Porcine circovirus type 2 (PCV2) causes some of the most significant economic losses in pig production. Several multisystemic syndromes have been attributed to PCV2 infection, which are known as PCV2-associated diseases (PCVDs). This study investigated the origin and evolution of PCV2 sequences in domestic pigs and wild boars affected by PCVDs in Croatia. Viral sequences were recovered from three wild boars diagnosed with PCV2-systemic disease (PCV2-SD), 63 fetuses positive for PCV2 DNA as determined by PCR, 14 domestic pigs affected with PCV2-SD (displaying severe interstitial nephritis) and five domestic pigs with proliferative and necrotising pneumonia. Seventeen complete PCV2 genomes were recovered. Phylogenetic and evolutionary analyses based on median-joining phylogenetic networks, amino acid alignments and principal coordinate analysis were performed using complete genomes, as well as complete and partial ORF sequences for ORF1 and ORF2. Two of the 17 PCV2 sequences belonged to PCV2a, 14 to PCV2b and one was unclustered. PCV2b was the predominant genotype in Croatia and has been linked to international trade as a route of introduction. Correlation between particular viral strains with PCVDs is lacking.

Torque teno virus (TTV) is ubiquitous and species-specific, and two different TTV species, Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2), have been described in domestic pigs and wild boars. Whether these two species are involved in clinical cases of porcine circovirus type 2-associated disease (PCVDs) remains unknown. This study investigates the presence of TTSuV in 85 fetuses, pigs and wild boars infected by PCV2 and affected by PCVDs other than postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome. It also explores the genetic diversity of identified strains using phylogenetic analysis. The presence or absence of TTSuV was determined in 85 samples of PCV2-containing organs from 85 infected animals using a specific, one-step PCR method. A nucleotide distance matrix between sequences was computed to infer phylogeny based on a median-joining (MJ) network. TTSuV2 showed high prevalence in animals affected by nephropathy and proliferative and necrotising pneumonia (PNP), and both TTSuV1 and TTSuV2 showed high prevalence in wild boars affected by PMWS. TTSuV1 showed low prevalence in animals affected by nephropathy and PNP, and both TTSuV1 and TTSuV2 showed low prevalence in animals with reproductive disorders. MJ network analysis revealed significant genetic diversity among Croatian isolates.

Acute hepatitis E virus (HEV) infection is a self-limiting symptomatic or asymptomatic disease. However, as recently observed, it can manifest itself as chronic hepatitis in patients receiving solid organ transplants as well as in patients with HIV infection or severe hematologic disorders.Here, we describe the clinical course of a 73-year-old male patient in whom HEV transmission occurred after receiving a HEV-infected liver from a donor with occult HEV infection, whereby the patient had tested negative for HEV RNA and anti-HEV antibodies shortly before explantation.Anti-HEV IgG, IgM, and HEV RNA were detected in the first tested serum sample of the liver recipient obtained 150 days after liver transplantation and remained positive (earlier samples after OLT were not available). Liver cirrhosis developed within 15 months and the patient died of septic shock. Based on phylogenetic analyses of the donor and recipient’s HEV strains, we were able to prove that the occult HEV infection was transmitted via the graft.

A comparative study of blood chemistry and histology was conducted on two groups of mullets (Mugilidae) living under different conditions with different feed sources. The aquaculture influenced mullet group (AIM), was collected near fish farms and the control group of mullet (CM) was caught in the waters without any aquaculture activities. Histological and biochemical procedures were employed to study liver histomorphology, plasma aspartate and alanine aminotransferase (AST, ALT), triglyceride (TRIG), cholesterol (CHOL), glucose (GLU) and total protein (TP) of both AIM and CM. Moderate histological changes (lipid infiltration) were observed in the liver of AIM. Significant changes in plasma variables were observed in AIM. Blood chemistry variables measured proved to be good indicators of artificial feed effects. Classical statistical approaches were applied to the blood chemistry and histopathology data. For the first time machine learning techniques were used to generate comprehensible classification models and to explore blood chemistry variable importance, strength, their mutual interactions or dependencies, and to investigate reliability of particular variables within the groups.

During the 10-year survey (1996–2005), a total of 868 blood samples from different game species in Croatia were analyzed for the presence of leptospiral antibodies. The specific antibodies (AB) were detected in 242 samples (27.88%). According to the species in red deer (Cervus elaphus), the antibodies against six different leptospiral serovars were found in 43 of 226 analyzed sera (19.02%). The most frequent antigen serovars in the deer population were Pomona and Ballum (with the same frequency of 23.6%), whereas the highest titer was recorded for serovar Sejroe (1:800). In the analyzed roe deer (Capreolus capreolus) serum samples, a low level of leptospiral antibodies (6.07%) was determined, with just two AB for antigen serovars—Australis and Sejroe. In wild boar (Sus scrofa), leptospiral antibodies were detected in 151 of 431 samples analyzed (35.03%), with AB for nine antigen serovars. The serovars most frequently found were Australis (48.70%) and Pomona (22.70%), and these serovars also recorded the highest titer (1:3,200). Among brown bear (Ursus arctos) samples, leptospiral antibodies were detected in 25.00% of the samples, with four AB for antigen serovars, of which the most frequent was Icterohaemorrhagiae (>40%). This serovar had the highest recorded titer (1:400). From 112 analyzed red fox (Vulpes vulpes) samples, leptospiral antibodies were found in 35 samples (31.25%). The determined antibodies were specific for four antigen serovars, of which the most frequent (46.2%) and with highest titer (1:1600) was serovar Australis. No antibodies (28/0) were recorded in mouflon (Ovis musimon). The most important game species from an epizootiological point of view in the studied area were certainly wild boar and red foxes. With strong serological reactions, these two species could be emphasized as important hosts for Leptospira interrogans sv. Australis in Croatia, but for their declaration as ‘maintaining hosts,’ isolation of sv. Australis is needed. According to aerial distribution, the highest number of positive samples from different game species was recorded in the central and eastern parts of Croatia, known as the ‘historical natural foci’ of leptospirosis—the regions of Posavina, Podravina, Slavonija, and Baranja. In contrast, the areas of Kordun and Gorski Kotar are declared as leptospira low-risk regions for the game species studied.

The presence of serum antibodies directed against classical swine fever (CSF) virus and other pestiviruses among the wild boar (Sus scrofa) population in Croatia was investigated. During 2003, serum samples from 214 wild boars were collected in 10 hunting areas in the continental part of the country. The sera were examined by enzyme immunoassay (ELISA) and in the virus neutralization test (VNT). Out of 214 sera tested 111 (51.87 %) were positive by ELISA and regarding neutralising antibodies, against CSFV 75 (35.05 %) samples were positive. In the VNT with the C-strain (conventional live vaccine strain China) and the strain Uelzen were used. Samples were also tested for neutralizing antibodies against border disease virus (BDV) using the strain 137/4 and against bovine viral diarrhoea virus (BVDV) using the NADL strain. Neutralizing antibodies against the C-strain were detected in 36 sera (16.82 %), against strain Uelzen in 17 sera (7.94 %) and in 22 sera (10.28 %) against both strains. In five sera (2.33 %) neutralizing antibodies against BVDV and BDV were found.

This report describes the first case of postweaning multisystemic wasting syndrome (PMWS) in wild boar in Croatia. During the winter season of 2004, eight wild young piglets (of approximately 2 to 5 months of age) were found dead in a fenced hunting area. Polymerase chain reaction (PCR) was carried out on mesenteric lymph nodes and all animals yielded positive results. In one of these animals diagnosis of PMWS was established based on the three key diagnostic criteria including the clinical manifestation, moderate lymphoid lesions consisting of lymphocyte depletion and granulomatous inflammation, and detection of the presence of PCV2 genome within the lymphoid lesions by in situ hybridisation (ISH). Three additional wild piglets had also mild PMWS-like lesions and a low amount of PCV2 was also found. No PMWS-like lesions or PCV2 genome were detected in the rest of the wild piglets studied. Three PCR-positive isolates were partially sequenced, which confirmed the diagnosis of PCV2 and demonstrated that the three sequences were genetically identical. The phylogenetic analysis of a representative PCV2 isolate indicated that its sequence (DQ875444) is grouped in a separate branch with Hungarian isolate (AY256460) and differs from any of the annotated sequences.

The insoluble immune complexes (ICs) were prepared under the conditions of double immunodiffusion in gel, using the suspension of the ultrasound treated PK-15 cell-line infected with porcine parvovirus (PPV) containing both viral particles and viral proteins, as well as pig or rabbit anti-PPV polyclonal immune sera. The immunodiffusion performed in an agarose gel allows only viral subunits with a molecular mass equal to or less than 1000 kDa, rather than the viral particles, to diffuse through the gel and reach the point where the immunoprecipitate is to be formed. The immunoprecipitation under the conditions of the diffusion ensures the optimal, i.e. equimolar ratio of both immunoprecipitating components, antibody/antigen in the IC. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blot analyses showed the ICs were composed of two proteins, a protein in which molecular mass corresponded to the VP2 of the PPV and a protein with a molecular mass of the IgG. This suggests that the ICs are mainly composed of the VP2 antigen and IgG class antibodies. The potency of the IC-vaccines prepared in the form of a water-in-oil-in-water emulsion was compared with that of a commercially available, inactivated oil vaccine. The vaccination of gilts, 6 weeks before mating, with the IC containing allogeneic pig antibodies, resulted in the development of high and long-lasting anti-PPV antibody titres, similar to those generated by the licenced vaccine (P > 0.01). The content of the virus material administered by the IC was twice lower than that in the licenced vaccine. Neither systemic nor local reactions were observed in the gilts during the period of the trial with the IC vaccine. The number of viable piglets per litter varied between 9 and 12 and no signs of the PPV infection were detected. Rabbits were used as one of the alternative laboratory animal models accepted for the testing of the vaccine against the PPV. The rabbit humoral immune response generated by the IC containing the allogeneic antibodies were higher than that generated by the ICs containing the xenogeneic pig antibodies. It was similar to that generated by two-times higher content of the virus material administered by a commercially available vaccine. The IC-based vaccines belong to non-replicating, subunit vaccines, which are both ecologically convenient and the safest vaccines of all.

Serologic evidence of exposure to porcine parvovirus (PPV) in the wild boar (Sus scrofa) in Croatia was investigated. Serum samples from 219 wild boars captured during 2003 from 12 different locations in the Republic of Croatia were tested by using a commercial enzyme-linked immunoassay (ELISA) and a hemagglutination inhibition (HI) test. Antibodies to PPV were detected in 91 (41.6%) of tested samples and positive results were detected in wild boar from all sample locations. Adults had a significantly higher prevalence (70%) than juveniles (31%; P < 0.01). Our results indicate that wild boar populations throughout the Republic of Croatia are exposed to PPV.

The objective of this study was to characterise porcine circovirus type 2 (PCV2) from pigs with naturally occurring postweaning multisystemic wasting syndrome (PMWS) in Croatia, and to determine the epizootiological, clinical and pathomorphological features of the disease. During a systematic health monitoring programme conducted in the period from January 2002 to June 2003, PMWS was suspected on eight different pig-producing farms in Croatia. The diagnosis of PMWS met all three key criteria: the presence of compatible clinical signs, the presence of the characteristic microscopic lymphoid lesions, and the detection of PCV2 within the lesions by polymerase chain reaction (PCR) and by in situ hybridisation (ISH). Moreover, PCV2 DNA from swine tissues was extracted and sequenced. The phylogenetic analysis of 4 Croatian PCV2 strains showed close relationship to PCV2 strains isolated in Slovenia, France, the Netherlands, the United Kingdom, China and Hungary. PCV2 was also demonstrated by electron microscopy in the lymph node of an affected animal. This is the first demonstration of PMWS in Croatia based on all scientifically accepted diagnostic criteria.

Histological and biochemical procedures were employed to study liver histomorphology and plasma levels of aspartate aminotransferases (AST), alanine aminotransferase (ALT), triglyceride (TRIG), cholesterol (CHOL), glucose (GLU) and total protein (TP) in cultured (CSB) and wild sea bass (WSB) (Dicentrarchus labrax) from the northern part of the Adriatic Sea. Histopathological changes in liver included varying degrees of infiltration and lipid degeneration of hepatocytes in examined cultured fish. No significant differences between median AST values of CSB (44 IU) and WSB (45 IU) were observed. Values of ALT were <5 IU in both groups. TRIG, CHOL, GLU and TP levels were higher in CSB (2.08 mmol/L, 3.67 mmol/L, 10.66 mmol/L and 49 g/L, respectively) than in WSB (0.67 mmol/L, 2.74 mmol/L, 3.68 mmol/L and 36 g/L, respectively). The study showed differences between plasma biochemical parameters and liver histomorphology of CSB and WSB. This can be explained as a consequence of different diets (artificial in contrast to natural foods), which influence energy metabolism.

PMWS was reported for the first time in Canada in 1996, while PDNS was first described in UK in 1993. During 2002 in several swine farms from different parts of Croatia, an extensive epidemiological, clinical and pathomorphological investiagtions were carried out.

During the spring of 1996 and autumn of 1997 unusual mortality outbreaks among rainbow trout fry and yearlings occurred at two different trout farms, resulting in mortality of 20 and 10 per cent, respectively. Generally, the affected fish, swimming at the water surface, were reluctant to eat and were dark pigmented with visible haemorrhages around and within the oral cavity. Bacterial isolates from moribund fish from both cases were identified as Yersinia ruckeri by standard biochemical tests and API 20E. The isolated strains were found to be sensitive to tetracycline, chloramphenicol, co-trimoxazole, nalidixic acid, flumequine, enrofloxacin, carbenicillin and gentamicin. Microplate agglutination assay confirmed that both isolates belonged to serotype O1. The pathogenicity of the isolated bacteria was confirmed by challenge experiment. Titres of specific antibodies were determined in the sera of survivors. The titre was highest on the 21st day postchallenge and was detectable until the 81st day.

In the period from 1995 to 1996, during systematic health monitoring program on four different pig producing farms in Croatia, reproductive and respiratory disturbances were recognized. The phenomenon was studied epizootiologically, clinically, pathomorphologically, bacteriologically and serologically. More than two thousand carcasses were examined. Based on a characteristic epizootiological situation, specific clinical, gross pathological and histopathological findings, as well as positive serological tests. the diagnosis of the porcine reproductive and respiratory syndrome (PRRS) was established. This is the first original report on PRRS in Croatia.

The immunoprophylaxis of mycoplasmal pneumonia of swine (MPS) caused by Mycoplasma hypopneumoniae was investigated for the first time in fattening pigs in Croatia. The incidence of MPS was monitored in pigs weighing on average 27.5 kg (12 weeks old) after immunization with a M. hyopneumoniae vaccine. Of 350 pigs in each group, in the nonvaccinated group 55 animals (15.7%) were affected by pneumonia and 11 (3.1%) died of consequences of pneumonia, whereas in the vaccinated group 20 pigs (5.7%) were affected by pneumonia without any death due to the infection. In the nonvaccinated group 44% more pigs were individually treated with antibiotic, and these animals received in-feed therapy for more than 1/4 of the fattening period. Vaccinated pigs gained weight faster, at the rate of 0.745 kg/day (or 82 g/day more) than control animals. The mean score of lung lesions due to M. hyopneumoniae was 10.51 in the control pigs and only 0.54 in the vaccinated animals. The total tissue alterations on lungs due to M. hyopneumoniae, Pasteurella multocida and/or Actinobacillus pleuropneumoniae expressed as the mean-score were 13.21 in the control group and 2.98 in the vaccinated group. According to the results of evaluation of the M. hyopneumoniae vaccine in the field, the vaccine appeared to provide an adequate immunity in fattening pigs but was less effective when administered to younger pigs at 1-3 weeks of age.