Parazitske bolesti čine više od dvije trećine svih bolesti divljih životinja. U tom odnosu nositelj se trajno nastoji riješiti parazita, dok parazit zauzvrat izbjegava imunosni odgovor. Važnu ulogu u obrani od parazita imaju geni glavnoga sustava tkivne podudarnosti (MHC) jer omogućavaju prezentaciju antigena parazita obrambenom sustavu nositelja, odnosno pokreću imunosni odgovor. Fascioloidoza kao parazitska bolest uzrokovana nezavičajnom vrstom metilja predstavlja idealno oboljenje za praćenje učinka parazita na nove nositelje. Veliki američki metilj se u Europi pojavio u drugoj polovici 19. stoljeća, a intenzivnije se počeo širiti dolinom Dunava krajem prošlog stoljeća te je u Hrvatskoj prisutan posljednjih dvadesetak godina. Kod istraživanja MHC gena u odnosu na uzročnike bolesti postoje dvije pretpostavke, jedna koja predmnijeva da su heterozigotne jedinke otpornije, dok druga govori da je otpornost, odnosno prijemljivost uvjetovana točno specifičnim alelima. Ciljevi ovog istraživanja su: i) utvrditi varijabilnost MHC gena kod jelena običnoga u Hrvatskoj, ii) utvrditi da li postoje razlike između različitih populacija i iii) utvrditi možebitnu povezanost alela u odnosu na invadiranost metiljem F. magna. Ukupno je prikupljeno 117 uzoraka jelenske divljači, od čega je 91 uzorak iz istočne Hrvatske gdje je metilj stalno prisutan u okolišu, dok je 26 uzoraka iz Gorskog kotara gdje metilj nikad nije utvrđen. Ukupna prevalencija fascioloidoze iznosila je 82,42%. Parazitološkom pretragom jetre utvrđen je stupanj invadiranosti, dok je koprološkom pretragom izmeta utvrđena prisutnost ostalih parazita u organizmu. Iz uzorka tkiva jetre izolirana je DNA te je lančanom reakcijom polimeraze umnožen odsječak genomske DNA (egzon 2 lokusa DRB MHC skupine II). Elektroforezom je potvrđena prisutnost PCR produkata koji su poslani na sekvenciranje u Macrogen servis. S obzirom da su utvrđene sekvence s više od 2 alela po jedinci, zaključeno je da je DRB lokus dupliciran u istraženih jedinki, te je provedeno daljnje sekvenciranje egzona 2 lokusa DRB tehnologijom sekvenciranja slijedeće generacije (NGS, engl. Next Generation Sequencing). Prema rezultatima Kruskal-Wallis testa
broj metilja po grlu je statistički znakovito najviši u fazi spolno zrelog metilja (22,3 metilja) u odnosu na ostale razvojne stadije ovog parazita. Analiziran je egzon 2 DRB lokusa MHC klase II odabranih 46 jedinki jelena običnoga. U istraženom uzorku utvrđeno je ukupno 44 DRB alela, a pojedina jedinka ima od jedan do četiri alela, što ukazuje na postojanje dvaju DRB lokusa kod jelena. Četiri alela otprije su poznata (DRB_NS36, DRB_NS38, DRB_ref01 i DRB_ref02), dok ostalih 40 do sad nije utvrđeno kod jelena. Pronađeni aleli translatiraju se u 43 jedinstvena aminokiselinska slijeda. Iste aminokiselinske produkte imaju aleli DRB_ref05 i DRB_NS47. Usporedbom prisutnosti alela DRB_ref02 i alela DRB_ref05 u nizinskoj i gorskoj populaciji nije utvrđena statistički znakovita razlika (DRB_ref05 - χ2 s Yatesovom korekcijom iznosi 0,0025, a p=0,96; DRB_ref02 - χ2 s Yatesovom korekcijom iznosi 0,0013, a p=0,97). S druge strane, usporedbom prisutnosti alela DRB_ref01 koji se javlja isključivo u nizinskom području dobivena je statistički značajna razlika između uzoraka s područja Lipovljana i Baranje, gdje se učestalije javlja u Lipovljanima (χ2 = 4,928, a p=0,026411). Alel DRB_ref02 se statistički znakovito više pojavljuje u jelena na području Lipovljana u odnosu na Baranju (χ2 = 3,931, p=0,0473). Nasuprot tome, alel DRB_ref03 se najčešće pojavljuje u jelena u Baranji, i to statistički znakovito u odnosu na jelene iz Lipovljana (χ2 = 5,1481, p = 0,023272) i Spačve (χ2 = 4,5365, p = 0,033). S obzirom na činjenicu da su aleli DRB_ref01 i DRB_ref06 utvrđeni isključivo u nizinskoj populaciji jelena, provedena je usporedba navedenih alela s obzirom na invadiranost metiljem F. magna. Usporedbom nalaza parazitološke pretrage jetre i prisutnosti pojedinih alela MHC DRB gena uočena je razlika u invadiranosti jelena ovisno o vrsti alela. Pri tome je razlika između invadiranosti jelena s DRB_ref01 i DRB_ref06 alelom statistički znakovita, gdje je invadiranost značajno manja kod jelena sa alelom DRB_ref06 (χ2 = 0,939, df = 1, χ2/df = 0,94, p = 0,332). Preostali aleli se pojavljuju uglavnom znatno rjeđe i ne mogu se dovesti u svezu s invazijom metiljem F. magna. Visoka raznolikost ukazuje na vitalnu populaciju jelena običnoga u Republici Hrvatskoj, a odnos alela DRB_ref01 i DRB_ref06
potencijalno na razvoj odnosa nositelj-parazit u slučaju fascioloidoze jelena običnoga. Činjenica da su svega tri alela zajednička jelenskim populacijama iz Gorskog kotara i nizinske Hrvatske ukazuje na moguće postojanje dvije subpopulacije.
|Abstract (english)|| |
INTRODUCTION: Parasitic diseases constitute almost 70% of all wildlife diseases, and therefore finding parasites in wild animals is the rule, rather than the exception. The term “parasitic disease” means a clinical disease, not just an infection. This coexistence between the host and a parasite species mostly results in establishing a balance, a dynamic relationship where the host tries to remove the parasite, and the parasite avoids the immune response. The entry of a new parasite species into a naive host population can lead to detrimental effects on the host’s health status, and even potentially compromise the existence of a certain species on a local level. Major Histocompatibility Complex (MHC) genes play a pivotal role in the host defence system by enabling the presentation of parasitic antigens to the immune system, thus initiating the immune response. As a parasitic disease caused by a non-native trematode species, fascioloidosis is an ideal model for monitoring the parasite impact on naive populations. Fascioloidosis was originally a disease of North American deer species, and European deer had no contact with this parasite until its first introduction to the northern part of Italy. In other words, from the example of this disease it is possible to create control and experimental groups under natural conditions, and with that gain insights into the evolution of the host-parasite relationship. The first stable foci of the large American liver fluke (Fascioloides magna) in Croatia was formed in the Baranja region. In contrast, red deer in the Gorski kotar region have never been in contact with this parasite species and they represent control/naive group. On the basis of all the above the primary goal of this research was to obtain data on the diversity of the MHC gene in red deer, as a precondition for assessing the vitality of the population and its relationship towards disease. It will also enable an insight into potential differences between red deer populations in Slavonija and Baranja, and the Moslavina and Gorski kotar regions, especially in relation to the hypothesis on the existence of two red deer subspecies (the eastern and western types).
REVIEW OF THE LITERATURE: The large American liver fluke was primarily a parasite of North American deer species, which appeared in the second half of the 19th century in Europe due to the uncontrolled introduction of exotic deer species. Later, migrations and translocations from enzootic regions enabled the spread of fascioloidosis to suitable habitats in Europe. Following the initial adaptation of flukes to new intermediate and final hosts, and hydrological and climate conditions, new permanent natural foci of fascioloidosis have been established – first in the Royal Park La Mandria in northern Italy, second in the Czech Republic, and third in the floodplains of the Danube River. In Italy, besides in red deer, cases of infection in dead-end hosts (cattle, horses, wild boar) and aberrant hosts (sheep, goat) have been detected. In the Czech Republic fascioloidosis has been detected in red deer, fallow deer, white-tailed deer, sika deer, roe deer and cattle. Molecular genotyping of flukes using mitochondrial markers (cox1) confirmed the close genetic relationship between flukes from Poland and the Czech Republic. In 2015 the presence of large American fluke was detected in farmed fallow deer in south-east Poland, close to the Slovakian border, which proved that the spread of fascioloidosis to new, non-infected areas is a dynamic and constant process. In neighbouring areas, fascioloidosis was confirmed in 1982 in Austria, followed by Slovakia, Hungary and Croatia. Almost ten years later the first cases were described in Serbia. At the end of 1999 hunters and foresters noticed a decline in the red deer population in the eastern part of Croatia. Liver analysis showed traces and pseudocysts filled with dark stained matter that resembled blood cloths. The liver of a red deer shot in the area of Šeprešhat (Baranja) was examined at the Veterinary Faculty University of Zagreb in January 2000. The analysis revealed 22 adult flukes, which was the first confirmation of the Fascioloides magna fluke in Croatia. This finding confirmed the presence of large American liver fluke in Croatia, with a tendency for the disease to spread throughout lowland areas. This trend is supported by the fact that fluke can release up to 4000 eggs per day, and by the migration characteristics of red deer and the transport of red deer from infected
to non-infected areas. By 2003, fascioloidosis was also present in the Vinkovci (P=12.5%) and Vrbanja (P=4.76%) regions, while the prevalence in Baranja reached 53.3%. The first cases of fascioloidosis between the Danube and Drava rivers were detected in 2004, showing that fascioloidosis was spreading towards the central part of Croatia. One of the striking characteristics of large American liver fluke is its large and thin body, which places it among the largest flukes. Adult flukes can reach up to 10 cm in length, 3.5 cm in width and up to 4.5 mm in thickness. The surface of the body is covered by cuticula, citoplasmatic sintitial tegument, whose primary function is to maintain the flukes’ integrity, like a skin-muscle envelope. However, it is also important in absorption and secretion. The tegument is covered by a layer rich in carbohydrates called glycocalix. Constant reparation and change of glycoproteins plays an important role in avoiding the host immune responses. Adult flukes are hermaphrodites, containing both male and female genital systems. The fluke's life cycle is divided into four phases, where the first phase begins with an adult fluke within the pseudocyst of the definitive final host. Secreted eggs reach the intestines via bile ducts and are shed into the environment with faeces. Following embrionation the miracidia actively search for water snails (Lymnaea truncatula) where they develop into sporocysts. Redia are developed from the sporocysts and cercaria from redia. Cercaria leaves the snail and searches for aquatic vegetation where it forms the next developmental stage called metacercaria. Metacercaria is the invasive stage for the final hosts. When ingested by final hosts, metacercariae reaches the intestine where immature flukes are liberated and actively penetrate the intestinal wall. Once in the abdominal cavity, immature flukes migrate through the ventral side of the abdominal cavity towards the liver where they penetrate Glisson's capsule, migrate through the liver parenchima and slowly develop into an adult flukes. In the livers of definitive hosts flukes are mainly in pairs, within thin-walled pseudocysts. Differently, thick-walled pseudocysts are found within dead-end hosts, while in aberrant hosts pseudocysts are not formed, which results in the permanent
migration of the fluke and consequent destruction of the liver parenchyma. Traces of dark pigment are visible on the surface of infected livers. This pigment is a bye-product of the fluke's metabolism and is called iron-porphirine. In order to react to infection caused by various pathogens, each organism has developed a system for recognition of foreign bodies. This system is based on complex molecules such as proteins, glycoproteins, lipoproteins, nucleic acids, etc., which are placed on the surface of each cell and the organism recognizes them as antigens. Tissue antigens are trans-membrane glycoprotins incorporated in the cell membrane, coded by the genes of the Major Histocompatibility Complex (MHC). MHC genes are divided into two main groups: MHC class I which codes glycoproteins of MHC class I, which presents viral and tumour antigens to cytotoxic T-cells, and MHC class II, which codes glycoproteins of the MHC class II that can be found on the surface of certain cells of the immune system (macrophages, plasma cells, dendritic cells). This group of glycoproteins presents antigens to T helper cells. The main characteristics of MHC genes in the majority of vertebrate species are polygeny and polymorphism. These parts of the genome are the most variable loci that provide instructions for protein synthesis in vertebrates. A great deal of polymorphism exists among species, but also within certain species, resulting in a potentially large number of alleles on each locus. A very important characteristic of MHC genes is their codominant expression, or in other words, glycoprotein receptors expressed on the cell surface are the result of alleles from both parents. The subregions of each chain are built from introns and exons. Exons represent the continuous coding parts of the DNA, while introns are non-coding parts between exons. Most research work in mammals has been directed towards exon 2 of the DRB subregion. This is an indicator of genetic variability, as the highest form of diversity between alleles and haplotypes is shown on this region. Research into MHC genes in wildlife is directed in two directions: analysis of variability and the relationship between variability and certain pathogens. Two main hypotheses propose that heterozygotes are more resistant to diseases than homozygotes, and
that resistance/susceptibility toward certain pathogens is influenced by specific alleles. So far, numerous studies on MHC variability in mammals have been conducted, including wild horses (Equus przewalskii), North-American bisons (Bison bison), oryx (Oryx leucoryx), European bison (Bison bonasus), cheetah (Aconyx jubatus), Asian lions (Panthera leo persica) and moose (Alces alces). Low MHC variability is not necessarily related to low genetic variability, as seen in European beavers (Castor fiber), Rudolph whales (Balaenoptera borealis), fin whales (Balaenoptera physalus) and southern sea elephants (Mirounga leonina). Habitat fragmentation in some species has resulted in small isolated populations, which still can have high MHC variability, i.e. in island foxes (Urocyon littoralis dickeyi) from the island of San Nicholas, the Malagasy giant rat (Hypogeomys antimena), or grey wolves (Canis lupus) in Croatia. Some research has revealed the existence of certain alleles related to resistance to infection with nematodes in sheep, European bisons, white-tailed deer (Odocoilues virginianus) and giant pandas (Ailuropoda melanoluca).
MATERIAL AND METHODS: For this research we used samples collected as part of the Croatian Science Foundation grant UIP 3421 ''Molecular epidemiology of selected parasitic diseases of wildlife''. In total we analysed 117 red deer, of which 91 samples was collected in the Posavina and Baranja regions (21 does, 34 males and for 36 animals no data were provided), while 26 were collected in the Gorski kotar region (5 does, 7 males and for 14 animals no data were provided). Locations were chosen according to the presence of Fascioloides magna. The State open hunting ground No. XIV/9: ''PODUNAVLJE-PODRAVLJE'' is located in the north-eastern part of the Danubian floodplain in Croatia, and spreads along the left and right banks of the River Danube. Inside this region is the ''Kopački rit'' Nature Park, which has a high influence on the epidemiology of fascioloidosis. The State open hunting ground No. III/39 - ''OPEKE II'' is a lowland area exposed to flooding, with altitudes up to 99 m.a.s.l. Territorially, the hunting ground is within the Sisačko-moslavačka County. Periods of high water levels occur twice per
year, in spring and autumn, when approx. 70% of the hunting ground is covered by water. Hunting ground No. VIII/110 „CRNA GORA“ is a mountain habitat located in the Gorski kotar region, in the northern part of the Primorsko-goranska County, near the border with Slovenia. Altitudes range between 380 and 1197 m.a.s.l. The only water streams in this habitat are Čabranka and Tršćanka Rivers. The collected red deer livers were analysed macroscopically for fibrous adhesions, altered colour and shape, and the presence of iron-porphyrin. Liver parenchyma was sliced transversally on approx. 2 cm thick layers and analysed. The observed changes were categorized according to their characteristics into immature and adult flukes, degraded flukes, flukes’ migratory channels and pseudocysts, which were further divided into young, old and degraded. All lesions were noted in a table for further analysis. A sample of the livers was taken for analysis of MHC genes in an Eppendorf tube with 96% alcohol, properly marked and stored at -20oC. Coprological analysis, which included sedimentation and flotation analysis, was applied to detect parasite eggs and larvae in faeces samples. Isolation of DNA was performed using a commercial Wizard Genomic DNA Purification Kit (Promega, USA), according to the manufacturer's instructions. Isolated DNA was stored at 4oC. A fragment of genomic DNA (exon 2 of the DRB MHC class II), 300-bp long, was replicated using PCR reaction. For that purpose a commercial HotStarTaqMaster Mix, containing buffer, dNTP and HotStarTaq DNA polimerase was used. The presence of PCR products was confirmed by gel electrophoresis. Successfully replicated PCR products were sent to Macrogen for sequencing. Since the presence of more than two alleles per sample was suggested, we concluded that the DRB locus was duplicated, and therefore further sequencing of the DRB exon 2 was carried out using Next Generation Sequencing (NGS). Nucleotide sequence, translation to amino acids, selection of the most appropriate substitution model and evolution distance were analysed using the MEGA program. Statistical differences between the level of infection, distribution of alleles and the relationship between alleles and the level of infection in the collected samples were
analysed using the Fisher exact test, the χ2 test and the McNemar χ2 test. Normality of distribution was analysed using Kolmogorov-Smirnov and Shapiro-Wilk tests. The relationship between variables was tested using the Spearmann rang correlation coefficient. Data among groups were tested using T-test and Kruskal-Wallis test.
RESULTS: For this research 117 red deer samples were collected (Gorski kotar (N=26), Lipovljani (N=28), Radinje (N=31), Vrbanja (N=8), Spačva (N=11), Tikveš (N=13)). The prevalence of F. magna positive samples was: Lipovljani (P=100%), Radinje (P=70.97%), Vrbanja (P=75%), Spačva (P=90.9%), Tikveš (P=30.77%). Total prevalence was 82.42%. Samples collected in the Gorski kotar region were free of large American liver fluke. Fisher's exact test showed that there were no statistical differences between the frequency of infection between males and females (p=0.0625), and that the prevalence of infected and non-infected animals was not identical (χ2=6.72; p<0.01 and McNemar χ2=15.57, p<0.0001). According to the Kruskal-Wallis test the number of flukes per animal was statistically higher in the case of adult flukes (22.3 flukes) when compared to other life stages. The reason for this could be the high variability of flukes per animal. The lowest variability was detected in the case of migratory flukes (CV=137), and the highest in the case of degrading flukes (CV=569). Relatively low variability was also observed in the case of adult flukes (CV=166). A positive correlation was also observed between most of the developmental stages. The highest correlation found was between the number of pseudocysts and the number of adult flukes (Rs=0.995), followed by the number of immature flukes and flukes in migration (Rs=0.805). The lowest correlation was detected between the number of immature flukes and degraded flukes (Rs=0,268). Exon 2 of the DRB MHC class II was analysed in 46 selected animals. A total of 44 different alleles were detected, with a maximum number of 4 alleles per animal, which suggests the existence of 2 DRB loci. Four alleles were previously known (DRB_NS36, DRB_NS38, DRB_ref01 i DRB_ref02), while other 40 alleles were detected for the first time.
The alleles found were translated into 43 amino acid sequences. The same products were detected for the DRB_ref05 and DRB_NS47 alleles. Comparison of the amino acid sequences revealed 43 mutable places. The presence of DRB_ref02 and DRB_ref05 alleles in lowland and mountain populations was not statistically significant (DRB_ref05 - χ2 with Yates correction is 0.0025, p=0.96; DRB_ref02 - χ2 with Yates correction is 0.0013, p=0.97). In contrast, the presence of the DRB_ref01 allele, found only in lowland populations, was statistically significant higher in the area of Lipovljani when compared to Baranja (χ2 = 4.928, p=0.026411). The number of DRB_ref02 alleles was significantly higher in the area of Lipovljani when compared to the Baranja region (χ2 = 3.931, p = 0.473). In contrast, the number of DRB_ref03 allele was significantly higher in the Baranja region when compared to Lipovljani (χ2 = 5.1481, p = 0.023272) and Spačva (χ2 = 4.5365, p = 0.033). Since DRB_ref01 and DRB_ref06 alleles were detected only in lowland populations, a comparison between these alleles and infection with F. magna was undertaken. Comparison of the presence of the respective MHC DRB alleles with parasitological findings revealed differences in the level of infection with respect to different alleles. The differences in infection between animals with DRB_ref01 and DRB_ref06 alleles were statistically significant (χ2 = 0.939, df = 1, χ2/df = 0.94, p = 0.332). The remaining alleles appeared rarely and cannot be related to F. magna infection in this research.
DISCUSSION: The high allelic diversity observed in this research is in accordance with other studies conducted on deer species. The fact that only three alleles are common in the red deer population from the Gorski kotar region and the lowland part of Croatia supports the hypothesis of the existence of two red deer subspecies – the western or Dinaric and the eastern or Pannonian deer. The relationship between the DRB_ref01 and DRB_ref06 alleles suggests the potential evolution of host-parasite interaction in the case of red deer fascioloidosis. Caution is needed when discussing this potential relationship since the level of infection is also influenced by disease control programmes, and therefore we suggest using the terminology “the reaction of
the organism to a novel parasitic disease” rather than “resistance to parasitic disease”. Further research is needed to include animals from lowland areas where fascioloidosis has not yet been established