Canine filarial infections in Tuscany, central Italy

Abstract

This study was conducted in order to investigate the epidemiology of filarial species in a region of central Italy (Tuscany) in dogs that did not undergo prophylaxis for filariasis. From 2007 to 2009, 630 blood samples were collected from 40 kennels throughout the regional territory. Samples were analysed with Knott's modified test and with an enzyme-linked immunosorbent assay (ELISA) for the detection of Dirofilaria immitis antigens, those positive for microfilariae were also subjected to histochemical staining for acid phosphatase activity to validate the identification of the species. An overall elevated prevalence of filariasis (23.2%) was found. Dirofilaria immitis and D. repens were found to be the dominant species, with similar prevalences (12.5% and 12.1% respectively), while Acanthocheilonema reconditum was less common (1.9%). Results of this study indicate that monitoring for filariasis should be kept at a high level. A constant monitoring of the canine sanitary status should also be carried out for the protection of human health, considering the zoonotic potential of filarial worms.

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CaninefilarialinfectionsinTuscany,centralItaly
M.Magi,L.Guardone,M.C.Prati,G.Tozzini,B.Torracca,G.MonniandF.Macchioni
JournalofHelminthology/Volume86/Issue01/March2012,pp113116
DOI:10.1017/S0022149X11000113,Publishedonline:04April2011
Linktothisarticle:http://journals.cambridge.org/abstract_S0022149X11000113
Howtocitethisarticle:
M.Magi,L.Guardone,M.C.Prati,G.Tozzini,B.Torracca,G.MonniandF.Macchioni(2012).Caninefilarialinfections
inTuscany,centralItaly.JournalofHelminthology,86,pp113116doi:10.1017/S0022149X11000113
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Canine filarial infections in Tuscany,
central Italy
M. Magi
1
, L. Guardone
1
, M.C. Prati
2
, G. Tozzini
1
, B. Torracca
1
,
G. Monni
1
and F. Macchioni
1
*
1
Dipartimento di Patologia Animale, Profilassi e Igiene degli Alimenti,
Pisa University, Viale delle Piagge 2, 56124 Pisa, Italy:
2
Scuola Normale
Superiore di Pisa, Italy; INFN Sezione di Pisa, Italy
(Received 17 September 2010; Accepted 1 March 2011; First Published Online 4 April 2011)
Abstract
This study was conducted in order to investigate the epidemiology of filarial
species in a region of central Italy (Tuscany) in dogs that did not undergo
prophylaxis for filariasis. From 2007 to 2009, 630 blood samples were collected
from 40 kennels throughout the regional territory. Samples were analysed with
Knott’s modified test and with an enzyme-linked immunosorbent assay (ELISA)
for the detection of Dirofilaria immitis antigens, those positive for microfilariae
were also subjected to histochemical staining for acid phosphatase activity to
validate the identification of the species. An overall elevated prevalence of
filariasis (23.2%) was found. Dirofilaria immitis and D. repens were found to be the
dominant species, with similar prevalences (12.5% and 12.1% respectively),
while Acanthocheilonema reconditum was less common (1.9%). Results of this
study indicate that monitoring for filariasis should be kept at a high level.
A constant monitoring of the canine sanitary status should also be carried out
for the protection of human health, considering the zoonotic potential of
filarial worms.
Introduction
Dogs can be infected by several species of filarial worms;
species found in Italy are Dirofilaria immitis,Dirofilaria
(Nochtiella)repens,Acanthocheilonema (syn. Dipetalonema)
reconditum and, less frequently, Acanthocheilonema
(syn. Dipetalonema)dracunculoides and Cercopithifilaria
(syn. Acanthocheilonema)grassii. These species can also
infect cats and wild animals, as well as foxes and wolves,
and occasionally humans. Filarial nematodes differ in the
localization of adult parasites: D. immitis adult stages live
in the right side of the heart and in the pulmonary
artery, D. repens lives in the subcutaneous tissue, while
A. reconditum, A. dracunculoides and C. grassii live in the
peritoneal cavity, in adipose tissue and in intramuscular
spaces, respectively. Their life cycle is indirect, with
definitive vertebrate hosts and arthropods as intermediate
hosts, also acting as vectors. After mating, adult female
worms release microfilariae into the haematic circulation
of the definitive host. Arthropods, in which first-stage
larvae complete their development to the infective
third stage, are mosquitoes belonging to genera Culex
and Aedes for Dirofilaria spp., and fleas (Ctenocephalides
spp.) and ticks (Rhipicephalus spp.) for Acanthocheilonema
spp. and C. grassii.
Dirofilaria immitis and D. repens are filarial species of
major concern in Europe for both canine and human
health (Simo
´net al., 2009). Dirofilaria immitis is the
aetiological agent of canine heartworm disease and it is
the most pathogenic species, as it can be lethal; in dogs
D. repens is considered less dangerous and causes
pruriginous dermatitis. In Italy current data on canine
dirofilariasis confirms the expanding trend of parasites as
observed in the rest of Europe (Genchi et al., 2009). Today
D. immitis is present, not only in the hyperendemic area of
the Po River Valley, but also in the northern part of Italy
(Lombardy and Friuli Venezia Giulia), in central Italy
(Umbria, Abruzzo and Tuscany), in the south (Campania,
Apulia and Calabria) and in the islands (Sicily and
*Fax: 0502216941
E-mail: fmacchion@vet.unipi.it
Journal of Helminthology (2012) 86, 113–116 doi:10.1017/S0022149X11000113
qCambridge University Press 2011

Sardinia). Dirofilaria repens is distributed in almost all
of the Italian territory.
Acanthocheilonema reconditum has a worldwide distri-
bution in dogs (Quinn et al., 1997). In Europe it has been
reported in Serbia (Tasic
´et al., 2008), in Spain (Aranda et al.,
1998), in Greece (Vakalis & Himonas, 1997), in Austria
(Hinaidy et al., 1987) and in Italy (Cringoli et al., 2001).
Acanthocheilonema dracunculoides has rarely been reported:
in Europe it has been found in dogs in The Netherlands,
Greece, Spain and Portugual (Bolio et al., 2004). In Italy it
was found in foxes in Latium and Tuscany (Cancrini, 1986;
Magi et al., 2009) and for the first time it has been reported
in a dog in Sicily (Giannetto et al., 2003). Cercopithifilaria
grassii was discovered in 1907 in a dog from Rome (Noe
`,
1908), subsequent sporadic reports have occurred (India:
Balasubramanian et al., 1975; Greece: Vakalis & Himonas,
1997). In Italy microfilariae were found in the blood of
a dog from Sicily (Giannetto et al., 2004) and of a cat from
the Aosta Valley (Tarello, 2004).
All canine filarial species have the potential to
infect humans; D. immitis can cause pulmonary nodules,
D. repens ocularand subcutaneous nodules and pruriginous
dermatitis (Pampiglione & Rivasi, 2007; Gonza
´lez-Miguel
et al., 2010) and A. reconditum can affect the eye (Huynh
et al., 2001). The aim of this study is to investigate the
prevalence and the distribution of different filarial species
in an area of central Italy (Tuscany).
Materials and methods
The study was carried out between April 2007 and
December 2009 in Tuscany, a region with natural breeding
sites for a great variety of mosquito species, such
as Culex pipiens,Culex modestus,Anopheles maculipennis,
Coquilletidia richardii and Aedes albopictus (Cancrini et al.,
2006), which have all proved to be suitable vectors of
dirofilarial nematodes (Cancrini & Gabrielli, 2007).
Blood samples were collected from 630 dogs; 40
kennels were involved in the study (see table 1). The
sample size was chosen so that the number of subjects
enrolled in each municipality was roughly proportional
to the surface area of the municipality itself. Animals
were housed in outdoor kennels located in different
towns in every district of Tuscany. They were all
autochthonous, older than 6 months of age and had not
undergone any prophylactic treatment for filariasis. Dogs
were sampled in kennels, as most privately owned dogs
currently undergo chemoprophylaxis. Individual data
about sex, age, breed, living habitat and geographical
origin were recorded. Each blood sample was divided
into two fractions: 1 ml of whole blood was mixed with
9 ml of 2% formaldehyde solution for the detection of
circulating microfilariae; and at least 2 ml were collected
in tubes with anticoagulant, in order to obtain plasma for
D. immitis antigen detection. Samples were submitted to
the Department of Animal Pathology, Prophylaxis and
Food Hygiene of Pisa University.
The identification of microfilariae species was carried
out using modified Knott’s technique (Lindsey, 1965).
In order to confirm the morphometric identification,
blood samples which were Knott’s positive were further
analysed using the naphthol AS-TR-phosphate method
for acid phosphatase activity as described by Barka (1966).
This staining distinguishes microfilariae of D. immitis,
D. repens,A. reconditum (Chalifoux & Hunt, 1971; Balbo &
Abate, 1972) and A. dracunculoides (Ortega-Mora et al.,
1989) by four different patterns, depending on the
different activity of the acid phosphatase enzyme:
D. immitis microfilariae have two red areas corresponding
to the excretory pore and the anal pore; D. repens
microfilariae only have one red area corresponding to
the anal pore; A. reconditum microfilariae show a reddish
colour on the whole body and A. dracunculoides micro-
filariae show a very intense red colour on the central part
of the body, at the anal pore and at the excretory pore.
Samples were also subjected to an enzyme-linked
immunosorbent assay (ELISA) for the detection of
antigens of D. immitis with the Canine Heartworm
Antigen kit (DiroCHEK
w
, Synbiotics, Kansas City,
Missouri, USA). The procedure was conducted following
instructions contained in the kit.
Results were subjected to statistical analysis, using the
x
2
test, and were considered significant if P,0.05.
Results and discussion
Amongst 630 samples tested, 146 were positive for
filariasis (prevalence 23.2%; 95% confidence interval:
19.9–26.5%). Dirofilaria immitis was found in 79 cases
(12.5%), D. repens in 76 (12.1%) and A. reconditum in 12 (1.9%).
Table 1. Dogs classified according to districts and detected filarial species; k¼number of kennels sampled; n¼number of dogs sampled.
Districts k n D. immitis D. repens A. reconditum
D. immitis
D. repens
D. immitis
A. reconditum
D. repens
A. reconditum
Arezzo 2 27 0 4 0 2 0 0
Firenze 8 162 11 13 4 5 0 1
Grosseto 7 141 22 20 0 4 0 0
Livorno 4 27 2 10 1 1 0 3
Lucca 4 26 2 4 0 0 0 0
Massa-Carrara 2 60 1 0 0 0 0 0
Pisa 5 55 10 0 0 0 3 0
Pistoia 4 41 7 0 0 0 0 0
Prato 1 31 5 3 0 0 0 0
Siena 3 60 2 4 0 2 0 0
Total 40 630 62 (9.8%) 58 (9.2%) 5 (0.8%) 14 (2.2%) 3 (0.5%) 4 (0.6%)
114 M. Magi et al.

Several cases of double infestations were found; the
detailed situations in each district are summarized
in table 1. The difference amongst the prevalence of the
first two species in the various districts is highly signi-
ficant (P,0.0001). No sex- and age-related differences
were observed.
Filarial infections in Italy have, in recent years, shown
an expanding trend, as reported for all Europe (Genchi
et al., 2005). The prevalence (23.2%) of filariasis in
untreated dogs found in this study, shows that in Tuscany
the parasitic pressure and the risk of infestation are high.
Dirofilaria immitis and D. repens were the dominant
species, showing widespread diffusion throughout the
region and similar prevalence values (12.5% and 12.1%,
respectively), whereas A. reconditum (1.9%) was less
common and was found in only three districts (Firenze,
Pisa and Livorno). Different intermediate hosts and
the easy control of fleas and ticks in contrast to
dirofilariasis vectors could explain the lower prevalence
of A. reconditum. The widespread diffusion in Tuscany
of mosquito species, potential vectors of dirofilariae
(Cancrini et al., 2006), including Ae. albopictus, might have
played a determining role in the presence of dirofilariasis.
The identification of species achieved with the modified
Knott’s technique was validated by the acid phosphatase
histochemical staining.
The ELISA test for D. immitis antigens was positive for
65 dogs, 42 of these were also found to be positive using
Knott’s modified test, while 23 were negative for the
presence of D. immitis microfilariae (occult filariasis): this
could be due to a unisex infection, a prepatent period or
an immune-mediated clearance of microfilariae. On the
other hand, 10 animals were found to be positive for
D. immitis microfilariae using Knott’s method and acid
phosphatase staining but negative for antigen detection;
this could be explained by the immune-mediated
clearance of antigen–antibody complexes.
This study shows that the monitoring of filariasis
should be kept at a high level because, despite the
common use of prophylaxis, parasites are widespread.
Dogs’ sanitary status should be constantly monitored, for
the protection of both animal and human health, taking
into consideration the zoonotic potential of filarial worms,
in particular D. repens. A complete diagnosis with the
correct species identification is needed in order not to
underestimate the presence of species that are not
detectable using serological tools. Finally, awareness of
prophylaxis, accurate diagnosis and correct treatment by
veterinary surgeons are important.
Acknowledgements
We would like to thank all veterinary surgeons who
kindly collaborated during the sampling.
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