This document provides information on parasitic foodborne diseases. It discusses several protozoan parasites that can cause foodborne illness, including Toxoplasma gondii, Entamoeba histolytica, Giardia intestinalis. For each parasite, it describes their life cycle, transmission, symptoms, diagnosis and treatment. It notes that foodborne parasites are a major public health problem worldwide and can cause disease ranging from mild diarrhea to more severe complications in vulnerable groups. Prevention focuses on proper hygiene, sanitation and food handling practices.
1. Presentation on
PARASITIC FOOD BORNE DISEASES
submitted to –DR.SIMRANPREET KAUR
submitted by- manjeet rathour (l-2017-v-04-d)
Guru Angad Dev Veterinary And Animal Sciences University,Punjab
2. • Food-borne parasitic zoonosis include both protozoan
and helminthic infections.
• Amongst one thousand five hundred known infectious
agents for human being, 66 are protozoa and 287 are
helminths
(Chomel, 2008; Taylor et al., 2001)
• Amongst them majority (60.3%) of the emerging
infectious diseases are zoonotic.
(Jones et al., 2008)
3.
4. Foodborne Disease
Illness caused by ingestion of contaminated food.
Symptoms often affecting stomach or intestinal tract including
i. Nausea and vomiting
ii. Diarrhea
iii. Abdominal pain
Sometimes nonspecific symptoms and symptoms outside GI
tract, depending on parasitic agent.
Young children, pregnant women, elderly and
immunocompromised persons at greatest risk for severe
illness
5. Transmission of foodborne diseases
Through
i. Raw or undercooked meat and meat products.
ii. Raw milk (that is, milk that has not been pasteurised or
sterilised)
iii. Food items contaminated with human faeces (directly or
indirectly)
iv. Raw vegetables contaminated with soil.
v. Food contaminated by chemicals, e.g. pesticides such as
malathion.
vi. Food prepared using contaminated water, e.g. for
washing vegetables and raw meat
vii. Food kept in an unsuitable condition for a long time
after preparation
7. Parasites
Organisms that obtain their food (meat ,milk or in vegetable) from other living
creatures
Smaller than their food source and this distinguishes them from predators which
also eat other living things.
Common food borne animal parasites – Protozoa and Worms
Worms include tapeworms (Cestodes), roundworms (Nematodes) and flukes
(Trematodes)
How are they transmitted?
They may be transmitted from host to host through consumption of contaminated
food and water, or by putting anything into your mouth that has touched the stool
(faeces) of an infected person or animal.
How do they vary?
Parasites are of different types and range in size from tiny, single-celled,
microscopic organisms ( protozoa) to larger, multi-cellular worms ( helminths)
that may be seen without a microscope. The size ranges from 1 to 2 µm
(micrometres) to 2 meters long.
8.
9. Protozoan Parasites
One-celled organisms but are larger and more complex than
bacteria
Generally not susceptible to antibiotics that kill bacteria but
there are effective drugs to treat some (not all) parasitic
infections
Most common types;
I. Toxoplasma
II. Entamoeba histolytica
III. Giardia
IV. Sarcocystis spp.
V. Cryptosporidium
VI. Trichinella spp.
10. Toxoplasma
Toxoplasma gondii
Obligate, intracellular- parasitic protozoan
causes toxoplasmosis
Infection in humans and other warm-blooded animals can occur
i. by consuming raw or undercooked meat containing T. gondii
tissue cysts
ii. by ingesting water, soil, vegetables, or anything contaminated
with oocysts shed in the faeces of an infected animal
iii. from a blood transfusion or organ transplant
iv. Transplacental transmission from mother to foetus, particularly
when T. gondii is contracted during pregnancy
Sexually reproduce only within the intestines of members of the cat
family (felids)
12. Epidemiology
• Most women were in the 18–25 years age group (48.3%), followed by 26–30 years
(28.2%) and 31–35 years (13.66). Few (45) women older than 35 yr. were included
• This pan-India study shows a prevalence rate of 22.4% with a wide variation in four
geographical regions ranging from as low as 8.8% to as high as 37.3%. The overall
IgM positivity rate was 1.43%, indicating that an estimated 56,737–176,882
children per year are born in India with a possible risk of congenital toxoplasmosis
(Singh S,2014)
• Antibodies to T. gondii have been demonstrated in 9.7 to 33.7 percent cases of
sheep, goats, pigs, cattle, buffaloes, camels, horses, cat and dogs .
(Chhabra et ai, 1985).
• Dubey (1987) stated that isolation of T. gondii from pig and sheep is very low and
clinical toxoplasmosis is not known in animals in India. A few cases of abortions
were, however, seen in goats with titers of 1:64 or above (IHA titers) and a neonatal
mortality was seen in goat born to dams with titers from 1:8 to 1:256 from Parbhani
(Marathwada)
(Singh and Msolla, 1986)
13.
14. • In human – T. gondii oocysts cause –no symptoms
Latent infection
• Domestic and wild cat –definitive host (for intestinal or sexual
phase)
• D.H. generally primary source of human toxoplasmosis
(shed faeces)
Transmission
• Cat to cat
• But all vertebrate susceptible to oocysts shed by cat
• ≥ 100 oocysts can produce toxoplasmosis
• Oocysts can survive over one year warm and moist environments
• In human source for toxoplasmosis – pig(raw meat)
17. Conti..
• In the USA, toxoplasmosis is the second leading cause of
foodborne illness related deaths and fourth leading cause of
foodborne illness related hospitalizations.
(Scallan et al., 2011).
18.
19. SYMPTOM, DIAGNOSIS AND TREATMENT
• Incubation -: 6-10 days
• SYMPTOMS
• Lymph node swelling
20. • Generally occur in immunocompromised host
• Trachzoites –Cross to placenta
Diagnosis
• Not diagnosed by stool (oocysts) present in cat
• Serological method- Acute Infection Detect , when 4
fold IgG antibodies found with convalescent serum
22. Conti..
• More rapid detection by IgM @ 1weeks and peak 2-4
week infection
• Some other method MB dye test , indirect
heamoagglutation method indirect
immunofluorescence resistance
23. Risk factors of toxoplasmosis and
preventive methods
Diminished vision or blindness after birth of child,
more severe effects include-hydrocephalus,
convulsions, and calcium deposits in the brain.
Responsible for the deaths of AIDS patients and
causes encephalitis in many immunosuppressed
Pregnant women and immunocompromised patients
should avoid the following:
i. Raw or undercooked meat or eggs
ii. Unpasteurized milk, particularly goat's milk
iii. Contact with cat faeces, including changing of cat
24.
25. Rx-
Sulfonamides and pyrimethamine (Daraprim) are two drugs widely
used to treat toxoplasmosis in humans
Spiramycin, a drug used in France to treat pregnant women to minimize
the effects of congenital toxoplasmosis (not useful in preventing relapse
of neurotoxoplasmosis in immunosuppressed patients
Control:-
i. Environment contamination with cat faeces
ii. Uncooked meat
iii. Cyst – 60*C (destroy)
iv. Irradiation -30 Krad
v. By freezing –inactivation of oocysts
27. Entamoeba
Entamoeba histolytica
An anaerobic, cause Amoebiasis
Transmission of the parasite occurs when a person
ingests food/water that has been contaminated with
infected faeces
Cysts of the parasite are the viable form outside the
host.
They can survive weeks in water, soils and on foods
under moist conditions.
An active Entamoeba infection will cause abdominal
pain, fever, severe diarrhoea, vomiting
28. Epidemiology
According to the WHO, E. histolytica is the second
leading parasitic cause of death (after malaria) and has
been estimated to infect 50,000,000 people worldwide
of whom 40,000 - 100,000 die yearly
(Babiker et al., 2009).
Amoebiasis is a disease caused by the
parasite Entamoeba histolytica. Only about 10% to 20%
of people who are infected with E. histolytica become
sick from the infection.
(http://www.cdc.gov , www.ncbi.nlm.nih.gov)
30. LIFE CYCLE
Ingestion of cysts
Passage of cysts through the stomach where gastric acid stimulates
therelease of the infectious trophozoites from the cysts
Trophozoites move to the duodenum where they divide
Trophozoites travels to the colon where they attach to colonic
epithelialcells
After attachment they produce a cytotoxin that kills epithelial cells so
theycan gain access to deeper tissues
Continue to divide in colon where amoeba/cysts are excreted in stool
Trophozoites invade the deeper mucousa and enter the peritoneal cavity
Trophozoites are carried in the circulation to the liver but can also
becarried to the lungs, brain and heart
31. DIAGNOSIS OF EXTRAINTESTINAL
AMOEBIASIS
• MICROSCOPY
•Pus aspirated from liver abscess-demonstrate
trophozoites of E.histolyticain less than 20% cases.
•Aspirate from the margin of the abscess show
trophozoites.
•Cysts are never seen in extra intestinal amoebiasis
32. LIVER BIOPSY
• Trozpozoites of Entamoeba histolytica may be
seen in liver biopsy specimen in case of
hepatic amoebiasis or amoebic hepatitis.
33. IODINE STAINED PREPARATION
i. Demonstration of cysts and trophozoites.
i. Stains yellow to light brown.
ii. Nucleus clearly visible with central karyosome
iii. . Cytoplasm smooth and hyaline appearance.
iv. Nuclear chromatin- Bright yellow Glycogen-Golden
brown
v. Chromatoids-No staining
34.
35. SEROLOGICAL TESTS
It has immense value in diagnosis of hepatic
amoebiasis.
1)Complement fixation testing
2)Indirect hemoagglutination(IHA)
3)Latex agglutination
4)Countercurrent immunoelectrophoresis(CIE)
5)Gel diffusion precipitation(GDP)
6)Cellulose acetate membrane precipitation(CAP)
7)ELISA
36. Symptoms
• The clinical spectrum ranges from asymptomatic infection, diarrhoea and
dysentery to fulminant colitis and peritonitis as well as extra-intestinal
amoebiasis.
• Acute amoebiasis can present as diarrhoea or dysentery with frequent,
small and often bloody stools.
• Chronic amoebiasis can present with gastrointestinal symptoms plus
fatigue, weight loss and occasional fever.
• Extra-intestinal amoebiasis can occur if the parasite spreads to other
organs, most commonly the liver where it causes amoebic liver abscess.
Amoebic liver abscess presents with fever and right upper quadrant
abdominal pain.
• Other organs can also be involved, including pleuropulmonary, cardiac,
cerebral, renal, genitourinary, peritoneal, and cutaneous sites. In developed
countries, amebiasis primarily affects migrants from and travellers to
endemic regions, men who have sex with men, and immunosuppressed or
institutionalized individuals.
37. Pathogenicity
• Human infection usually begins with the ingestion of the cyst in
food or water contaminated with human faecal material.
• Cysts survive the acidic pH of the stomach and pass into the
intestine where the cysts undergo excystment and mature into
trophozoites which are passed to the colon.
• In the intestine, many of the trophozoites encyst and both
trophozoites and cysts are excreted along with the faeces; cysts can
survive for prolonged periods outside the host while the
trophozoites survive only for a few hours.
• Infections can range from non-invasive intestinal diseases which are
often asymptomatic to invasive where the trophozoites penetrate
the intestinal mucosa to other organs and produce an
extraintestinal amoebiasis which are usually more serious and life
threatening
(Acker and Mirelman, 2006)
38. Treatment and prevention of E. histolytica
• Metronidazole—penetrates deeper tissues and
destroys amoeba present in liver, brain, lungs etc.
• The organism’s metabolism converts the drug into
its lethal form
• A second drug is used to eradicate the amoeba
present in the intestinal lumen (paromomycin)
Prevention:
• When traveling to areas where E. histolytica is
epidemic or endemic
• Thoroughly wash unpeeled fruits and raw
vegetables
39. Giardia
Giardia lamblia
Single celled, flagellated, microscopic parasite that can live in the intestines of
animals and people
Cause giardiasis
Invade in gastrointestinal tract ,present in primary cell -upper small intestine
(virulence factor)
not phagocytic ,not penetrating
nutrition by absorption
Giardiasis does not spread via the bloodstream, nor does it spread to other parts
of the GI tract
Giardia is worldwide in distribution and considered as most common intestinal
protozoan parasites of man and animals (Thompson, 2011).
Due to host specificity of G. duodenalis, its role in zoonoses is controversial.
40. How do people get giardiasis?
Frequently associated with drinking contaminated
water, but some people might get infected by
consuming uncooked meat also contaminated with
G. lamblia cysts (the infective stage of the
organism)
By putting anything into mouth that has touched
contaminated surfaces or the stool of a person or
animal with giardiasis(young live)
Foodborne giardiasis can result from the use of
contaminated water for irrigating or washing fruits
and vegetables
41. Symptoms of Giardiasis
• Incubation-: 1-4 weeks
• Cyst appear in stool after 3-4 weeks
• In human sympom-: several month to year or more(9*108 cyst/ Day)
• The clinical signs of giardiosis in man vary greatly (Thompson, 2011) and
some of them are acute short-lasting diarrhoea or chronic syndromes
associated with nutritional disorders, malabsorption of fat and weight loss.
• A chronic syndrome is more common and supposed to cause more harm.
• Most common symptoms- Diarrhea, abdominal
• cramps, gas, and nausea
• Chronic infection might lead to dehydration and
• severe weight loss
Molecular studies support the zoonotic transmission of giardia from dog
(Leonhard et al., 2007).
42. Risk factors
• The giardia parasite is a very common intestinal parasite.
• Children. Giardia infection is far more common in children than it is in adults.
(contact with faeces), especially if they wear diapers, are toilet training or spend
time in a child care centre.
• People without access to safe drinking water.
Complications
• Giardia infection is almost never fatal in industrialized countries, but it can cause
lingering symptoms and serious complications, especially in infants and children.
The most common complications include:
• Dehydration. Often a result of severe diarrhea,
• Failure to thrive. Chronic diarrhea from giardia infection can lead to malnutrition
and harm children's physical and mental development.
• Lactose intolerance. Many people with giardia infection develop lactose
intolerance — the inability to properly digest milk sugar. The problem may persist
long after the infection has cleared.
43. Diagnosis and Treatment
Diagnosis - Accurate diagnosis requires an antigen test or, if that is
unavailable, an ova and parasite examination of stool.
Multiple stool examinations are recommended, since the cysts and
trophozoites are not shed consistently.
Given the difficult nature of testing to find the infection, including many false
negatives, some patients should be treated based on symptoms.
Wet mount or stained specimen
ELISA may also use
Treatment :-Nitroimidazole medication is used such as metronidazole,
tinidazole, secnidazole or ornidazole.
Quinacrine( Acridine derivative)
The CDC recommends hand-washing and avoiding potentially contaminated
food and untreated water.
44. Prevention
• No drug or vaccine can prevent giardia infection. But common-sense precautions
can go a long way toward reducing the chances that you'll become infected or
spread the infection to others.
• Wash your hands. This is the simplest and best way to prevent most kinds of
infection. Wash your hands after using the toilet or changing diapers and before
eating or preparing food. When soap and water aren't available, alcohol-based
sanitizers are an excellent alternative.
• Purify wilderness water. Avoid drinking untreated water from shallow wells,
lakes, rivers, springs, ponds and streams unless you filter it or boil it for at least 10
minutes at 158 F (70 C) first.
• Keep your mouth closed. Try not to swallow water when swimming in pools,
lakes or streams.
• Use bottled water. When traveling to parts of the world where the water supply is
likely to be unsafe, drink and brush your teeth with bottled water that you open
yourself. Don't use ice, and avoid raw fruits and vegetables, even those you peel
yourself.
https://www.mayoclinic.org/diseases-conditions/giardia-infection/symptoms-
causes/syc-20372786
46. Cryptosporidium
Mainly Cryptosporidium parvum (ZOONOTIC SPP.)
Obligate intracellular coccidian parasite
4-5 micrometre size
One host required
Present in small intestine and free sporozoites
Inter microvillus region of host enterocyte
Sexual reproduction zygote form
Host cell actin polymerization t/p lead to infection
80% zygote sporuled in host cell
(inthick wall)envivoment resistant oocysts shed in feces other host (by ingested)
47. EPIDEMOLOGY
• 1st notified -1907 (asymptotic mice) , in human 1984 in Braun Station Texas due to
consumption of artesion well water
• World wide prevelance-1-4% among with diarrhea
• Serious infection in the immunocompromised patients
• There have been no reports of Cryptosporidium infections in cattle or buffalo
calves or any other farm animals in India.
• There are 16 species of Cryptosporidium species and 33 genotypes, but only few of
them are zoonotic
(Xiao and Fayer, 2008).
• Human cases have been reported, however, with symptoms of diarrhea,
particularly in bottle-fed children, from Vellore (13 percent) (Mathan et al, 1985),
Calcutta (Das et al. 1987), Varanasi (Singh et al, 1988), Bombay (Saraswathi et al,
1988), Kashmir (Navash and Wattal, 1988) and from Chandigarh (Malla et al, 1987;
Mahajan et al. 1988; Malla et al, 1989).
• There have been other reports from Bangalore and Madurai
(Parija, 1990)
48. Conti..
The following groups have an elevated risk of being exposed to
Cryptosporidium:
People who handle infected cattle – People who eat contaminated food;
meat, fish, milk, fruits and vegetables
– People who swim regularly in pools with insufficient sanitation
– Parents of infected children
– People who take care of other people with cryptosporidiosis(cross
contamination)
– People who drink untreated water
49. Conti..
• In humans, C. hominis (anthroponotic origin) and C. parvum (zoonotic
origin) are responsible for more than 90% of cryptosporidiosis which
accounts for more than 3.1 million deaths each year among children less
than 15 years of age (Fayer, 2004).
50. LIFE CYCLE
The ingested parasite in the form of oocytes, excyst
in the gastrointestinal tract and release infective
sporozoites
Attach to the apical membrane of the host epithelial cells
mature-merozoites (asexual reproduction)
The merozoites (+ intestinal lumen)
infect other epithelial cells or mature into gametocytes
Releases the oocysts which are excreted in faeces into
the environment to start another life cycle (Morgan et al.,
2002).
51.
52. Cryptosporidiosis Symptoms
Cause cryptosporidiosis, a parasitic disease of the
mammalian intestine tract
Incubation -: 6-14 days symptom last 9-23 days
• Primary symptoms - acute, watery, and non bloody
diarrhoea
• Other symptoms -anorexia, nausea/ vomiting and
abdominal pain
• The diagnosis of C. parvum consists of serological tests
and microscopic evaluation of oocysts in stools using
Kinyoun acid-fast staining
53. Diagnosis Treatment
• Diagnose – oocysts in stool
• Staining method negetive staining modefied acid ,sugar
flotation and direct immunoflorosence test
• Rx- Spiramycin, fluconazole ,Amphotericin B
54. Prevention
• C. parvum -4 sporozoites ,highly resistant ,can
survive in moist an cold condition for month
• Can be destroyed by 50%ammonia 10% formalin
@30 min
• Milk – HTST
• Destroy infectivity - @45*C for -20 min
55. Cyclospora
Cyclospora cayetanensis
Cause cyclosporiasis
8-10 micrometer , 2 sporocytsis (each sporocystes -: 2
crescent shaped sporozoited)
By consuming food or water contaminated with C.
cayetanensis oocysts (the infective stage of the organism)
By putting anything into mouth that has touched the stool of
a person or animal with cyclosporiasis
56.
57. Symptoms
• watery diarrheal (sometimes explosive)
• loss of appetite
• Bloating
• stomach cramps
• nausea vomiting
• muscle aches
• low-grade fever
• fatigue
Cyclospora organisms have also been isolated from patients
suffering with AIDS and chronic diarrhoea
(Long et al., 1990; Ortega et al., 1993; Pape et al., 1994; Cox, 2002; Orlandi et al.,
2002; Vuong et al., 2007)
58. Diagnosis and tretment
• Microscopic examination: -direct microscopy
and concentration procedures(formalin-ethyl acetate sedimentation and
Sheather’s flotation procedure)
• Stained smears (using modified acid-fast stain or a modified safranin stain)
• UV fluorescence microscopy
• Sporulation assay- 2.5% potassium dichromate
• Molecular methods- 2.5% potassium dichromate ,frozen without fixation
Treatment:- Trimethoprim and sulfamethoxazole
59. • 1ST case found-1835(J. Paget) London
• 1st -1859 (in human) – Germany
• The first serious outbreak @Pauri and Tehri of
(Uttarakhand)- :undercooked/roasted wild boar meat
(Sethi et al. 2010)
The outbreak caused high mortality (26.2%)
A sero-prevalence study of major parasitic zoonoses,
conducted in Punjab, revealed 5.73% of human subjects
as seropositive for trichinellosis
(Singh et al. 2015).
60. Trichinella spiralis
• 1.4 and 1.6 mm long, and are more flat anteriorly than
posteriorly.
• Pork worm" due to it being typically encountered in
undercooked pork products
• An ovoviviparous, occurring in rodents, pigs, horses, bears,
and humans.
• The smallest nematode parasite of humans, have an unusual
lifecycle
61. • There were a few reports of the occurrence of
T. spiralis in some mammalian hosts, namely,
from a civet cat in Calcutta (West Bengal)
(Schad andChowdhury, 1967);
• Trichinella spiralis is a foodborne roundworm
that can cause parasitic infection.
• Host to host
(No free living stage exit)
62. Conti..
(both larva and adults stage in same host)
Common foods People get trichinellosis by consuming
raw or undercooked meats such as pork, beef cara
beef marine ,bear, cougar, fox, wolf or dog infected
with Trichinella larvae.
In human –consumed Trichnae infested flesh/meat
Trichinella spiralis –female ;- 3-4mm , Male 2-1.5mm
Female produces 1500 egg (larva 0.1 mm in leanth)
66. Symptom and treatment
Survive -10 years in living host
1-2 days after- penetrate the intestinal mucosa(symptom show)
Incubation period :-30 days
Persist several days after initial symptoms
≥100 gm larva –symptoms show
≤10 gm- no symptoms
≥1000gm –serous and acute consequences
Muscle pain (myalgia) breathing difficulty
After 6 month – tissue pain ,swelling and fever t/p
67. Diagnosis and Treatment
Diagnosis
ELISA –positive for IgG/IgM antibody to trichinella
serum reaction
Immunological method including Bintonite
flocaution , CLF
A bentonite titer of 1:5 is significant(not positive up
to 3 weeks after infection)
Rx:-Thiabentazol , Membendazole
68. Prevention and control
Trichinella spiralis affected all animal but avian to be resistant
Transmission in foods This parasite is inherently carried by animals
It is generally thought that a possible route to the animal is through
consumption from eating infected tissues from other animals.
Prevention Cook pork until there are no signs of pink and always
cook meats properly.
Curing and smoking:- Gamma rays
After curing the meat hung for 30 days
69. Anisakis and Pseudoterranova
(Sealworm,Codworm)
Anisakiasis was first recognized as a human disease about
forty years ago
Found with fish
Have intermediate host ,more than one definitive host
Human -: accident host (not a definitive host)
Marine live – definitive host
Chub mackerel and flying squid in Japan and pickled
anchovies, raw sardines, cold smoked salmon, raw or
pickled herring are some vectors
Other fish, including whiting, mackerel, pollack, and
flounder, may also contain these parasites with anisakid
larvae
70. Life cycle
Faeces egg smallcrustaceous(copepode)(ingested) large crustace
intermediated host for 2nd mold (L2 & L3) Fish or squid (L3) L4 &L3 in
fish adult and motile some larva at fish bell flap
71. Varies by season and increases with fish size
Water temperatures and seal populations may
also affect the abundance of these parasites
1st case –Netheland 1955
72. Diseases symptom
Activity of juvenile worms
Harmful A. simple≥ P. decipen (due to present in mucosal
lining )
P. decipiens – pass in faeces(Vomited and cough –irritation
to muscle)
Symptom
4-6 hrs after consumption –infested fish
Epigamic pain , nausea and vomiting
Body swelling –after 7 days infection
When parasite in mucus – peritonitis
73. Diagnosis and Treatment
Endoscopy ,CFT and Indirect IFT
Rx:-Thiabendazole
Prevalence
i. Avoid raw fish (Sushi , Ceriche,Sashaini)
ii. Cooking meat /fish @60* C
iii. -20*C @60 hrs (larva infected)
74. Conclusion
Outbreaks caused by FBP continue to be difficult to identify and
even more challenging to investigate. The weakest link in current
practice for delayed or missed recognition of foodborne outbreaks
by parasite.
At consumer’s level, foodborne illnesses can be reduced by proper
cooking of meat, poultry and eggs to temperatures that will kill
parasite ; although, several stage are heat resistant; steaming under
pressure, grilling, roasting and frying of foods can destroy.
Additionally, refrigerating leftovers promptly and storing foods at
recommended temperatures; avoiding cross-contamination of
cooked and raw foods; washing of utensils and surfaces before and
after use with hot, soapy water; and frequently washing hands
and/or using gloves when preparing food are all recommended
(Gashaw et al., 2008).