| Abstract (english) | INTRODUCTION: The Eurasian wild boar is widespread in Europe and Asia, and to a
lesser extent in Africa and North America. In most of Europe, as well as in the Republic
of Croatia, the wild boar population size has been increasing constantly over the recent
years. The exception is currently Eastern Europe itself, where a significant drop in
numbers has occurred locally due to the outbreak of African swine fever. In other parts
of Europe, on the other hand, wild boar are coming closer and closer to settlements,
and are even found in some larger cities. This fact is extremely important given that
the wild boar is a potential reservoir of numerous viral and bacterial diseases
transmitted not only to domestic pigs, but also to other domestic animals and humans.
It is in recent times that African swine fever has focused research on the epidemiology
of wild boar diseases, primarily due to the potential spread to domestic pigs and the
incalculable damage that can therefore occur in pig production. In this study, the
emphasis is on Aujeszky's disease. This disease can cause great economic damage
in domestic pig production and is therefore controlled by preventive vaccination and
the "stamping out" method. As the disease is enzootic in wild boar (RUIZ-FONS, 2017),
it poses a constant threat to domestic pig production. On the other hand, the risk for
the population of hunting dogs, which often come into contact with the virus during
regular hunting activities or through feed with raw meat and offal of shot wild boars,
should be emphasized. In Croatia, interspecific transmission of the virus has been
proven, and virus strains isolated from domestic, wild boar and dogs are genetically
highly matched (KEROS et al., 2015). As the previous research was mainly focused
on seroprevalence, comparison of serological findings and virus detection by molecular
methods on samples originating from the same wild boar was performed in this
research. Also, a comparison of demographic indicators of the population with the
obtained search results was performed, as well as a comparison of the findings with
the characteristics of environmental factors, peculiarities in light of different rating
classes, total hunting area and hunting productive areas. In this way, we tried to
determine which indicators may indicate an increased risk of developing this disease
in the wild boar population. Finally, as wild boar have a complex social structure, it was
necessary to take those factors into consideration.
REVIEW OF THE LITERATURE: Wild boars inhabiting the European continent belong
to one species, the Eurasian wild boar (Sus scrofa L). In the Republic of Croatia, wild
boar is present in all its parts except certain Adriatic islands. The wild boar genotype
in these areas has been preserved to date with a limited effect of hybridization on
genetic structure (ALEXANDRI et al., 2012). In the period immediately after the Second
World War, in the Republic of Croatia, the number was estimated at only 300
individuals (DARABUŠ AND JAKELIĆ, 1996), and the reason for such a small
population are war casualties, large numbers of predators and the presence of
classical swine fever virus. The wild boar population is constantly growing not only in
Croatia, but also in the rest of Europe after the Second World War (SÁEZ-ROYUELA
and TELLERÍA, 1986). According to official records, the number of wild boars in the
Republic of Croatia ranges from 30,000 to 60,000, while hunting bag in the past few
years ranges from 29,500 to 30,000 per year (ANONIMUS, 2018). Pigs are extremely
social animals, except for boars that leave the herd at the age of two. The herd is led
by an old sow, and consists of several sows, and piglets (JANICKI et al., 2007). Wild
boars suffer from the same diseases as domestic ones, so it is necessary to single out
some of the most common diseases such as brucellosis, classical swine fever, African
swine fever, trichinosis, hepatitis E, Aujeszky's disease, parvovirus infection, circovirus
infection of pigs type 2, reproductive and respiratory pigs (JEMERŠIĆ et al., 2019).
Furtheromore, it is important to note that wild boar are a possible reservoir of numerous
viral infections such as reproductive and respiratory syndrome in pigs, porcine
circovirus infections type 2 and parvovirus infections (ROIĆ et al., 2005, 2006, 2012).
Wild boar have important role in the spread of various diseases, some of which are
zoonoses, and poses a potential risk to the health of domestic and feral pigs, and in
some situations to human health. In epidemiological terms, the fact that the wild boar
is a social animal, with the exception of adult males, its role as a reservoir of a number
of different pathogens is growing (ACEVEDO et al., 2007) due to such living conditions
with its current distribution and number. Aujeszky's disease or pseudorabies is an
acute viral infection of domestic and wild animals. This disease was first mentioned in
the United States in 1813 under the name "Mad Itch", and in Europe in 1889 in
Switzerland. The first record in Croatia dates from 1904 (CVETNIĆ, 1997). Aujeszky's
disease virus is swine herpesvirus 1 (Suid herpesvirus-1 or SuHV-1). It is a DNA virus
belonging to the family Herpesviridae, the subfamily Alphaherpesvirinae and the genus
Varicellovirus. The virus is easily maintained in an environment that can serve as a
permanent source of infection, as evidenced by the fact that airborne transmission has
been reported between Germany and Denmark in areas of high pig density
(CHRISTENSEN et al., 1990, 1993). Also, it is important to note that it is rapidly
inactivated by ultraviolet rays as well as pH lower than 6 and higher than 11. A total of
four major genotypes of Aujeszky's disease virus in wild boar have been demonstrated,
of which Type I was found in the US and Central Europe, Type II and Type III in Central
and Northern Europe, and Type IV in Asia (MÜLLER et al., 2011). The virus has
tropism on the respiratory and nervous systems, multiplying in the body of a large
number of animals, including almost all mammals, which serve as the ultimate host
(METTENLEITER, 2000).The disease occurs mainly in pigs, and sporadically in other
species. In domestic pigs, it is mainly transmitted by droplet infection, primarily due to
the density of pigs on the farm (PACINI et al., 2020), and is retained for up to 6 months
after recovery. If sows are infected, they can spread the virus via milk and uterus. Wild
boars mainly transmit the virus sexually during mating (ROMERO et al., 2001),
although droplet transmission within the herd during the year should not be neglected.
It can also be found in the lungs of healthy animals, in which, after a drop in immunity,
it multiplies rapidly and its virulence increases. This phenomenon makes it difficult to
find the source of the infection (CVETNIĆ, 1997). PACINI et al. 2020. isolated the virus in
the fetus of seropositive feral pigs in the endemic region of Italy, which suggests
possible new ways of transmitting the virus and the pathogenetic role of the virus in
wild boar. Carnivores contribute to the spread of the virus, by spreading the virus
further with carcasses and their parts. Carnivores become infected mainly orally by
eating raw meat and offal. The virus initially multiplies in the nasopharynx and is
present in the nasal discharge for the first ten days after infection. The virus penetrates
the lymph nodes of the pharynx and nose, from where it reaches the nerves in the pons
and medulla. After penetrating the central nervous system, it spreads rapidly and can
be found in the lumbar and sacral segments of the spinal cord. What is especially
important to state is that the pig is the only host that can survive the infection and serve
as a reservoir for the virus. Infection with this virus, as in the case of other
herpesviruses, can lead to permanent infection in pigs (METTENLEITER et al., 2012),
or to viral latency (WIDEN et al., 2012), which is mainly established in the cells of the
trigeminal and sacral ganglia. (ROMERO et al. 2003; METTENLEITER et al., 2012).
This also tells us that the cells of the central nervous system neurons are the sites of
latent infection with this virus (BROWN et al., 1995). Due to the action of certain
stressors, the latent virus can be reactivated in the tonsils and other tissues
(WITTMANN et al., 1983; METTENLEITER et al., 2012). Aujeszky's disease virus can
be confirmed in live pigs in swabs of the nose and tonsils and in oropharyngeal fluid.
Postmortem examination can also confirm it in peripheral organs such as the brain,
spleen and lungs. In domestic pigs that are latently infected, the virus can be confirmed
in the trigeminal ganglia, while in wild pigs it can also be confirmed in the sacral ganglia.
Aujeszky's disease can result in trade restrictions as well as significant economic
losses. Eradication programs can successfully keep the disease under control, but the
virus is still maintained in the wild boar population and thus poses a constant threat to
the domestic pig population, and can sporadically cause the disease in other animals,
such as hunting dogs. European countries have very different data on the
seroprevalence of Aujeszky's disease in the wild boar population, and research shows
that this virus is widespread. The available data show that seroprevalence varies
considerably between different regions of Europe, and also within the areas of low
seroprevalence, areas with moderate or low seroprevalence of Aujeszky's disease
virus have been identified (MEIER et al., 2015). Thus, according to available data, high
seroprevalence of Aujeszky's disease virus was found in feral pigs in the
Mediterranean countries as well as in Central and Eastern European countries, while
moderate to low seroprevalence was found in Central and Northern European
countries. In the Republic of Croatia, previous studies have found a high
seroprevalence of 54.54% in the area of Moslavačka gora (ŽUPANČIĆ et al., 2002)and
38.5% in 4 localities of the continental wild boar habitat (ROIĆ et al., 2012). . In
addition, the virus was successfully isolated (JEMERŠIĆ et al., 2012; KEROS et al.,
2014). High serporevalence in certain European countries poses a risk for further
spread of Aujeszky's disease in the wild boar population, but also domestic, despite
efforts to eradicate this disease. Seroprevalence ranges in certain countries, primarily
France, Spain, Italy and Croatia, indicate heterogeneity within different wild boar
habitats and population characteristics.The characteristics of Aujeszky's disease virus
infection in feral pigs have been investigated in several countries, and uneven results
in clinical signs and mortality of piglets have been demonstrated, while the level of
seroprevalence was related to age and sex (MÜLLER et al., 2011). CASADES-MARTI
et al. (2019) found that a high proportion of seropositive individuals in the population
increases the likelihood of infecting wild boar that have not previously been in contact
with the virus and that the only factor that can assess infection pressure is the
emergence of new infections during the first year of wild boar life (piglet category). For
Croatia, it is important to note that the transmission of the virus from species to species
(wild boar - dog) for which a high genetic match with the strain of wild and domestic
swine virus has been established has been proven (KEROS et al., 2015).
MATERIAL AND METHODS: Sampling was carried out in the continental, lowland and
mountain habitats of the Republic of Croatia. Samples were collected in 10 hunting
grounds and in the area of the Medvednica Nature Park (a total of 11 localities). The
sites are located in five counties and the City of Zagreb. Specifically, sampling was
carried out in two hunting grounds in Zagreb County, four hunting grounds in SisakMoslavina County, one hunting ground in Karlovac County, one hunting ground in
Varaždin County, two hunting grounds in Osijek-Baranja County and in the
Medvednica Nature Park (City of Zagreb). This selection of sampling sites included
three different rating classes, as well as three habitat types (lowland, hilly and
mountainous). The total area of all sampling sites is 101817 ha. At the same time,
sampling included wild boar populations in the wild (open hunting grounds) and in
fenced hunting grounds. A total of 222 wild boar were sampled. Wild boars were shot
according to the availability of samples with subsequent distribution by sex and age
categories. The age of wild boar was estimated according to the criteria given by
WAGENKNECHT (1984), with subsequent classification into three categories, piglets,
yearlings and boar (from two years of age and older). In this way, the possibility of error
in the classification of pigs by age is reduced compared to regular categorization into
offsprings, yearlings, boar (ANONIMUS, 2006).
Immediately after wild boar were shot , blood, spleen, cranial lobe of the lung and
olfactory bulb were sampled directly in the field. Blood samples (3-5 ml) were sampled
with a syringe and needle directly from the heart of a wild boar, and in cases where
there was no blood in the heart, it was taken from large blood vessels, directly or after
decapitation. The sample of the spleen and the front part of the lung were sampled "in
situ", measuring about 5x5x5 cm. Samples of olfactory bulbs were sampled in such a
way that the skull bones were cut with three incisions. The collected blood samples
were screened by an enzyme-linked immunosorbent assay for the presence of
antibodies to gpI protein of Aujeszky's disease virus in wild boar serum to determine
seroprevalence. At the same time, in samples of the spleen, anterior lobes of the lungs
and olfactory bulbs, in which longer virus retention is expected, a polymerase chain
reaction test for viral DNA was performed. This study was cross-sectional, performed
at a certain time, excluding the time component from research. Sampling is random
and the size of the population according to each hunting ground is determined on the
basis of data from the Central Hunting Records, which primarily for this work include
data on breeding stock and hunting and hunting productive area for wild boar. From
the obtained data, the prevalence and prevalence of the appearance ratio of positive
versus negative herds were determined. Differences in the incidence of Aujeszky's
disease between the sexes were examined by Fisher's exact test (ZAR, 1999), χ2 test
by McNemar χ2 test. The software tool Akaike Information Criterion (AICc BURNHAM
and ANDERSON, 2002) was used in the analysis and selection of the model. Logistic
stepwise regression was used to calculate the prediction of Aujeszky's disease virus
infection ELISA AB gpI IDEXX (1 = negative ELISA AB gpI IDEXX finding or 0 = positive
ELISA AB gpI IDEXX finding) (HOSMER and LEMESHOF, 2000). A log probability
ratio test was used to determine the statistical significance of each model. The
significance of the coefficients of the dependent variables (predictors) is based on χ2
Wald statistics (Wald's χ2).
RESULTS: Samples of olfactory bulbs, spleen or cranial lung were not positive by PCR
screening for Aujeszky’s disease virus, while the overall prevalence of 33.78% of
serologically positive wild boar for Aujeszky’s disease virus. According to gender, the
prevalence in males is 25.26% and in females 40.15%. The odds ratio in this case is
1.9852, which indicates that females are 1.9 times more likely to be positive than
males. Additionally, the sex classes were broken down into age categories, so a
seroprevalence in piglets of 10%, in the category of boar from 1 to 2 years 27.53%,
and in boar older than two years 65.75% was determined. Statistically significant
values in yearlings and boar in the second year are three times more likely to be
positive for females, while in the category of piglets and boar older than two years, the
values are not statistically significant.The obtained results observed by the McNemar
ꭕ2 test indicate that at the level of the compared individuals there is a statistically
significant identity between the expected frequencies of negative boars and positive
sows. Examination of differences between age classes within each sex showed far
greater statistically significant differences between groups, so that boars are
significantly more often positive than yearlings, while sows are more often positive than
yearlings and piglets. Tests have shown that in both sexes the frequency of positive
piglets does not affect the frequency of positive yearlings, while in all other
combinations the frequencies show correlation. Observed according to the location of
sampling by comparing positive and negative heads in the area of Zagreb and SisakMoslavina counties and in the area of Osijek-Baranja and Sisak-Moslavina counties, a
statistically significant difference was obtained.According to the criterion of ΔAIC <2
units, 21 models of assessment (prediction) of Aujeszky's disease virus infection were
singled out. With the proposed models, it is possible to estimate an infected or
uninfected animal with an average accuracy of 77.12 to 78.67%. The accuracy of the
assessment of positive individuals is about 10% lower than the accuracy of the
assessment of negative individuals and ranges from 70.15% (model 14) to 74.63%
(models 5, 6 and 8). On the other hand, the accuracy of estimating negative individuals
ranges from 82.46% (models 5, 6, and 8) to 85.09% (models 4, 7, 9, and 15). Models
that estimate positive wild boar positive individuals (5, 6, and 8) at the same time are
less accurate in estimating negative individuals.12 predictors were used in the
development of 21 models. They can be divided into population predictors (age
classes, sex, population density and relative hunting quota), hunting technique
predictors (hunting technique) and habitat predictors (altitude and share of individual
land use categories). The most common predictors are age classes, which are included
in all models. In second place is the population density, which is not included only in
model 12, but it is also a model that gives low accuracy of estimation of positive
(71.64%) and negative heads (83.33%), ie in general it is a model with the lowest
reliability (-2LL = 170.73). Gender predictors and relative culling quota occur relatively
rarely in the model. The signs of the coefficients obtained by logit regression indicate
the final value of the dependent variable. The age class coefficient has a positive sign,
while the coefficients of grassland share, arable land share and population density
have a negative sign. This indicates that with increasing age of the individual, the
probability that the individual in the population will be positive for Aujeszky's disease
increases. On the other hand, the smaller the proportion of grassland and arable land
and the lower the density of the feral pig population, the more likely it is that individuals
in the population will be negative for Aujeszky's disease.
DISCUSSION AND CONCLUSION: Determined average seroprevalence was 33.78%
which is consistent with the findings from Slovenia and Spain. The prevalence
increases with the age, being almost identical in females and males. Furthermore, the
prevalence is influenced by geografical location and type of breeding. The statistically
significant difference was determined in prevalence between Sisačko-moslavačka
County and Zagrebačka or Osječko-baranjska County. Animals kept in fenced
breeding have higher prevalence compared to free-living ones. According to obtained
dana it can be concluded that majority of infections in wild boar are latent ones.
However, increased stress during mating season and collective hunting are potential
triggers for virus activation and consequent transmission to other individuals.
Therefore, and based on detected seroprevalence, the role of boars in virus
transmission is highly emphasized. The applied models indicate that decreasing
population size results in decreasing prevalence, on the other hand increasing
population size does not result in proportional increase in prevalence. Higher ration of
forest stands is related to higher prevalence, probably due to the higher habitat quality
and consequently larger wild boar population. PCR analysis did not confirm presence
of the virus |
