United States, and • Dengue rebounding in Brazil and other parts of

South America and working its way into the Caribbean.

persist

3

Deliberately emerging

• Deliberately emerging diseases are those that are intentionally introduced.

• 1995 – “The apocalyptic religious sect Aum Shinrikyo released saringas in a Tokyo subway, killing 12 commutes and injuring thousands. The cult also had enlisted Ph.D. scientists to launch biological attacks.

• Between 1993 and 1995, Aum Shinrikyo tried as many as 10 times to spray botulinum toxin and anthrax in downtown Tokyo (NOVA Online, 2002).”

• 2001 – Just one week after the terrible September 11th, terrorist attack, letters containing anthrax spores were sent to Tom Brokaw at NBC News in New York, the New York Post, and Senator Tom Daschle in Washington, D.C. Twenty-two individuals were made sick by the spores, five of which died of inhalation anthrax (NOVA Online, 2002).

4

Factors that are involved in re-emerging and emerging of pathogens

Ecological changesHuman demographics, behaviour

International travel and commerce

Technology and industry

Microbial adaptation and change

Breakdown in public health or control

Breakdown of the host’s defenses

5

Gastrointestinal infections• Gastrointestinal infection

(infectious intestinal disease) can be caused by a variety of communicable diseases and infections, which gain entry by and/or affect the gastrointestinal tract.

• Symptoms of gastrointestinal infection, which are not necessarily confined to diarrhoea and vomiting, are caused by the organisms themselves or by the toxins that they produce.

6

PathogensPathogens

7

E. Coli

• Is one of the main species of bacteria living in the lower intestines of mammals, known as gut flora.

• When located in the large intestine, it actually assists with waste processing, vitamin K production, and food absorption.

• Gram-negative bacterial rod occurs as a Vero cytotoxin-producing species (VTEC), mostly of serogroup 0157

• The cause of outbreaks of haemorrhagic colitis since the early 1980s,

• A not infrequent complication is the haemolytic uraemic syndrome (HUS)

8

Source of contamination with E. coli

Water contamination with E. coli O157:H7

• The presence of E. coli in water is a strong indication of recent sewage or animal waste contamination.

• Sewage may contain many types of disease-causing organisms

• E. coli comes into waterduring rainfalls, snow melts, or other types of precipitation, E. coli may be washed into creeks, rivers, streams, lakes, or ground water.

• When these waters are used as sources of drinking water and the water is not treated or inadequately treated, E. coli may end up in drinking water.

9

Salmonella enteritidis

• Egg-associated salmonellosis is an important public health problem

• Salmonella enterica are involved in causing diseases of the intestines (enteric means pertaining to the intestine).

• The three main serovars of Salmonella entericaare Typhimurium, Enteritidis, and Typhi

S. Typhi• This bacterium is the causative agent of typhoid fever.

• It is very common in under-developed countries, and causes a serious, often fatal disease.

• Symptoms of typhoid fever include nausea, vomiting, fever and death.

• S. Typhi can only infect humans, and no other host has been identified.

• The main source of S. Typhi infection is from swallowing infected water.

• Food may also be contaminated with S. Typhi, if it is washed or irrigated with contaminated water.

10

S. Typhimurium

• Until recently the most common cause of food poisoning by Salmonella species was due to S. Typhimurium.

• As its name suggests, it causes a typhoid-like disease

• The disease is characterized by diarrhea, abdominal cramps, vomiting and nausea, and generally lasts up to 7 days not normally fatal

S. Enteritidis• has become the single most common cause of food poisoning

• causes a disease almost identical to the very closely related S. Typhimurium.

• is particularly adept at infecting chicken flocks without causing visible disease, and spreading from hen to hen rapidly.

• mass production chicken farms.

• Salmonella infection can rapidly spread throughout the whole food chain.

• chickens from a single farm may be distributed over many cities, and even states, and hence Salmonella infections can be rapidly dispersed through millions of people.

11

Enteroviruses

• Over 100 types in human and animal waste

• To environment via waste water, aerosols fromWWTP, rivers,…

• Transmission: foecal-oral

• In the sludge 10 5 - 1011 virus particles/g

Characteristics• Intracellular pathogens

• Very resistant in the environmetn (pH, temp., chlorine, UV)

• Cause of gastroenteritis, respiratory diseases, conjuctivitis, aseptic meningitis, encephalitis, paralyses…

• Low infectious dose

• Risk in water 10- do 10.000 x higher than for pathogenicbacteria

12

Survival• More resistant than bacteria

• Survival classical desinfection techniques (chlorine, filtration…)

• Lipid solubilisers resistant

• pH = 3 to 10

• Long lasting low temperatures

• 120 days in waste water, 100 days in soil at temperatures 20-30°C

Enteroviruses• Picornaviridae

• Caliciviridae

• Adenoviridae

• Reoviridae

• Poliovirus, Hepatitis virus, echovirus,…

• Norvovirus, kalicivirus, astrovirus,…

• Adenovirus

• Reovirus, rotavirus

Emerging waterborne pathogens!!

13

Hepatitis E virus

• 20% of people through the world have been already contaminated

• Mortality rate about 1 to 3% (up to 25% for pregnant women)

• Non-enveloped, positive sense, single-stranded RNA virus from Hepeviridae virus family

HEV

11

22

3344

Liver

Plants

Water

Faeces Contaminatedanimals

ContaminationContamination

14

A) Age and sex distribution of acute hepatitis E patients, 2003-2005 (n = 604). B) Age, sex distribution, and travelassociated with indigenous acute hepatitis E case-patients (n = 143). NR, not recorded.

• Avian influenza

Avian influenza, or "bird flu", is a contagious disease caused by Influenza A viruses that normally infect only birds and, less commonly some mammals such as pigs.

• Avian influenza viruses can be highly species-specific, but have, on occasions, crossed the species barrier to infect humans and other mammals.

• The currently circulating H5N1 viruses represent a previously unrecognized type of avian influenza that is causing fatal infections in wild birds, domestic poultry, mammals like cats, and occasionally humans on a broad geographic scale.

15

Scheme of avian influenza pathogenesis and transmission

16

Bacterial indicator as enterococci, coliforms and faecal

coliforms are not the ONLY INDICATORS OF MICROBIAL

QUALITY OF WATER!!

17

Focus:Focus:AssessmentAssessment ofof microbialmicrobial

qualityquality ofof wwastewaterastewaters s andand disposeddisposed sludge sludge thatthat cancan bebe ofof potentialpotentialuse in agriculture. use in agriculture.

Objectives:Objectives:•• To To investigateinvestigate the the

presencepresence ofof potential potential humanhuman pathogens pathogens (viruses, bacteria) in (viruses, bacteria) in wastewater and wastewater and disposed sludge. disposed sludge.

•• To To assessassess thethe mmicrobiicrobialalriskrisk ofof wastewaterswastewaters andanddisposeddisposed sludgesludge..

EXAMPLEEXAMPLE• TWO WASTE WATER TREATMENT PLANTS

• Samples were taken on exact estimated point;

(a) at point where wastewater is pumped into tank where biological treatment starts (20l)

(b) at the end of the process where the water is pumped to hydro – channel (20l)

(c) sample of sludge after centrifugation process and dried sludge at the end of the process (10 l and 100g)

18

19

20

MethodsMethods usedused

• Water: filter adsorption-elution method

• Sludge: centrifugation-elution method

- eluate (-70°C / onto cellmonolayers)

CellCell infectioninfection assayassay• CLAB cell line derived from porcine small intestine• chicken small intestine cells line B

-CPE or cytopathogenic effect was observed in the case of all applied samples 24h to 96 hours after infection ofcell monolayers.

21

Dot Dot blotblot assayassay

321

Dot blot positive samples on HEV:

1. output from Mb. on Caco-2 cells and CLAB cells;

2. output from Lj. on CLAB cells;

3. active sludge from Mb. on Caco-2 cells, CLAB and C3A cells.

1 2

Dot blot positive samples on influenza matrix protein:

1. active sludge from Mb. on Caco-2 cells;

2. active sludge from Lj. on Caco-2 and CLAB cells.

!!! weakly positive signal was obtained with dotblot assay

performed before infecting cells with raw sample of active sludge

from Lj. (data not shown).

Dot-blot

321

Dot blot positive for HEV:

1. Efflux Mb.2. Efflux Lj.3. Active sludge Mb.

Dot blot positive for Rotavirus:

Efflux MbActive sludge MbActive sludge LjDry sludge Mb

22

CPE CPE observationobservation

Monolayerdestruction

Fig. 1.Control CLAB cells 48 h Fig. 2. Sample of dry sludge on CLAB cells 48 h

Monolayerdestruction

Fig. 3. Control B cells 24 h Fig. 4. Sample of dry sludge on B cells 48 h

23

Monolayerdestruction

Monolayerdestruction

Fig. 5. Control PoM2 cells 24h Fig. 6. Sample of dry sludge on PoM2 cells 24h

Fig. 7. Control PSI cells 24 h Fig. 8. Sample active sludge on PSIcells 24 h

Dot Dot blotblot assayassay1

2 3 4

Dot blot positive samples on HEV:

1. raw sample of active sludge from Mb

2. active sludge from Mb. on MA104 andPoM2 cells

3. active sludge from Lj. on MA 104, PoM2 and PSI cl1 cells

4. dry sludge from Mb. on MA104, PoM2, PSI cl1 and CLAB cells

24

UTILISATION OF PURIFIED WASTE UTILISATION OF PURIFIED WASTE WATER (WATER (andand sludgesludge))

FOR AGRICULTURE AND INDUSTRYFOR AGRICULTURE AND INDUSTRY

Nothing in the world of living things is permanently fixedNothing in the world of living things is permanently fixed..

Hans Hans ZinnserZinnser. Rats, Lice and History (1935). Rats, Lice and History (1935)