Detection of trace amounts of abamectin used as an antiparasitic agent in fallow deer tissues.

A sensitive and reliable method for the determination of trace amounts of abamectin in muscles, kidneys and fat tissue of fallow deer is presented. Abamectin was extracted from the tissues with acetonitrile and the extract was cleaned up on a C8 solid-phase extraction cartridge. Abamectin residue was derivatised with trifluoroacetic acid anhydride and 1-methylimidazole, and determined using reversed-phase high-performance liquid chromatography under isocratic conditions and fluorescence detection. The recoveries of the method were high and consistent, ranging from 78% to 90%. The limit of detection of the method was below 1 microg/kg when analysing muscle, kidney and fat tissue. Matrix-matched calibration was used in order to obtain accurate values and to avoid matrix interference.

Identification of major immunogenic proteins of Mycoplasma synoviae isolates.

Mycoplasma synoviae isolates differ in patterns of immunogenic proteins, but most of them have not been identified yet. The main aim of this study was their identification in two closely related M. synoviae isolates, ULB 02/P4 and ULB 02/OV6, recovered recently from chickens in Slovenia. N-terminal sequencing identified 17 M. synoviae proteins. Amongst them were 14 major, highly expressed but previously unidentified proteins, including enzymes, chaperones and putative lipoproteins. ULB 02/P4 proteins with increasing molecular weight (M(w)) in the region above the lipoprotein MSPB (approximately 40 kDa) were elongation factor EF-Tu, enolase, NADH oxidase, haemagglutinin MSPA, ATP synthase beta chain, trigger factor, pyruvate kinase and chaperone DnaK. Enolase (approximately 47 kDa) seemed to be immunogenic for chickens infected with M. synoviae, whereas EF-Tu, which might cross-react with antibodies to the P1 adhesin of Mycoplasma pneumoniae, was not. ULB 02/OV6 synthesized several immunogenic proteins and those with M(w) of approximately 70, 78, 82, 90, 110 and 160 kDa, cross-reacted with antibodies to Mycoplasma gallisepticum. They remain to be identified, because besides putative lipoproteins, protein bands of 78, 82, 85 and 110 kDa contained also dehydrogenase PdhD, elongation factor EF-G, enzyme PtsG and putative neuraminidase, respectively.

Control of rabies in Slovenia.

Red foxes (Vulpes vulpes) are the main reservoir of rabies in Slovenia, whereas cases of rabies in other wildlife species occur sporadically. In 1995, a program of oral vaccination of wildlife in Slovenia was initiated; baits with oral vaccine were distributed by air at a density of 20 baits/km(2). During 1995, when the oral vaccination program was started, 1,089 cases of rabies (including both wild and domestic animals) were reported. Five years later (1999), only six positive animals were detected among 1,195 tested (0.5%). Despite an increase in bait density (25 baits/km(2)) during the years 2000 and 2001, reported rabies cases increased to 115 and 135, respectively. In 2003, following initiation of a new bait-dropping strategy, which incorporated perpendicular rather than parallel flight lines, the number of rabies cases decreased to eight.

Time profile of abamectin and doramectin excretion and degradation in sheep faeces.

We studied abamectin and doramectin excretion and their degradation in sheep faeces under field conditions on pasture after a single subcutaneous dose (0.2mg/kg body weight). In the excretion experiment, maximal abamectin concentration (1277 ng/g dry faeces) was detected on day 3, while doramectin concentration showed two peaks (2186 and 1780 ng/g dry faeces on days 2 and 5, respectively). Both avermectins were excreted at approximately the same rate (k=0.23 day(-1) for abamectin and 0.19 day(-1) for doramectin). In the field, a rapid loss of abamectin and doramectin from sheep faeces was seen during the first 32 days after which concentrations remained constant at approximately 77 ng/g and 300 ng/g, respectively. The half life values (DT(50)) for abamectin and doramectin dissipation from sheep faeces were 23 and 22 days, respectively, during the first 32 days. Dissipation of both avermectins was strongly correlated with moisture content of the faeces.

Isolation of Mycoplasma capricolum-like strains from chickens.

Members of the genus Mycoplasma infect a wide range of hosts, but individual Mycoplasma species tend to exhibit a considerable degree of host specificity. We characterized Mycoplasma strain 700, isolated from a kidney of a layer hen in Spain and Mycoplasma strains ULB-A and ULB-B, isolated from the air sac and from the bile of stunted broiler chickens in Slovenia. The serologic examination showed that these three strains are antigenically unrelated to all of the recognized Mycoplasma species of avian origin, but closely related to the ruminant mycoplasma Mycoplasma capricolum subspecies capricolum (M. capricolum). The comparison of their 16S rRNA gene sequences with the sequence of M. capricolum (California kid) revealed 99.66% sequence identity for the strain 700 and 99.59% identity for strains ULB-A and ULB-B. Moreover, the predicted DnaK sequences of the M. capricolum-like strains, isolated from chickens, were identical to DnaK sequences of M. capricolum. Comparison of their dnaK gene sequences with M. capricolum showed 99.64% sequence identity for strain 700 and 99.27% identity for strains ULB-A and ULB-B. In the flock from which M. capricolum-like strains ULB-A and ULB-B were isolated, the majority of chickens (83% of the chickens examined) raised antibodies reacting with M. capricolum antigens. Notably, the infection of chickens with M. capricolum-like strains represents an unusual exception to the range of Mycoplasma species host specificity.

Cervids as Babesiae hosts, Slovenia.

We describe cervids as potential reservoir hosts of Babesia EU1 and B. divergens. Both babesial parasites were found in roe deer. Sequence analysis of 18S rRNA showed 99.7% identity of roe deer Babesia EU1 with the human EU1 strain. B. divergens detected in cervids was 99.6% identical to bovine B. divergens.

Transfer of maternal immunoglobulins and antibodies to Mycoplasma gallisepticum and Mycoplasma synoviae to the allantoic and amniotic fluid of chicken embryos.

Maternal antibodies can protect avian embryos against vertically transmitted pathogens during embryogenesis and also young birds after hatching. In contrast to the well-known transfer of maternal immunoglobulin (Ig) G (also termed IgY) from the yolk to embryonic blood, information about the transfer of IgA, IgG and IgM from the egg albumen to the extra-embryonic fluids is very limited. In our study, IgA, IgG and IgM to Mycoplasma gallisepticum and Mycoplasma synoviae were detected in oviduct washings of naturally infected hens and in the corresponding egg albumen samples. In their progeny embryos, IgA, IgG and IgM antibodies to these Mycoplasma species were detected in the allantoic fluid (ALF) and amniotic fluid (AMF) on day 14 of embryonic development (ED). Examination of embryos from chickens immunized with antigens of M. synoviae revealed that the appearance of IgA and IgG and of antibodies to M. synoviae in ALF could vary even among embryos of the same dam. However, IgA, IgG and IgM were detected as early as day 7 of ED in ALF and AMF in certain embryos from hens infected with M. synoviae. In five groups of embryos examined on day 7, IgG to M.synoviae was found in 51% of ALF and 33% of AMF samples. M. synoviae was isolated from 2.3% of ALF samples. IgA to M. synoviae appeared in ALF and AMF on day 12 of ED, and could be found in the majority of AMF samples examined from day 14 onwards. IgM to M. synoviae appeared in AMF on day 13 and in ALF on day 14, but was detected in those fluids less frequently than IgA or IgG.

Presence of antibodies against Aujeszky's disease virus in wild boar (Sus scrofa) in Slovenia.

Serum samples from 427 hunter-killed wild boar (Sus scrofa) from Slovenia were tested for antibodies to Aujeszky's disease virus (ADV). Samples were collected throughout Slovenia and corresponded to 6.2% of the total harvest. Antibodies against ADV were detected in 111 sera (26%) using a commercial enzyme-linked immunosorbent assay (ELISA). Antibody prevalence increased significantly with age. This report describes the first evidence of ADV infection in wild boar populations in Slovenia.

Infection with Anaplasma phagocytophila in cervids from Slovenia: evidence of two genotypic lineages.

OBJECTIVE: Human granulocytic ehrlichiosis (HGE) was recently recognized as an emerging tick-borne infection in Europe. The disease is caused by Anaplasma (previously Ehrlichia) phagocytophila. The first confirmed acute human disease caused by A. phagocytophila was reported from Slovenia in 1998. The tick Ixodes ricinus was identified as the likely vector for this pathogen of humans and animals in Europe. In order to assess the possibility that roe and red deer in Slovenia serve as potential reservoir hosts for A. phagocytophila, materials from both species were examined. METHODS: Samples were obtained from 32 red deer (Cervus elaphus) and 56 roe deer (Capreolus capreolus). Polyvalent antibodies to the USG3 isolate of Anaplasma phagocytophila were detected by indirect immunofluorescence assay (IFA). DNA was extracted from spleen tissue. The 16S rRNA gene and a portion of the groESL heat shock operon were used for PCR detection and subsequent direct sequencing of amplified products. RESULTS: Serological and PCR results indicated that high proportions of roe and red deer were infected with A. phagocytophila. Infection was confirmed in 74% of the animals by IFA and in 86% of animals by PCR. While similar prevalences by PCR were seen in the two species (approximately 86%), the prevalence of antibodies was much higher in roe deer (94% vs. 35% in red deer). Sequence analysis of a 1256-bp fragment of the groESL operon revealed genetic diversity among collected samples. Identity of sequences ranging from 98% to 100%. None of the A. phagocytophila groESL and 16S rRNA gene sequences from red or roe deer were identical to the sequences previously obtained from human patients with ehrlichiosis from Slovenia or elsewhere in the world. All red deer sequences clustered with those obtained from humans, whereas all but two sequences from roe deer clustered separately. CONCLUSIONS: The results of our study indicate that a high percentage of red deer and roe deer in Slovenia are infected with A. phagocytophila. Analysis of groESL and 16S rRNA gene sequences revealed two distinct genetic lineages. Among deer, one variant was primarily associated with roe deer. Although none of the sequences from red deer was identical to those found in humans, they were more closely related.