Distribution and elimination of levamisole in eggs and tissues after oral administration to laying hens, determined by LC-MS/MS.

Levamisole was administered to laying hens, and concentrations in eggs and tissues (thigh muscle, breast muscle, liver and kidney) were determined by a newly developed liquid chromatography tandem mass spectrometry method, which allowed trace level quantification of levamisole. The adopted analytical method showed good sensitivity, repeatability and percentage of recovery from spiked matrices. Maximum concentrations of levamisole were found on the first day after the administration (531.1 µg/kg in liver, 164.3 µg/kg in egg yolk, 130.7 µg/kg in kidney, 78.0 µg/kg in breast muscle, 70.7 µg/kg in thigh muscle and 64.0 µg/kg in egg white), after which there is a decline. The compound was rapidly eliminated from eggs, with a half-life of 1.3 days. Elimination appeared to be slower in thigh muscle (3.5 days), breast muscle (3.4 days) and liver (3.3 days). According to this experiment, the levamisole withdrawal periods calculated for eggs, liver, kidney, breast muscle and thigh muscle in laying hens were 14.1, 6.1, >4.0, 14.5 and 13.0 days, respectively. The longest time for levamisole residues to be completely released from tissues was seen in liver samples (37.4 days), followed by thigh muscle, breast muscle and kidney. Elimination from eggs was fastest (16.4 days for levamisole residues to drop below the method quantification limit).

Mucocutaneous Inflammatory Pseudotumours in Simultaneous Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis Infection in a Cat.

Mycobacterial spindle cell 'pseudotumour' has been described only once in cats. This unique proliferation of spindle-shaped histiocytes containing Mycobacterium avium is associated with extensive subcutaneous lesions. We report mycobacterial pseudotumour with invasion of muscular and subcutaneous tissues in a 1-year-old female domestic longhair cat. Lesions involved the facial muscles and nasal cavity, making surgical excision impossible. Necropsy examination revealed additional nodules in the subcutis and muscles of the trunk and submandibular lymph nodes. Genotyping of organisms within these lesions revealed simultaneous infection with Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis. The microscopical appearance of the granulomas was identical, regardless of the strain of bacterium or anatomical location.

Brucella suis infection in domestic pigs and wild boar in Croatia.

Between the years 2000 and 2004, 93,107 sera from 1,997 pig herds in 11 regions of Croatia were tested for the presence of antibodies against brucellosis. Positive results were observed in 67 herds from seven regions (mean individual prevalence: approximately 1%; herd prevalence: 3.4%). The herds from all but two of the infected farms were reared outdoors and thus almost certainly came into contact with wildlife. From 2003 to 2004, 424 sera, which were randomly collected from hunted wild boar (Sus scrofa), were also tested and shown to have a mean seroprevalence of 27.6%. Brucella was isolated from 88 out of 151 serologically positive pigs (58.3%) and 7 of the 93 (7.5%) wild boar which were randomly submitted for bacteriological study. All but three isolates were Brucella suis biovar 2; the others being biovar 3. These results suggest that brucellosis is enzootic in Croatian populations of wild boar. These populations represent a potential disease reservoir for free-range pig farms, as they do in other countries of Central and Western Europe. This is the first report of B. suis biovar 3 in swine and wild boar in Europe, which is an issue of serious concern for public health.

Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems.

We describe the development of a novel PCR assay for the rapid detection of members of the Brucella genus, and the differentiation between six recognized Brucella species. The assay has proven to be highly specific with the additional advantage of being suitable for use with both conventional and real-time PCR.

Mycobacterium caprae in cattle and humans in Croatia.

SETTING: This paper describes an outbreak of human and related bovine tuberculosis (TB) caused by Mycobacterium caprae in Croatia. A 13-year-old boy clinically presented enlargement of cervical lymph node with consecutive isolation of M. caprae. His 7-year-old sister, who had no clinical signs of disease, hyper-reacted to the purified protein derivative (PPD) test (>25 mm) and peribronchial infiltration was found by radiology. The children came from a family that ran a small-sized cattle dairy farm. DESIGN: All cattle on the farm were subjected to cutaneous TB testing: six of the 14 reacted positive, while three were suspicious. The entire herd was slaughtered, their carcasses examined and collected material subjected to pertinent diagnostic procedures. RESULTS: Gross examination findings consistent with TB were observed in the PPD-positive cows. Mycobacteria isolated from the boy and cattle were identified by classical and molecular methods, confirming M. caprae as the causative agent. CONCLUSION: Although not bacteriologically proven, consumption of raw milk or non-pasteurised milk products from infected dairy cattle was suspected as the source of infection in humans. Our findings confirm the domination of M. caprae among cattle in Croatia and represent the first evidence of M. caprae infection in humans in Croatia.

Mycobacterial infection of pigs in Croatia.

During a five-year period (2000 to 2004) 74,342 pigs were tested by the intradermal tuberculin test in Croatia. Of them, 248 (0.33%) pigs were positive and 91 (0.12%) were found to be suspicious in 7 out of the 13 farms included in the study. Gross pathological changes characteristic of tuberculosis were observed in tuberculin-positive and/or suspicious swine. Mycobacterium was isolated from the lymph nodes of 183 out of 234 swine (78.2%). For better epidemiological understanding, isolates were typed by conventional methods, PCR and hybridisation. The results show that most of the isolates belonged to the Mycobacterium avium complex (175 isolates, 95.7%). Other isolates belonged to M. fortuitum (6 isolates, 3.3%), M. chelonae (1 isolate, 0.5%), and M. peregrinum (1 isolate, 0.5%). Isolated strains of the M. avium complex were identified as M a. avium (37 isolates, 21.1%) and M. a. hominissuis (138 isolates, 78.9%).

Outbreak of tuberculosis caused by Mycobacterium caprae in a zoological garden.

In the autumn of 2004, tuberculosis caused by Mycobacterium caprae occurred in a zoo in Slovenia. A dromedary camel (Camelus dromedarius) was killed after a history of progressive emaciation. Necropsy findings indicated disseminated tuberculosis, which was confirmed by cultivation of M. caprae. Consequently, a tuberculin skin test was performed in all epidemiologically linked animals and another dromedary camel and six bison (Bison bison) were positive and killed. Mycobacterium caprae was isolated from two bison while M. scrofulaceum and Mycobacterium spp. were found in two other bison, respectively. The second dromedary camel was found to be negative for mycobacteria under both microscopic and culture tests. The isolates were investigated with commercial identification kits, IS6110 PCR, IS6110 restriction fragment length polymorphism analysis, spoligotyping and mycobacterial interspersed repetitive units typing. Genotyping results revealed that the dromedary camel and the two bison were infected by the same M. caprae.

An outbreak of Salmonella Abortusovis abortions in sheep in south Croatia.

The aim of the study was to give an account of the epidemic of abortions in sheep caused by Salmonella enterica ssp. enterica serovar Abortusovis, which occurred in Dalmatia, south Croatia, in winter 2003-2004. Five sheep flocks with rate of abortion ranging from 22% to 38% during the last-third of gestation were examined. Salmonella Abortusovis was isolated from 13 vaginal smears and two fetuses. Direct inoculation was found to be superior to pre-enrichment and enrichment in selective broth for Salmonella Abortusovis isolation. The isolates were biochemically identified, and characterized by serotyping and polymerase chain reaction based on the amplification of the IS200 sequence specific for Salmonella Abortusovis. A fragment of 900 bp was detected in all Salmonella Abortusovis isolates. The sensitivity testing of the isolates, carried out by the disk diffusion method and the determination of the minimal inhibitory concentrations, resulted in a high sensitivity to almost all antimicrobials used. Only two isolates were moderately sensitive to oxytetracycline, whereas one isolate showed resistance to streptomycin. Campylobacter fetus ssp. fetus and Listeria monocytogenes were excluded as causative agents of abortion in sheep by culture testing, and brucellosis, leptospirosis, Q fever and chlamydiosis by serological testing.

Leukocyte subsets and specific antibodies in pigs vaccinated with a classical swine fever subunit (E2) vaccine and the attenuated ORF virus strain D1701.

Total white blood cell (WBC) counts and percentages of CD4a+, CD8a+, CD5a+, CD45RA+, CD45RC+, wCD21+ and SWC3a+ cells in the peripheral blood of pigs were analysed in this study. Blood samples were collected before and on days 4, 10, 21 and 28 after vaccination. Group 1 pigs were vaccinated with a subunit E2 vaccine (gp E2 32 microg/dose), and Group 2 received a subunit vaccine combined with an attenuated ORF virus strain D1701 10(6.45) TCID50/dose. Control pigs received a placebo. The total WBC count and percentage of particular cell types were within the normal range in vaccinated and control pigs. Although the mechanism of attenuated ORF virus activity is not clear, changes were observed in CD4a+, CD5a+, CD8a+, CD45RA+ and CD45RC+ cells in pigs that received the combination of a subunit vaccine and ORF virus. However, the percentage of wCD21+ and SWC3a+ did not differ significantly from that recorded in pigs given only the subunit vaccine. At days 4 and 10 the number of pigs positive to E2 antibodies was higher in the group that received the subunit vaccine and ORF virus than in pigs vaccinated with the subunit vaccine only. A higher percentage of memory cells (CD45RC+) as well as Th and Tc lymphocytes in pigs that received the ORF virus and the subunit vaccine could be ascribed to a nonspecific influence of the ORF virus on the development (through cognate interactions between T and B cells) and the duration (presumed according to the finding of the clonal expression of memory cells) of humoral immunity (assessed by a higher number of seropositive pigs in this group). This seems likely since the proportion of these cells was found to be lower in the pigs that received E2 vaccine only.

Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia.

Porcine circovirus type 2 (PCV2) from the Circoviridae family has recently been associated with two serious diseases of swine, post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). During 2002, several outbreaks of clinical disease in pigs with weights ranging from 10 to 70 kg occurred on four farms in different locations in Croatia. The signs were consistent with PMWS and PDNS. Apart from progressive weight loss, pneumonia and/or diarrhoea, multifocal erythematous skin lesions and dermal necrosis were also observed. The PCR results obtained from PCV2 specific oligonucleotide primers confirmed a PCV2 infection. In addition, archive samples that were classical swine fever virus positive and derived from domestic pigs during an outbreak in 1997 were included in this study and one out of the three isolates was found to be positive for PCV2. For a better epizootiological understanding, genetic typing of representative isolates was carried out and compared with available isolates reported in the GenBank databases.

Serological evidence of bluetongue and a preliminary entomological study in southern Croatia.

In December 2001, bluetongue (BT) was confirmed serologically by the Croatian Veterinary Institute using the competitive enzyme-linked immunosorbent assay (c-ELISA). Results of the serological testing of blood samples from ruminants in the Dubrovacko-Neretvanska County are presented (3,318 sera of ruminants from 53 herds were examined). In total, 357 bovine sera (178 or 49.9% positive), 1,268 ovine sera (174 or 13.7% positive) and 1,693 caprine sera (270 or 15.9% positive) were tested. Antibodies to BT virus serotype 9 were detected in 212 of the positive sera by serum neutralisation. A preliminary light-trap survey for midges of the Culicoides genus was also performed in the Dubrovacko-Neretvanska County. Fourteen light-trap collections from seven locations were examined and yielded a total of 4,872 Culicoides of which 4,492 (92%) belonged to the Obsoletus Complex (including C. obsoletus and C. scoticus).

Wild boars (Sus scrofa) as reservoirs of Brucella suis biovar 2 in Croatia.

This work presents the results of findings for brucellosis in wild boars and domestic swine in two regions of Croatia. In the region of Djakovo the blood samples of 211 wild boars were analysed and in 29.4% of the samples serologically positive reactions were established. In the same region the blood samples of 1080 domestic swine on pastures were also analysed and positive serological reactions were established in 12.3%. In the regions around Lonjsko Polje the blood samples of 53 wild boars were analysed and in 22.6% of them positive serological reactions were established. On several locations around Lonjsko Polje the blood samples of 901 domestic swine were serologically analysed and 13.5% of the swine were found to be seropositive. Bacteriological analyses of submitted materials from 24 wild boars resulted in isolation of Brucella from seven (29.2%) samples, and from 43 samples originating from domestic swine that had aborted and had been serologically positive, Brucella were isolated from 25 (58.1%) swine, as well as from 10 (62.5%) out of 16 aborted piglets. In all the isolates Brucella suis biovar 2 was identified. Wild boars are carriers and reservoirs of Brucella suis biovar 2 in Croatia.

An overview of the epidemiology and epizootology of brucellosis in selected countries of Central and Southeast Europe.

The objective of this paper is to give an overview of the epidemiologic and epizootic status of brucellosis in selected countries of Central and Southeast Europe (Balkan region). Based on dimension of the disease problem, there is a need to establish collaboration in the eradication and prevention of brucellosis between all countries in the region. Although there were no readily accessible data concerning epidemiology and epizootology of brucellosis in these countries, the limited official and published data were analyzed. The incidence of brucellosis caused by Brucella melitensis in sheep, goats and humans is a very significant problem in Macedonia and Greece. In Greece, cattle are also affected either by B. melitensis or B. abortus. The disease is an endemic problem in some regions of Yugoslavia and includes B. suis biovar 2 in pigs and in Croatia, B. melitensis in sheep, goats and human is found occasionally. No problem appears to exist with brucellosis in Bulgaria. Financial well-supported brucellosis control programs of the European Union that will include all countries, regardless of the magnitude of brucellosis incidence, are needed for eradication and control of brucellosis.

Production of cyclopiazonic acid by aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus.

96 strains of Aspergillus flavus isolated from samples of stored grain and smoke-dried meat products were examined for ability to produce cyclopiazonic acid and aflatoxins, grown on mycological broth medium and malt extract agar. Five strains produced cyclopiazonic acid in the range of 0.5-30 mg/kg and 9 produced aflatoxin B1 (0.1-14.8 mg/kg) but none of them produced both cyclopiazonic acid and aflatoxins.

An outbreak of abortion in mares associated with Salmonella abortusequi infection.

An abortion outbreak occurred in a herd of 38 horses, 26 of which were pregnant mares. Twenty-one mares aborted between 5-10 months of gestation. In no case were there indications of impending abortion. Pathoanatomical, histopathological, virological and bacteriological examinations were carried out on 4 aborted fetuses. Histopathology identified Gram-negative bacteria compatible with salmonella in all 4 placentae. By subsequent bacteriological examination Salmonella abortusequi was isolated as the single causative agent in each case. Nonmotile Salmonella abortusequi with antigenic formula 4,12:-:- was isolated from one of the 4 fetuses. The described episode of equine abortion clearly indicates that Salmonella abortusequi has not been eradicated from Europe as previously thought.

Serum biochemical changes associated with cystic ovarian degeneration in pigs after atrazine treatment.

Biochemical and histopathological parameters of the ovarian function were observed to assess the toxic effect of low dose of atrazine, an s-triazine herbicide, in female pigs (gilts) undergoing intensive breeding. Female pigs (cross-bred between Swedish and German landrace) received 2 mg atrazine kg-1 body wt. in feed daily during 19 days of the oestrous cycle. The last treatment day corresponded to day -3 of the onset of the next expected oestrus. Blood samples were collected 3 times daily at 3-h intervals on the first 5 post-treatment days. Serum 17 beta-oestradiol (17 beta-E) and progesterone (P) concentrations were determined. A significantly higher (P < 0.05 and P < 0.001, respectively) serum 17 beta-E concentration was recorded 48 and 24 h before the onset of the next expected oestrus in atrazine-treated pigs, as compared to intact pigs. The onset of the next expected oestrus failed to occur, but no other adverse clinical reactions associated with the treatment were recorded. Histopathological examination of the ovaries of treated pigs indicated multiple ovarian follicular cysts and persistence of corpus luteum. Biochemical and histopathological findings showed that subacute exposure of female pigs to low dose of atrazine prolonged their oestrous cycle.

Use of UV photography to identify aflatoxin-producing strains of Aspergillus flavus and A. parasiticus.

UV photography in glucose, yeast extract (GY) agar medium was tested as a simple and rapid method for the distinction of aflatoxin-positive from aflatoxin-negative strains of Aspergillus flavus and A. parasiticus. In the UV photographs aflatoxin-producing moulds were identified as grey or black colonies, whereas aflatoxin-nonproducing moulds appeared as white colonies. Of the aflatoxin-positive strains detected by the UV photographic method, 10% was confirmed by extraction of the GY agar medium and mould mycelium in chloroform, extracts which were analysed subsequently using thin-layer chromatography. Confirmation of aflatoxigenic strains was achieved by biosynthesis on liquid medium yeast extract sucrose (YES) broth.

Aflatoxin-producing potential of Aspergillus flavus and Aspergillus parasiticus isolated from samples of smoked-dried meat.

Over a period of three years 420 samples of various smoke-dried meat products, collected from individual households in different region of Croatia were analysed for the presence of aflatoxigenic strains of the Aspergillus flavus group. Strains of A. flavus and A. parasiticus were present in 17.8% of the samples, and aflatoxin-producing ability was tested in 75 strains. In relation to sequential method of aflatoxin detection, 5 of 8 isolates were found in the first step (fluorescence in aflatoxin-producing ability medium--APA) and all of them in the second step (extraction method from syntheses on moist shredded wheat--SW). A. flavus strains produced mainly aflatoxin B1, and had various levels of toxigenicity (1.4-3.12 mg/kg). Some strains of A. parasiticus produced all four aflatoxins B1 B2 G1 G2, while the other ones produced AF B1 + G1 only, with concentrations of aflatoxins from 0.1 to 450 mg/kg.

Ochratoxinogenicity of Aspergillus ochraceus strains from nephropathic and non-nephropathic areas in Yugoslavia.

Mycological analyses of 855 samples of stored grains and dried meat collected in period 1980-1987 from individual households in the nephropathic and wider non-nephropathic area in SR Croatia in Yugoslavia showed 10% of samples to be contaminated with Aspergillus ochraceus. Ability to produce ochratoxin A (OA) was tested in 70 samples (27 from nephropathic areas and 43 from non-nephropathic areas). The detection was carried out under UV-light (365 nm) (light blue fluorescence) and 6 OA-producers were found. A biosynthetic procedure on liquid nutritional substrate with saccharose and yeast extract as well as a method using wet crushed wheat revealed that 37% of the samples from a nephropathic area, and 35% of the samples from a non-nephropathic area produce OA. In the nephropathic area 1/10 strains was a strong producer of OA (concentration crushed wheat 135 mg/kg, and 240 mg/l on YES liquid substrate), 1/10 strains was a moderate producer (concentration 16.6 mg/l and 0.07-7.0 mg/l and 0.1-10.4 mg/kg). Among the strains isolated from a wider non-nephropathic area no strong producers of OA were found, but 2/15 strains were moderate producers of OA (concentration of OA 20.4-27.0 mg/l and 15.0-33.7 mg/kg). The other strains, 8/10 on the crushed wheat and 13/15 on the liquid substrate, were weak producers of OA with concentrations of OA between 0.2-9.0 mg/l and 0.2-10.0 mg/kg with the two methods respectively.