| Abstract | Virusni arteritis konja (VAK) je zarazna bolest kopitara proširena diljem svijeta koja se
očituje različitim kliničkim oblicima koji variraju od subkliničkog do teških respiratornih
infekcija s mogućim smrtnim ishodima u ždrebadi. Uz to virus virusnog arteritisa konja
(EAV) smatra se jednim od najznačajnijih uzročnika pobačaja u konja što bolesti daje
izniman gospodarski i uzgojni značaj. Prvi dokaz cirkulacije EAV na području Republike
Hrvatske (RH) datira iz 2005. godine, a od 2009. godine je propisano obvezno nadziranje
i suzbijanje bolesti.
Kako bi se istražila uspješnost mjera nadzora te odredile epizootiološke značajke VAK
na području RH, u istraživanju je određivano kretanje seroprevalencije infekcija u prvih
šest godina provođenja nadzora. Istražena je i mogućnost provođenja serološke
dijagnostike komercijalnim imunoenzimnim kompletima (ELISA) te molekularna
epizootiologija VAK na području RH. U tu svrhu analizirani su podaci serološkog
pretraživanja 4445 konja s područja RH u razdoblju od 2010. do 2015. godine. Uz to 94
seruma konja su usporedno pretražena virus neutralizacijskim testom (VN-test) te s dva
komercijalno dostupna ELISA kompleta. Načinjena je i filogenetska analiza 16 EAV
dokazanih na području RH.
Rezultati su potvrdili značajno smanjenje seroprevalencije u istraživanom razdoblju tako
da je svake slijedeće godine vjerojatnost za serološki pozitivan nalaz bila značajno manja
(OR=0,77, 95% CI 0,7-0,84, p<0,001). Ustanovljeno je da je cirkulacija EAV
najizraženija u Istočnoj Hrvatskoj (OR=1,82, 95% CI 1,62-2,06, p<0,001) te da su
infekcije češće u ženskih životinja (OR=1,96, 95% CI 1,44-2,68, p<0,001). Uz navedeno
najznačajniji čimbenik rizika je viša životna dob, posebno u skupini životinja iznad deset godina (OR=16,53, 95% CI 10,6-26,65, p<0,001), a infekcije su najčešće u konja
lipicanske pasmine (OR=6,46, 95% CI 4,17-10,31, p<0,001). Kroz kretanje
seroprevalencije u istraživanom razdoblju, osim stalnog prisustva uzročnika, dokazane su
i neprijavljene epizootije u Istočnoj i Središnjoj Hrvatskoj s različitim epizootiološkim
značajkama, najvjerojatnije uvjetovane pasminskom strukturom te načinom držanja i
korištenja konja. Također je dokazano da se za serološko pretraživanje konja na VAK
može koristiti određeni komercijalni ELISA komplet ali samo nakon validacije u
lokalnim epizootiološkim prilikama. Molekularnom tipizacijom EAV dokazanih na
području RH potvrđena je kocirkulacija najmanje dva soja EAV od kojih je jedan u skladu
s globalnom cirkulacijom virusnih sojeva dok drugi ima neovisnu evoluciju i predstavlja
soj prisutan više desetljeća lokalno na prostoru RH i susjednih država.
Cjelokupnim rezultatima istraživanja potvrđena je opravdanost uvođenja nadzora VAK
na području RH kao i učinkovita usmjerenost propisanih mjera te optimalan odabir
serološke dijagnostičke metode u njihovoj provedbi. Dokaz nepotpunosti i nedosljednosti
provođenja mjera nadzora i suzbijanja, uz istovremeno stalno prisustvo EAV u populaciji
konja RH te kocirkulaciju različitih sojeva EAV, naglašava nužnost kontinuiteta
provođenja mjera. Temeljem analize čimbenika rizika posebno intenzivne mjere moraju
biti u konja lipicanske pasmine u Istočnoj Hrvatskoj. Navedenim se može jamčiti
očuvanje zdravlja životinja i uspješna provedba uzgojnih programa. Ovo je temeljni
preduvjet za daljnji razvoj konjogojstva RH, ali i očuvanje nacionalne baštine koju
predstavlja uzgoj hrvatskih autohtonih pasmina konja. |
| Abstract (english) | Introduction
Equine viral arteritis (EVA) is one of the most important equine viral diseases both
because of the clinical presentation of the disease and as one of the single most important
causes of abortion in mares. Despite the global widespread distribution of the disease, the
first evidence of the equine viral arteritis virus (EAV) circulation in the Republic of
Croatia was confirmed in 2005. The confirmation of positive animals in different parts of
Croatia in 2008 led to the introduction of mandatory disease control measures in 2009,
which have been in place ever since. For disease control purposes, the frequent
asymptomatic infections that are dominant among the carrier stallions who can remain
lifelong virus carriers, have been the biggest challenge. Therefore, the control measures
are based on the serological testing of animals and the exclusion of carrier stallions from
the breeding programme. In line with the recommendation of the World Organisation for
Animal Health (OIE), serological diagnostics is based on the virus neutralisation test
(VN-test). As this method is very demanding in practice, sometimes the enzyme-linked
immunoassay (ELISA) is used as an alternative. However, the reliability of the use of the
ELISA method has to be assessed against the area-specific individual epizootiological
situation. The epizootiological characteristics of the EVA in an area depend on the breed
structure, the methods of keeping and use of horses, as well as the circulating virus strains,
which will ultimately determine the clinical presentation of the disease. In Croatia, horsebreeding is characterised by the indigenous horse breeding programmes, with the most
dominant being the coldblooded breeds such as the Croatian Posavina horse and the
Croatian Coldblood horse, which are mostly bred in Central Croatia and often by using
extensive farming methods; and the lipizzaner horse, which is the most widely bred horse breed in Eastern Croatia. An analysis of these two biggest groups of horses already clearly
shows that their respective epizootiological characteristics are mutually very different.
Additionally, we also have to take into account the growing population of warmblood
horses, which are kept for sports and leisure, and have a different set of epizootiological
characteristics.
The epizootiological characteristics of the EVA have not been examined in detail in
Croatia so far, and neither has been evaluated the effect of the mandatory measures
despite the fact that they have been implemented for more than a decade. Furthermore,
the diagnostic potential of the commercial ELISA assays in our epizootiological situation
has not been sufficiently examined and no molecular typing of the circulating EAV strains
has been carried out to the present day. These existing gaps, in addition to the fact that
the disease is present in Croatia, were the main reasons that lead to this survey with the
aim of investigating of the epizootiological situation, determining the risk factors,
evaluating the effectiveness of the mandatory control measures, examining the
serological diagnostic alternatives and expanding the knowledge with respect to the EVA
molecular epizootiology; all against the backdrop of the specific epizootiological features
of the horse breeding in Croatia.
Methods
In order to achieve the objectives of this study, the test results for a total number of 4445
horses from across the Croatian territory that had been serologically tested by VN-test in
the period 2010-2015, were analysed. For each individual sample, the analysis included
the year of sampling, the area where the tested animal was kept, the breed, age, sex and
category of the animal, as well as VN-test result. Based on the above, the seroprevalence was established for the reference period, as well as for each individual year that fell within
the temporal scope of the survey at the level of the entire country and then at the level of
the three individual regions (Eastern, Central and Coastal Croatia). These regions were
selected for the survey based on the grouping of the counties with similar epizootiological
situations, so as to obtain a satisfactory sample for determining the risk factors. Due to an
imbalanced sample, such an analysis could not be conducted at the county level. The data
that were thus grouped were then analysed to determine the differences and risk factors
in relation to the region, age, sex, gender, category and the year covered by the survey.
All data were statistically processed to determine the significance of the obtained result
and the risk factors and their significance were determined by establishing the odds ratio
with a confidence interval of 95% and p<0.05. Additionally, the correlation was
determined by using the Pearson correlation coefficient (r) and the results were shown as
r and a confidence interval of 95%. A logistic regression analysis was also carried out, as
well as an analysis of the relative significance of individual factors in the outcome of the
VN-test.
With the aim of analysing the possibilities of using commercial ELISA test kits for the
serological diagnostic purposes, 94 horses were tested using VN test and two commercial
immunoenzyme tests: ID Screen® Equine Viral Arteritis Indirect (ID.Vet, Grabels,
France) and INgezim Arteritis 2.0 (Eurofins Technologies Ingenasa, Madrid, Spain). The
sensitivity and specificity, as well as the reliability of the commercial ELISA tests, were
determined by comparing the test results.
A total of 16 archived RNAs isolated from the viruses confirmed in the Osijek-Baranja
County and the Brod-Posavina County were used for the molecular epizootiology
examination. For molecular typing, the nucleotide sequences of the EAV ORF5 gene were analysed. After the successful multiplication of the ORF 5 gene confirmed by gel
electrophoresis, the nucleotide sequences were determined in Macrogen Europe
(Amsterdam, the Netherlands) and obtained sequences were phylogenetically compared
with the 138 nucleotide sequences of the same EAV gene isolated in the world with the
known year and country of isolation, and which were freely available in the gene bank
(GenBank).
Results
The seroprevalence in the territory of Croatia decreased from 21.3% to 9.7% in the 2010-
2015 period. It was demonstrated that with every successive year, the odds of infection
for horses was 0.77 lower (OR=0,77, 95% CI 0,7-0,84, p<0,001). Eastern Croatia is the
area with the statistically significantly higher seroprevalence of EAV infections and there
is a high correlation between the seroprevalence in this area and the seroprevalence at the
level of Croatia (r=0.86; 95%CI=0.16-0.98; p=0.03). The same was demonstrated for the
seroprevalence of stallions in the same region (r=0.88; CI 95% 0.22-0.99; p=0.02).
Among the risk factors, the most impactful on the seropositivity throughout the period is
the rising age of the animal so that, when compared to the animals below five years of
age, the probability of the positive result increases by factor 3.99 in animals aged 5-10
years (OR=3,99, 95% CI 2,55-6,43, p<0,001), and by as much as factor 16.5 in animals
older than 10 years (OR=16,53, 95% CI 10,6-26,65, p<0,001). Female animals are 1.96
more likely to have a positive test result (OR=1,96, 95% CI 1,44-2,68, p<0,001), as well
as the horses of lipizzaner breed, who are 6.46 times more often positive than other
warmblood horses (OR=6,46, 95% CI 4,17-10,31, p<0,001).
The scope of application of the mandatory control measures were from 60.3% to 91.5%
at the level of the breeding animals, and from 18.6% to 23.6% at the level of the general
animal population. In addition, incomplete epizootiological data was present in 23.9-
45.9% of the cover letters received with the laboratory sample.
Serological diagnosis of EVA monitoring in the equine population in the Republic of
Croatia can be carried out with specific ELISA tests but only after validation in local
epizootic conditions.
The phylogenetic analysis confirmed a close link between the EAV strains isolated in the
territory of the Osijek-Baranja County and they belong to the EU-1 virus group. The
strains confirmed in the territory of the Slavonski Brod-Posavina County were mutually
matching and phylogenetically distant from the strains found in the Osijek-Baranja
County. These viruses, together with the strain isolated in the Republic of Serbia and the
strain isolated in the United States in 1963, form a separate branch on the phylogenetic
tree.
Conclusions
Equine viral arteritis is widespread across the Republic of Croatia with the highest
seroprevalence in Eastern Croatia. After the introduction of the mandatory control
measures, the seroprevalence has been continuously decreasing, and an even better result
could be achieved by the full implementation of the mandatory measures. The most
significant risk factor is the increasing age of the animal, and female animals are more
often positive than male animals. In addition, the infections are significantly more
frequent in horses of lipizzaner breed. Seroprevalence results analysis indicate two previously unreported epizootics of the EVA in Central and Eastern Croatia, which
additionally emphasises the deficiency in mandatory measures conducting.
In the serological diagnostics of EVA, the method of choice can be ELISA tests but only
after validation in local epizootic conditions. Although the ID Screen® Equine Viral
Arteritis Indirect test has a higher matching rate of results in terms of routine laboratory
diagnostics, it is not acceptable due to the potential false-negative results.
In view of all of the above, and the proven co-circulation of at least two different virus
strains of EAV in Croatia, it is necessary to continue the implementation of the control
measures to the fullest extent, placing an additional emphasis on the lippizaner horse
populations in Eastern Croatia in order to successfully control and eradicate this disease
that has a significant impact, both economically and in terms of the breeding programmes.
This is the only way to ensure the future development of the horsebreeding in Croatia and
protect the national heritage that is the Croatian indigenous breeds. |
