Population structure and virulence gene profiles of Streptococcus agalactiae collected from different hosts worldwide.

Streptococcus agalactiae is a leading cause of morbidity and mortality among neonates and causes severe infections in pregnant women and nonpregnant predisposed adults, in addition to various animal species worldwide. Still, information on the population structure of S. agalactiae and the geographical distribution of different clones is limited. Further data are urgently needed to identify particularly successful clones and obtain insights into possible routes of transmission within one host species and across species borders. We aimed to determine the population structure and virulence gene profiles of S. agalactiae strains from a diverse set of sources and geographical origins. To this end, 373 S. agalactiae isolates obtained from humans and animals from five different continents were typed by DNA microarray profiling. A total of 242 different S. agalactiae strains were identified and further analyzed. Particularly successful clonal lineages, hybridization patterns, and strains were identified that were spread across different continents and/or were present in more than one host species. In particular, several strains were detected in both humans and cattle, and several canine strains were also detected in samples from human, bovine, and porcine hosts. The findings of our study suggest that although S. agalactiae is well adapted to various hosts including humans, cattle, dogs, rodents, and fish, interspecies transmission is possible and occurs between humans and cows, dogs, and rabbits. The virulence and resistance gene profiles presented enable new insights into interspecies transmission and make a crucial contribution to the identification of suitable targets for therapeutic agents and vaccines.

New polymorphisms within the variable number tandem repeat (VNTR) 7 locus of Mycobacterium avium subsp. paratuberculosis.

Variable number tandem repeat (VNTR) is a frequently employed typing method of Mycobacterium avium paratuberculosis (MAP) isolates. Based on whole genome sequencing in a previous study, allelic diversity at some VNTR loci seems to over- or under-estimate the actual phylogenetic variance among isolates. Interestingly, two closely related isolates on one farm showed polymorphism at the VNTR 7 locus, raising concerns about the misleading role that it might play in genotyping. We aimed to investigate the underlying basis of VNTR 7-polymorphism by analyzing sequence data for published genomes and field isolates of MAP and other M. avium complex (MAC) members. In contrast to MAP strains from cattle, strains from sheep displayed an "imperfect" repeat within VNTR 7, which was identical to respective allele types in other MAC genomes. Subspecies- and strain-specific single nucleotide polymorphisms (SNPs) and two novel (16 and 56 bp) repeats were detected. Given the combination of the three existing repeats, there are at least five different patterns for VNTR 7. The present findings highlight a higher polymorphism and probable instability of VNTR 7 locus that needs to be considered and challenged in future studies. Until then, sequencing of this locus in future studies is important to correctly assign the underlying allele types.(1).

Improvement of sensitivity for Mycobacterium avium subsp. paratuberculosis (MAP) detection in bovine fecal samples by specific duplex F57/IC real-time and conventional IS900 PCRs after solid culture enrichment.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants and a probable pathogen of Crohn's disease in humans. Accurate, cost-effective, and time-relevant diagnostics are the basis for efficient control programs. This study was conducted as an attempt to re-evaluate MAP detection improvement by coupling solid media enrichment to a more specific IS900 conventional PCR and a very specific F57/IC real-time PCR. In a spiking experiment, we investigated the improvement of molecular-based MAP detection in feces after a culture-based enrichment step into Herrold's egg yolk media with mycobactin J (HEYM-MJ) for different time intervals, when compared to traditional culture. Detection limit of culture was 0.33 × 10(4) bacteria × g(-1) (33 CFU g(-1)), while that of IS900 PCR when coupled with an enrichment step for 2, 4, and 6 weeks was 0.33 × 10(5) (0.33 × 10(3) CFU g(-1)), 0.33 × 10(4) (33 CFU g(-1)), and 33 (>3.3 CFU g(-1)) bacteria × g(-1), respectively. Whereas the detection limits of F57/IC real-time PCR after the enrichment step for the same time intervals were 0.33 × 10(5) (0.33 × 10(3) CFU g(-1)), 0.33 × 10(3) (3.3 CFU g(-1)), and 33 (>3.3 CFU g(-1)) bacteria × g(-1), respectively. Altogether, enrichment of bovine fecal samples into solid media increased the sensitivity of specific molecular detection of MAP using IS900 conventional PCR and duplex F57/IC real-time PCR and offers an expedited and accurate alternative for MAP detection in bovine feces. Validation of these results is further recommended using field bovine fecal samples.

Arcanobacterium phocisimile sp. nov., isolated from harbour seals.

A polyphasic taxonomic study was performed on two previously unidentified Arcanobacterium-like Gram-positive strains isolated from harbour seals. Comparative 16S rRNA gene sequencing showed that both bacteria belonged to the genus Arcanobacterium and were most closely related to Arcanobacterium haemolyticum CIP 103370(T) (98.4% 16S rRNA gene sequence similarity), A. canis P6775(T) (97.4%), A. phocae DSM 10002(T) (97.4%), A. pluranimalium M430/94/2(T) (95.7%) and A. hippocoleae CCUG 44697(T) (95.5%). The presence of the major menaquinone MK-9(H4) supported the affiliation of the isolates with the genus Arcanobacterium. The polar lipid profile consisted of major amounts of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified phospholipid and two unidentified glycolipids. The major fatty acids were C16:0, C18:0, C18:1ω9c and summed feature 5 (comprising C18:2ω6,9c and/or anteiso-C18:0). Physiological and biochemical tests clearly distinguished the isolates from other members of the genus Arcanobacterium. Based on the common origin and various physiological properties comparable to those of A. phocae, it is proposed that the isolates are classified as members of a novel species with the name Arcanobacterium phocisimile sp. nov. The type strain is 2698(T) (=LMG 27073(T) =CCM 8430(T)).

Boot swabs to collect environmental samples from common locations in dairy herds for Mycobacterium avium ssp. paratuberculosis (MAP) detection.

The aim of the present study was the examination of the boot swab sampling technique for the collection of environmental material in order to identify Mycobacterium avium ssp. paratuberculosis (MAP)-infected herds. Eight dairy herds were included into the study. Four of them had a well-known history of MAP-infection from a herd surveillance programme conducted since 2006. Cows in these herds were repeatedly tested positive in Pourquier® MAP-ELISA (Pourquier, Montepellier, France); in some MAP could be isolated in individual faecal culture despite that symptoms of paratuberculosis were never reported. In four presumably negative herds nearly all cows were repeatedly tested serologically negative for MAP. The pathogen was never isolated from faecal samples of cows by culture. The study was initiated with the aim of standardising environmental samples as a herd diagnostics, in which overall 130 pairs of boot swab samples from the cows' surroundings were taken In 58 of 64 swab samples (90·6%) from confirmed MAP-infected herds the organism could be isolated by mycobacterial culture of the boot swab. Contrarily, in 66 samples from presumably MAP-negative herds only one swab was positive (1·5%). The utilisation of boot swabs as a standardised technique for environmental sampling offers an effective and inexpensive tool for identifying herds infected with MAP. This is the first report of using boot swabs for the collection of environmental samples for MAP- detection in cattle herds. This easy to perform technique enables the economical detection of MAP herd status.

Mycobacterium avium subspecies paratuberculosis: an insidious problem for the ruminant industry.

Mycobacterium avium subspecies paratuberculosis is considered as one of the most serious problems affecting the world's ruminant industry due to its significant impact on the global economy and the controversial issue that it may be pathogenic for humans. M. avium subspecies paratuberculosis is the causative agent of Johne's disease in animals and might be implicated in cases of human Crohn's disease. We provide an insight into M. avium subspecies paratuberculosis from some bacteriological, clinical, and molecular epidemiological perspectives.

Isolation of potentially novel Brucella spp. from frogs.

Bacterial isolates from frogs were phenotypically identified as Ochrobactrum anthropi, but 16S rRNA sequencing showed up to 100% identity with Brucella inopinata. Further analysis of recA, omp2a, omp2b, bcsp31, and IS711 and multilocus sequence analysis (MLSA) verified a close relationship with Brucella, suggesting the isolates may actually represent novel members of this growing genus of zoonotic pathogens.

Arcanobacterium canis sp. nov., isolated from otitis externa of a dog, and emended description of the genus Arcanobacterium Collins et al. 1983 emend. Yassin et al. 2011.

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(H(4)) 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 C(14 : 0), C(16 : 0), C(18 : 0), C(18 : 1)ω9c and C(18 : 2)ω6,9c/anteiso-C(18 : 0) (detected as a summed feature). C(10 : 0) and C(12 : 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 P6775(T) (= CCM 7958(T) = CCUG 61573(T) = CIP 110339(T)). An emended description of the genus Arcanobacterium is also provided.

Actinomyces weissii sp. nov., isolated from dogs.

Two Gram-positive, rod-shaped, non-spore-forming bacteria were isolated from the oral cavities of two dogs. On the basis of 16S rRNA gene sequence similarities both strains were shown to belong to the genus Actinomyces and were most closely related to Actinomyces bovis (97.3% and 97.5%, respectively). The polyamine profile of the two isolates and Actinomyces bovis DSM 43014(T) was composed of spermidine and spermine as the major components. Menaquinone MK-9 was the major compound in the quinone system of the two strains and Actinomyces bovis. The polar lipid profiles of strains 2298(T) and 4321 were almost identical, containing diphosphatidylglycerol as the major compound, and moderate to trace amounts of phosphatidylcholine, phosphatidylinositol, phosphatidylinositol-mannoside, phosphatidylglycerol and several unidentified lipids. A highly similar polar lipid profile was detected in Actinomyces bovis DSM 43014(T) supporting the affiliation of strains 2298(T) and 4321 to the genus Actinomyces. The typical major fatty acids were C(16:0), C(18:0) and C(18:1)ω9c. Fatty acids C(14:0) and C(18:2)ω6,9c were found in minor amounts. The results of physiological and biochemical analyses revealed clear differences between both strains and the most closely related species of the genus Actinomyces. Thus, strains 2298(T) and 4321 represent a novel species, for which the name Actinomyces weissii sp. nov., is proposed, with strain 2298(T) ( = CIP 110333(T) = LMG 26472(T) = CCM 7951(T) = CCUG 61299(T)) as the type strain.

The effect of dry cow antibiotic with and without an internal teat sealant on udder health during the first 100 d of lactation: a field study with matched pairs.

The objective of this field study was to compare the udder health status as well as the clinical mastitis rate during the first 100 d of lactation in cows that received long-acting dry cow antibiotic alone (group AB) or in combination with an internal teat sealant (group AB + OS). The study was conducted during a 9-month period and included 136 Holstein cows from 12 dairy farms in Hessia, Germany. Between days 1 and 5 after calving a California mastitis test (CMT) was performed. Milk-samples were collected for bacteriological culture before drying off, between days 6 and 14 and days 35 and 56 of lactation. Additionally the cows were monitored for the occurrence of clinical mastitis events until 100 d post partum. Within the 12 herds cow-pairs were formed on the basis of age, milk yield and SCC. A cow-pair consisted of one cow from group AB and one cow from group AB + OS. For statistical analysis within every cow-pair one quarter that has been dried off with internal teat sealant and dry cow antibiotic (group AB + OS) was compared with one quarter that has been dried off with dry cow antibiotic (group AB) alone. As criterion for the matching process of udder quarters the cytobacteriological udder health status before drying off was used. A total of 544 quarters (136 cows) were used in this analysis. In the first 5 d after calving, group AB had significantly more quarters with a positive CMT reaction than group AB + OS (85 vs. 57; P <0·001), and in the first 100 d of lactation, group AB had more quarters with clinical mastitis than group AB + OS (25 vs. 15; P = 0·03). In the time periods 6-14 and 35-56 d of lactation, there were fewer quarters in group AB + OS populated with Corynebacterium spp. (days 6-14, P = 0·05; days 35-56, P = 0·02) and aesculin-positive streptococci (days 35-56, P = 0·02). The internal teat sealant was a promising tool for the prevention of new intramammary infections (IMI) of dry cows with environmental udder pathogens as expressed during early lactation.

Microscopic differential cell counts in milk for the evaluation of inflammatory reactions in clinically healthy and subclinically infected bovine mammary glands.

Somatic cell count (SCC) is generally regarded as an indicator of udder health. A cut-off value of 100×10(3) cells/ml is currently used in Germany to differentiate between normal and abnormal secretion of quarters. In addition to SCC, differential cell counts (DCC) can be applied for a more detailed analysis of the udder health status. The aim of this study was to differentiate somatic cells in foremilk samples of udder quarters classified as normal secreting by SCC <100×10(3) cells/ml. Twenty cows were selected and 72 normal secreting udder quarters were compared with a control group of six diseased quarters (SCC >100×10(3) cells/ml). In two severely diseased quarters of the control group (SCC of 967×10(3) cells/ml and 1824×10(3) cells/ml) Escherichia coli and Staphylococcus aureus were detected. DCC patterns of milk samples (n = 25) with very low SCC values of ≤6·25×10(3)cells/ml revealed high lymphocyte proportions of up to 92%. Milk cell populations in samples (n = 41) with SCC values of (>6·25 to ≤25)×10(3) cells/ml were also dominated by lymphocytes (mean value 47%), whereas DCC patterns of milk from udder quarters (n = 6) with SCC values (>25 to ≤100)×10(3)cells/ml changed. While in samples (n = 3) with SCC values of (27-33)×10(3) cells/ml macrophages were predominant (35-40%), three milk samples with (43-45)×10(3) cells/ml indicated already inflammatory reactions based on the predominance of polymorphonuclear leucocytes (PMN) (54-63%). In milk samples of diseased quarters PMN were categorically found as dominant cell population with proportions of ≥65%. Macrophages were the second predominant cell population in almost all samples tested in relationship to lymphocytes and PMN. To our knowledge, this is the first study evaluating cell populations in low SCC milk in detail. Udder quarters classified as normal secreting by SCC <100×10(3) cells/ml revealed already inflammatory processes based on DCC.

Phenotypic and genotypic characterization of Arcanobacterium haemolyticum isolates from infections of horses.

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.

[Two-stage control of paratuberculosis: Herd-status surveillance as the basis for operational measures to reduce the prevalence. - Experiences from Lower Saxony, Hesse, Thuringia and Tyrol]. / Zweistufige Paratuberkulosebekämpfung in der Praxis: Überwachung auf Herdenebene als Basis für betriebliche Maßnahmen zur Prävalenzsenkung.

Economic losses caused by paratuberculosis (Johne's disease) can be high in infected herds. A universally accepted concept for the surveillance or control of paratuberculosis in cattle herds has not yet been established.In the course of the program for the reduction of MAP (Mycobacterium avium subsp. paratuberculosis) infections in Lower Saxony, Germany, dairy farms are obliged to test bulk tank milk samples for the presence of MAP-antibodies every 6 months. In case of a non-negative result, testing is required at the single animal level. Farmers can than decide whether they join a program to control MAP-infections in their herd. Within the voluntary certification program for paratuberculosis in Hesse, Germany, the MAP-herd status is evaluated using boot swab sampling. On positive farms, animals are tested at 6-month intervals by milk or blood serology with timely culling of positive individuals. The program for the abatement of paratuberculosis in cattle herds in Thuringia, Germany, is based on a yearly fecal examination for MAP-shedding of all adult cattle within a herd. Fecal MAP-positive animals should be culled as soon as possible. The basis of the surveillance and control program for MAP in Tyrol, Austria, is the biennial survey of the MAP-herd status by boot swab sampling. Farms with a MAP-positive boot swab result have the option to have their adult animals tested for MAP by single animal fecal sampling. On the basis of the results of these samples, farmers can decide whether they wish to join the MAP-control program.The programs presented above show that a two-stage approach consisting of the evaluation of the MAP-herd level, followed by the testing of single animals, represents a feasible approach for the surveillance and control of paratuberculosis in cattle herds.

Comprehensive literature review of the sources of infection and transmission routes of Coxiella burnetii, with particular regard to the criteria of "evidence-based medicine".

The present review aims to compile the currently available literature since 1936 according the sources of infection of the Q fever pathogen (Coxiella (C.) burnetii) as well as the transmission from animal to man and also from human to human. In terms of quality and validity, the existing publications were reviewed systematically. For this purpose, firstly a structured literature search was carried out using various databases and search engines supplemented by a manual literature search. For critical appraisal, 1444 relevant publications were identified for the moment and evaluated. A total of 73 publications describing a transmission of C. burnetii from animals to man or a human-to-human transmission were discovered. The identified publications are 29 case series, two case reports, 21 cohort studies and 21 case-control studies. With regard to the sources of infection, 25 publications describing the transmission of C. burnetii from sheep to humans could be identified.

Genotyping methods and molecular epidemiology of Mycobacterium avium subsp. paratuberculosis (MAP).

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) which affects mainly ruminants and is characterized by chronic diarrhea and emaciation. Johne's disease is highly prevalent in many countries around the world and leads to high economic losses associated with decreased production. Genotyping of the involved pathogen could be used in the study of population genetics, pathogenesis and molecular epidemiology including disease surveillance and outbreak investigation. Principally, researchers have first assumed the presence of two different MAP strains that are associated with the animal host species (cattle and sheep). However, nowadays MAP characterization depends mainly upon genetic testing using genetic markers such as insertion elements, repetitive sequences and single nucleotide polymorphisms. This work aims to provide an overview of the advances in molecular biological tools used for MAP typing in the last two decades, discuss how these methods have been used to address interesting epidemiological questions, and explore the future prospects of MAP molecular epidemiology given the ever decreasing costs of the high throughput sequencing technology.

Genotyping of Streptococcus uberis isolates in healing process of bovine clinical mastitis.

In the present work, macrorestriction analysis was applied to characterize 44 S. uberis field strains isolated from lactating cows suffering from mastitis in three dairy herds in Hesse State, Germany. Analysis of the obtained data by Pulse-Field Gel Electrophoresis (PFGE) showed that most of the isolates originating from different herds and cows were not related to each other. However, identical macrorestriction patterns were noted in 12 of 13 mastitic quarters in healing process, in three quarters even over the whole sampling period indicating persistent infection. In the present work, PFGE could detect variable levels of similarity ranging from 76 to 100%. The macrorestriction analyses revealed the presence of 10 S. uberis PFGE pattern with more than four bands difference. PFGE profiles with minor differences (only one to three bands) were considered to be subtypes. The use of sensitive genotyping methods like macrorestriction analyses by PFGE enables the differentiation among new and persistent infections. Nevertheless minor changes in macrorestriction profiles could occur which are clearly distinguishable from totally unrelated strains.

Trueperella pyogenes isolated from a brain abscess of an adult roebuck (Capreolus capreolus).

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.

Detection of canine adenovirus 1 in red foxes ( Vulpes vulpes) and raccoons ( Procyon lotor) in Germany with a TaqMan real-time PCR assay.

We developed a real-time (rt)PCR assay based on TaqMan probe technology for the specific detection of canine adenovirus 1 (CAdV-1). The assay is able to detect three 50% tissue culture infectious dose/mL in CAdV-1-containing cell culture supernatant. Viral genomes were not amplified of canine adenovirus 2 or of several bovine, porcine, and avian adenoviruses. In silico analysis provided no indication of amplification of other heterologous genomes. The sensitivity of the real-time assay exceeded that of a conventional gel-based CAdV-1 PCR by a factor of 100. Following the integration of the novel PCR into the Hessian wildlife-monitoring program, CAdV-1 DNA was detected in none of the tested raccoons ( n = 48) but in 11 of 97 foxes.

Streptococcus agalactiae in elephants - A comparative study with isolates from human and zoo animal and livestock origin.

Streptococcus (S.) agalactiae represents a significant pathogen for humans and animals. However, there are only a few elderly reports on S. agalactiae infections in wild and zoo elephants even though this pathogen has been isolated comparatively frequently in these endangered animal species. Consequently, between 2004 and 2015, we collected S. agalactiae isolates from African and Asian elephants (n=23) living in four different zoos in Germany. These isolates were characterised and compared with isolates from other animal species (n=20 isolates) and humans (n=3). We found that the isolates from elephants can be readily identified by classical biochemistry and MALDI-TOF mass spectrometry. Further characterisations for epidemiological issues were achieved using Fourier transform-infrared spectroscopy, capsule typing and molecular fingerprinting (PFGE, RAPD PCR). We could demonstrate that our elephant isolate collection contained at least six different lineages that were representative for their source of origin. Despite generally broad antimicrobial susceptibility of S. agalactiae, many showed tetracycline resistance in vitro. S. agalactiae plays an important role in bacterial infections not only in cattle and humans, but also in elephants. Comparative studies were able to differentiate S. agalactiae isolates from elephants into different infectious clusters based on their epidemiological background.