Abstract A monitoring programme aimed at the diagnosis of subcutaneous dirofilariasis and heartworm disease in working (police and military) dogs in Slovakia has been performed during th

A Pavlačka

D Antolová

J Németh

M Miterpáková

P Dubinský

Z Hurníková

CiteSeerX

Dirofilaria infections in working dogs
in Slovakia
M. Miterpáková1*, D. Antolová1, Z. Hurnı́ková1, P. Dubinský1,
A. Pavlačka2 and J. Németh3
1Parasitological Institute SAS, Hlinkova 3, Košice, Slovak Republic:
2Canine and Equine Division Police Corps Presidium, Mlynská dolina 35,
Bratislava, Slovak Republic: 3Health Service of the Army Headquarters,
Gen. Miloša Vesela 21, Ružomberok, Slovak Republic
(Accepted 21 July 2009; First Published Online 4 September 2009)
Abstract
A monitoring programme aimed at the diagnosis of subcutaneous dirofilariasis
and heartworm disease in working (police and military) dogs in Slovakia has
been performed during the period of September 2007 to February 2008. In co-
operation with the Ministry of the Interior and the Ministry of Defence, in total,
710 dogs (591 police dogs and 119 military dogs) were investigated for the
presence of microfilariae in blood. All police and military dogs in active service
held on the territory of Slovakia were included. Microfilariae were detected in 118
(20.0%) police dogs and 10 (8.4%) military dogs. The most infected individuals
originated from southern parts of Slovakia (Trnava region 53.6% and Nitra region
39.6%); the prevalence was low in northern regions (Žilina 3.1% and Prešov 6.6%).
In several districts of southern Slovakia, the prevalence of subcutaneous
dirofilariasis in working dogs exceeded 40%. In all infected animals, the
autochthonous origin of the disease was confirmed; however, due to the frequent
movement of working dogs, it was not possible to identify the exact locality of
infection. At present, a dog living in Nemšová village in Trenčı́n district (north-
western part of the country) is regarded as the northernmost localized
autochthonous case of subcutaneous dirofilariasis in Slovakia. In three dogs,
co-infection of Dirofilaria repens and Dirofilaria immitis was detected. High
prevalence rates in working dogs and the zoonotic characteristic of the disease
represent an undoubtedly important veterinary and medical problem that
requires the urgent introduction of prophylactic and control measures.
Introduction
Dirofilariasis is a parasitic infection caused by
helminths of the genus Dirofilaria. In Europe, the most
important causative agents of disease are Dirofilaria repens,
a parasite of subcutaneous tissues, and Dirofilaria immitis
which causes the pulmonary and heart form of disease.
Definitive hosts of both species are dogs and other
carnivores and several mosquito species play the role of
both intermediate host and vector of the parasite.
Nowadays, subcutaneous dirofilariasis is regarded to be
an arthropod-borne disease with the fastest spread in
Europe, representing a high infection risk for both animals
and humans. An increase of prevalence and new endemic
foci have been ascertained in several eastern and central
European countries, such as Bulgaria (Georgieva et al.,
2001), Serbia (Dimitrijevic et al., 2007), Hungary (Fok et al.,
2007) and also Austria (Duscher et al., 2009). The main
reasons for such an expansion are in particular climatic
changes, which have occurred in central Europe in recent
years, accompanied by global warming and frequent
floods, resulting in an increased abundance of mosquitoes
(Genchi et al., 2005; Rinaldi et al., 2006).*E-mail: miterpak@saske.sk
Journal of Helminthology (2010) 84, 173–176 doi:10.1017/S0022149X09990496
q Cambridge University Press 2009
In the Slovak Republic, canine subcutaneous dirofilar-
iasis was recorded for the first time in 2005 (Svobodová
et al., 2005). Within the primary monitoring of dirofilar-
iasis that has been started in southern parts of Slovakia in
February 2007, in total, 287 dogs of different age, sex,
breeds and use were examined for the presence of
microfilariae. Infection was diagnosed in 99 animals
(32.1%). The prevalence was the highest in the group of
police dogs, with microfilariae detected in 46 out of 90
examined individuals (51%; Miterpáková et al., 2008).
Therefore, the aim of the present study was to ascertain
the prevalence of dirofilariasis in working dogs in
Slovakia.
Materials and methods
Study area and dog origin
The survey of dirofilariasis in working dogs took place
in between September 2007 and February 2008. Within
the co-operation of the Ministry of the Interior and the
Ministry of Defence, blood samples from 710 working
dogs (591 police and 119 military dogs) from kennels in
different districts of Slovakia were collected and
examined. While police dogs are used in almost all
districts, military dogs serve in only ten districts.
Sample collection and questionnaire
Sampling was done by police, military and/or contract
veterinarians. All veterinarians were asked to fill out a
questionnaire regarding the breed, age, sex, kennel
locality, localities visited by dogs and eventual health
problems of each dog.
Histological and molecular procedures
Larval stages of dirofilariae and microfilariae were
detected in total blood using a modified Knott (1939) test.
Briefly, 1 ml blood was mixed with 9 ml of 2% buffered
formalin and centrifuged at 2000 rpm for 5 min. The
supernatant was removed and sediment was stained with
1% methylene blue. The stained sediment was examined
under the light microscope at 150 £ magnification.
For species determination, positive samples were
analysed using histochemical staining based on the
different distribution of acid phosphatase activity using a
commercial kit test (Leucognost-SP, Merck, Darmstadt,
Germany) according to Peribáñez et al. (2001). Micro-
filariae of D. immitis show two acid phosphatase activity
spots localized around the anal and excretory pores,
whereas D. repens shows only one acid phosphatase
activity spot localized around the anal pore.
DNA was extracted from positive samples using a
DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany)
for polymerase chain reaction (PCR) analyses. The
amplification of DNA fragments for cytochrome oxidase
subunit 1 gene was performed with specific D. repens and
D. immitis pairs of primers according to Rishniw et al.
(2006). All PCRs consisted of a denaturing step at 948C for
2 min and 35 cycles of denaturing for 30 s at 948C,
annealing for 30 s at 578C and extension for 30 s at 728C,
with final extension for 7 min at 728C. The PCR products
were visualized by 1.5% agarose gel electrophoresis.
Data analysis
Questionnaire data were analysed with respect to
relative dependency of infection incidence and different
epidemiological indicators, especially the kennel locality.
A non-parametrical test (x 2 test with Yates correlation)
was used for statistical comparison using Statistica 6 Base
(StatSoft, Inc., Tulsa, Oklahoma, USA).
Results
Dirofilariasis in police dogs
Microfilariae were detected in 118 blood samples out of
591 police dogs, which represents an overall prevalence of
20.0% (table 1). Dogs originated from 55 districts in
Slovakia, and the parasite was diagnosed in animals from
28 districts. Statistically significant differences in infection
occurrence were found between individual regions
(x 2 test: x 2 ¼ 83.0304; df ¼ 7; P ¼ 0.0000). The majority
of infected police dogs had been bred in southern regions
of Slovakia (Trnava region 53.6% and Nitra region 39.6%).
The incidence was the lowest in northern parts of
Table 1. Dirofilariasis in working dogs in individual regions of Slovakia (N, number examined;
%, prevalence).
Police dogs Military dogs
Region N (%) ^95% CI N (%) ^95% CI
Bratislava 63 23.1 11.3 4 25.0 79.6
Trnava 56 53.6 13.5 4 0 0
Nitra 48 39.6 14.54 5 20.0 55.5
Trenčı́n 42 9.5 9.3 55 7.3 7.1
Žilina 64 3.1 4.4 21 0 0
Banská Bystrica 71 14.1 8.3 11 18.2 27.2
Košice 125 22.4 7.4 – – –
Prešov 122 6.6 4.5 19 10.5 15.2
Slovak Republic 591 20.0 3.2 119 8.4 5.1
Bold values are the total values for the Slovak Republic.
174 M. Miterpáková et al.
Slovakia, in the region of Žilina (3.1%) and Prešov (6.6%).
Dogs born between years 1996 and 2003 were infected
more frequently than younger animals born between 2004
and 2007; however, the difference was statistically
insignificant.
D. repens was detected in all infected police dogs and,
in three individuals, co-infection with D. immitis was
present. The mixed infection occurred in one dog from the
south-western Nitra region and in two dogs bred in
the south-eastern part of Slovakia in the village Velké
Slamence on the frontier with Ukraine.
Dirofilariasis in military dogs
A total of 119 military dogs were examined from ten
districts of Slovakia. The prevalence of dirofilariasis in
military dogs was significantly lower compared with
police dogs (x 2 test: x 2 ¼ 8.9616; df ¼ 1; P ¼ 0.0028).
Infection was confirmed in ten animals from seven
districts. The average prevalence in military dogs was
8.4% (table 1). The infection was present in nine dogs born
in the years 1995–2003 and in one dog born in 2005. In all
infected military dogs, simple infection with D. repens was
confirmed by PCR analysis.
Discussion
In the present study, we aimed to establish dirofilariasis
prevalence in working dogs bred in the territory of
Slovakia and to identify the localities where the disease
could originate.
The results of the first reported investigation of
dirofilariasis in working dogs were published from
southern France, where in 1983, in total, 207 military
dogs and, 5 years later, 180 dogs utilized by the army were
examined for dirofilariasis. Chauve (1997) recorded a
high prevalence in both years under study (46.8 and
37.2%, respectively).
Our results point to a frequent occurrence of infection
in police dogs; the prevalence rates in some districts
exceeded 40.0%. The infection in military dogs was
significantly lower with an average prevalence 8.4%. The
number of microfilariae in 1 ml of blood varied between
one up to several hundreds of specimens.
Based on the evaluation of questionnaire data, we
consider all infections to be autochthonous (all dogs
became infected in Slovakia). Nevertheless, the exact
determination of the locality where the disease was
acquired and the designation of the origin of individual
infection cases are very difficult. The reason is that almost
all police dogs spend several weeks per year in a training
centre located in Záhorie (Bratislava and Trnava regions),
which is considered to be an endemic area of subcu-
taneous dirofilariasis. Also, a police dogs’ kennel is
situated in this region. The stay in endemic localities is
believed to be the main cause of high infection prevalence
in dogs working in northern parts of Slovakia with
climatically less favourable conditions, as well as the
possible reason for big difference in numbers of infected
police dogs when compared with military dogs. Out of
ten infected military dogs, we were able to identify the
source of infection only in one individual. This dog lived
in the cadastre of Nemšová village (latitude 488570000N;
longitude 188100000E) in Trenčı́n district and is regarded as
the northernmost localized autochthonous case of
subcutaneous dirofilariasis in Slovakia (fig. 1). Despite
the impossibility of localizing exactly the origin of
infection, infected dogs become the source of parasite
and, in suitable climatic conditions, they may be
considered as source for new foci of dirofilariasis.
The high prevalence of infection recorded in police
dogs represents a considerable veterinary problem, as the
disease (in particular, the pulmonary and heart forms
caused by D. immitis) and its pharmacotherapy may cause
thrombosis and heart failure, especially in animals
exposed to physical labour.
Fig. 1. Dirofilariasis in Slovakia (regions: BA, Bratislava; TT, Trnava; NR, Nitra; TN, Trenčı́n; ZA, Žilina; BB, Banská Bystrica; PO, Prešov;
KE, Košice); dogs not examined (W), uninfected ( ), infected ( ), human cases (S).
Dirofilaria infections in working dogs in Slovakia 175
Another potential risk is the transplacental transfer
of microfilariae from the female dog to fetuses. The
possibility of intrauterine infection has being discussed
for several decades and this transmission pattern has
been proved experimentally in gravid female dogs.
Mantovani & Jackson (1966) found microfilariae in
amniotic fluid, in fetuses and in the blood of newborn
whelps. In Todd & Howland (1983) investigated the
transmission of microfilariae from three gravid bitches to
their offspring. They found microfilariae in nine whelps
from one mother; in other female dogs, the transmission
was not proved. The numbers of microfilariae in newborn
whelps varied between 1 and 39 specimens per individual.
Regarding the zoonotic character of infection, the risk
of human infection is also of importance, in particular, in
endemic localities and in regions with high mosquito
populations. Humans represent a ‘dead end’ of infection
because they are not suitable definitive hosts for the
parasite, and larvae develop only from infective L3 stage
to pre-adult L5 larvae. To date in Slovakia, two
autochthonous human dirofilariasis cases with the
subcutaneous form of infection have been reported in
2007 (Jalili et al., 2007) and in 2008 (Ondriska et al., 2008).
Both cases were reported from western Slovakia, in the
regions of Bratislava and Trnava (fig. 1).
The high prevalence of subcutaneous dirofilariasis in
working dogs from Slovakia, with 40% of dogs infected in
some regions, requires the urgent introduction of
prophylactic and control measures. In endemic localities,
prevention includes the administration of selamectin-,
ivermectin- or moxidectin-based preparations monthly
throughout the mosquito season and for 1 month after.
Preventive administration of these anti-parasitic drugs is
effective on L3 and L4 larvae and inhibits their develop-
ment to the adult stage. A single dose of drug should also
be administered to dogs travelling into endemic localities.
When a longer stay is expected, monthly administration
during the entire period of residence is recommended. The
use of repellents and anti-parasitic collars is also an
alternative protective measure.
Acknowledgements
The investigation was supported by the Science Grant
Agency VEGA 2/7186/27.
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Dirofilaria infections in working dogs in Slovakia

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Dirofilaria infections in working dogs in Slovakia

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