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In the present study a Trueperella (T.) pyogenes strain isolated from an abscess on the left side of the face of a six year old grey slender loris (Loris lydekkerianus nordicus) could successfully be identified phenotypically, by MALDI-TOF MS analysis and genotypically using T. pyogenes superoxide dismutase A encoding gene sodA and T. pyogenes 16S-23S rDNA intergenic spacer region specific oligonucleotide primers. 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, nanH encoding neuraminidase NanH and the genes fimA, fimC, fimE encoding the fimbrial subunits FimA, FimC and FimE but not the genes cbpA and nanP encoding collagen-binding protein CbpA and neuraminidase NanP, respectively. The present data give the first information about properties of T. pyogenes isolated from a monkey.

Comparable to previously conducted phenotypical and genotypical investigations (Hijazin et al., 2011c), three strains of the newly described species Actinomyces weissii, isolated from infections of the oral cavity of three dogs could be classified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and by sequencing the target genes 23S rDNA and cpn60 as novel species of genus Actinomyces. The detection of peptidic spectra and both genotypic approaches might help to identify A. weissii in future and elucidate the role this species plays in infections of dogs.

In the present study A. (T.) abortisuis isolated from pigs and bovines could be reliably identified by determination of phenotypic properties, genotypically by polymerase chain reaction with the help of A. (T.) abortisuis 16s-23S rDNA intergenic spacer region specific oligonucleotide primer and by Matrix-Assisted Laser Desorption Ionization-Time Of Flight mass spectrometry (MALDI-TOF MS). The latter appeared to be a promising tool for fast and cost effective identification of this species and might help to elucidate the role A. (T.) abortisuis plays in infections of pigs, bovines, possibly other animals or humans.

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.

In the present study, the 16S-23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.

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.

In the present study, we report data on virulence determinants of Staphylococcus aureus from stable nasal carriers, emphasizing on the genes encoding fibronectin (fnbA, fnbB) and collagen (cna) adhesive molecules. Of the 44 S. aureus isolates included, 32 isolates (16 pairs) were cultured from the anterior nares of 16 healthy carriers, eight isolates (four pairs) were collected from the nose of four patients with recurrent skin infections and four isolates were obtained from the infection site of these patients. The period between the two nasal swabs taken was 3-5 days. The persistency of carriage could be demonstrated by the indistinguishable genotypic characteristics of the S. aureus isolates in each pair. This could be shown by determination of gene polymorphisms of coa gene and the X-region and IgG-binding region encoding segments of spa gene. In addition, the isolates within the pairs showed identical toxin patterns. This was determined by PCR amplification of the genes encoding staphylococcal enterotoxins (SEA to SEJ) and TSST-1. The genotypic properties also yielded an identity between persistent nasal carriage isolates and the corresponding skin infection isolates of the four patients. In addition, all S. aureus nasal and skin infection isolates were positive for gene fnbA, fnbB and cna could be found with a high frequency. Among the 44 isolates investigated, 16 isolates (36.7%) harbored gene fnbB and 21 isolates (47.7%) gene cna. The data in the present study showed a relatively wide distribution of the genes fnb and cna among the investigated isolates, indicating that the persistent carriage of strains harboring these virulence determinants may increase the risk for subsequent invasive infections in carriers.

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.

In the present study three phenotypically CAMP-negative Streptococcus agalactiae, isolated from three cows with mastitis, were characterized by molecular analysis. An identification of the S. agalactiae was performed by conventional methods and by PCR amplification of species specific parts of the 16S rRNA gene and the 16S-23S rDNA intergenic spacer region. In addition all three phenotypically CAMP-negative isolates harboured a normal sized CAMP-factor encoding cfb gene indicating a reduced expression of CAMP-factor or a gene defect elsewhere along the pathway of expression. The clonal identity of the three isolates could be demonstrated by macrorestriction analysis of their chromosomal DNA.

In the present study, 103 Staphylococcus aureus strains isolated from milk samples from 60 cows with mastitis from eight different farms in seven different locations in one region of Germany were compared pheno- and genotypically and by identification of various toxins. On the basis of culture and hemolytic properties and by determination of the tube coagulase reaction, all of the isolates could be identified as S. aureus . This could be confirmed by PCR amplification of species-specific parts of the gene encoding the 23S rRNA. In addition, all of the S. aureus isolates harbored the genes encoding staphylococcal coagulase and clumping factor and the genes encoding the X region and the immunoglobulin G binding region of protein A. These four genes displayed size polymorphisms. By PCR amplification, the genes for the toxins staphylococcal enterotoxin A (SEA), SEC, SED, SEG, SEI, SEJ, and TSST-1 but not those for SEB, SEE, SEH, and the exfoliative toxins ETA and ETB could be detected. To analyze the epidemiological relationships, the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNAs. According to the observed gene polymorphisms, the toxin patterns, and the information given by macrorestriction analysis of the isolates by pulsed-field gel electrophoresis, a limited number of clones seemed to be responsible for the cases of bovine mastitis on the various farms.

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.