| Abstract | Rast ljudske populacije i sve veće iskorištavanje prirodnih resursa uzrokovali su
promjene ekoloških uvjeta za različite vrste divljih životinja. Mnoge od tih vrsta prilagodile su
se na život u blizini ljudi, odnosno na život u urbanim staništima koristeći razne antropogene
resurse za preživljavanje. Pripadnici porodice kuna (Mustelidae), poput kune bjelice (Martes
foina), primjeri su koji pokazuju izrazitu prilagodljivost u pronalaženju skrovišta i hrane u
ljudskim naseljima i nastambama, pa čak i u razmnožavanju u gradskim jezgrama. U nekoliko
europskih zemalja zabilježeno je progresivno seljenje i smještanje kune bjelice u urbanim
područjima. Usporedno s time, kao negativni aspekti rastućeg broja divljih životinja u
gradovima navode se pojačani rizik od prijenosa zaraznih i invazijskih bolesti, mogući napadi
na ljude i domaće životinje, oštećenja imovine, te prometne nezgode. Pored toga, promjene
društvenih odnosa takvih jedinki razlog su veće aglomeracije na malom prostoru te podržavanja
bliskog kontakta među pripadnicima iste vrste, što povećava rizik od održavanja i prijenosa
pojedinih uzročnika bolesti u gradskim i prigradskim područjima. Upravo navedeno, kao i
činjenica da je poznat veliki broj zoonotskih bolesti u divljih mesoždera, ukazuje na nužnost
nadziranja populacije divljih životinja u svrhu procjene trenutnog zdravstvenog statusa i
predviđanja uloge u prijenosu uzročnika bolesti. Kada govorimo o leptospirozi divljih životinja,
glodavci se smatraju primarnim rezervoarom leptospira u ruralnim i urbanim sredinama. U
kontekstu složenog istraživanja ekologije prirodnih ognjišta leptospiroze u Hrvatskoj naglasak
je stavljen na mišolike sisavce kao primarne rezervoare leptospira u prirodi. Međutim, činjenica
da su štakori rezervoari bolesti ne objašnjava raznovrsnost seroloških skupina koje su
identificirane u ljudi i domaćih životinja inficiranih leptospirama. Stoga, postoji osnovana
sumnja da i druge divlje životinje igraju značajnu ulogu u ciklusu prijenosa leptospira, a na što
upućuje i njihova česta seroreaktivnost na leptospire, utvrđena u mnogim zemljama. Virus štenećaka rasprostranjen je po cijelome svijetu, a sposoban je inficirati brojne vrste
životinja unutar porodica Canidae, Procyonidae, Mustelidae, Hyaenidae, Mustelidae,
Procyonidae, Ursidae, Viverridae i Felidae. U Europi je bolest prvi puta utvrđena u Španjolskoj
davne 1761. godine, odakle se proširila na ostale europske zemlje. Danas je štenećak
rasprostranjen po cijelome svijetu i predstavlja značajnu zaraznu bolest domaćih i divljih
mesojeda s visokom stopom smrtnosti. Po značaju za mesojede svrstava se odmah iza bjesnoće.
Iako je štenećak dugo godina smatram primarno bolešću mesojeda, u novije vrijeme je
ustanovljeno kako je u stvarnosti riječ o virusu s vrlo širokim rasponom potencijalno
prijemljivih vrsta. Održavanje uzročnika bolesti u prirodi podržava se kroz prijenos između
različitih vrsta životinja. Pri tome se pasažama kroz organizme u kojih prethodno nije
zabilježena pojava štenećaka povećava mogućnost za prijelaz vrsne barijere. U ovome radu
analizirani su uzorci krvi, slezene i jetara 64 kune bjelice s područja Zagrebačke i Bjelovarskobilogorske županije na protutijela na bakteriju Leptospira spp. pretragom MAT, te na virus
štenećaka PCR analizom. Utvrđena je prevalencija protutijela na bakteriju Leptospira spp. u
iznosu od 37,5%. Pri tome je dokazano 8 serovarova, od kojih najviše sv Australis i Bratislava
(po 17%) te sv Icterohaemorrhagiae (14%). Nisu utvrđene statistički znakovite razlike u
ovisnosti o spolu, dobi (izuzev za serovar Icterohaemorrhagiae) i lokaciji. Usporedbom prema
serovaru omjer izgleda (OR) govori da je 1,51 puta veća vjerojatnost da će se u kunama utvrditi
protutijela na serovar Australis ili Bratislava negoli na serovar Icterohaemorrhagiae. Relativni
rizik (RR) za pojavu serovara Australis ili Bratislava (zasebno gledano) iznosi 1,22, dok je isti
za serovar Icterohaemorrhagiae 0,58. Relativni rizik od infekcije nešto je viši u mužjaka i iznosi
1,2, dok je u ženki 0,76. Omjer izgleda za infekciju mužjaka u odnosu na ženke iznosi 1,32.
Prema modelu, porastom vrijednosti temperature za jednu jedinicu indeksa postoji čak 2,6%
veća vjerojatnost pozitivnog nalaza. Utjecaj količine oborina je još veći tako da porastom SPI
za jednu jedinicu indeksa vjerojatnost infekcije je 2,88 puta veća. Model za sv Australis i Bratislava govori kako u slučaju porasta srednje vrijednosti temperature za jednu jedinicu
indeksa postoji oko 3,8% manja vjerojatnost infekcije serovarom Australis/Bratislava. U
slučaju porasta srednje vrijednosti oborinskog indeksa za 1, vjerojatnost od infekcije serovarom
Australis je 46,1% manja, a serovarom Bratislava je 37,9% veća. Pretraga na virus štenećaka
nije utvrdila pozitivne nalaze. Dobiveni rezultati su u skladu s prijašnjim istraživanjima
leptospiroze u divljih životinja, s izuzetkom sv. Bratislava koji do 2010. godine nije bio u panelu
antigena za MAT. Zanimljiv je negativan nalaz pretraga na virus štenećaka, koji zasigurno
cirkulira među divljim mesojedima i prema brojnim studijama se smatra jednom od njihovih
važnijih zaraznih bolesti. |
| Abstract (english) | INTRODUCTION: Human population growth and increasing exploitation of natural resources
have caused changes in ecological conditions for different species of wildlife. Many of these
species have adapted to living near humans, i.e. to living in urban habitats using a variety of
anthropogenic resources for survival. Typical adjustments include increasing social groups and
increasing population density, reducing living space, and changing eating habits. Members of
the marten family (Mustelidae), such as the beech marten (Martes foina), are examples that
show remarkable flexibility in finding shelter and food in human settlements and dwellings,
and even in breeding in urban cores. In several European countries, progressive migration and
placement of the beech marten (Martes foina) in urban areas has been recorded. Negative
aspects of the growing number of wild animals in the cities include the increased risk of
transmitting infectious diseases, possible attacks on humans and domestic animals, property
damage and traffic accidents. In addition, changes in social relations are the reason for greater
agglomeration of individuals in a small area and maintenance of close contact among members
of the same species, which increases the risk of maintaining and transmitting certain pathogens
in urban and suburban areas. The above-mentioned, as well as the fact that a large number of
zoonotic diseases are known in wild carnivores, indicates the need to monitor wildlife
population in order to assess current health status and predict the role in disease transmission.
REVIEW OF THE LITERATURE: The marten family appeared in the late Eocene and early
Oligocene periods when members of this family inhabited Europe and North America. Their
migrations to the south of the continents were recorded first in Africa during the early Miocene
and then in South America in the Quaternary. The marten family consists of five subfamilies:
Mustelinae (weasels, minks, ferrets and martens), Melliovorinae (honey badger), Melinae (badgers), Mephitinae (American ferrets) and Lutrinae (otters). According to the usual
classification, the beech marten (Martes foina) is classified as a mammal of the order Carnivora,
the family of marten (Mustelidae) and the subfamily of marten (Mustelinae). The genus marten
(Martes) consists of 7 species, most of which are animals of tropical, temperate and boreal
forest zones of North America, Europe and Asia. The distribution and abundance of martens is
strongly influenced by changes in habitat caused by the implementation of forestry and
agricultural measures. Newer classifications based on molecular analyses of the martens are
included into the magnorder Boreoeutheria, the superorder Laurasiatheria and the order
Carnivora. In addition to these, in some places between the superorder of Laurasiatheria and
the order of Carnivora, an unranked part of Ferae or in translation “of the beast” is also
mentioned. The beech marten lives in habitats from Mongolia and the northern Himalayas to
most of Europe. It is relatively rare on the islands. The northern boundary of the habitat is
Denmark. The prevalence of beech marten is growing in most European countries. In the
Republic of Croatia, the beech marten is a native species that inhabits the entire continental
part, but it is also found on the islands. It is more numerous in karst habitats, whereas in highland
and mountain areas it is found during the summer, and at the beginning of winter it usually
migrates to lower areas (example of vertical migration). It often lives near human settlements,
mostly next to outbuildings, backyards, under piles of stones, branches, etc. Beech marten is a
typical inhabitant of open areas, but is also fond of woodland edges, rocky terrain and
abandoned quarries.
The beech marten is 70 to 80 cm long, and 20 to 25 cm of that length is the tail. Their height is
about 25 cm, while body weight ranges from 1.5 to 2 kilograms. The body is elongated and
flexible. Their skull is shaped in such a way that it can pass through small openings, searching
for holes in the ground and openings in trunks. Their ears are short and the eyes are large. The
claws of a beech marten are long and sharp, helping them climb and catch prey. They have a
small amount of white hair on their throat, neck and chest, after which the beech marten got its
name. Their diet varies and comprises food of both plant as well as animal origin; therefore,
it can be said that they belong to the group of opportunistic carnivores. In accordance with the
aforesaid, beech martens usually take the food that is most available, while the prey that is
harder to catch is chosen in times of crisis. They hunt mostly in wooded pastures and rocky
areas. Often in the absence of prey they crawl into fowl houses and chicken coops where they
can do great damage.
Leptospirosis is a bacterial infectious disease that affects domestic and wild animals and
humans, resulting in a significant public health problem. The causes of leptospirosis are various
serotypes of the bacterium classified within the species Leptospira spp. Small rodents are
natural reservoirs of leptospira and can be lifelong carriers of the pathogen. Geographically,
leptospirosis is widespread throughout the world, but can generally be said to be most common
in areas with alkaline soils, especially in seasons when soils are warm and moist and when there
is abundance of surface water. Leptospirae are thin, flexible, filamentous bacteria, 6 to 20 μm
long (sometimes longer), about 0.1 μm in diameter, slightly spiral, and curled at one or both
ends in a liquid medium. Leptospirae are obligate aerobes with optimal growth at temperatures
from 28 to 30 ° C. Until 1989, the genus Leptospira was divided into two species, L.
interrogans, which included all pathological strains, and L. biflexa, which included all
saprophytic strains. Serovars that are antigenically linked are grouped within serological
groups. The phenotypic classification of leptospire has been replaced by genotypic, in which
all serovars are included in numerous genomic species. Based on their genetic characteristics,
we now know at least 19 species of bacteria from the genus Leptospira, 6 of which are
saprophytic and 13 pathogenic.
In each country, depending on its climatic, geological and ecological relations, appropriate
enzootic / endemic areas can be identified, and in the epizootiology / epidemiology of leptospirosis, the urinary excretion of leptospira (leptospiruria) of infected animals (especially
pigs) plays a decisive role, as well as long-term survival of pathogens outside the body. When
mentioning wild-type leptospirosis, rodents are considered the primary reservoir of leptospirae
in rural and urban areas. In the context of a very complex research on the ecology of natural
foci of leptospirosis in Croatia, emphasis has been placed on mouse-like mammals as primary
reservoirs of leptospira in nature, and rodents have been determined as reservoirs of leptospira
in the Sava and Drava river valleys.
Canine distemper is a very contagious, often deadly, multisystem disease of susceptible
carnivores. The causative agent of canine distemper is the RNA virus (CDV is an abbreviation
for “canine distemper virus”) with a size of 100 to 250 nm, and it is classified in the genus
Morbillivirus and the family Paramyxoviridae. This virus is widespread throughout the world
and is capable of infecting numerous species of animals within the families Canidae,
Procyonidae, Mustelidae, Hyaenidae, Mustelidae, Procyonidae, Ursidae, Viverridae and
Felidae. Today, canine distemper is widespread throughout the world and represents a
significant infectious disease of domestic and wild carnivores with a high mortality rate.
According to its importance for carnivores, it is immediately behind the rabies virus. Although
canine distemper has been considered a primary carnivorous disease for many years, it has
recently been established that it is in fact a virus with a very wide range of potentially
susceptible species. Dogs are considered the primary reservoir of disease for wildlife. Likewise,
certain species of wild animals (e.g., raccoons) may be reservoirs of disease for a susceptible
population of dogs. The maintenance of pathogens in nature is supported through transmission
between different species of animals. At the same time, passages through organisms in which
the appearance of canine distemper has not been previously recorded increase the possibility of
crossing the top barrier. When it comes to martens, canine distemper is considered one of their
most important infectious diseases. Thus, in Central Europe, Austria and Germany the disease has been reported in martens, ferrets, weasels and Eurasian badgers. Except for the martens,
epidemiological characteristics of canine distemper in wild animals have also been studied on
the example of Ethiopian wolves, lions, raccoon dogs, foxes and raccoons.
MATERIAL AND METHODS: In total 64 stone martens were collected (32 males and 32
females). The processed martens were delivered from the area of northern Croatia to the
Croatian Veterinary Institute (HVI) and the Faculty of Veterinary Medicine, University of
Zagreb. Each marten was dissected at the dissection room of the Department for Pathological
Morphology of the Croatian Veterinary Institute or at the dissection hall of the Faculty of
Veterinary Medicine, University of Zagreb. During the examination of the carcass, the sex was
determined, and after the opening of the thoracic and abdominal cavities, blood was sampled
directly from the heart or from large blood vessels, depending on the possibilities. The latter
primarily depended on the location of the hit and the application of a pellet or carbine bullets.
Of all the other organs the liver and spleen were sampled for the purpose of canine distemper
analysis. A part of the spleen and liver was stored in a plastic bag, properly labeled and stored
in the freezer until sent for analysis. For the purpose of determining the age of the marten, the
lower jaw was taken, stored in a marked plastic bag and frozen until analysis. A blood sample
was screened for antibodies to Leptospira spp. using a microscopic agglutination method
(MAT). The MAT method is a reference serological method for the diagnosis of leptospirosis
that determines the presence of antibodies to leptospirae in the examined serum. The reaction
of antigens and antibodies (IgM and / or IgG) results in the formation of insoluble agglutinate
complexes, which can be performed and read in microtiter plate with wells. The test result is
read under a dark field microscope in which agglutination is most visible. The method of
microscopic agglutination is a qualitative and quantitative procedure because it determines the
presence and quantity (titer) of antibodies at the same time. In the qualitative part of the test, it is determined whether there are antibodies for a certain serovar of leptospirae in the basic
dilution of the tested serum. In case of a positive test result, a quantitative MAT is undertaken,
which determines the amount of antibody - titer. Samples of brain, liver, and spleen will be
screened for canine distemper virus by the polymerase chain reaction method. RNA Rneasy
Midi Kit (Qiagen, Hilden, Germany) will be used to extract RNA from 51 samples of
homogenized spleen tissue according to the manufacturer's instructions.
Spleen and liver samples were analyzed for presence of canine distemper virus according to
CASTILHO i sur. (2007.). For RNA extraction RNA Rneasy Midi Kit (Qiagen, Hilden,
Njemačka) was applied in accordance with manufacturer’s instructions. Following RNA
extraction, it was used as substrate for reverse transcriptase M-MuLV (Fermentas, Burlington,
Kanada). A total of 20 μL of reaction mixture contains: 5 μL of extracted RNA (cca 500 ng),
5µl of extracted RNA was added to reverse transcriptase (C-DNA synthesis), containing 1x
First Strand Buffer, 40U RNAseOUT. 1.3Mm of each Dntp, 50pmols of each primer. 8.5 mM
DTT, 200U Superscript II Reverse Transcriptase and RNAse/DNA-se, water till 47µl of volume
and was exposed to 42°C during 60 min. For PCR amplification 10µl cDNA was added to
solution containing 1 x PCR Buffer, 50pmols of each primer, 0.2Mm Dntp, 2.4 Mm Mg Cl2 ,
2.5 U/µl Taq DNA polymerase Recombinant i RNAse/DNAse up to volume of 102 µl and
exposed to initial denaturation at 94°C during 5 mins. 35 cycles at 94°C during 45s, 48°C during
45s and 72°C during 90s followed, with final extension at 72°C during 10min. PCR products
were analyzed on 1% agarose gel stained with ethidium bromide. Amplified fragments of DNA
were purified with GFX PCR DNA and Gel Band Pirification Kit, and subjected to bidirectional sequencing using DYEnamic ET Dye Terminator, in accordance to manufacturers
instructions. Final sequence of each sample of 447 bp was analyzed using CLUSTAL/W
method and Bioedit software (HALL, 1999), with homologous sequences obtained from the
GenBank. Obtained data were processed by descriptive statistical methods and regression analysis in STATISTICA 12 program, and the probability ratio in Win Episcope 2.0. The
Kruskal-Wallis ANOVA test and the χ2 test were used to test the significance of the differences.
Linear regression model was applied to predict relation between positive findings and increase
in ambiental temperature or precipitation.
RESULTS: Out of 64 analyzed animals 24 reacted positively to at least one of the Leptospira
spp. Serovars, giving the prevalence of 37.5%. A total of 8 serovars were detected, presented
here in decreasing order Australis (17%), Bratislava (17%), Icterohaemorrhagiae (14%),
Pomona (9%), Saxkoebing (9%), Sejroe (6%), Poi (3%) and Haebdomadis (2%). Odds ratio
shows 1.27 higher possibility to detect sv Australis or sv Bratislava, compared to sv.
Icterohaemorrhagiae. According to the gender there was no statistically significant difference
(p=0.598). Odds ratio shows 1.32 higher possibility for males to be positive, compared with
females. According to age Mantel-Haenszelov test showed increasing trend, but no statistically
significant difference. Statistically significant difference was found in the case sv
Icterohaemorrhagiae positive samples and age (p=0.038) with statistically significant relation
according to the contingency coefficient (0.341; p=0,038). No statistically significant relation
was detected between sampling location and positive samples neither for subadult (χ2=1.32;
p=0.251) or adult individuals (χ2=0.09; p=0.764). Linear regression model showed 2.6% higher
possibility of infection if ambiental temperature becomes higher for 1 index unit, in the same
time with increasing precipitation for 1 index unit probability of infection is 2.88 times higher.
Analyzing the same effects on presence of sv Bratislava and sv Australis, model predicts
approx. 3.8% higher possibility of infection in case of increasing ambiental temperature for 1
unit, or 1.37 times higher possibility of infection with sv. Bratislava in case of increasing
precipitation. All samples were negative for CDV.DISCUSSION:
Determined prevalence of 37.5% is lower than previously detected prevalence in Croatia.
However, those studies were based on small sample size. Detected serovars are in general in
accordance with previous findings, with exception of sv Bratislava that was added to the antigen
panel much later. On the European level reported prevalence ranged from 0% to 86%. It is also
necessary to mention that only one study analysed 96 samples, other were mainly up to ten
animals. Serovars Bratislava and Australis are confirmed as serovars related to wildlife, while
serovar Icterohaemorrhagiae may be explained by the fact that stone martens utilizes suburban
areas and are frequently visiting human settlements with increasing rat population. Observed
problems with studies on marten population include inadequate samples (due to exsanguination,
freezing, haemolysis, etc) and small sample size due to decreasing hunting pressure on this
species. Interestingly, none of the analysed samples were positive for CD virus. Further analysis
are required to give better insight about role of stone martens in maintaining and spreading of
leptospirosis in different habitats. Also, further research is needed to understand role of urban
stone martens as source of CDV. |
