| Sažetak (engleski) | INTRODUCTION: Aujeszky's disease is a viral disease of many species of domestic and
wild animals. The natural host of the Aujeszky virus is pig, however, Aujeszky's disease
infects many vertebrates, e.g., cattle, sheep, goats, dogs, cats, rodents, and others.
Infection with the Aujeszky virus in animals other than pigs almost always results in the
death of the infected animal. Aujeszky's disease is endemic in many parts of the world,
but there are areas and countries that have successfully eradicated the disease. Aujeszky's
disease causes great economic damage, not so much because of the direct effect but
because of the damage caused by trade barriers. Although Aujeszky's disease has been
eradicated in most European Union Member States, there are areas and countries where
Aujeszky's disease still persists. As the most probable reason for the re-emergence of
Aujeszky's disease in the previously free area is the circulation of Aujeszky's disease virus
in feral pigs. In Croatia, the disease has not yet been eradicated from the domestic pig
population, although the eradication program has been implemented since 2013 and to
date only 27,672 out of a total of 72,144 farms have achieved Aujeszky's disease-free
status. This fact calls into question the current strategy for the eradication of this disease
in Croatia since many farms still have an unknown status of Aujeszky's disease.
Therefore, this paper conducted an analysis of previous results and data on farms to assess
the actual prevalence in the population and to assess and determine risk factors such as
locations, categories of pigs kept housing practices and applied biosecurity measures. The
results of the research are used to devise a new effective strategy and policy in the
approach to controlling Aujeszky 's disease in Croatia with the aim of eradicating it.
REVIEW OF THE LITERATURE: According to historical data, it can be concluded that
Aujeszky's disease was widespread. Namely, records found in America and Europe from
the 19th century describe an animal disease that was later determined to be Aujeszky’s
disease. Since signs of the disease were observed in different species of animals,
Aujeszky's disease was initially associated with the disease in cattle, dogs, and cats, and
only later it was proven that pigs are the only natural hosts of this disease. In Europe, the
first etiological evidence of Aujeszky's disease was recorded in 1902 by the Hungarian
pathologist Aujeszky Aladár, after whom the disease was later named (AUJESZKY, 1902). The first record of Aujeszky's disease in Croatia dates from 1904 (CVETNIĆ,
1997). ZWICK and ZELLER (1911) studied a disease they called pseudorabies, and
MAREK (1904) investigated a similar disease called acute bulbar paralysis. STREBEL
(1889) described the onset of a disease characterized by severe itching in four cattle from
a farm in Switzerland, however, without a clear etiology. In the mid-twentieth century,
based on immunological studies, the creation of intranuclear inclusions, and ether
sensitivity, the virus was classified as the Aujeszky's disease virus (pseudorabies - PrV)
from the herpesvirus group (SABIN, 1934; KAPLAN and VATTER, 1959). Given the
different signs of the disease in pigs from those in other species, pigs were initially only
sporadically associated with the onset of Aujeszky's disease, and it was not until the late
first half of the twentieth century that pigs were found to be reservoirs of the disease.
Aujeszky's disease is caused by swine alphaherpesvirus 1 (abbreviations: SuAHV1;
PRV) from the genus Varicellovirus, a subfamily of Alphaherpesvirinae, family
Herpesviridae, as classified by the International Committee on Taxonomy of Viruses
(International Committee on Taxonomy of Viruses – ICTV; Database issue
https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=10345
&lvl=3&lin=f&keep=1&srchmode=1&unlock) (LEFKOWITZ et al, 2018; GATHERER
et al, 2021).
Herpesviruses are highly adapted to their hosts, numerous mammals, birds, and reptiles.
After a primary infection, these viruses can cause a lifelong latent infection. Severe
clinical signs of disease after herpes virus infection usually develop only in very young
or immunocompromised infected individuals, in fetuses, or in individuals who are not
natural hosts of the virus (GATHERER et al., 2021). Despite significant homology with
human alphaherpesviruses and a wide range of hosts, Aujeszky’s disease virus is not
transmitted to humans (POMERANZ et al., 2005). There are sporadic cases of the disease
in humans that have been linked to Aujeszky’s disease virus, but it is generally considered
that Aujeszky’s disease is not a zoonosis (EFSA, 2017). The genome spans approximately
140-143 kpb and contains at least 72 open reading frames (ORFs) encoding 70 different
proteins. A linear double-stranded molecule is composed of two components, L and C.
Double-stranded DNA is encased in a protective capsid that forms a nucleocapsid, protein
matrix, or viral tegument that separates the nucleocapsid from the outer lipoprotein
envelope made up of 11 viral glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, gN, gL, gL, gL, gL, gL,) (POMERANZ et al., 2005). Infection of cells with herpesviruses begins with
the attachment of free virions to target cells, followed by the fusion of the viral envelope
and the cell cytoplasmic membrane and replication. The main sites of Aujeszky's disease
virus latency are the trigeminal ganglia, olfactory bulb, and tonsils. In these organs, viral
DNA can be detected even when no infectious virus is produced, and the fragment
responsible for latency can also be detected by highly sensitive methods such as real-time
polymerase chain reaction or RT-PCR. It is hypothesized that after oronasal infection,
Aujeszky's disease virus first multiplies in epithelial tissue and can enter directly into the
terminals of nasopharyngeal sensory neurons. After this first multiplication, the virus
multiplies abundantly, leading to increased infection of primary neurons
(METTENLEITER, 2000). One of the key proteins for neuroinvasiveness of Aujeszky's
disease virus is gE, and its removal strongly weakens (attenuates) the virus, while the
ability of its primary multiplication remains preserved. Removal of gE affects the
inhibition of transinaptic transmission to other neurons thereby substantially reducing its
neuroinvasiveness (METTENLEITER, 2000). Aujeszky's disease virus is resistant to
external conditions, which largely depends on the level of pH, humidity, and temperature.
Heat treatment of meat, sausages, and ham at a temperature of 80 ° C destroys the
Aujeszky's disease virus. In manure the virus can survive for a month in summer and two
months in winter, while in soil it can survive for five to six weeks. It can be kept on hay
and straw for 15 days in summer and 40 days in winter, and on other surfaces (bags,
wooden surfaces) it can survive for 10 days in summer and 15 days in winter (EFSA,
2017). Heating kitchen waste at 70 ° C destroys the virus in 10 and 5 minutes at 80 ° C,
respectively (WITTMANN, 1991). Inactivation of Aujeszky's disease virus in aerosol
depends on temperature and relative humidity, and generally about 50% of the virus is
destroyed within 24 hours (SCHOENBAUM et al., 1990).
Aujeszky's disease virus is sensitive to disinfectants based on orthophenolphenate
compounds, peracetic acid, formalin, 2% sodium hydroxide, trisodium phosphate iodide,
1-2% quaternary ammonium compounds, hypochlorite, and chlorine (chlorhexidine)
(BERAN, 1991). Aujeszky's disease virus has spread almost all over the world. The
disease occurs in the population of pigs, domestic and wild, and sporadically in other
species of animals, most often carnivores and ruminants. There is ample evidence of virus
confirmation in wild animals, especially in wild pigs, which are the main reservoir of the virus, whose circulation in wild pigs has also been confirmed in Croatia (KEROS et al.,
2014). Pigs (Sus scrofa) are the only natural hosts of Aujeszky's disease virus and are
considered a reservoir of this virus (METTENLEITER et al., 2019). Pigs usually do not
show clinical signs, except in younger age groups and newborn piglets, but pigs remain
permanently infected after recovery and may occasionally shed the virus. It is this ability
of the Aujeszky’s disease virus to latency in seemingly healthy pigs that is the reason for
the long-term survival of the virus in the population, especially in the feral pig population,
which can be a permanent source of re-infection of domestic pigs. In countries with
developed intensive pig production, Aujeszky's disease was successfully brought under
control by the implementation of eradication during the second half of the twentieth
century, mainly in combination with a vaccination strategy. Aujeszky's disease is spread
by direct contact with an infected animal or indirect contact with contaminated objects,
air (aerosol) (VANNIER, 1989), food (STOIAN et al., 2020), litter or surfaces
contaminated with contaminated secretions (LI et al., 2020). Aujeszky's disease virus is
excreted in saliva, nasal secretions, urine, feces, vaginal secretions, and pig semen, and
can also be transmitted to suckling piglets by colostrum-infected sows (BERAN, 1991).
Aujeszky's disease virus enters the body horizontally through the nose and mouth
(oronasal), sexually after mating or insemination, and vertically transplacental. In pig
farming, Aujeszky's disease most often enters by introduction an infected animal where
it spreads rapidly within the naive population.
In the last ten years, the occurrence of Aujeszky's disease in intensive domestic pig
breeding in China has been associated with the spread of a new variant of Aujeszky's
disease virus in the vaccinated pig population (XIA et al., 2018). The manifestation of
signs of Aujeszky's disease in infected pigs depends on several factors, the category and
number of pigs, their immune status, and the way they are kept. The primary infection
takes place in the upper respiratory tract after the Aujeszky’s disease virus enters the pig’s
body through the mucous membranes of the eye and nose. In the nasal septum, tonsils,
nasopharynx, trachea, and lungs, it causes infection of epithelial cells, which causes the
destruction and erosion of the epithelium. At this stage, the infection is manifested by
mild signs by the respiratory system, such as sneezing, coughing, shortness of breath, and
nasal discharge. These signs appear three to six days after the infection and can last up to
ten days. Infected pigs generally recover very quickly, unless they develop pneumonia due to secondary bacterial infections. Virus excretion can be detected from the first to the
14th day after inoculation. To detect Aujeszky's disease virus in animals with a clinical
picture, virus isolation methods and viral genome detection by PCR method are used. The
PCR method for the detection of Aujeszky's disease virus DNA in organ samples is
considered the method of choice. Serological diagnosis is applied to detect antibodies to
Aujeszky's disease virus mainly in subclinical or latent infected pigs by ELISA tests. The
immune response in infected and vaccinated pigs is very rapid but the rate of immune
response depends on the strain of the virus, the route of entry and the immune status of
the animal. Specific serum antibodies are present in the blood of pigs already at the
appearance of clinical signs of the disease (KRETZSCHMAR, 1970). By highly sensitive
serological methods such as the ELISA test, antibodies can be detected as early as 5 to 7
days after inoculation, and the by the virus neutralization test (VNT) only from the 12th
day. In accordance with European legislation and WOAH recommendations and based
on the experience of countries that have successfully eradicated Aujeszky’s disease from
the domestic pig population, in addition to vaccination against Aujeszky's disease, other
control measures should be applied to protect pig farms from infection. The methods of
keeping pigs, hygiene and biosecurity measures are a prerequisite for successful
protection of pig breeding from Aujeszky's disease. In case of disease, infected pigs that
show clinical signs are killed, and latent infected (seropositive) pigs are sent for slaughter.
Healthy pigs must be kept separate with all biosecurity measures in place to prevent the
spread of the virus within the holding. In case of high prevalence in pig farms or in a
certain area, DIVA vaccines can be used in the first phase of eradication to minimize the
further spread of the disease, followed by testing and removal of the remaining
seropositive pigs. In the final stage of eradication, vaccination is generally stopped, and
after successful eradication, depopulation of breeding with seropositive pigs is carried out
as a final measure. Direct losses sampled due to Aujeszky's disease can be very large if a
virulent virus is introduced into the naive pig population. However, in pig production,
losses are not so much related to direct damage due to death, abortion, or clinical
manifestation but to indirect economic costs incurred due to control measures and trade
reasons. In the European Union, Aujeszky's disease is on the list of diseases that must be
officially reported to the competent animal health authority of each member state and to
the European Union in accordance with European legislation. European Union legislation prescribes disease control measures and conditions for the movement of pigs in respect
of Aujeszky's disease. The Animal Health Law (Regulation (EU) 2016/429) and
Implementing Regulation 2018/1882 (ANONIMUS, 2016, ANONIMUS, 2018) classify
Aujeszky's disease as a Category C disease called Aujeszky's disease virus infection.
World Organisation for Animal Health (WOAH) classifies Aujeszky's disease on the list
of diseases as a disease of several species of animals, which must be reported. RES in
Chapter 8.2. The Manual for Terrestrial Animals (WOAH, 2021) provides
recommendations for the trade of pigs and products of pig origin. Aujeszky's disease has
spread throughout the world but there are countries and areas that are considered free.
However, in these areas or countries, the virus may be present in the feral pig population,
which poses a constant risk of recurrence of Aujeszky's disease in domestic pigs. There
are only a few regions in the world where Aujeszky's disease has never become endemic,
and these are Norway, Australia, and most of the south-eastern islands of Asia. During
2021, Aujeszky's disease reoccurred in both Hungary and France. In Croatia Aujeszky's
disease was confirmed in 2004 and again in 2011, since when it has been continuously
confirmed every year. During the period of implementation of the control and eradication
program from 2013 to 2020, Aujeszky's disease was confirmed in latent infected pigs by
serological testing. The progress of the eradication program and the assessment and
analysis of applied strategies for the control and eradication of Aujeszky's disease have
been described using various epidemiological and statistical methods (MARTINI et al.,
2003; ALLEPUZ et al., 2009). In free countries and territories, the research mainly
concerns the assessment of the risk of introduction of Aujeszky's disease virus by swine
trafficking or contact with feral pigs and the identification and assessment of risk factors
(MÁRTINEZ-LÓPEZ et al., 2009) by using of different mathematical models.
MATERIAL AND METHODS: To be able to conduct a comprehensive analysis and
draw conclusions, the materials used in this paper are divided into several groups, i.e.,
levels, adapted to the objectives of the research:
- population: animals and farms in the period from 2013 to 2021
- laboratory testing for Aujeszky's disease in the period from 2013 to 2021
- comparative data on categorized farms and testing for Aujeszky's disease on 31.1.2021. The analysis was done within each group separately, and the result of separate analyzes
in each group/level were discussed, compared for the purpose of conclusions, and
providing basis for guidelines for developing a new more effective strategy and policy in
the risk-based approach to Aujeszky's disease control in Croatia. The population covered
by this research consists of domestic pigs kept on farms from the entire territory of Croatia
divided by counties. Population of domestic pigs in Croatia on 31.12.2021. counts
1,257,753 pigs on 72,144 farms. The results of serological tests by ELISA Pseudorabies
Virus gpI Antibody test Kit, IDEXX PRV/ADV gI for 219,974 blood samples of pigs
taken from 36,255 farms within the active surveillance were analyzed. The assessment of
risk factors for Aujeszky's disease included an analysis of 30,687 categorized farms on
which at least one Aujeszky's disease test was conducted in 15 counties. Due to the small
number of tested farms (less than 10), four counties were not considered, namely
Primorje-Gorski Kotar, Split-Dalmatia, Šibenik-Knin and Lika-Senj County. In analysis
statistical methods and metaregression model were used. For data processed at the sample
level, the exact binomial test estimated confidence limits considering the 95% confidence
interval (CI 95%) and the differences at P <0.05 were considered significant. population,
an exact binomial test calculated the confidence limits considering the confidence level
of the 95% confidence interval (CI 95%). Data on testing were spatially and temporally
processed and presented by counties for each year in the period from 2013 to 2021
according to the determined prevalence. To show the spatial distribution of positive farms
in Croatia, using the ArcGIS system, maps were made for each year of the research
period. To assess the interaction of risk factors (variables), a model was developed which,
by modeling the original data for monitoring the situation on farms in the period from
2013 to 2021 and through multiple visits (maximum 9), enabled easy reading of the
impact of individual variables and interactions between a total of 84 independent
variables from the questionnaire.
RESULTS: Croatian pig population counts 72144 farms with 1.257.753 distributed by
the counties namely on the continental part of Croatia. The largest total number of farms
is in Osijek-Baranja and Krapina-Zagorje counties. Considering categories of farms
depending on biosecurity level distribution the largest number of category 2 farms is in
Krapina-Zagorje County, while Osijek-Baranja and Vukovar-Srijem counties have the largest number of category 3 farms. A total of 219,974 samples were serologically tested
out of which 3,815 samples were positive. The share of positive farms above 5% was
recorded in Varaždin County (10.28%), Split-Dalmatia County (7.69%) and OsijekBaranja County (5.93%), while in other positive counties was below 5%. Last
confirmation of the Aujeszky’s virus by PCR was in 2013 in two domestic pigs. The
assessment of the relationship between the exposure of the holding to one of the analyzed
risk factors was based on a comparison of the results of positive (at least one serologically
positive pig found) and negative farms (farms where no seropositive pig was found).
Logistic regression analysis was used to test for differences, and the strength of the
association was described by the probability of exposure to infection associated with
positive farms - OR (odds ratio). The average prevalence in the analyzed farms at the
country level was 4.09%. Lower-than-average prevalence was found in 7 (46.67%)
counties: Krapina-Zagorje 0.15%, Karlovac 0.24%, the City of Zagreb 1.10%, PožegaSlavonia 1.54%, Virovitica-Podravina 2, 42%, Brod-Posavina 2.28% and Zagreb 2.60%.
The highest prevalence of Aujeszky's disease at the farm level was significantly higher in
Varaždin County (12.14%) compared to other counties where it ranged from 0.15%
(Krapina-Zagorje) to 7.46% (Sisak-Moslavina) <0.01). Testing the difference showed
that the largest difference in the number of farms in categories 1 and 2. The City of
Zagreb, Varaždin, Krapina-Zagorje and Međimurje counties have the same distribution
of categories. Compared to others, these counties have a significantly smaller number of
farms in category 1 (from 22.10% to 24.65%), and a significantly larger number of farms
in category 2 (from 53.88% to 68.29%). Also, in Varaždin County and the City of Zagreb,
and then in Vukovar-Srijem and Sisak-Moslavina counties, a significantly larger number
of farms is in category 3. Comparing the number of farms by categories in Varaždin and
Krapina-Zagorje counties with the prevalence of Aujeszky's disease in these counties,
despite the equal distribution of categories, they have a statistically significantly different
prevalence and Krapina-Zagorje County the lowest (0.15%) and Varaždin the largest
(12.14%) (P <0.05). The results of categorization, i.e., the answers to the questions from
the questionnaire were analyzed as individual variables divided into groups. All the
results obtained in the total sample were analyzed to find the main factors that could be
associated with Aujeszky’s disease-positive farms and to assess the strength of the
association of these factors with positive farms. Significant variables influencing the prevalence of Aujeszky's disease were marking pigs with tattoos, deaths in the last 3
months, the presence of boars on the farm, keeping pigs outdoors, purchasing animal feed
from other producers and producing litter on their own farm. The model determined the
reduction of positive farms in successive testing, which indicates the effectiveness of
control measures on infected farms. The model for estimating the interaction of variables
has determined that individual variables can have a synergistic effect that can reduce or
increase the likelihood of infection with Aujeszky's disease virus.
DISCUSSION: The prevalence of Aujeszky's disease at the level of farms by years of the
study period ranges from 4.66 to 0.67%, while the prevalence of Aujeszky's disease varies
by counties from 0.15% to 12.14% and by categories from 3.44% in Category 0 to 15.00%
in Category 4. The highest prevalence of Aujeszky's disease was found in Varaždin
County (12.14%), and the lowest in Krapinsko-Zagorje County (0.15%). Based on data
from testing performed in the study period, it is concluded that the prevalence of
Aujeszky's disease decreased from 4.66% in 2014 to 1.03% in 2021 by applying an
eradication strategy in such a way that positive pigs were culled (test and slaughter
strategy). However, it is important to mention that free farms were not included in the
regular annual active surveillance of Aujeszky's disease during 2018-2021, which may
represent a gap in the knowledge of the real status of free farms in relation to Aujeszky's
disease. It is known that in parallel with a decrease in prevalence, the susceptibility of the
population to Aujeszky's disease virus infection increases and the virus can spread again
in free areas (PENSAERT and MORRISON, 2000). Continuous sporadic confirmation
of Aujeszky's disease in previously free states supports this finding (ANONIMUS, 2020).
Detection of new, potentially more virulent strains of Aujeszky's disease virus, such as
MdBio wild-type virus isolated in Serbia (ZSOLT et al., 2019) is also an additional risk
and a new source of infection for the domestic pig population.
The analysis of risk factors revealed significant variables that also differ within the
categories of farms, and most of the variables also differ by counties. This was also
confirmed by the assessment of the risk exposure of individual areas / counties in relation
to the county with the lowest prevalence (Krapina-Zagorje, 0.15%). The prevalence of
less than 1% is usually the designed prevalence in disease eradication programs, i.e. the
establishment of disease-free areas (ANONIMUS, 2020). The exposure estimate was from 6.52 (Varaždin County) to 44.13 (Osijek-Baranja) times higher than in KrapinaZagorje County, while the exposure estimate depending on the category was significantly
higher for category 4 in relative to category 0. To analyze risk factors in more detail and
their effects over time, an applied mathematical model was developed based on the
dynamics of the number of positive farms over time and the presence of the examined
risk factor. The model compared the performance of factors and positive farms that were
tested multiple times (in the model called visits). The number of positive farms in some
counties has increased significantly over time, which is especially evident in Varaždin,
Sisak-Moslavina, Bjelovar-Bilogora and Koprivnica-Križevci counties, which indicates
that in these counties the activity of some risk factors more significantly than in others.
The probability of infection on farms where tattooing was applied on positive farms was
2.77 times higher (OR = 2.77 (CI 95% 1.47-5.23, P = 0.0016) compared to negative farms.
For the association of pig deaths on positive farms, it was found that on farms where
deaths were reported the probability of infection is 1.73 (CI 95% 1.33-2.25, P <0.0001)
times higher than on negative farms. The presence of boars on farms increases the
probability of infection on positive farms by 1.85 (CI 95% 1.60-2.14, P <0.0001) times
compared to negative farms. Outdoor pig keeping increases the probability of infection
by 2.13 (CI 95% 1.29-3.51, P = 0.0031), which means that the probability of Aujeszky's
disease on positive farms due to keeping pigs outdoors is 2.13 times larger than if pigs
are kept indoors in closed facilities. The probability of Aujeszky's disease infection on
farms that purchased food from other producers is 1.47 (CI 95% 1.31-1.65, P <0.0001)
times higher than on farms that do not. Litter production on own farm has proven to be
one of the important factors associated with positive farms. The probability of Aujeszky's
disease on farms that produce the own litter is 1.88 (CI 95% 1.31-2.69, P = 0.0006) times
higher than on farms that do not produce it. Each of the risk factors were confirmed by
model as well. A model that enabled the analysis of two variables and their mutual
interaction was developed. Based on the results, individual variables, if they act
simultaneously, can lead to a decrease, or increase in infection on the farm. Thus, natural
mating has been shown to increase the spread of infection, pest control and regular
cleaning and disinfection reduce the possibility of infection spreading between farms,
although another factor present on the holding at the same time contributes to infections.
On the other hand, if there is a satisfactory factor such as a neatly fenced holding with controlled entry of persons and vehicles into the facility, and if disinfection is not carried
out, the infection spreads. Furthermore, feeding food containing waste from the food
industry has been shown to reduce the spread of Aujeszky’s disease. Deviations by
categories were also evident. The model analyzed all variables and determined an
unexpected result for individual variables in the sense that the desired treatment affects
greater exposure to infection. This may lead to conclusion that the questions in the
questionnaire are designed so that the answers can be vague and non-specific, i.e., biased,
which leads to this result of the analysis. In conclusion, the model found a reduced number
of infected farms during successive testing, which indicates the effectiveness of control
measures implemented on positive farms. However, the prevalence has been shown to be
different in different areas, which indicates the need to define monitoring and eradication
programs for individual areas (counties) according to prevalence. Farms exposed to these
factors should be the first to be considered and further testing is advisable. At the same
time, it is advisable to reduce the incidence of risk factors at the same time, which can
also lead to a reduction in the prevalence of Aujeszky's disease at the animal level, and
thus at the farm level. Since the eradication of Aujeszky's disease is envisaged by
European legislation in a way that covers the entire population of domestic pigs, the risk
factors found in this paper can be used when planning risk-based surveillance of free
farms (areas). At the same time, the questionnaire should be revised in accordance with
the analyzed risk factors in such a way that the questions are adjusted so that certain risk
factors are further examined, and indefinite and non-specific answers are avoided in order
to reduce the possibility of bias. Simultaneously with the implementation of sampling and
testing of farms for Aujeszky's disease, it is necessary to carry out activities aimed at
eliminating or reducing risk factors identified as significant for Aujeszky's disease. |
