Application of commercial enzyme linked immunosorbent assays (ELISA) for the detection of antibodies for foot-and-mouth disease virus in wild boar and red deer

For detecting antibodies towards foot and mouth (FMD) virus in sera collected from red deer hinds (Cervus elaphus) and wild boars (Sus scrofa), three commercially available enzyme-linked immunosorbent assays (ELISA) were used. Two ELISA kits (PrioCHECK FMDV NS and CHEKIT FMD-3ABC) were used for the detection of antibodies towards non-structural proteins of FMD virus and one assay was based on the detection of antibodies for serotype O (PrioCHECK FMDV type O). All of the sera tested in our study were negative for antibodies against FMD virus. The aim of this study was to investigate the usefulness of commercially available ELISA kits given for marketing authorization in Croatia in testing the prevalence of FMD antibodies in wild boar and red deer populations. Since the producers of ELISA kits used in our study did not declare wild animals as a target species, we hypothesised that the same kits could be used for serological diagnosis of FMD in red deer and wild boars. Our study confirmed that the kits used are acceptable for detecting antibodies in both species tested, however, the investigation highlighted the problem of validating the kits due to the absence of available positive sera originating from red deer, as well as other susceptible species, especially artiodactyls

The Centenary Progress of Molecular Genetics. A 100th Anniversary of T. H. Morgan’s Discoveries

A century ago, Thomas Hunt Morgan, the American scientist, studied the cytogenetic changes of drosophila and came to cytogenetic explanation of Mendel’s basic laws of genetic heredity. These studies resulted in today’s Mendel-Morgan chromosomal theory of heredity. On the occasion of the hundredth anniversary of this important discovery the authors have decided to give a review of the most significant achievments in the field of molecular genetics until the completion of the Human Genome Project. The most important points concerning the technology of DNA recombination and genetic engineering are also presented. The final section discusses the significance of previous achievements of molecular genetics in biomedicine and other related fields. There is also a tabular presentation of the sequence of the most important findings in the field of molecular genetics through time

Characterisation of pseudorabies virus in domestic pigs and wild boars in Croatia

Serological data imply that pseudorabies (Aujeszky’s disease) is present in domestic pigs and wild boars in Croatia. Therefore, this study included testing of brain tissue samples collected from 200 domestic pigs and 105 wild boars originating from seventeen districts of Croatia. The presence of pseudorabies virus (PrV) DNA was confirmed in samples originating from six domestic pigs (3%) and one wild boar (0.95%). Positive samples were sequenced and analysed on the basis of a gC genome fragment. PrV strains have shown to be genetically identical and they are strongly related to some representative strains in the relatively heterogeneous Clade A. The results clearly show that PrV is still circulating among the domestic pig population in Croatia. Furthermore, the presence of pseudorabies virus in wild boars underlines the importance of this species as a PrV reservoir. Continued surveillance is necessary to track the viral spread in order to achieve final eradication of the disease

Utjecaj infekcije virusom AEK na proizvodnju mlijeka francuske alpske koze u Hrvatskoj

We have investigated the influence of infection with Caprine Arthritis Encephalitis Virus (CAEV), developed clinical arthritis and recorded subclinical mastitis on the length of lactation, milk yield and its composition per lactation. The study was carried out on the sample of 808 goats of French Alpine breed from production farms in Croatia. Samples of blood were taken for serological tests and milk samples for subclinical mastitis detection. Presence of clinical arthritis was noted by inspection and palpation of the joints. Lactation and production data were collected by Croatian Agricultural Agency. The influence of infection status for CAEV and clinical arthritis, separately, were tested on milk yield, length of lactation and milk composition (fat, protein and lactose) using GLM procedure for models including the year of measurement, lactation number of the animal and subclinical mastitis record. CAEV was serologically confirmed in 53.72 % of the total number of goats. From the total sample 23.08 % were infected with CAEV with confirmed subclinical mastitis of bacteriological etiology. In addition, 22.47 % of the total sample had clinical arthritis. Of the total sample 16.95 % were CAEV infected and with diagnosed clinical arthritis. Influence of virus infection was confirmed on all of the analyzed traits, with lower production results. Clinical arthritis had significant influence on decrease in most of analyzed traits, exceptions being total milk, fat amount (%) and lactose content (%) and amount (kg). Diagnosed subclinical mastitis was not found to be significant influence in models of duration of lactation, or fat, protein and lactose content (%).Istraživan je utjecaj infekcije virusom artritis encefalitisa koza (AEK), kliničkog artritisa i subkliničkog mastitisa na trajanje laktacije, ukupno mlijeko i sastav mlijeka. Istraživanje je provedeno na uzorku od 808 koza pasmine francuska alpska koza s proizvodnih mliječnih farmi u Hrvatskoj. Uzeti su uzorci krvi za serološka testiranja i uzorci mlijeka za detekciju subkliničkog mastitisa. Prisutnost kliničkog artritisa dijagnosticirana je pregledom i palpacijom zglobova. Podaci o laktacijama i proizvodnji preuzeti su od Hrvatske poljoprivredne agencije (HPA). Zasebno su testirani utjecaji AEK infekcije i kliničkog artritisa na ukupno mlijeko, trajanje laktacije i sastav mlijeka (mast, protein, laktoza) pomoću GLM procedure za modele koji su uključivali godinu mjerenja, broj laktacije i subklinički mastitis. Infekcija AEK virusom potvrđena je u 53,72 % koza od ukupnog broja koza. Od ukupnog uzorka 23,08 % inficirano je s AEK i imalo subklinički mastitis bakterijske etiologije. Nadalje 22,47 % od ukupnog broja imalo je klinički artritis. Od ukupnog broja 16,95 % bilo je AEK pozitivno i imalo klinički artritis. Utjecaj infekcije virusom AEK potvrđen je za sva analizirana svojstva u smislu smanjenja proizvodnih rezultata. Klinički artritis je pokazao značajan utjecaj na smanjenje većine proizvodnih vrijednosti osim na ukupno mlijeko, udio (%) masti laktoze i ukupnu laktozu (kg). Subklinički mastitis nije značajno utjecao na trajanje laktacije, kao ni udio (%) i udio masti, proteina i laktoze

The wild boar (Sus scrofa) – victim and ally of the most important viral infections in europe

Divlja je svinja jedna od najstarijih preživjelih vrsti životinja na euroazijskom području. Danas je proširena u Europi, Aziji, Africi, a uvedena je i na područje Sjeverne Amerike. Zbog svoje visoke prilagodljivosti, klimatskih promjena, načina gospodarenja lovištima i malog broja prirodnih neprijatelja, divlja je svinja najmanje ugrožena vrsta životinja i njezin broj sustavno raste. Danas ona predstavlja ugrozu poljoprivrednicima kojima nanosi štete, a zbog hrane ulazi i u prigradska te gradska okružja. Prepoznata je za jednog od najznačajnijih rezervoara virusnih infekcija životinja, ali i nekih zoonoza. Naime, divlja svinja može doći neposredno i posredno u dodir s domaćim životinjama te na taj način prenijeti uzročnike virusnih infekcija. Obzirom da je lov jedna od važnijih turističkih ponuda Hrvatske, a divlja svinja jedna od najtrofejnijih divljih životinja, dodir divlje svinje s ljudima može ugroziti i zdravlje ljudi. U ovom smo radu opisali ulogu divljih svinja pri prijenosu najznačajnijih virusnih infekcija u svinjogojskoj industriji Europe, poput šireće afričke svinjske kuge te nama poznate klasične svinjske kuge i za druge vrste životinja poput bolesti Aujeszkoga. Opisali smo i značenje divlje svinje kao rezervoara virusa hepatitisa E, danas sve značajnije zoonoze. Ujedno je dat osvrt na moguću pojavnost navedenih infekcija ili njihovu prisutnost u divljih svinja s područja Republike Hrvatske.The wild boar is one of the oldest animal species in Eurasia. Currently, it is spread throughout Europe, Asia, Africa, and was introduced into North America in the 1920s. Due to its great adaptability, hunting ground management, its lack of natural enemies, and favorable climate changes, the wild boar is an animal species whose conservation status is of least concern and which is increasing in numbers. However, it presents a threat to farmers due to the losses caused by it eating and damaging crops. Furthermore, the wild boar is entering the suburbs or even centers of cities, and since it is known that wild boars may be sources of infectious diseases for different animal species, as well as numerous zoonoses, direct and indirect contact may cause interspecies transmission and the spread of viruses. Hunting may also be a risk factor in viral spread, since wild boar hunting is a significant tourist attraction in Europe, including Croatia, and wild boars are a prestigious trophy. Hunted wild boar carcasses and blood may be sources of infection not only for animals but for humans as well. This is a review regarding the role of wild boars as reservoirs of the most important viral infections in the swine industry in Europe, such as African swine fever (ASF), classical swine fever (CSF) and Aujeszky’s disease (AD). Even though ASF has never been reported in Croatia, its continuous spread throughout Europe is cause for concern. Currently, the infection is 200 km from Croatia’s borders, and early preparedness measures have been taken. CSF has been one of the most devastating diseases in swine production in Croatia. The last outbreak was recorded in 2006-2008. However, surveillance programs are still being implemented to ensure the healthy status of swine and wild boars. AD has been diagnosed in wild boars, and sporadic cases of infection in hunting dogs have occurred in the last decade. Therefore, the infection is still a threat to the swine industry. We have also described the importance of wild boars as reservoirs of hepatitis E (HE), an emerging public health problem and zoonosis. The Croatian Veterinary Institute has been involved in epidemiological studies of HE since 2007. However, a thorough investigation involving the testing of domestic pigs, wild boars and other wild and domestic animal species started in 2009. Currently, the domestic pig and wild boar are the only animal reservoirs of HE in Croatia, but wild boar may be chronically infected and spread the virus for months

Filogenetska analiza goveđeg herpesvirusa-1 izdvojenoga u Hrvatskoj.

In the present study, we report the detection, isolation, molecular characterisation and phylogenetic analysis of bovine herpesvirus type 1 (BoHV-1) in nasal swabs, sera and tissue samples derived from naturally infected cattle. In the spring of 2008, clinical symptoms of the respiratory disease were observed in three Croatian dairy herds. Nasal swabs and sera were collected on the 5th and 27th days after appearance of symptoms. A tissue sample (lungs) from an eight week old calf was also collected. Specific antibodies against BoHV-1 were not found in sera samples collected after the first sampling, but all sera samples were positive after the second sampling. The cytopathic effect on the bovine embryo kidney (BEK) cells was visible after 72 hours for the nasal swab samples, and after 48 hours for the lung tissue sample. Sensitive PCR assays specific for BHV-1 glycoproteins gB and gC were also performed. All nasal swab samples and the lung sample were positive with both sets of PCR primers. The gC PCR product obtained from one sample from each farm as well as from the lung sample DNA were sequenced. Phylogenetic analysis of gC gene sequence clustered our isolates with BoHV subtype 1.1.U radu je opisano dokazivanje, izdvajanje, molekularna karakterizacija i filogenetska analiza goveđega herpesvirusa-1 (GHV-1) iz nosnih briseva i organa prirodno zaraženih goveda. U proljeće 2008., u tri domaća stada mliječnih krava pojavili su se klinički simptomi dišne bolesti. Peti i 27. dan nakon pojave simptoma sakupljeni su uzorci krvi i nosnih briseva kao i uzorak pluća uginuloga teleta u dobi od od 8 tjedana. Svi uzorci seruma prikupljeni prvim uzorkovanjem dali su negativan rezultat na prisutnost specifičnih protutijela za GHV-1, a pozitivan nakon drugoga uzorkovanja. Citopatski učinak na stanicama bubrega goveđega zametka bio je vidljiv nakon 72 sata u slučaju infekcije stanica inokulumom pripravljenim od nosnoga brisa te nakon 48 sati u slučaju suspenzije organa. Napravljena je i lančana reakcija polimerazom (PCR) upotrebom specifičnih početnica za gene gB i gC GHV-1. Svi uzorci nosnih briseva kao i pluća dali su pozitivnu reakciju s oba para početnica. Nakon PCR, odsječcima gC DNA određen je nukleotidni slijed, a filogenetska analiza GHV-gC sekvencija svrstala je naš izolat u podtip 1.1

Supklinički mastitis i klinički artritis u francuskih alpskih koza iz sjeverozapadne Hrvatske serološki pozitivnih na artritis-encefalitis virus

The prevalence and aetiology of subclinical mastitis in goats seropositive for Caprine Arthritis Encephalitis Virus (CAEV) and the connection with the occurrence of clinical arthritis were investigated on 543 French Alpine goats from intensive production farms in North West Croatia. The aim was to determine if seropositivity to CAEV is connected with susceptibility to subclinical mastitis of bacterial aetiology, and to examine if CAEV is the main reason for the occurrence of clinical arthritis in the investigated sample. All the goats were examined clinically and the presence of arthritis was noted. The blood samples were tested for CAEV antibodies using the immunoenzyme test. Milk samples from each udder half were examined for the bacteriological cause of mastitis. All the collected data were cross-classified in two-way contingency tables. Of the total number of goats, CAEV was serologically confirmed in 50.8%. Subclinical mastitis was confirmed in 52.3% of the goats. Both subclinical mastitis and seropositivity to CAEV were diagnosed in 30% of the total number. Of the total sample 21.9% were seropositive to CAEV and with clinical arthritis. Statistical tests confirmed a positive association for subclinical mastitis occurrence and seropositivity to CAEV. A similar result was obtained for the association between clinical arthritis and seropositivity to CAEV. A weak positive association between subclinical mastitis and clinical arthritis occurrence was also recorded. These results indicate that a developed clinical manifestation of the disease could also influence higher manifestation of subclinical mastitis. Results also suggest that CAEV infection could be one of the main reasons for clinical arthritis occurrence in the investigated herdsIstražena je povezanost prevalencije i etiologije supkliničkog mastitisa s pojavnošću kliničkog artritisa u 543 francuske alpske koze serološki pozitivne na virus artritisa-encefalitisa (AEK) iz proizvodnih farmi sa sjeverozapada Hrvatske. Cilj je bio odrediti je li serološka pozitivnost na AEK povezana s pojavnošću supkliničkog mastitisa bakterijske etiologije i istražiti je li AEK glavni uzrok pojavnosti kliničkog artritisa u pretraženih koza. Sve su koze bile pregledane na pojavu kliničkog artritisa. Uzorci krvi iz jugularne vene bili su testirani na AEK imunoenzimnim testom. Uzorci mlijeka, uzeti iz svake polovine vimena zasebno, pretraženi su na prisutnost bakterijskih uzročnika mastitisa. Svi prikupljeni podatci unakrsno su klasificirani u kontingencijskim tablicama. Od ukupnog broja koza, AEK je serološki potvrđen u 50,8%. Supklinički mastitis je potvrđen u 52,3% koza. Istodobna pojava supkliničkog mastitisa i AEK zabilježena je u 30% od ukupnog broja, dok je 21,9% koza s kliničkim artritisom bilo serološki pozitivna na AEK. Statističke su analize potvrdile pozitivnu povezanost pojave supkliničkog mastitisa i AEK seropozitivnosti. Slični su rezultati dobiveni i za povezanost pojavnosti kliničkog artritisa i AEK seropozitivnosti. Također je zabilježena slaba pozitivna povezanost između supkliničkog mastitisa i kliničkog artritisa. Rezultati upućuju na to da bi AEK mogao utjecati na učestalost supkliničkog mastitisa. Rezultati također nalažu dodatno istraživanje AEK infekcije kao glavnog uzroka pojave kliničkog artritisa u istraženim stadima

INFLUENCE OF INFECTION WITH CAPRINE ARTHRITIS ENCEPHALITIS VIRUS ON MILK PRODUCTION OF FRENCH ALPINE GOATS HERDS FROM NORTHWEST CROATIA

Istraživanje je provedeno sa svrhom utvrđivanja utjecaja infekcije virusom artritis encefalitisa koza (AEK) na ukupnu količinu i sastav mlijeka u trajanju od jedne laktacije. Istraživanje je provedeno na 259 francuskih alpskih koza iz komercijalnih farmi sjeverozapadne Hrvatske. Koze su držane i hranjene na sličan način. Kontrola i uzorkovanje mlijeka provedeno 2009. godine ICAR4 metodom. Istovremeno su prikupljeni uzorci krvi za serološko testiranje na prisutnost AEK antitijela koje je provedeno imunoenzim-nim testom (ELISA). Utjecaj infekcije na ukupno mlijeko , trajanje laktacije i sastav mlijeka (mast, protein i laktozu) testirano je analizom varijance (PROC GLM). Utjecaj kliničkoh artritisa na laktaciju također je testiran. AEK je serološki potvrđen u 37% koza. Nadalje, 24% AEK pozitivnih koza je imalo klinički artritis. Potvrđen je utjecaj serološkog statusa na sva istraživana svojstva. Klinički artritis utjecalo je na količinu (%) proteina i masti u mlijeku.The objective of this study was to investigate the influence of infection with Caprine Arthritis Encephalitis Virus (CAEV) on estimated milk yield per lactation and its composition. The study was carried out on 259 goats of French Alpine breed from production farms in NW Croatia. All goats were kept under similar intensive feeding and management conditions. Milk control was preformed and milk samples were collected using the ICAR method A4 during the year 2009. At the same time, blood samples were collected and presence of CAEV antibodies in the sera was detected using enzyme-linked immunosorbent assay (ELISA). The influence of infection status on milk yield, length of lactation and milk composition (fat, protein and lactose) were tested by analysis of variance (PROC GLM). Influence of clinical form of arthritis on lactation was also investigated. CAEV was serologically confirmed in 37% of goats. In addition, 24% of CAEV positive animals were expressing clinical symptoms of arthritis. Strong influence of serological status on all recorded traits has been confirmed.Presence of clinical arthritis has confirmed influence only on protein and fat content in milk

Arthritis Encephalitis Virus in goats in the Republic of Croatia in the period from 2012 to 2019: serological and molecular research

Artritis-encefalitis koza (AEK) je neizlječiva virusna bolest proširena u većini zemalja koje se bave intenzivnim uzgojem koza. Ekonomsko značenje ove bolesti očituje se uginućem jaradi, smanjenim prirastom i mliječnošću, prijevremenim lučenjem iz uzgoja. U razdoblju od 2012. do 2019. godine na području 16 županija u Republici Hrvatskoj pretraženo je 10 715 uzoraka krvi, a tijekom tog osmogodišnjeg razdoblja specifična protutijela za virus AEK bila su potvrđena u 4 353 uzoraka krvnog seruma pa zaključno možemo govoriti o seroprevalenciji od 40,62 %. Rezultati genotipiziranja odabranih sojeva LVMP ukazuju na cirkulaciju genetske skupine A i B u populaciji koza i to su prva takva istraživanja provedena u Hrvatskoj. Dokaz prisutnosti genotipa A u stadu koza u kojem je istovremeno dokazan i genotip B ukazuje na činjenicu da sojevi LVMP nisu ograničeni na jednu vrstu već da sojevi MVV koji se uglavnom nalaze u ovaca, cirkuliraju i u koza. Rezultati višegodišnjeg istraživanja provedenog na Hrvaskom veterinarskom institutu ukazuju na široku rasprostranjenost virusa AEK u uzgojima koza u Republici Hrvatskoj. Ako znamo da jednom zaražena jedinka ostaje doživotni nositelj i prenositelj virusa postavlja se pitanje uspješnosti cijele proizvodnje pa je stoga jedini dugoročno održiv način uzgoja uzgoj slobodan od AEK.Caprine arthritis and encephalitis (CAE) is a contagious and an economically important viral disease of goats. Caprine arthritis encephalitis virus (CAEV) is a member of the small ruminant lentiviruses (SRLV) that is distributed worldwide, including Croatia. Common symptoms resulting from CAEV infection include arthritis, pneumonia, indurative mastitis and encephalitis. One of the first and most common signs of CAEV infection is weight loss. However, many goats will not show any symptoms. Goats that do show symptoms may display swollen knees or carpal joints, exhibit reduced body condition, and develop a rough hair coat. Pregnant and nursing does display a hard udder that is firm and swollen, and produce very little milk. During 2012 to 2019, a total of 10,715 blood serum samples were collected in 16 counties in Croatia and examined for the presence of CAEV specific antibodies using ELISA. Antibodies against CAEV were confirmed in 4353 (40.62%) of samples. As this is the first such study to be carried out in Croatia, the results of molecular testing of selected SRLV strains indicate the circulation of the A and B genotypes in the Croatian goat population. The simultaneous presence of both genotypes A and B in a goat herd suggests that SRLV strains are not limited to one species, and that Maedi visna virus (MVV) strains, which are mainly detected in sheep, also circulate in goats. The results of this eight-year study conducted by the Croatian Veterinary Institute (CVI) indicate the widespread prevalence of SRLVs in goat breeding in Croatia. It is known that once an infected, the individual remains a lifelong carrier and can spread the virus. This raises the issue of the success of entire production. In conclusion, the only long-term sustainable goat breeding is on CAEV-free goat farms