Zoonotic potential of the soil-transmitted helminths...

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Stefano D’Amelio – Dept. of Public Health and Infectious Disease

Zoonotic potential of the soil-transmitted

helminths Ascaris and Trichuris

Twelfth Workshop of National Reference Laboratories for Parasites

Rome, 18-19 May, 2017

Soil-transmitted helminthes (STHs)

• Soil-transmitted helminthes are

important parasites of humans,

domestic and wild animals. Some

species have a severe economic

impact.



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Soil-transmitted helminthes (STHs)

Disease Aethiological agent Occurrence Endemic regions

Ascarosis Ascaris lumbricoides 807- 1221 millions Asia, Africa and

Latin America

Trichurosis Trichuris trichiura 604- 795 millions Asia, Africa and

Latin America

Ancylostomos

is

Necator americanus

Ancylostoma duodenale

576- 740 millions Asia, Africa and

Latin America

Strongylosis Strongyloides stercoralis 30- 100 millions Asia, Africa and

Latin America

WHO lists STHs as neglected diseases



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Deaths due to

ascariasis



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Absolute Poverty

(up to $2 a day)

Deaths due to

ascariasis





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Ascariasis world map-DALYs

per million persons

Deaths due to

ascariasis



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STHs in Italy

The San Gottardo epidemics

STHs in Italy

Palma di Montechiaro

«Il figlio di Biagio è morto, non si capiva di che, e poi gli

sono usciti i vermi dal naso. Quando i vermi sono nel

sedere, si tirano con le mani che quelli mordono, fanno

male. Ce n’è di corti, lunghi come una mano, quanto un

braccio. Si tirano anche dalla bocca. Siamo pieni di vermi

tutti».

An international congress was organized in Palma, by Silvio

Pampiglione and Danilo Dolci, chaired by Carlo Levi, attended

by Jean Paul Sartre, Pier Paolo Pasolini, Giorgio Napolitano,

Leonardo Sciascia and many others.

Morphology

• Ascarids are large nematodes, 5-30

cm long, and the body is usually

stout.



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TAXONOMY AND

LIFE CYCLE Phylum: Nematoda

Class: Secernentea

Order: Ascaridida

Family: Ascarididae

Genus: Ascaris

Species: A. lumbricoides Linnaeus 1758

A. suum Goeze 1782



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During lung migration, Ascaris provokes an

allergic reaction (Loeffler’s syndrome)



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In the intestine, massive Ascaris infections

my determine obstruction



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Grey’s Anatomy – season 13

Ascaris sp. is able to migrate and invade

the bile duct



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Food as a source for infective stages







Ascaridoid nematodes tend to be highly

conserved in their morphological

characters, producing a number of

virtually identical species



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Cryptic species are genetically

differentiated biological species that

show no evident differences at

morphological level

Sibling species are cryptic species that

cluster together at the most precise

detectable level in phylogenetic trees



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Species A and C are sibling

Species A and C are cryptic



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Hypotheses to explain Ascaris spp. origin in their respective hosts (Leles et al., 2012):

QUESTIONS

• Ascaris lumbricoides (usually infecting humans) and Ascaris suum (recorded mostly from pigs) are both valid species

• A. lumbricoides in humans is derived directly from A. suum , with A. suum then existing as a persistent ancestor. A. lumbricoides could have arisen by an allopatric event of host-switching (pig to human)

• Ascaris suum is derived directly from A. lumbricoides, with the persistent ancestor being A.

lumbricoides

• A. lumbricoides and A. suum are conspecific

• Are A. lumbricoides and A. suum representing distinct species?

• Is this really relevant to describe the zoonotic potential?



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EPIDEMIOLOGY of ASCARIASIS

Hall A, Horton S, de Silva N - (2009)

AFRICA GUATEMALA CINA

- Human-human

transmission (A.

lumbricoides)

- A.suum in pigs

USA EUROPE JAPAN

-A.suum in pigs and in humans

Cross-infection and

hybridization

Non endemic Endemic Mixed model



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Poorly supported phylogeny

may lead to unreliable pictures



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AIMS OF OUR STUDY Genetically characterize :

- Italian and Slovak nematodes from pigs and humans

- Human nematodes from Colombia

Evaluation of zoonotic potential of Ascaris sp. in non endemic

regions



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MATERIALS AND METHODS N HOST (N of

specimens)

GEOGRAPHICAL

ORIGIN (N of

specimens)

316

Pig (187)

Human (13) Italy (197)

Pig (94)

Human (15) Slovakia (109)

Human (8) 8 Colombia (8)

Human (1) 1 Pakistan

Human (1) 1 Romania

Molecular characterization:

-nuclear marker: PCR-RFLP ITS region– HaeIII

-mt DNA marker : cox1 sequencing,

parsimony network and phylogenetic analyses

101

Pig (45 )

Human (9) Italy (54)

Pig (33)

Human (6) Slovakia (39)

Human (7) Colombia (7)

Human (1) Pakistan (1)

Human (1) Romania (1)

168

Pig (93 ) Endemic (7)

No endemic (86)

Human (73) Endemic (51)

No endemic (22)

-AMOVA: Host affiliation and Endemicity

DATASET 1:

DATASET 2:



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ITS-RFLP RESULTS

N HOST (n of

specimens)

POSITIVE PATTERN

ITS-RFLP

319

Pigs (284)

222

183 Pig A. spp

29 Hybrids

10 Human A. spp

Humans (35)

23

5 Pig A. spp

17 Human A. spp

1 Hybrid

PATTERN LENGTH

Human 610 bp; 370 bp

Pig 610 bp; 230 bp; 140 bp

Heterozygote 610 bp; 370 bp; 230 bp; 140 bp

PIG

82,4 %

13,1 %

4,5%

Hu P He

Human Ascaris spp. Pig Ascaris spp. Hybrids

HUMAN

73,9 %

21,7%

4,3%



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Slovakia and Colombia

human

Human

Ascaris

spp

ITS-RFLP RESULTS N ORIGIN (n of

specimens)

PATTERN

ITS-RFLP

252

Italy (162)

130 Pig A. spp

25 Hybrids

7 Human A. spp

Slovakia (80)

59 Pig A. spp

8 Hybrid

13 Human A. spp

Colombia (8) 8 Human A. spp

Italy-human

55 %

11,1%

33,3%

Italy- pig

82,3 %

15,7 %

1,9% Slovakia- pig

79,4 %

11 %

9,6%

HW not significant

p= 0,16

HW significant

p< 0,05

Italian

pig farmer

Patients from Italy

and Romania

From Pakistan,

unknown and one

Roma Gypsy

Human Ascaris spp. Pig Ascaris spp. Hybrids



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Pig pattern in pig host

Human pattern in human host

Human pattern in pig host

Pig pattern in human host

Heterozygote pattern in pig host

A

B

C

Hap12 UC=2

UI=2

HapN

SI

HapN

si

Hap N UC

Hap15

Si

HapN

SS

Hap 6

UI=1 SI=3

Hap14

SI

HapN

SI

HapN

SI

HapN

U=1

SS=1

Hap21

UP

Hap12

SS=2

Hap13

SS

Hap 3 SS=7

Hap 4 U=1

SI=4 SS=10

Hap N

SS

Hap 5 UI=4

SI=25

SS=8 UR=1

Hap 1 UC=4

SI=6 US=6

SS=2

RESULTS – mtDNA cox1 sequences: DATASET1

31/16

Cross

infection

Hybridization



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Human host

Pig host

cox1 RESULTS – DATASET2

A2

B

C

A 1



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Host affiliation

- ITS-RFLP

cox1 RESULTS:

DATASET2



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A2

A1

B

C

Hap5

Hap1

Hap4

cox1 RESULTS

DATASET2



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AMOVA- DATASET2

SOURCE OF

VARIATION

VARIANCE

COMPONENTS

VARIATION

%

Among groups 0.22283 6.15

Among

populations

within

groups

0.93121 25.71

Within

population 2.46830 68.14

HOST AFFILIATION

SOURCE OF

VARIATION

VARIANCE

COMPONENTS

VARIATION

%

Among groups 0.36799 9.79

Among

populations

within

groups

0.69239 18.43

Within

populations 2.69675 71.78

ENDEMICITY

HIGHER INFLUENCE OF THE EPIDEMIOLOGICAL CRITERION IN

MODULATING THE ACCUMULATION OF VARIABILITY



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• Human specimens from Italy show A. suum genotype (italian and romanian

nationalities) and the human genotype A. lumbricoides (unknown

nationality + Pakistan + Roma gypsy), so pigs is the main source of

infestation in Italy

•All human infestations in Slovakia and Colombia are due to A. lumbricoides

genotype, mirroring high endemicity epidemiological scenario

•All genotypes circulate in italian and in slovak pig hosts

•Cluster C seems basal in the tree (later studies revealed it can be the most

probable ancestor)



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Regno: Animalia

Phylum: Nematoda

Class: Adenophorea

Order: Stichosomida

Family Trichuridae

Genus Trichuris Roederer, 1761

TAXONOMY OF TRICHURIS



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MORPHOLOGY

(a) T. trichiura; female

(b) T. trichiura male

T. trichiura egg



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SOME HISTORICAL FACTS…

1740 – first detection (Morgani);

1761 – morphological description (Roederer);

1771 – named as Ascaris trichiura (Linneo);

1886 – description of the life cycle (Calandruccio);

1941 – the American Society of Parasitologists

attributes the worm to the genus Trichuris .



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Human Reservoir hosts

Environment (Soil)



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Human dog, pig, monkey

Environment (Soil)





An egg measured with an ocular micrometer (A) and adult worms (B)

of Trichuris vulpis from a 9-year-old girl in Mexico.

(Marquez-Navarro et al 2012)

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From Traversa 2011

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(da Callejion et al., 2012)

Taxonomic status of the genus Trichuris

(da Callejion et al.,

2012)

Humans and other primates



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A genetic analysis of Trichuris trichiura

and Trichuris suis from Ecuador

(Meeksum et al 2015)



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Fig. 2. Gel picture from the PCR-RFLP of the ITS-2 region of pig- and human-derived Trichuris. The gel

shows the band pattern from the PCR-RFLP the using the BseL I on the ITS-2 region of Trichuris derived from

humans and pigs.

Sofie Nissen, Azmi Al-Jubury, Tina V.A. Hansen, Annette Olsen, Henrik Christensen, Stig M. Thamsborg,

Peter Nejsum

Genetic analysis of Trichuris suis and Trichuris trichiura recovered from humans and pigs in a

sympatric setting in Uganda

Veterinary Parasitology, Volume 188, Issues 1–2, 2012, 68–77



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Fig 2. Inferred phylogenetic relationship among Trichuris spp. using concatenated amino acid

sequences and Maximum Likelihood (ML) and Bayesian Inferences (BI).

Hawash MBF, Andersen LO, Gasser RB, Stensvold CR, Nejsum P (2015) Mitochondrial Genome Analyses Suggest Multiple

Trichuris Species in Humans, Baboons, and Pigs from Different Geographical Regions. PLOS Neglected Tropical Diseases 9(9):

e0004059. https://doi.org/10.1371/journal.pntd.0004059

http://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0004059





Hawash et al 2016

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Fig 3. Inferred phylogeny among Trichuris spp. recovered from pigs and primates based on

partial cox1 sequences and NJ clustering.

Hawash MBF, Andersen LO, Gasser RB, Stensvold CR, Nejsum P (2015) Mitochondrial Genome Analyses Suggest Multiple

Trichuris Species in Humans, Baboons, and Pigs from Different Geographical Regions. PLOS Neglected Tropical Diseases 9(9):

e0004059. https://doi.org/10.1371/journal.pntd.0004059




Study area



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Data Ordine Specie ospite Feci Contenuto intestinale

2012 PROBOSCIDAE Elephas maximus 2 -

2011 PRIMATI Lemur catta 8 -

2011 Eulemur macaco 4 -

2011 Callithrix pygmaea 1 -

2011 Saguinus imperator 1 -

2011 Saguinus oedipus 1 -

2011 Cercocebus sp. 3 2

2011 Chlorocebus aethiops 2 2

2011 Macaca fuscata 10 4

2011 Mandrillus sphinx 6 1

2011 Pan troglodytes 16 -

2012 Pongo pygmaeus 7 -

2011 LAGOMORPHA Oryctolagus cuniculus - 2

2011 RODENTIA Dolichotis patagonum - 1

2012 Octodon degus - 1

2012 CARNIVORA Lynx lynx 3 -

2012 Panthera leo 6 -

2011 Panthera pardus saxicolor 12 1

2012 Panthera tigris altaica 6 -

2011 Canis lupus 6 -

2012 Lycaon pictus 14 2

2011 Nasua nasua 10 -

2012 Ursus arctos 3 -

2012 Halichoerus grypus 2 -

2012 PERISSODACTYLA Equus hemionus kulan 2 1

2012 Equus grevyi 10 1

2012 Tapirus terrestris 3 1

2012 ARTIODACTYLA Hippopotamus amphibius 4 -

2012 Hexaprotodon liberiensis 2 -

2011 Camelus bactrianus 11 -

2012 Cervus nippon 2 1

2012 Giraffa camelopardalis 1 1

2012 Bos javanicus 4 1

2012 Bison bonasus 3 -

2012 Taurotragus oryx 1 -

2011- Addax nasomaculatus 4 -

2012 Capra aegagrus 3 -

2011 Capra hircus 6 -

2011 Hemitragus jemlahicus 1 -

2012 Ovis orientalis 1 1

2012 Kobus megaceros 3 3



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FIRST RESULTS

Two well separati clusters, supported by

high bootstrap values.

ITS DATASET 1

Chlorocebus aethiops

Macaca fuscata



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Chlorocebus

aethiops

Macaca fuscata



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A.lumbricoides 99

99

99 99

99

99

85

MCTV 1-15 from Macaca

(Bioparco)

+ AB586133 T. sp.

T. trichiura human (GenBank)

CH 1-8 from Chlorocebus

(Bioparco)

+ T. colobae (GenBank)

99 51

Macaca fuscata

Chlorocebus aethiops

ITS-2

T. skrjabini

T. leporis

T. discolor T. ovis

T. vulpis

T. muris T. arvicolae

DATASET 1, DATASET 2, DATASET 3 E DATASET 4

+ T. suis (GenBank)

+ JN181814, JN181822, JN181826 , JN181829

T. trichiura



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Trichuris in NHP – Testing Phylogeny Trichuris from Grivet

Trichuris from Macaque

Trichuris from M. fuscata (designated as

subclade MF) showed a close relationship

with subclade DG which contains T. trichiura from humans from China and from Papio

ursinus (cape baboon) from South Africa

Nomascus gabriellae

(yellow-cheeked gibbon)

Trichuris from C. aethiops

(designated as subclade CA) and two

sequences of T. trichiura, one from a human case from Cameroon and one

from P. ursinus from South Africa,

formed a separated group related to

T. suis from pigs and humans

Colobus guereza

kikuyuensis (mantled guereza)

Analyses performed on two datasets (entire ITS and only ITS2) using two methods (NJ and Bayes): same topology – high statistics

support



Trichuris from Macaque and from Grivet form two well supported and separated

clades

Different taxonomic entities associated to several primates species

No clear monophyly either of human-derived and of NHP derived Trichuris

Is T. trichiura a complex of cryptic species with a certain degree of host preference?

route of parasites introduction in an isolated structure/group of hosts

murine/human/pig Trichuris through rats or contaminated food ? NO

management measures to prevent cyst/eggs transport from

one enclosure to another within the zoological garden ?

MORE INVESTIGATION NEEDED

zoonosic risk? POSSIBLE

(Trichuris from C. aethiops and humans in the same clade)

EVIDENCES FROM PHYLOGENETIC ANALYSIS

RECOVERY OF PARASITES IN ZOOLOGICAL GARDENS



Exhibit reproducing natural habitats

TWO MAIN INTERESTING

ASPECTS

High frequency of contacts with humans



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“...Trichuris from humans and pigs were

genetically very distinct with independent

demographic histories..”

“.. Evidence for an African origin of T.

trichiura which were then transmitted with human

ancestors to Asia and further to South

America...A host shift to pigs may have

occurred in Asia from where T. suis seems to

have been transmitted globally by a combination

of natural host dispersal and anthropogenic

factors...” Hawash et al 2016

The presence of different Trichuris lineages in humans and other primates makes for

a taxonomic conundrum in phylogenetic analysis, because it is

unclear which of these taxa represents T. trichiura as originally described, and

which are undescribed or possibly cryptic species.



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Zoonotic potential of Trichuris must be taken

seriously into account

• Helminthic therapy is currently being studied as

a treatment for several (non-viral) autoimmune

diseases in humans including celiac disease,

Crohn's disease, multiple sclerosis, ulcerative

colitis and atherosclerosis

• Most autoimmune disorders are believed to

involve hyperactive Th1 or Th17 immune

responses that are down regulated by the

promotion of a Th2 response by helminths



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ZOONOTIC RISK OF

HELMINTHIC THERAPIES

T. Suis eggs IBD

T. trichiura

T. vulpis T. suis



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Zoonotic potential is not necessarily bad

• Animal can serve as models for studying

several aspects

• Animal can serve as refugia to fight drug

resistance caused by massive treatments in

humans



Treated

Untreated

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Stefano D’Amelio

Serena Cavallero

Federica Berrilli

David Di Cave Claudio De Liberato

Klaus Friederich Viliam Snabel



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