Josip Madić

Prevalence of antibodies to IBR and BVD viruses in dairy cows with reproductive disorders

Abstract We determined the prevalence of antibodies to infectious bovine rhinotracheitis virus (IBRV) and bovine viral diarrhea virus (BVDV) in sera of dairy cows on 4 different farms in the Republic of Croatia. A high percentage (60.8%) of cows had various reproductive disorders. The results showed that seroprevalence of infectious bovine rhinotracheitis (IBR) was 85.8% and that of bovine viral diarrhea (BVD) was 79.2% in tested cows. Antibodies to both viruses were found in 80.8% of cows with reproductive disorders but in only 46.8% of cows without reproductive disorders. This difference was statistically significant (P<0.01), and indicated a connection between reproductive disorders and simultaneous infections with IBR and BVD viruses in dairy cows.

Serological evidence for the presence of A/equine-1 influenza virus in unvaccinated horses in Croatia.

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Vaccine failure caused an outbreak of equine influenza in Croatia.

In April 2004 an outbreak of equine influenza occurred at the Zagreb hippodrome, Croatia. Clinical respiratory disease of the same intensity was recorded in vaccinated and non-vaccinated horses. The equine influenza vaccine used in Croatia at the time of the outbreak contained the strains A/equine/Miami/63 (H3N8), A/equine/Fontainebleau/79 (H3N8) and A/equine/Prague/56 (H7N7). At the same time, the usual strains in vaccines used in Europe were, in accordance with the recommendation of the World Organisation for Animal Health (OIE) Expert Surveillance Panel on equine influenza, A/equine/Newmarket/1/93 (H3N8) and A/equine/Newmarket/2/93 (H3N8). At the same time, some current vaccines in the USA contained A/equine/Kentucky/97 (H3N8). Genetic characterization of the HA1 portion of the haemagglutinin (HA) gene of virus isolated from the outbreak indicated that the isolate (A/equine/Zagreb/04) was an H3N8 strain closely related to recent representative viruses of the American lineage Florida sub-lineage. In comparison with both H3N8 vaccine strains used in horses at the Zagreb hippodrome, A/equine/Zagreb/04 displayed amino acids changes localised to 4 of the 5 described antigenic sites (A-D) of subunit protein HA1. Comparison of the amino acid sequence of the HA1 subunit protein of the outbreak strain with that of A/equine/Newmarket/1/93 displayed three amino acids changes localised in antigenic sites B and C, while antigenic sites A, D and E were unchanged. The Zagreb 2004 outbreak strain had the same amino acids at antigenic sites of the HA1 subunit protein as the strain A/equine/Kentucky/97. Amino acid changes in antigenic sites between HA1 subunit of the outbreak strain and the strains used in the vaccines likely accounted for the vaccine failure and the same clinical signs in vaccinated and unvaccinated horses. Use of a recent strain in vaccines should limit future outbreaks.

Serologic survey for selected viral and rickettsial agents of brown bears (Ursus arctos) in Croatia

Sera from 22 (13 wild and nine captive) European brown bears (Ursus arctos) from Croatia were tested to 18 viral and rickettsial agents. Serologic evidence of exposure was found to the following agents (number positive/number examined): Bhanja virus (3/15), Tahyna virus (3/15), West Nile virus (4/15), Naples sandfly fever virus (1/15), human adenovirus (1/22), influenza A (1/22) and B (1/22) virus, cytomegalovirus (1/22), parainfluenza virus 1 (2/22), Chlamydia psittaci (1/22), Coxiella burnetii (2/22), and canine parvovirus 2 (CPV-2) (7/22). Evidence of exposure to arboviruses was found exclusively among free-living bears. Evidence of exposure to agents usually transmitted directly was predominant among captive bears. Canine parvovirus 2 antibodies were the most frequently found antibodies and the only antibody common to both groups of bears. This may be the first report of antibodies to CPV-2 in bears.

Spreading of West Nile virus infection in Croatia.

West Nile virus (WNV) is an emerging zoonotic pathogen with rapid global expansion. The virus circulation is confirmed in many countries of Mediterranean Basin and Southern and Central Europe. In our study detection of specific WNV antibodies was performed in horses and cattle sera samples collected from October 2010 to April 2011. Serum samples were randomly taken from different parts of Croatia and tested by IgG and IgM ELISA. Positive serological results were confirmed by virus neutralization assay (VN-assay) and plaque reduction neutralization test (PRNT). Results showed that WNV antibodies were present in 72 out of 2098 horse sera (3.43%) and 3 of 2695 cattle sera (0.11%). The highest seroprevalence was found in Eastern Croatia in counties next to Hungarian, Serbian and Bosnia and Herzegovinian state borders. In Adriatic part of Croatia positive animals were found only in the westernmost county, near Slovenian and Italian borders. Geographic distribution and number of positive horses indicated that WNV is highly present in Croatia and spreading from East to West. However, positive horses in westernmost part of country indicate possible second origin of spreading. Location of serological positive cattle supports the hypothesis that seropositive cattle could be indicators of high WNV activity in the respective geographic regions.

Two outbreaks of neuropathogenic equine herpesvirus type 1 with breed-dependent clinical signs

Equine herpesvirus type 1 (EHV-1) is a worldwide spread pathogen of horses. It can cause abortion, respiratory and neurological disease and consequentially significant economic losses in equine industries. During 2009, two outbreaks of EHV-1 were confirmed in two stud farms in Eastern Croatia. The first outbreak occurred in February following the import of 12 horses from USA, serologically negative to EHV-1 before transport. Four mares aborted in the late stage of pregnancy and one perinatal death was recorded. Other six mares showed clinical signs of myeloencephalopathy with fatal end in four. One month later, the second EHV-1 outbreak was confirmed in stud farm about 100 km further with 17 abortions, three perinatal deaths and one mild neurological case. Epidemiological data showed that the disease was probably introduced in the first stud farm during international transport. The second outbreak started with the introduction of clinically healthy stallion from the first stud farm. Molecular characterisation and phylogenetic analysis confirmed that, despite different clinical signs, the identical virus caused both outbreaks. Both horse populations were free from EHV-1 infection before the outbreak and had not been vaccinated. Significant difference in clinical signs could be explained by different breed-related risk factors.

Serological evidence for West Nile virus infection in horses in Croatia

WEST Nile virus (WNV) is an arthropod-borne virus belonging to the family Flaviviridae, genus Flavivirus. WNV is included in the Japanese encephalitis virus group, together with the Japanese, St Louis and Murray Valley encephalitis viruses (Heinz and others 2000). Its natural life cycle involves birds and mosquitoes, particularly Culex species and Aedes/Ochlerotatus species (Hubalek and Halouzka 1999, Kulasekera and others 2001). Many species of wild birds may act as vertebrate hosts (Komar and others 2001); human beings, horses and other mammals are only incidental hosts. Although infection of human beings in endemic areas is common, such infection usually causes only an asymptomatic or mild illness. However, severe neurological diseases, mostly associated with people over 50 years of age, as well as high rates of mortality among birds and horses have recently been observed (Makar and Stowell 2004). Bridge vectors for equine infection are primary Aedes/ Ochlerotatus species. Since the mid-1990s, outbreaks of WNV infection have been reported with increasing frequency in human beings, birds and horses in many countries such as Romania (1996), Morocco (1996), Tunisia (1997), Italy (1998), Russia (1999), USA (from 1999), Israel (1999), France (2000, 2004), Canada (2002) and Mexico (2002) (Hubalek and Halouzka 1999, Murgue and others 2001, Ostlund and others 2001, Petersen and Roehrig 2001, Estrada-Franco and others 2003). No data on the prevalence and spread of WNV in horses in Croatia are available, although haem-inhibition antibodies in human beings have been detected (VesenjakHirjan and others 1991). This short communication describes the survey of a population of horses in Croatia for the presence of WNV antibodies in an attempt to provide information for veterinary epidemiologists. Between May 2001 and October 2002, 980 equine serum samples were collected by the Croatian Veterinary Services. Horses were randomly selected from eight Croatian regions: 119 from Buje, 81 from Ðakovo, 69 from Garesnica, 46 from Labin, 66 from Nasice, 398 from Sisak, 82 from Sibenik and 119 from Pula (Fig 1). Sampling was carried out at the time of the equine infectious anaemia national monitoring plan. Serum samples were first screened using a home-made immunoglobulin G (IgG) ELISA, according to the method described by Tsai and others (1998). Plates were coated with WNV antigen prepared on Vero E6 cells, ATCC CRL-1586 VERO C1008 (Early and Johnson 1988) according to the method used by the Unite des Arbovirus et des Fievres Hemorragiques, Institut Pasteur, and IgG antibodies were revealed by peroxidase antibody (Sigma). Sera were considered positive if the optical density was more than three standard deviations above the negative mean, and the positive results were confirmed using serum neutralisation (Anon 2004). The serology was performed at the Instituto Zooprofilattico Sperimentale ‘G. Caporale’, Teramo, Italy. In the statistical analysis of the results, the total prevalence of WNV in horses was calculated by a Bayesian approach using the beta (s+1, n–s+1) distribution, where s is the total number of positives and n is the total number of tested animals. The probability distribution of the percentage of positive animals shows not only the most probable value of prevalence, but also the level of uncertainty due to sample size. Of the 980 serum samples tested, four were found to be positive for WNV antibody by the home-made IgG ELISA. All four were confirmed by serum neutralisation. Neutralising antibody titres ranged from 1:10 to 1:80. The overall prevalence was 0·4 per cent (95 per cent confidence interval 0·17 to 1·06). All positive animals came from a stud farm located in the Ðakovo region, which was home to 160 animals. The stud farm considered animals from different locations under the guidance of a selection centre. A total of 81 randomly selected horses from the Ðakovo region were examined in the present study, and the four positive animals identified were the only animals positive for WNV antibody (Table 1). Fig 2 shows the most probable values of WNV prevalence in horses from Croatia and the Ðakovo region. No clinical signs were observed in horses at the time of sampling. In laboratory diagnosis, WNV infection can be detected by IgG ELISA; however, the assay cannot readily differentiate between WNV and other members of the Flavivirus genus. Serological confirmation of WNV infection is possible only by the detection of WNV-specific neutralising antibodies by the serum neutralisation assay or plaque reduction neutralisation test (PRNT) in which titres equal to or greater than 1:10 in a single serum sample are considered positive (Ostlund and others 2001). Horses with subclinical infections developed neutralising antibody titres of at least 1:10 (Bunning and others 2002). Thus, as demonstrated in other similar studies, the combination of both ELISA and PRNT or the serum neutralisation assay, the tests applied in this study, were the most reliable methods of detecting and confirming cases of WNV (Ostlund and others 2001). FIG 1: Locations of equine serum sampling for West Nile virus detection in Croatia. Inset: Migratory routes of birds from northern Europe to South Africa, modified from Vuilleumier (2001)

Comparison of Antibody Values in Sera of Pigs Vaccinated with a Subunit or an Attenuated Vaccine against Classical Swine Fever

Ten pigs, aged 85 days, were vaccinated with a subunit vaccine containing 32 μg of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (104±0.15 TCID50/ml), produced by amplification in minipig kidney (MPK) cell culture (group 2). Nine non-vaccinated pigs served as a control group (group 3). Serum samples were collected before (day 0) and at 4, 10, 21 and 28 days after vaccination and were analysed by two commercially available enzyme immunoassays and by a neutralizing peroxidase-linked assay (NPLA). At the same times, peripheral blood was taken for determining the total leukocyte count and the body temperature was taken daily. Antibodies were not detected in serum samples collected before vaccination (day 0), and no side-effects that could be connected with vaccination were observed during the trial. Ten days after vaccination 6/10 pigs vaccinated with the subunit vaccine were seropositive. On days 21 and 28, the ratios of serologically positive to vaccinated pigs were 9/10 and 10/10, respectively. Four of the ten pigs that were vaccinated with the C strain vaccine were positive on day 21 and 9/10 on day 28. However, the results of the NPLA showed that only 4/10 pigs had an antibody titre >1:32 at the end of the trial in both the vaccinated groups, even though the subunit vaccine initiated an earlier and higher level of neutralizing antibodies than the vaccine produced from the C strain. Challenge was performed 28 days after vaccination on four randomly selected pigs from both vaccinated groups. The pigs survived the challenge without showing any clinical signs of classical swine fever (CSF), while two nonvaccinated control pigs died on the 10th and 12th days after infection.

Susceptibility to Antimicrobial Agents of Streptococcus suis Capsular Type 2 Strains Isolated from Pigs

The minimal inhibitory concentrations (MICs) for thirty-three epidemiologicaly unrelated clinical isolates of Streptococcus suis capsular type 2 were determined in relation to ampicillin, ampicillin-sulbactam, amoxicillin, clavulanate-amoxicillin, penicillin G, cephalexin, gentamicin, streptomycin, erythromycin, tylosin and doxycycline, using the microtitre broth dilution procedure described by the U.S. National Committee for Clinical Laboratory Standards (NCCLS). Gentamicin was the most active compound tested, with an MIC for 90% of the strains tested (MIC(90)) of 0.4 mg/L. Overall, 70% of strains were resistant to doxycycline (MIC(90) > or = 100.0 mg/L), followed by penicillin G (51% of strains) (MIC(90) + or = 100.0 mg/L). Resistance to amoxicillin and ampicillin was 36.4% (MIC(90) 12.5 mg/L) and 33.3% (MIC(90) 50.0 mg/L), respectively. 15.2% of S. suis strains were resistant to streptomycin, tylosin and cephalexin with MIC90 values of 25.0 mg/L, 12.5 mg/L and 25.0 mg/L, respectively. A combination of ampicillin and sulbactam (MIC(90) 6.3 mg/L) and a combination of amoxicillin and clavulanate (MIC(90) 3.1 mg/L) as well as erythromycin (1.6 mg/L) were of the same efficacy, with a total of 9.1% resistant S. suis strains. This high percentage of resistance to doxycycline and penicillin G precludes the use of these antibiotics as empiric therapy of swine diseases.

Prevalence of Antibodies to Porcine Parvovirus in Wild Boars (Sus scrofa) in Croatia

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.