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Cryptosporidium spp. and Giardia spp. in oysters, cockles, seawater and waters from natural

sources in Brazil.

PhD. Regina Maura Bueno Franco

Protozoology Laboratory – Health Ministry Collaborator Center for Water and Parasites

Department of Animal Biology – Biology Institute.

Campinas State University –UNICAMP.

V Fórum da Sociedade Paulista de

Parasitologia

Campinas, 13 de outubro de 2009.

Cryptosporidium spp. and Giardia spp.: Public Health Relevance

• These protozoan parasites are major causes of diarrheal disease in humans and have been recognized as the predominant causes of protozoan waterborne diseases worldwide (n=325) (Karanis et al., 2007):

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Cryptosporidium

spp., 50,8%

Giardia spp.,

40,6%

E. histolytica,

2,8%

Cy. cayetanensis,

1,8%

T. gondii, 0,9%

I. belli, 0,9%

Blastocystis,

0,6%

Outros, 0,3%

Demais, 7,3%

Environmental Dispersion of Cryptosporidium and Giardia.

• Their life cycles are monoxenous, with transmission by the fecal-oral route; cysts and oocysts are produced in large numbers and are infectious when excreted (1 x 109-10

g/feces/week).

• These protozoa have low infective doses (9-1,042 oocysts), and have potential to be transmitted from non-human to human hosts; cysts and oocysts shows a marked resistance to environmental stressors and water treatment processes.

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Academic Investigation on Protozoology Laboratory, Campinas State University:

Environmental Samples Analyses

•Partnership: SANASA (Campinas) ; ** FEM / UNICAMP. Financial support: FAPESP

Raw sewage*

Backwashing waters*

(FAPESP 02/11343-1)

Natural source waters

Activated sludge*(FAPESP 02/12845-0)

Hospital raw sewage**

Biossolids

Superficial raw waters*

Bivalve mollusks

Irrigation Waters Field Samples

Molecular characterization

(CNPq)

Occurrence of Cryptosporidium and Giardiain Atibaia River Surface Natural Waters

(South-east Brazil), 2004.

• Atibaia river water is used for domestic, industrial andirrigation purposes and is highly affected by discharge ofdomestic and industrial effluents around Campinas city(CBH, 2001).

• Two point sample collection were selected for the analysis of water:

• A1: upstream Pinheiros Stream (“montante”);

• A2: downstream Pinheiros Stream (‘jusante”).

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Pinheiros Stream

Atibaia River Point A2Point A1

Concentrations of Protozoa in Atibaia River Water Samples by Membrane Filtration

Method* (Franco et al., 2001).

POINT A1

• Giardia spp. cysts were found in 52.0%

of water samples (mean concentration:

25 cysts/liter; range:16-135).

• Oocysts of Cryptosporidium were not

detected in these samples.

• Recovery Efficiency rate of protozoan

parasites achievable by MF method*:

65.1% and 72.2% for oocysts and cysts.

POINT A2

• Giardia spp cysts were detected in

76.0% of the raw water samples

(mean concentration: 64 cysts/liter;

range: 14 – 240)

• Cryptosporidium spp oocysts were

detected in only 0.04% of the raw

water samples (mean

concentration: 0.8 oocysts/l).

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Density of Giardia spp. cysts in Atibaia River Samples: Influence of Pinheiros Stream in

the Water Quality.

Evaluation of Activated Sludge Treatment of Protozoa Removal in Samambaia WWTP,

Campinas city.

• The Samambaia Wastewater TreatmentPlant (WWTP) uses a conventionalsewage treatment system based onrailing, aeration and biological oxidationthrough activated sludge, secondaryclarification and disinfection of thesecondary effluent with a dose of 25-30mJ/cm2 (medium-pressure UV) and anoutflow of 7L/sec.

• Three sampling points were selected:– (1) influent– (2) treated effluent without UV

disinfection– (3) treated effluent with UV disinfection

• During two years, samples were collected once in two weeks.

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Occurrence of Giardia spp. Cysts and Cryptosporidium spp. Oocysts in Samambaia

WWTP, South-east Brazil*.

*Cantusio Neto R; Santos LU; Franco RMB. Water Science and Technology 54(3): 89-94, 2006.

Sample ProtozoaPositive Samples

(%)

Mean and Standard

Deviation

Influent

Giardia 90.5M =1.0 x 105 (± 8,7)

cyst/L

Cryptosporidium 6.4M=6.0 x 104 (± 2,8)

oocysts/L

EffluentGiardia 96.2 M=1.0 x 103 (± 1,0) cyst/L

Cryptosporidium 0.0 __

Molecular Data: confirmed Giardia duodenalis,based on Giardin gene.

Efficiency of Protozoan Removal in Samambaia WWTP by Activated Sludge

Process (2003-2005)

360.000

109.434

40.000

6.153 1.191 2855.143

1.159 153

0

100000

200000

300000

400000

Me

an

Cy

st/

L

Influent Effluent -UV Effluent +UV

Max. Mean Min

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ERv Giardia : 98.9%

Fig.1. Average cyst of Giardia spp recovered from wastewater samples in wastewater treatment facility in

Brazil, during 2 years.

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Animal Infectivity Assay to Evaluate the Inactivation of Giardia Cysts in UV-Treated

Effluent Samples

From day 5-15 p.i.:Feces examined by zinc

flotation; mucosal scrapings of upper duodenum

and ileum.

Animal Model: BALB/c nude

Protocol

Positive Effluent Samples treated by UV light (14

cysts)

All samples were sanitized with sodium

hypochlorite at 1% (60 min; 4°C)

Intragastric inoculation

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Presence of Cysts in Feces and Trophozoites in Intestinal Scrapings of Mouse Infectivity for

determining Giardia spp. Cysts’ Inactivation by UV Light

Groups Presence of Cysts Presence of Trophozoites

Effluent + UV + (3/3) + (1/3)

Efluent - UV + (3/3) - (0/3)

Efluent Filtrated (control group) - (0/3) - (0/3)

Sentinel - (0/3) - (0/3)

Cantusio Neto R; Santos, L.U.; Franco, R.M. B. Water Science & Technology v.54, nº.3, pp.89-94, 2006.

Occurrence of Oocysts and Cysts in Waters from Natural Springs of Campos do Jordão, a

tourist city, São Paulo State.

• Analysis concerning the microbiological quality of the water from natural springs revealed that 13 of the total samples analyzed (n=22) presented standard of quality inadequate for the human consumption:– Spring water samples collected at the point where water flows

naturally to the earth’s surface for parasitological analysis.

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Detection of Pathogenic Protozoa using Membrane Filtration Method

• From June 2003 to May 2004, Cryptosporidium or Giardiawere detected in 25% (3/12) of springs, with concentrations that varied from 0.2 to 0.3 oocyst/L or 0.1 cyst/L. When considered the location of the springs, the positivity for those belonging to the urban areas was 33.3% (2+/6) while the ones from rural area was of 16.7% (1+/6).

14Cryptosporidium spp Giardia spp

Evaluation of Photoeletrochemical Process for Wastewater Treatment (Campinas State

University Clinic Hospital)

• The discharge of hospital sewagewithout treatment can exercisenegative effects on the receivingwaters.

• The design and operation ofwastewater treatment systemsfor hospital is a challenge forwastewater engineers andbiologists. In this study, a pilot-scale photoeletrochemicalreactor for hospital wastewatertreatment was evaluatedconsidering protozoa removal,after 30, 60 and 90 minutes ofexposure.

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Morphological Damage was evaluated by M.E.V. and Phase Contrast Microscopy

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Fig.1: T=0 min. (reactor).

(5.000x)Fig.2: T=30 min. (reactor).

(5.000x)

• Effect of photoeletrochemical treatment after different

exposure time: 0’, 30’ and 90’: Cryptosporidium (Fig. 1

and 2); Giardia cysts (Fig. 3)

Fig.3: T=90 min. (reactor).

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Decrease of the Protozoa Number

after Exposure to the

Photoelectrochemical Reactor.

Cryptosporidium: after 60’ (3 independent trials)

Giardia cysts: after 90’ (3 independent trials)

Recovery and Bioaccumulation of Cryptosporidium spp. by Oysters and Cockles

in Camaroeiro Beach, Brazil.

• The amount of partially treated oruntreated wastewater beingdischarged into the oceans isparticularly important if they aredischarged into coastal waters closeto shellfish habitats, especially if thelocal population has the habit ofeating raw shellfish.

– Bivalve mollusks are great filterfeeders that can accumulate andconcentrate protozoa in theirtissues. These animals may transmitCryptosporidium whenever they areingested in a raw or lightly cookedform.

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Parasitological Analysis of Shellfish Tissues and Seawater

• The aim of this study was to evaluate the natural contamination ofoocysts and cysts of protozoan parasites in bivalve mollusks located inan estuarine environment in the Northern coast of São Paulo Stateand detect these protozoa in seawater.

• Shellfish were processed through the homogenization of bivalvetissues (gill and gastrointestinal tract) and submitted to centrifugalconcentration with ether-PBS; water samples were analyzed bymembrane filtration technique. Protozoa were visualized with DirectImmunofluorescence Reaction and monoclonal antibodies.

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Protozoa in an Estuarine Environment (Camaroeiro Beach), Brazil.

Cryptosporidiumoocysts visualized in seawater of Camaroeiro Beach (600x)

Fig. 2: Fig. 3:

Giardia cysts detected in seawater (600x)

Giardia cyst visualized in gill pool homogenate of oysters.

(400 x)

Fig. 1:

Protozoa in an Estuarine Environment (Camaroeiro Beach), Brazil: Molecular

Characterization.

• Line 1: Cysts in tissue homogenate pool of oysters;

• Line 2 and 3: Cysts in Ipiranga raw river water;

• Line 4: Cysts in Camaroeiro beach (seawater).

Giardia duodenalis

( 217 pb bands)

Confirmed Genera:Giardia (174 pb bands)

Quantitative Assessment of Pathogenic Protozoa in Oysters, Cockles and Seawater

1. Both pathogens were detected in seawater: Cryptosporidium were present in 40.0% (45-80 oocysts/L) and Giardia in 20.0% (50-95 cysts/L) of all samples analyzed.

2. Oocysts were found in both bivalve species: they were detected in three of the six gill pools of cockles (60 oocysts/cockle) and in one of twelve gill pools of oysters (12 oocyst/oyster).

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This is the first time thatCryptosporidium oocystswere found in shellfish fromthe coastal region of Brazil.

The number of oocysts found inthese bivalve mollusks issufficient to cause an infection,if they are viable.

Leal DAG; Pereira MA; Franco RMB; Branco N; Cantusio Neto R. Journal of Water andHealth, 2008 .

Molecular Characterization of Giardiaduodenalis cysts from Clinical Samples of

Campinas State University Hospital.

• This study was conducted to determine the assemblages level of Giardia duodenalis isolates from clinical samples to assess the epidemiological implications in the city of Campinas, SP State.

– Two gene fragments were amplified*:• The filamentous protein giardin is associated with microtubules in

Giardia. The identification of their sequence allowed the development of specific primers for the identification of genera and species of G. duodenalis (174 pb and 217 pb).

• The analysis of a sequence of 220 bp of glutamate dehydrogenase gene confirmed the divergence between A and B assemblages (Nested-PCR).

• Succcessful PCR was performed with DNA cysts from 13 patients of a total of 20 samples (65%).

– PCR was conducted according Mahbubani etal. protocol. J. Clin. Microbiol. 30 (1):74-78, 1992.

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Molecular Characterization of Giardia duodenalis cysts from Clinical Samples of

Campinas State University Hospital.

• The PCR products were purified using a GFX PCR DNA kit and were sequenced with the Big-Dye-Terminator cycle sequencing kit at the CBMEG/UNICAMP with ABI PRISM 377 sequencer.

• The obtained sequences were compared with reference sequences using Clustal W Software and submitted to Basic Local Alignement Search Tool (BLAST).

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Agarose gel (PCR of G and E reactions forsamples D, E e C). # shows a 200pb ladderband.

Giardia G. duodenalis

Alignement

• Reference lineages:

Molecular Characterization of Giardiaduodenalis cysts from Clinical Samples of

Campinas State University Hospital.

• Results:– Sample 16: was found similar to L40508 sequence (assemblage B) in the Clustal W

software (score = 1438). When used BLAST algorithm, was detected similarity with sub-genotype B2 – human specific (Acess number: DQ923586);

– Sample 13: was similar to L40509 sequences (Assemblage A2; human-specific). (Acess number: DQ 923587.1)

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Conclusions

1. The results showed that Cryptosporidium oocysts were present in few of the analyzed samples but with significant oocyst densities while Giardia cysts were detected in more samples with higher densities.

2. The UV disinfection was not completely efficient in the case of wastewater treatment plant studied.

3. Giardia cysts showed greater resistance to photoelectrochemical treatment than Cryptosporidium oocysts.

4. Cryptosporidium oocysts and Giardia cysts were detected in bivalve tissues of oysters and seawater from Camaroeiro Beach located on the north coast of the São Paulo state.

5. The human-specific A2 and B2 sub-genotypes account for some giardiasis cases detected at Campinas State University Hospital.

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Thank You!Agradecimentos aos meus Orientandos:

Paula Vilma de Oliveira (Mestre)

Luciana Urbano dos Santos (Doutor)

Nilson Branco (Mestre)

Rita Borges França (Mestre)

Diego A. G. Leal (Mestre)

Maurício Durigan (Bolsista IC– CNPq)

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Profa. Regina Maura Bueno FrancoE-mail: mfranco@unicamp.br