Microbiological identification and analysis of swine tonsils collected from carcasses at slaughter

The primary objective of this 7-month study was to determine the prevalence of porcine pathogens of the tonsil of the soft palate of swine at slaughter. Additional objectives were to determine if sampling the carcasses of normal or abnormal hogs provided different microbiological profiles and if the slaughter plant provides a feasible sampling frame and environment for detecting and monitoring important pathogens in tonsils that have health implications for both swine and humans. A total of 395 samples were collected from 264 farms. Of these, 180 tonsils were collected from normal carcasses and 215 tonsils were collected from carcasses that were diverted to the hold rail. Laboratory testing included bacteriological culture and identification as well as real time-polymerase chain reaction (PCR) testing for porcine reproductive and respiratory syndrome virus (PPRSV) and immunohistochemistry (IHC) for porcine circovirus-2 (PCV-2). The most commonly isolated bacteria included: Streptococcus suis (53.7%), Arcanobacterium pyogenes (29.9%), Pasteurella multocida (27.3%), and Streptococcus porcinus (19.5%). Virus screening revealed evidence of PRRSV and PCV-2 in 22.0% and 11.9% of the samples, respectively. Salmonella Typhimurium and Yersinia enterocolitica were isolated in 0.5% and 1.8% of the samples, respectively. Tonsils collected from the hold rail were more likely to be positive for Staphylococcus hyicus [odds ratio (OR) = 7.51, confidence interval (CI) = 2.89 to 19.54], Streptococcus porcinus (OR = 9.93, CI = 4.27 to 23.10), and Streptococcus suis (OR = 2.16, CI = 1.45 to 3.24). Tonsils collected from abnormal carcasses were less likely to be positive for Staphylococcus aureus (OR = 0.05, CI = 0.005 to 0.482).     

Antimutagenic effects of extracts from sage (Salvia officinalis) in mammalian system in vivo

Naturally occurring antimutagenic factors, especially those of plant origin, have recently become a subject of intensive research. Antimutagenic properties of terpenoid fractions of sage (Salvia officinalis) were tested in mammalian system in vivo through examining the ability of sage to decrease the frequency of aberrant cells induced by a potent mutagen. First, groups of mice were treated with three concentrations of sage alone and it was established that the frequency of aberrant cells after treatment with a concentration of 25 microL/kg was not significantly different from the negative control (olive oil), while that found after treatment with the 50 microL/kg concentration differed significantly (chi2(1) = 4.05, p < 0.05). Sage used at a concentration of 100 microL/kg was cytotoxic. Mitomycin C (MMC), known as a potent mutagen, was used for induction of chromosome aberrations. Post-treatment with sage suppressed the effects of MMC significantly. Both concentrations (25 microL/kg and 50 microL/kg) produced a significant decrease in the frequency of aberrations relative to MMC (chi2(1) = 5.42, p < 0.02, chi2(1) = 14.93, p < 0.001, respectively). The percent of aberrations decreased with increasing concentrations of sage. Only nontoxic concentrations of sage without mutagenic effects can be recommended for use as inhibitors of mutagenesis or carcinogenesis.     

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 g of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (10^4±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.     

Antimicrobial susceptibility of Pseudomonas aeruginosa isolates from dogs with otitis externa

Pseudomonas aeruginosa is a common cause of otitis externa in dogs, and treatment of these infections is becoming problematic because of the increasing number of multiresistant strains. The aim of the present study was to compare the in vitro activities of cefepime, ceftazidime, enrofloxacin, ciprofloxacin, gentamicin and ticarcillin/clavulanic acid against 104 strains of P aeruginosa isolated from dogs with otitis externa. Antimicrobial susceptibility and minimum inhibitory concentrations, in μg/ml, were evaluated by the E test (bioMérieux). The most active compound was ceftazidime, with 100 per cent efficiency. The majority of tested strains were susceptible to ticarcillin/clavulanic acid (89.4 per cent), followed by ciprofloxacin (88.5 per cent) and cefepime (60.6 per cent). The highest resistance was observed to enrofloxacin (51.9 per cent) and gentamicin (43.3 per cent). Large numbers of strains were intermediately susceptible to antibiotics registered for use in veterinary medicine in Croatia--enrofloxacin (47.1 per cent) and gentamicin (41.3 per cent).     

Comparison of mouse and rabbit model for the assessment of strong PGM-containing oil-based adjuvants

Peptidoglycan monomer (PGM) is an adjuvant active molecule with potential for use in human and veterinary vaccine. PGM's action is short-lived in mammals hence its effects might be limited. Novel PGM-containing oil-based formulations have been developed recently by incorporation of PGM into Montanide ISA720 and ISA206 adjuvants with the aim to prolong and improve immunostimulating activities of PGM. In the present work we studied the efficacy of such novel adjuvant formulations using two different antigens, ovalbumin and snake venom, respectively. Novel formulations were also tested in two experimental models, mice and rabbits. In rabbits the incorporation of PGM into oil-based adjuvants led to overall improvement of antigen-specific IgG response. However, in the mouse model, under experimental conditions used, it was not possible to distinguish differences in antigen-specific IgG response among several strong oil-based adjuvant formulations.     

High prevalence of intestinal zoonotic parasites in dogs from Belgrade, Serbia--short communication

To identify areas of risk for canine-related zoonoses in Serbia, the aim of this study was to provide baseline knowledge about intestinal parasites in 151 dogs (65 household pets, 75 stray and 11 military working dogs) from Belgrade. The following parasites, with their respective prevalences, were detected: Giardia duodenalis (14.6%), Ancylostomatidae (24.5%), Toxocara canis (30.5%), Trichuris vulpis (47.0%) and Taenia-type helminths (6.6%). Of all examined dogs, 75.5% (114/151) were found to harbour at least one parasite species. Of these, mixed infections with up to four species per dog occurred in 44.7% (51/114). Infections with all detected species were significantly higher (p < 0.05) in military working (100%) and stray dogs (93.3%) versus household pets (50.8%). Among all parasites, agents with zoonotic potential including Giardia, Ancylostomatidae and Toxocara were detected in 58.3% (88/151) of all examined dogs with a significant difference (p < 0.05) among the subgroups (100%, 62.7% and 46.2% for military working dogs, stray dogs and household pets, respectively). The high prevalence of zoonotic parasites registered in the dog population from a highly urban area in south-eastern Europe indicates a potential risk to human health. Thus, veterinarians should play an important role in helping to prevent or minimise zoonotic transmission.     

Plasma parathyroid hormone-related peptide and bone metabolism in periparturient dairy cows

The first weeks of lactation in dairy cows are characterised by elevated bone resorption. The connection between lactation and bone metabolism is still much discussed. In this work, changes in the concentration of plasma parathyroid hormone-related peptide (PTHrP) and markers of bone metabolism were studied in Holstein cows and heifers in the dry period and early lactation to determine the role of PTHrP in the relationship between the rate of bone remodelling and the onset of lactation in dairy cows. Blood samples were taken 14 days before calving ('D-14', n = 23) and then on day 10 ('D+10', n = 21) and day 30 after calving ('D+30', n = 23). Using enzyme immunoassay (EIA), the concentrations of PTHrP, parathyroid hormone (PTH), carboxyterminal cross-linked telopeptide of type I collagen (CTX) and oestradiol and the activity of bone specific alkaline phosphatase (BSALP) were determined. The results showed a statistically significant increase in plasma PTHrP (p < 0.005) and CTX (p < 0.0001) in cows on 'D+10' as compared to 'D-14' and CTX on 'D+30' as compared to 'D-14' (p < 0.0001). Significant negative correlations were found between the concentrations of PTHrP and oestradiol (r = -0.29, p < 0.05) and those of CTX and oestradiol (r = -0.54, p < 0.0001). In nonpregnant heifers (n = 6), the concentration of CTX and the activity of BSALP were significantly higher (p < 0.0001) than in dry cows. The observed increments of PTHrP and bone resorption after parturition reveal adaptations of bone metabolism to lactation in dairy cows.     

Sequence analysis of both genome segments of three Croatian infectious bursal disease field viruses

In order to determine the mutations responsible for virulence, three Croatian field infectious bursal disease viruses (IBDV), designated Cro-Ig/02, Cro-Po/00, and Cro-Pa/98 were characterized. Coding regions of both genomic segments were sequenced, and the nucleotide and deduced amino acid sequences were compared with previously reported full-length sequenced IBDV strains. Phylogenetic analysis, based on the nucleotide and deduced amino acid sequences of polyprotein and VP1, was performed. Eight characteristic amino acid residues, that were common to very virulent (vv) IBDV, were detected on polyprotein: 222A, 256I, 294I, 451L, 685N, 715S, 751D, and 1005A. All eight were found in Cro-Ig/02 and Cro-Po/00. C-Pa/98 had all the characteristics of an attenuated strain, except for glutamine on residue 253, which is common for vv, classical virulent, and variant strains. Between less virulent and vvIBDV, three substitutions were found on VP5: 49 G --> R, 79 --> F, and 137 R --> W. In VP1, there were nine characteristic amino acid residues common to vvwIBDV: 146D, 147N, 242E, 390M, 393D, 511S, 562P, 687P, and 695R. All nine residues were found in A-Ig/02, and eight were found in B-Po/00, which had isoleucine on residue 390. Based on our analyses, isolates Cro-Ig/02 and Cro-Po/00 were classified with vv IBDV strains. C-Pa/98 shared all characteristic amino acid residues with attenuated and classical virulence strains, so it was classified with those.     

Comparison of bacterial culture and real-time PCR for the detection of Salmonella in grow-finish pigs in Western Canada using a Bayesian approach

The study objective was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) and a culture protocol used to detect Salmonella in the faeces of grow-finish pigs using a Bayesian approach. The RT-PCR was invA-gene-based assay, while the culture protocol included pre-enrichment in buffered peptone water, selective enrichment in tetrathionate and Rappaport-Vassiliadis broths, and isolation on semi-solid (modified semi-solid RV) or solid (XLT4, Rambach) agar plates. Bayesian analysis was performed using a two-test, two-population model with dependence between culture and RT-PCR and compared to a second model with conditional independence between these two tests. Two hundred and ninety three individual faecal and 294 pooled pen samples from grow-finish pig collected from 10 farms were tested and results were divided into two groups according to herd size (five herds <250 sows, five herds with >400 sows). In the dependence model, RT-PCR sensitivity (Se) and specificity (Sp) were estimated to be 90% (95% probability interval 74, 97) and 99% (98, 99), respectively. Culture Se was 92% (75, 99), while culture Sp was considered 100% as all culture-positive samples were confirmed by serotyping. In the conditional independence model, RT-PCR Se and Sp, and culture Se, were 96% (93, 98), 99% (98, 100) and 97% (94, 100), respectively. The dependence model resulted in posterior estimates of Se that were lower and with broader probability intervals than the independence model, indicating that when RT-PCR and culture are evaluated relative to each other, the correlation between these tests is an important source of bias and should be adjusted for during analysis. The RT-PCR evaluated in this study performed almost comparably to culture; given the cost savings associated with using this test and more timely results, the RT-PCR may be a useful alternative to culture for screening large numbers of samples, particularly when Salmonella prevalence is low.     

Distribution and Genotypic Characterization of Campylobacter jejuni Isolated from Poultry in Split and Dalmatia County,Croatia

Consumption of poultry contaminated with Campylobacter jejuni has been recognized worldwide as the leading cause of campylobacteriosis. Therefore, the aim of our study was to investigate the prevalence and genotype diversity of Campylobacter jejuni in poultry meat intended for consumption in Split and Dalmatia County, which is the second biggest County in Croatia. Furthermore, we also wanted to discover possibly stable clones of C. jejuni appearing in different samples and periods of time, which would indicate their ability to persist in or adapt to poultry. In the period from March 2008 until June 2010, 834 samples of poultry from various sources were examined using a surface swab technique. Isolation of C. jejuni was performed by Preston broth and Karmali agar. Identification of the isolates was carried out using biochemical tests. C. jejuni was found in 84 of 574 chicken samples (14.6%) and in nine of 260 samples of turkey (3.5%). Pulse‐field gel electrophoresis (PFGE) was used to analyse 61 obtained isolates using SmaI and KpnI. Of 22 different macrorestriction profiles (MRP) that were found, five were detected in poultry from both different locations and periods of time. Samples from 11 locations were found to be contaminated with more than two different genotypes of C. jejuni. Interestingly, the same MRP were found both in poultry declared to be of domestic origin and in the poultry imported from abroad. The prevalence of C. jejuni in poultry samples was in accordance with previously reported results. Genotypic analysis indicated that the population of C. jejuni in Split and Dalmatia County was diverse and that multiple strains of C. jejuni could be found in the same poultry samples. Furthermore, the same genotypes were identified from the samples obtained from different locations and periods of time, which could support the theory of a global existence of certain MRP that are able to persist in or adapt to poultry.