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A polyphasic taxonomic study was performed on an unidentified Arcanobacterium -like Gram-stain-positive bacterium designated strain C605018/01/1T isolated from a milk sample collected from the udder of a cow at post mortem. Comparative 16S rRNA gene sequencing showed that the bacterium belonged to the genus Arcanobacterium and was most closely related to the type strain of Arcanobacterium pluranimalium (99.76 %); sequence similarities to all other Arcanobacterium species were below 97 %. The wet-lab DNA–DNA hybridization values among strain C605018/01/1T and A. pluranimalium DSM 13483ᵀ were low, 16.9 % (reciprocal, 49.8 %). Pertaining to the whole genome sequence with a total length of 2.02 Mb and 1654 protein counts, the novel strain C605018/01/01T displayed a G+C content of 51.6 % mol%. The presence of the major menaquinone MK-9(H4) supported the affiliation of this strain to the genus Arcanobacterium . The polar lipid profile consisted of the major components diphosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylinositol-mannoside and unidentified glycolipid and aminophospholipids. Based on these results it is proposed that strain C605018/01/1T should be classified as representing a novel species, Arcanbacterium bovis sp. nov. The type strain C605018/01/1T (CCUG 45425T=DSM 107286T=BCCM/LMG 30783T)

This experiment was conducted to study the effect of using biological treatment by (Trichoderma reesei), to reduce the content of antinutritional factors in watermelon vines and improvement their nutritive value and the possibility of using it in animal nutrition and its effect on productive performance of growing lambs. Fifty male Barki lambs at 6 to 8 months of age with an average live body weight 24.49 ±0.2 Kg were divided into five similar groups according to their live body weight (10 lambs for each). They were randomly assigned to receive the five experimental rations where control group was fed 50% concentrate feed mixture (CFM) plus 50% berseem hay (BH ) on dry DM basis (R1) and the other four groups R2, R3, R4 and R5 were fed rations containing 25 or 50 % untreated watermelon vines hay (WMVH) and treated with Trichoderma reesei fungi (WMVF), respectively. Animals were fed the five respective rations in groups. The growth experiment lasted for sex months. Results showed that degradation of antinutritional factors was more efficient when WMV hay biologically treated with fungi (Trichoderma reesei).The values of antinutritional factors of the watermelon vines reduced as follows: total phenols by 53.43%, total tannins 71.38%, saponins 63.67%, alkaloids 44.02% and flavonoids 16.98%, compared with untreated vines. Control ration (R1) and the tested ration (R4) had higher DM intake than the other tested ones (R2, R3 and R5), being ration R3had the lowest value. Animal fed R4 showed more soluble, degradable, less undegradable fractions and more effective degradability. The values of final body weight, total body weight gain and daily gain were significant lower for tested rations R2, R3 and R5 than those of control one (R1), while the tested ration R4 was similar in these values with those of control one, being the highest values among all tested rations. The feed conversion as (g DMI/g gain) was worse with all tested rations related to that of control one, whereas R4 had the best feed conversion value among all tested ones. The values of feed conversion recorded 6.93 and 7.05 for R1 & R4 and 8.22 & 8.73 for R2 and R5, respectively, while the worst value was recorded with R3 (8.96). The feed cost was decreased by 11.98 and 25.62 for R2 & R3 (rations which contained 25 or 50% untreated watermelon vines) and 11.78 & 24.59 for R4 and R5 (rations which contained 25 or 50% watermelon vines treated with fungi) respectively, compared with the figure of control one (R1). The economic efficiency improved by 23.64% for R4 compared with control group (R1) and the other tested rations (R2, R3 and R5). The results could be indicated the possibility of introducing biologically treated watermelon vines with fungi in growing lambs rations up to 25% (i.e. 50% in replacing of berseem hay) to reduce the feed costs without harmful effects on their performance and health.

A taxonomic study using a polyphasic approach was performed on an unidentified Arcanobacterium -like Gram-stain-positive bacterium isolated from the genital tract of a rhinoceros. Comparative 16S rRNA gene sequencing showed that the bacterium belonged to the genus Arcanobacterium and was most closely related to the type strains of Arcanobacterium canis (98.8 % 16S rRNA gene sequence similarity), Arcanobacterium phocisimile (97.8 %), Arcanobacterium phocae (97.7 %), Arcanobacterium haemolyticum (97.4 %), Arcanobacterium hippocoleae (96.6 %), Arcanobacterium pinnipediorum (96.4 %) and Arcarnobacterium pluranimalium (95.4 %). DNA–DNA hybridization values between strain 647T and Arcanobacterium canis DSM 25104T were very low, 13.4 % (reciprocal 15.9 %). The genomic DNA G+C content of strain 647T was 58.7 mol%. The presence of the major menaquinone MK-9(H4) supported the affiliation of this strain to the genus Arcanobacterium . The polar lipid profile consisted of the major components diphosphatidylglycerol, phosphatidylcholine and an unidentified phosphoglycolipid. The results of physiological and biochemical testing clearly distinguished the unknown bacterium from other species of the genus Arcanobacterium . Based on these tests, it is proposed that the unknown bacterium should be classified as a representative of a novel species of the genus Arcanobacterium named Arcanobacterium wilhelmae sp. nov. The type strain is 647T (=DSM 102162T=LMG 29418T).

Aims The aim of this study was to design an assay for the identification of Mycobacterium avium subsp. paratuberculosis (MAP) to be used in faeces and milk samples of small ruminants with a loop-mediated isothermal amplification (LAMP) system, as a time-saving and user-friendly method in contrast to real-time PCR. Methods and results For the detection of MAP in milk and faeces of small ruminants, we developed a set of primers, specific for the target gene ISMap02. The analytical sensitivity of LAMP, when targeting ISMap02, showed a DNA detection limit of 10 fg μl-1 . After performing spiking experiments with two MAP reference strains, DSM 44133 and ATCC 19698T , the limit of detection, using the LAMP protocol described herein were 3·8 MAP CFU per ml milk and 12·5 MAP CFU per gram faeces. All LAMP results during the establishment of the assay were compared to those of the real-time PCR results. An internal amplification control was incorporated into the assay to exclude false-negative results produced and had no significant negative impact on the analytical sensitivity. Validation of the assay was confirmed by testing field samples of faeces and revising the results with real-time PCR. Conclusion Our study conducted the first MAP detection system with a LAMP targeting ISMap02. Due to the positive results we encourage the use of LAMP in combination with ISMap02, when detecting MAP in faeces samples, as an alternative to targeting other genes as f57 or IS900. Further research on MAP detection in different matrices like raw milk, tissue or sperm with this system is recommended. Significance and impact of the study This study provides new achievements in MAP diagnostic. Especially small ruminants do not show signs of diarrhoea until the terminal stage of the illness. The greatest task in fighting MAP is to rule out animals, which shed MAP with faeces and milk before showing symptoms of Johne's disease. Worldwide there is a need to eradicate animals, which are low MAP shedders to stop the illness spreading in animal holdings. MAP detection with LAMP is time saving, easy to use, does not need expensive equipment, as, for example, PCR kits and can be used without access to laboratories. The target gene ISMap02 was shown to be a specific insertion element for MAP and is a reliable aim in future MAP detection studies.

The standard procedure for the improved cultural recovery of viable Mycobacterium spp. from diverse samples mainly depends on reducing the viability of background microbiota using different chemical compounds. This study was designed to i) evaluate the efficacy and comparison between N-Acetyl-l-Cysteine-Sodium hydroxide (NALC-2% NaOH) and hexadecylpyridinium chloride (0.75% HPC) treatment and exposure time on reducing the viability of undesirable microorganisms with minimal impact on colostrum consistency; and ii) assess the impact of NALC-2% NaOH on improved and enhanced recovery of Mycobacterium avium subsp. paratuberculosis (MAP) in spiked postpartum colostrum samples and consistency of colostrum. A total of 40 samples, each treated with NALC-2% NaOH for 15 min or 0.75% HPC for 5 h, were investigated for total mesophilic aerobic bacteria (MAB) and enterobacteria (EB) (CFU mL-1). The results showed that treatment of colostrum samples with NALC-2% NaOH completely eliminated EB and significantly reduced MAB (3.6 log10 CFU mL-1). Conversely, samples treated with 0.75% HPC produced a complex mixture following interaction with the colostrum protein and showed non-significant and variable results. In addition, the spiked colostrum treated with NALC-2% NaOH for 15 min revealed recovery of viable MAP cells with a minimum limit of detection of 1.36 log10 CFU 10 mL-1 where no change in the consistency of colostrum was observed. In conclusion, 15-min NALC-2% NaOH treatment of colostrum may significantly reduce the viability of undesirable microorganisms and help to enhance the efficient recovery of MAP without impacting the consistency of high quality postpartum colostrum. This rapid procedure is suitable for efficient recovery and early detection of MAP as well as preventing its transmission to neonates and young calves in MAP infected herds.

The present study was designed to identify nine Arcanobacterium phocae strains isolated from cases of mink dermatitis of a single farm in Finland and characterize the strains for epidemiological relationships. All nine strains and previously described A. phocae used for comparative purposes were identified and further characterized phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), by Fourier Transform Infrared Spectroscopy (FT-IR) and genotypically by detection of phocaelysin encoding gene phl with a previously developed loop-mediated isothermal amplification (LAMP) assay and by sequencing 16S rRNA gene and gene phl, the elongation factor tu encoding gene tuf and the β subunit of bacterial RNA polymerase encoding gene rpoB. Genetic relatedness among isolates was determined using whole-genome single nucleotide polymorphism (wgSNP) analysis. The wgSNP results, partly the MALDI-TOF MS and FT-IR analyses and sequencing of the genes, revealed that the nine A. phocae strains recovered from a single farm showed close sequence similarities among each other and differed from previously investigated A. phocae strains isolated from other farms and animals in Finland and from the A. phocae type strain. This indicated a close epidemiological relationship of the A. phocae strains isolated from a single farm and that the nine A. phocae strains of the present study might have developed from a common ancestor.

In the present study 12 Arcanobacterium phocae strains isolated from fur animals in Finland, including foxes, minks and Finnraccoons, could successfully be identified phenotypically, by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and phocaelysin (PHL) encoding gene phl. The PHL of all 12 A. phocae strains in the present study and reference strains A. phocae DSM 10002T and A. phocae DSM 10003 displayed, as typical members of the cholesterol dependent cytolysin-group of toxins, the variant undecapeptide sequence EATGLAWDPWW which appeared to be most closely related to arcanolysin of Arcanobacterium haemolyticum and pyolysin of Trueperella pyogenes. In addition, gene phl could be determined with a newly designed loop-mediated isothermal amplification (LAMP) assay. The detection of mass spectra by MALDI-TOF MS and the LAMP assay based on gene phl might help to reliably identify A. phocae in future and also elucidate the role this species plays in infections of fur animals.

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S–23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.

In the present work a total of 143 Mycobacterium avium subsp. paratuberculosis (Map) field isolates recovered from faeces of dairy cattle in Hessen State, Germany were investigated. Different Short Sequence Repeats (SSR), Mycobacterial Interspersed Repetitive Units (MIRU) and Variable Number Tandem Repeats (VNTR) were applied to classify these cattle-group field isolates into subgroups. When combining the results obtained from SSR sequencing together with the data obtained from other genotyping PCR techniques applied in the current study, 78 different Map profiles were detected. One profile dominates over the investigated field isolates. This profile was detected in 24 isolates (16.8%) with 11 G (SSR); amplicon size of 280 bp, 300 bp, 200 bp, 200 bp, 210 bp, 350 bp, 210 bp, 300 bp and 300 bp for the primer MIRU 2, MIRU 3, X3, Primer 3, Primer 7, Primer 25, Primer 47, 292 and VNTR 1658, respectively. One profile was represented by eight isolates. Two profiles were represented by six members each. The remaining isolates were subdivided into smaller groups (2 profiles each 4 isolates, 3 profiles each 3 isolates, 13 profiles as 2 isolates each and finally 56 profiles were represented by a single strain for each profile).

The present study was designed to identify phenotypically and genotypically 61 Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins. The A. pyogenes isolates showed the typical cultural and biochemical properties of this species and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the superoxide dismutase A encoding gene sodA of reference strains representing 8 species of genus Arcanobacterium and subsequent design of A. pyogenes sodA gene-specific oligonucleotide primer. The A. pyogenes sodA gene-specific oligonucleotide primer allowed, together with previously described A. pyogenes 16S-23S rDNA intergenic spacer region-specific oligonucleotide primer, a reliable molecular identification of all 61 A. pyogenes of various origins. The additionally performed PCR-mediated amplification of 5 known and putative virulence factor encoding genes revealed that 100, 20, 87, 75, and 98% of the A. pyogenes carried the genes plo, cbpA, nanH, nanP, and fimA, which allowed an individual strain characterization. This might help to elucidate the role the putative virulence factors play in bovine mastitis and in various other infections caused by this bacterial pathogen.

The present study was designed to characterize phenotypically and genotypically seven Arcanobacterium haemolyticum strains obtained from infections of six horses. All seven strains showed the cultural and biochemical properties typical of A. haemolyticum and were susceptible to most of the antibiotics tested. The species identification could be confirmed by amplification and sequencing of the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region and by PCR amplification of species-specific parts of the gene encoding phospholipase D in A. haemolyticum . Use of the latter could possibly improve future identification of this generally human pathogenic bacterial species which, according to the present results, seems to occur also in infections of horses.

A species specific PCR test, based on manganese-dependent superoxide dismutase A encoding gene sodA, was developed for the identification of Staphylococcus hyicus, an important bacterial pathogen in pigs. The designed primers allowed a rapid and reliable identification of phenotypically characterized S. hyicus, isolated in Russia, Germany and Denmark. No cross reactivities could be observed investigating staphylococcal reference strains representing 18 different species and subspecies. The use of the described primers might improve a future diagnosis of this bacterial pathogen.

Streptococcus canis was isolated from 31 milk samples from 11 cows in a dairy herd (with 49 lactating cows) affected by subclinical mastitis in north Rhine-Westphalia, Germany. Thirty-one isolates from the infected udder quarters were further characterized for their phenotypic and molecular properties. Most isolates (83.9%) produced α-galactosidase, and all were negative for β- d -glucuronidase. Amplification of the 16S rRNA gene by the PCR method and digestion with the restriction enzymes RsaI, MspI, and AvaII yielded species-specific patterns. Additional identification by species-specific amplification of the 16S rRNA gene, the 16S-23S rRNA gene intergenic spacer region, the CAMP factor-encoding gene cfg , and the internal fragments of the sodA gene was consistent with S. canis . Macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis showed that the S. canis isolates originated from a single clone or were very closely related.

Streptococcus dysgalactiae serogroup C, G and L strains were investigated by polymerase chain reaction (PCR) using oligonucleotide primers designed according to species-specific parts of the 16S-23S rDNA intergenic spacer region. The oligonucleotide primers with specificity for the 16S-23S rDNA intergenic spacer region allowed a correct identification of all S. dysgalactiae serogroups C, G and L strains investigated. No cross-reactivities could be observed with any of the control strains indicating the usefulness of PCR-technology to identify the serologically heterogeneous species S. dysgalactiae.

Summary The 16S–23S rDNA intergenic spacer regions (ISR) of different streptococcal species and subspecies were amplified with primers derived from the highly conserved flanking regions of the 16S rRNA and 23S rRNA genes. The single sized amplicons showed a uniform pattern for S. agalactiae , S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis , S. phocae , S. uberis , S. parauberis , S. pyogenes and S. equi subsp. equi , respectively. The amplicons of S. equi subsp. zooepidemicus , S. porcinus and S. suis appeared with 3, 5 and 3 different sizes, respectively. ISR of selected strains of each species or subspecies investigated were sequenced and multiple aligned. This allowed a separation of ISR into regions, with 7 regions for S. agalactiae , S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis , S. phocae , S. pyogenes and S. suis , 8 regions for S. uberis and S. parauberis and mostly 9 regions for S. equi subsp. equi , S. equi subsp. zooepidemicus and S. porcinus. Region 4, encoding the transfer RNA for alanine (tRNA Ala ), was present and identical for all isolates investigated. The size and sequence of ISR appears to be a unique marker for streptococci of various species and subspecies and could be used for bacterial identification. In addition the size and sequence variations of ISR of S. equi subsp. zooepidemicus , S. porcinus and S.suis allows a molecular typing of isolates of these species possibly useful in epidemiological aspects.

Streptococcus uberis and Streptococcus parauberis reference strains and isolates obtained from routine diagnostics were investigated by PCR with oligonucleotide primers designed according to species-specific parts of the 16S rRNA gene, the 23S rRNA gene, and the 16S-23S rRNA intergenic spacer region of both species. All three primer pairs allowed an identification of 67 isolates as S. uberis and 4 isolates as S. parauberis .

An Arcanobacterium haemolyticum strain isolated from a postcastrational lesion of a horse was identified phenotypically and genotypically. The latter was performed by sequencing the 16S-23S rDNA intergenic spacer region (ISR), by amplification of the gene encoding A. haemolyticum phospholipase D, by amplification of A. haemolyticum specific parts of ISR-23S rDNA and by amplification of the newly described CAMP factor family protein encoding gene of A. haemolyticum. This indicates (as described previously for seven additional A. haemolyticum strains; Hassan et al. 2009) that A. haemolyticum seems to occur also in infections of horses.

In the present study, the CAMP-factor (cfb) gene of streptococci of serological group B (Streptococcus agalactiae) and the CAMP-factor (cfu) gene of S. uberis could be amplified by polymerase chain reaction. A cfb specific amplicon could be observed for all 128 phenotypically CAMP-positive S. agalactiae, for the phenotypically CAMP-negative S. agalactiae strain 74-360, and for 2 S. difficile reference strains. A cfu specific amplicon could be observed for all 7 phenotypically CAMP-positive S. uberis. Four S. agalactiae strains isolated from 4 cows with mastitis appeared to be phenotypically CAMP-negative and negative in the cfb gene PCR. The CAMP-positive and CAMP-negative isolates, including both S. difficile, could be identified as S. agalactiae by amplification of a S. agalactiae specific part of the V2 region of the 16S rRNA and a species-specific part of the 16S-23S rRNA intergenic spacer region. Amplification of an internal fragment of the cfb gene with a reduced annealing temperature yielded positive reactions not only for CAMP-positive S. agalactiae, but also for phenotypically CAMP-positive S. pyogenes (n = 4), S. canis (n = 28), and S. uberis (n = 7), indicating a close relation of the CAMP genes of these 4 species. The relation could be further demonstrated by sequencing the internal fragment of the CAMP-factor (cfg) gene of S. canis and comparing the sequence with those of S. agalactiae, S. pyogenes, and S. uberis.

A total of 57 bacteria representing eight species of genus Arcanobacterium (A.) were investigated for hemolytic properties on blood agar containing sheep and rabbit blood and for CAMP-like reactions. An enhanced hemolysis on blood agar containing rabbit blood compared to sheep blood could be observed for A. haemolyticum, less pronounced for A. hippocoleae and A. pluranimalium. A synergistic hemolytic reaction with staphylococcal beta-hemolysin appeared to be constantly visible for A. hippocoleae, A. pluranimalium and A. pyogenes, with Streptococcus agalactiae for A. phocae and A. haemolyticum, with Rhodococcus equi for A. phocae, A. haemolyticum, A. pluranimalium and A. pyogenes and with A. haemolyticum for A. hippocoleae, A. pluranimalium and A. pyogenes, respectively. A reverse CAMP-reaction in the zone of staphylococcal beta-hemolysin could be observed for A.phocae and A.haemolyticum. In addition, a novel CAMP-like reaction could be noted between Psychrobacter phenylpyruvicus, identified by 16S rDNA sequencing, and A. phocae and A. haemolyticum. These synergistic or antagonistic hemolytic properties could possibly be used as additional criteria for identification of bacteria of genus Arcanobacterium.

A polyphasic taxonomic study was performed on an unidentified Arcanobacterium -like Gram-stain-positive bacterium isolated from otitis externa of a dog. Comparative 16S rRNA gene sequencing showed that the bacterium belonged to the genus Arcanobacterium and was most closely related to the type strains of Arcanobacterium haemolyticum (97.2 %), Arcanobacterium hippocoleae (96.5 %) and Arcanobacterium phocae (96.4 %). The presence of the major menaquinone MK-9(H4) supported the affiliation of this strain to the genus Arcanobacterium . The polar lipid profile contained the major lipids phosphatidylcholine, diphosphatidylglycerol, phosphatidylinositol mannoside and an unidentified phospholipid (PL2). Major fatty acids were C14 : 0, C16 : 0, C18 : 0, C18 : 1ω9c and C18 : 2ω6,9c/anteiso-C18 : 0 (detected as a summed feature). C10 : 0 and C12 : 0 were present in minor amounts. The results of physiological and biochemical testing clearly distinguished the unknown bacterium from other species of the genus Arcanobacterium . Based on these tests, it is proposed that the unknown bacterium should be classified in the novel species Arcanobacterium canis sp. nov. The type strain of Arcanobacterium canis is P6775T ( = CCM 7958T = CCUG 61573T = CIP 110339T). An emended description of the genus Arcanobacterium is also provided.

In the present study matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for species identification of 98 bacteria previously classified phenotypically and genotypically to genera Arcanobacterium and Trueperella . Species identification was carried out by comparing the main spectra of each strain with the main spectra of reference strains of both genera and 3740 database entries included in the MALDI Biotyper 2.0 software package (Bruker Daltonik GmbH, Bremen, Germany). MALDI-TOF MS correctly identified (log (score) values ≥ 2.0) all investigated strains of the species A. ( T. ) bialowiezense ( n = 3), A. ( T. ) bonasi ( n = 7), A. haemolyticum ( n = 10), A. pluranimalium ( n = 1) and A. ( T. ) pyogenes ( n = 77). According to the present results MALDI-TOF MS had a comparable discriminating power than previously conducted tests on DNA level. Further studies with strains isolated from human infections would show the robustness of MALDI-TOF MS for identification of bacteria of these genera.

The present study was designed to characterize phenotypically and genotypically nine Arcanobacterium abortisuis strains collected from specimen of pigs in a period of nine years. All nine A. abortisuis strains and A. abortisuis reference strain DSM 19515 displayed a synergistic hemolytic reaction with Staphylococcus aureus β-hemolysin, Rhodococcus equi, and Arcanobacterium haemolyticum indicator strains and showed the typical biochemical properties of this species. The species identity could be confirmed by identification and sequencing of the 16S-23S rDNA intergenic spacer region (ISR), which appeared to be a useful target for genotypic characterization of this bacterial species. The A. abortisuis strains of the present study were isolated from specimen of pigs together with various other bacterial species indicating that the pathogenic importance of this newly described species remains to be elucidated.

The aim of the present study is to evaluate the efficiency of three methods to determine the molecular diversity of 34 Mycobacterium avium subsp. paratuberculosis (MAP) strains isolated from 17 cattle herds. The applied methods included the analysis of sequence polymorphism of the mononucleotide (G1 and G2) and trinucleotide sequences (GGT) of the Short Sequence Repeats (SSR) and the determination of size polymorphism of 9 different Mycobacterial Interspersed Repetitive Units (MIRU) and 6 Variable Number Tandem Repeats (VNTR). Sequence analysis of SSR of 34 isolates showed 4, 6, and 2 alleles of G1, G2, and GGT repeats, respectively. The amplification of the investigated 9 MIRU units revealed only two discriminatory genotyping systems (MIRU2 and MIRU3). Out of 6 VNTR PCR differentiation methods, only one method could be recommended for genotyping purposes. The profile 7g-12g-4ggt-II-b-2 of the combination systems G1-G2-GGT-MIRU2-MIRU3-VNTR1658 dominates among the examined isolates and was detected in 14.7% of the isolates. The use of certain repetitive loci of SSR, MIRU, and VNTR techniques in this study showed greater potential than others for the characterization of MAP isolates. The recommended loci can be used for the epidemiological tracing of MAP field strains and to determine the relationships between isolates in different herds.

Goats' milk cheeses (n=181) from the Hessian market (retail shops, weekly markets, farm markets) were quantitatively analysed for Staphylococcus (S.) aureus, and 14 were found positive. From these samples, 64 isolates of S. aureus were characterized biochemically and genetically, including their potential to produce staphylococcal enterotoxins (SE). SE genes sea to selo was studied by PCR and gene expression was evaluated by reverse transcriptase (RT)-PCR. SEA-SEE production in culture was determined by enzyme immunoassay (EIA). One isolate produced SEA, 18 isolates (from 4 samples) produced SEC, while SEB, SED, and SEE were not found. Toxin production was in agreement with PCR and RT-PCR results for the presence and expression, respectively, of the corresponding toxin genes. Trans-SE genes seg, sei, selm, seln, and selo were detected in 14 isolates from 4 cheese samples, exclusively as clusters. These samples were all from small-scale producers which directly or indirectly market their products regionally. No isolate was positive for seh or sej. RT-PCR detected the presence of the corresponding mRNA for all genes except selo, further indicating the possibility that respective proteins indeed have been produced in culture. These results suggest that S. aureus in goats' milk cheese potentially produces SE like proteins, besides SEA and SEC.

To determine the occurrence of Salmonella and Shigella in infant formula from Southeast Asia, 74 packages of dehydrated powdered infant follow-on formula (recommended age, > 4 months) from five different manufacturers, four from Indonesia and one from Malaysia, were analyzed. None of the 25-g test portions yielded Salmonella or Shigella. However, further identification of colonies growing on selective media used for Salmonella and Shigella detection revealed the frequent occurrence of several other Enterobacteriaceae species. A total of 35 samples (47%) were positive for Enterobacteriaceae. Ten samples (13.5%) from two Indonesian manufacturers yielded Enterobacter sakazakii. Other Enterobacteriaceae isolated included Pantoea spp. (n = 12), Escherichia hermanii (n = 10), Enterobacter cloacae (n = 8), Klebsiella pneumoniae subsp. pneumoniae (n = 3), Citrobacter spp. (n = 2), Serratia spp. (n = 2), and Escherichia coli (n = 2). To our knowledge, this is the first report to describe the contamination of dehydrated powdered infant formula from Indonesia with E. sakazakii and several other Enterobacteriaceae that could be opportunistic pathogens. Improper preparation and conservation of these products could result in a health risk for infants in Indonesia.

The gene encoding the 16S rRNA of Enterobacter (E.) sakazakii (ATCC 29544, plus four strains isolated from powdered infant formula) was studied, and the sequence compared with those of other Enterobacteriaceae in aspects of genetic variability. Sequence differences between E. sakazakii and other Enterobacteriaceae within the hypervariable regions V1, V2, and V3, respectively, were used to develop two PCR methods for E. sakazakii. PCR1 employed a primer pair located in V1/V2, while PCR2 utilized a primer pair located in V1/V3, respectively. The two PCR methods were tested against a set of 57 E. sakazakii and 148 non-E. sakazakii isolates. PCR1 gave an amplicon with a size of 406 bp and resulted in 100% positive results for E. sakazakii, but also detected Citrobacter koseri/amalonaticus and all nine tested Salmonella enterica serovars. In contrast, PCR2 (amplicon size of 952 bp) gave positive results only for E. sakazakii, thus allowing specific identification of this species.

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.

The present study was designed to identify and compare 32 beta-hemolytic streptococci isolated from 28 different harbor seals of the German North Sea during the phocine distemper outbreak in 2002. The bacteria were identified as Streptococcus equi subsp. zooepidemicus based on cultural, biochemical, serological and molecular studies. Epidemiological investigations by PCR restriction fragment length polymorphism analysis of the 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 32 strains appeared to be identical. These results indicate that a single bacterial clone seemed to be distributed among the harbor seal population of the German North Sea during this outbreak.

Streptococcus uberis, a well-known bacterial pathogen associated with bovine mastitis, appears to be biochemically and serologically almost indistinguishable from the closely related species Streptococcus parauberis. In the present study, species-specific oligonucleotide primers were designed using internal parts of the genes sodA, encoding superoxide dismutase A, and cpn60 encoding chaperonin 60 of S. uberis and S. parauberis, respectively. The two oligonucleotide primer pairs allowed a rapid and reliable PCR-mediated identification and differentiation of both species. These studies, performed with S. uberis and S. parauberis reference cultures and clinical isolates from routine diagnostics, revealed that the occurrence of S. parauberis as causative agent of bovine mastitis appears to be rare. In addition the sodA and cpn60 sequence data confirmed that both species could taxonomically be classified to the pyogenic group of genus Streptococcus.

Streptococcus canis isolates, also including S. canis of artificially contaminated milk, could be identified by polymerase chain reaction (PCR) amplification using oligonucleotide primers designed according to species-specific parts of the 16S rRNA gene and, after sequencing, according to S. canis-specific parts of the 16S–23S rDNA intergenic spacer region and with oligonucleotide primers detecting an internal fragment of the group G streptococcal CAMP factor gene cfg. The 16S rRNA gene- and CAMP factor gene cfg-specific oligonucleotide primers could be used together in a multiplex PCR. No cross-reactivities could be observed with other group G streptococcal isolates or with any of the other control strains of various streptococcal species and serogroups. The PCR methods presented in this study allowed a rapid and reliable identification of S. canis and might help to improve the diagnosis of this bacterial species in animal and human infections.

The present study was designed to identify phenotypically and genotypically two Arcanobacterium (A.) pyogenes strains isolated by post mortem examinations of a bearded dragon and a gecko. The A. pyogenes strains showed the typical biochemical properties and displayed CAMP-like synergistic hemolytic activities with various indicator strains. The species identity could be confirmed genotypically by amplification and sequencing of the 16S rDNA gene and, as novel target gene, by sequencing of the beta subunit of RNA polymerase encoding gene rpoB, of both strains and of reference strains representing nine species of the genus Arcanobacterium. The species identity of the two A. pyogenes strains could additionally be confirmed by PCR mediated amplification of species specific parts of the 16S-23S rDNA intergenic spacer region, the pyolysin encoding gene plo and by amplification of the collagen-binding protein encoding gene cbpA. All these molecular targets might help to improve the future identification and further characterization of A. pyogenes which, as demonstrated in the present study, could also be isolated from reptile specimens.

Crohn’s disease (CD) that becomes a public health concern in developed countries shows similarities in clinical signs and pathological features with Johne’s disease (JD) in ruminants infected by Mycobacterium avium subspecies paratuberculosis (MAP). Few researches conducted in Europe, the USA, and Australia showed relationships between MAP, CD, JD and dairy products. Indonesians consume milk and diary products from domestic and imported source. Adji in 2004 found some domestic dairy cows that were seropositive for MAP, and this could be a serious problem in dairy farm animals and human health in the future. The aim of this study was to detect MAP in the growing up formula milk. Fifty samples from five established factories were taken from supermarkets in Bogor. Polymerase chain reaction method (PCR) with insertion sequence (IS) 900 as primer and culture in Herrold’s egg yolk media with mycobactin J (HEYM J) as a gold standard were used in this study. Neither MAP grew up in HEYM J medium after 20 weeks of culture period nor positive samples by PCR IS 900 were found. Although there were no positive samples found in this study, further extensive and comprehensive studies on MAP should be done with more and varied samples, as well as in human to provide data on MAP in Indonesia. (Med J Indones 2008; 17: 183-7) Keywords: Crohn’s disease, dairy cow, growing up formula milk

The economic and zoonotic importance of infections caused by Mycobacterium avium complex (MAC) strains in human, animals and birds are increasing. At present, few data are available about the genetic diversity of field isolates of M. avium subsp. hominissuis (MAH) and subspecies avium (MAA). The close relationship between human and swine isolates indicates a possible zoonotic role for such strains. In the present work 73 M. avium field strains isolated from feces and lymph nodes of diseased/slaughtered animals in Hesse State, Germany were investigated. Forty eight primers were used for the confirmation, differentiation and finally the genotyping of the isolates based on the presence of polymorphism of different repetitive loci. These include the Large Sequence Polymorphism (LSP), the Mycobacterial Interspersed Repetitive Units (MIRU) and Variable Number Tandem Repeats (VNTR). The genotyping of MAA ( n = 27) and MAH ( n = 16) isolates revealed 33 different genotypes (18 MAA, 14 MAH and 1 shared profile). The described methods show great potential for epidemiological mapping of M. avium subspecies.