Ovine pedomics : the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H), interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified due to mismatches in the 16S rRNA universal forward primer (27F). A specific real time PCR assay was used to demonstrate the presence of D. nodosus which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (104-109 cells/g tissue) than those with H or VFR feet

Identification of Arcanobacterium pyogenes isolated by post mortem examinations of a bearded dragon and a gecko by phenotypic and genotypic properties

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

Properties and antimicrobial susceptibility of Trueperella pyogenes isolated from bovine mastitis in China

Trueperella (T.) pyogenes is an opportunistic pathogen that causes suppurative diseases in domestic animals. In this work, the properties, pathogenesis and phenotypic diversity of T. pyogenes isolates from bovine mastitis were studied. Both pyolysin (plo) and collagen-binding protein (cbp) virulence factor genes were detected by PCR in all T. pyogenes isolates (n = 50). Using the tissue culture plate method, 90% of T. pyogenes isolates were able to form biofilms. The minimum inhibitory concentrations (MICs) of 13 antimicrobials against T. pyogenes isolates were determined. High susceptibility was observed to rifampin (96%), ampicillin (94%), ciprofloxacin (94%), and penicillin (92%), while low susceptibility was found to trimethoprim-sulphamethoxazole (10%) and bacitracin (2%). The intracellular assay revealed that T. pyogenes isolates had different cytopathogenic effects on cells. The high percentage (28.6%) of T. pyogenes isolates suggests that this bacterium is an important contributor to mastitis. Moreover, the high occurrence of multidrug resistance, biofilm production, intracellular survival, and the temporal dynamics of T. pyogenes interactions are key factors for a better understanding of how immunity acts on infections with these bacteria and how they evade immune surveillance, thus highlighting the need for the prudent use of antimicrobial agents in veterinary medicine

Pathogenesis and antigenic characterization of a new East European subtype 3 porcine reproductive and respiratory syndrome virus isolate

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is divided into a European and North American genotype. East European PRRSV isolates have been found to be of the European genotype, but form different subtypes. In the present study, PRRSV was isolated from a Belarusian farm with reproductive and respiratory failure and designated "Lena". Analyses revealed that Lena is a new East European subtype 3 PRRSV isolate. The main purpose of this investigation was to study the pathogenesis and antigenic characteristics of PRRSV (Lena). Results: Obvious clinical and virological differences were observed between the animals inoculated with a recent European subtype 1 PRRSV isolate (Belgium A) and animals inoculated with PRRSV (Lena). Three out of six pigs inoculated with PRRSV (Belgium A) had anorexia and low fever at 3, 4 and 5 days post-inoculation (dpi). High fever, anorexia and depression were prominent signs in most pigs inoculated with PRRSV (Lena) between 2 and 28 dpi. Four pigs out of ten died during the experiment. Arcanobacterium pyogenes was isolated from lungs of one animal that died, and Streptococcus suis was isolated from lungs of one animal that was euthanized. The difference in viral titres in sera from PRRSV (Belgium A) and PRRSV (Lena)-infected pigs was statistically significant (p < 0.05) at 7, 10, 14 and 21 dpi. The highest viral titres in sera ranged from 10(4.8) to 10(6.1) TCID50/ml for PRRSV (Lena) whereas they ranged from 10(3.1) to 10(4.8) TCID50/ml for PRRSV (Belgium A). The replication of PRRSV (Lena) was further studied in depth. Viral titres ranged from 10(2.5) TCID50/100 mg to 10(5.6) TCID50/100 mg in nasal secretions between 3 and 14 dpi and from 10(2.8) TCID50/100 mg to 10(4.6) TCID50/100 mg in tonsillar scrapings between 3 and 21 dpi. High viral titres were detected in lungs (10(2.3)-10(7.7) TCID50/g tissue), tonsils (10(2.0)-10(6.2) TCID50/g tissue) and inguinal lymph nodes (10(2.2)-10(6.6) TCID50/g tissue) until 35, 28 and 35 dpi, respectively. To examine the antigenic heterogeneity between the East European subtype 3 isolate Lena, the European subtype 1 strain Lelystad and the North American strain US5, sets of monospecific polyclonal antisera were tested in immunoperoxidase monolayer assays (IPMAs) with homologous and heterologous viral antigens. Heterologous antibody titres were significantly lower than homologous titres (p = 0.01-0.03) for antisera against PRRSV (Lena) at all sampling time points. For antisera against PRRSV (Lelystad) and PRRSV (US5), heterologous antibody titres were significantly lower than homologous titres at 14 and 21 dpi (p = 0.01-0.03) and at 10 and 14 dpi (p = 0.04), respectively. Conclusions: Lena is a highly pathogenic East European subtype 3 PRRSV, which differs from European subtype 1 Lelystad and North American US5 strains at both the genetic and antigenic level

Arcanobacterium pyogenes: Virulence factors, importance in mastitis etiology and therapeutic (im)possibilities

Arcanobacterium pyogenes is an opportunistic pathogen, a causative agent of suppurative infections of organs and tissues in economically important livestock species. Most frequently this bacteria is isolated from inflamed lung lesions in pigs and cattle, in samples of uterine mucus of cows with endometritis and milk from cows with clinical mastitis. A. pyogenes possesses a number of virulence factors: cholesterol-dependent cytolysin (pyolysin), two neuraminidases, several proteases, extracellular matrix-binding proteins, DNases, fimbriae. The virulence factors are well studied in laboratory conditions, but the role of these factors in the pathogenesis of A. pyogenes infections remains to be elucidated. Lately, the ability of A. pyogenes to form biofilm in vivo has also been implicated as a virulence factor and a possible cause of therapeutic failure. Despite the fact that A. pyogenes milk isolates in cows with mastitis in vitro are very sensitive to β-lactam drugs and tetracycline, experience has shown that therapy is usually ineffective, prognosis is poor and the affected quarter is lost for milk production

ِDiagnosis of causes of suppurative arthritis in sheep in Mosul, Iraq

The prevalence of suppurative arthritis in one hundred sheep from both sexes and different ages were studied. Aspiration of synovial fluid from knee joint of animals showed signs of lameness with reluctant to move in addition to decrease in the body weight and loss of appetite. Physical and bacterial examination of the synovial fluid was revealed green color, turbid and containing pus. Mucin clot showed difference in degree of clotting, and increased number in leukocyte, neutrophile but decreased in lymophocytes and monocyte. The result showed that Streptococcus dysagalactiae was predominate 61.1% while Arcanobacterium pyogenes and Pseudomonas aeruginosa were the less 5.5%, also the result showed that all the strains were more sensitive to the Ciprofloxacin. This study is considered to be the first attempt to isolate bacteria from Sheep joints in Mosul-Iraq

Arcanobacterium haemolyticum associated with pyothorax: case report

Arcanobacterium haemolyticum has an established role in the etiology of human pharyngitis. There are increasing reports of systemic infections caused by this organism. From India, we report the first case of Arcanobacterium haemolyticum causing pyothorax in an immunocompetent adolescent male patient. The probable mode of infection is also discussed. The role of A. hemolyticum as an animal pathogen needs further study

Trueperella pyogenes endocarditis in a Swiss farmer: a case report and review of the literature.

BACKGROUND Trueperella pyogenes (T. pyogenes) is a bacterium that colonizes the skin and mucosal surfaces of various domestic and wild animals. It rarely leads to infections in humans, with only a few descriptions available in the literature. CASE PRESENTATION A 71-year-old Swiss farmer with a history of recurring basal cell carcinoma and metastasized pancreatic neuroendocrine tumor presented with signs of sepsis after a three-day history of general weakness, malaise and fever. Clinical and echocardiographic findings, as well as persistent bacteremia were consistent with mitral valve endocarditis caused by T. pyogenes. The patient's condition gradually improved under antibiotic treatment with piperacillin/tazobactam (empiric therapy of sepsis), and later penicillin G based on resistance testing. He was discharged after 13 days and continued outpatient antibiotic therapy with ceftriaxone, resulting in a total antibiotic treatment duration of six weeks. This is the first literature review of T. pyogenes endocarditis in humans. Among nine cases of T. pyogenes endocarditis, three patients had documented contact with farm animals and five had an underlying condition that compromised the immune system. While antibiotic resistance of T. pyogenes is an emerging concern, susceptibility to beta-lactam antibiotics seems to persist. The mortality of T. pyogenes endocarditis described in the literature was high, with 66% of patients not surviving the disease. CONCLUSIONS T. pyogenes is a rare causative organism of infectious endocarditis in humans and descriptions are mainly restricted to case reports. In our review of the literature, we found that both an impaired immune system and contact with farm animals might be risk factors. Growth of T. pyogenes in blood cultures is unlikely to be missed during routine analysis, as it shows marked beta-hemolysis on blood agar culture plates, which generally leads to further characterization of the bacteria. Susceptibility to penicillin, ceftriaxone, and macrolides seems to be retained and the reported mortality in the few patients with T. pyogenes endocarditis is high

description of two novel species Arcanobacterium bialowiezense and Arcanobacterium bonasi

Deckblatt-Impressum persönlicher Dank Inhaltsverzeichnis Einleitung Literaturübersicht Problemstellung und Ziele Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Abkürzungsverzeichnis Danksagung SelbständigkeitserklärungDer Białowieża Nationalpark erstreckt sich zu beiden Seiten der Grenze zwischen Polen und Weißrußland. Nach dem zweiten Weltkrieg wurde er in zwei Teile geteilt. Heute gehören zum polnischen Teil 594 km2 und zu dem weißrussischen 874 km2. ImBiałowieża Nationalpark lebt die weltweit größte freilebende Wisentpopulation. In dieser Population erkranken seit 1980 regelmäßig eine große Zahl der Bullen an einer chronisch nekrotisierenden Entzündung des Präputiums und des Penis (Balanoposthitis). Der Wisent wird innerhalb der Familie Bovidae (Gray, 1872) in die Subfamilie Bovinae (Gray, 1821) eingeordnet. Er gehört zu der Gattung Bison, welche die beiden Arten Bison (Bison bison, Linnaeus, 1758) und Wisent (Bison bonasus, Linnaeus, 1758) umfasst. Der Wisent ist eine bedrohte Tierart (Appendix III (geschützte Tierart) der Berner Konvention zum Schutz der Lebensräume wildlebender Pflanzen und Tiere, bedrohte Art auf der IUCN roten Liste der bedrohten Tierarten) und die Balanoposthitis bedeutet ein ernst zu nehmendes Problem für die in Białowieża existierende Wisentpopulation (Anonymous, 2002). 1980 wurde erstmalig eine chronische Erkrankung des äußeren Geschlechtsapparates bei Wisentbullen beschrieben (Kita et al., 1994). Charakteristisch für fortgeschrittene Erkrankungsstadien sind die Ödematisierung der Haut um die Präputialöffnung herum, und die Ansammlung von eitrigem Exsudat sowie nekrotischem Gewebe in der Präputialhöhle. Im Spätstadium kann es zu Paraphimose, Verklebung von Präputium und Penis sowie Autamputation der Penisspitze kommen. Histopathologisch wird das nekrotische Gewebe durch einen Demarkationswall aus Entzündungszellen und Granulationsgewebe vom darunterliegenden Bindegewebe abgetrennt (Jakob et al., 2000). Im Spätstadium der Balanoposthitis erkrankte Bullen können durch die entzündlichen und nekrotischen Veränderungen an ihrem Präputium, die auch auf den Penis übergreifen, nicht mehr am Reproduktionsgeschehen teilnehmen. Durch den regelmäßigen Ausfall der an Balanoposthitis erkrankten Bullen (zwischen 5 und 10% pro Jahr) wird das genetische Potential der Białowieża Population vermutlich weiter eingeschränkt. Daher war es Ziel dieser Arbeit die ursächliche oder sekundäre Beteiligung bestimmter Bakterien an der Balanoposthitis abzuklären. Weiterhin sollte ein Überblick über die bakteriologische Normalflora sowohl des männlichen als auch des weiblichen Geschlechtstrakts der Wisente in Białowieża gewonnen werden. Zu diesem Zweck wurden Tupferproben von gesunden und erkrankten Wisentbullen sowie Wisentkühen entnommen und bakteriologisch untersucht. Aus Tupferproben an Balanoposthitis erkrankter Bullen wurden dabei zwei Bakterien isoliert welche der morphologischen Beschreibung von A. pyogenes entsprachen. Der Stamm W3/01 und der Stamm W106/04 zeigten aber keine Serolyse auf Löffler Agar und unterschieden sich auch in den enzymatischen Reaktionen von A. pyogenes. Eine vergleichende 16S rDNA Analyse ordnete die Isolate W3/01 und W106/04 eindeutig in die Gattung Arcanobacterium ein. Die höchste Ähnlichkeit bestand zu A. pyogenes (96,1% und 96,4%) und A. bernardiae (95,5% und 95,8%). Untereinander waren die Isolate zu 97,2% identisch. Das Isolat mit der Stammbezeichnung W3/01 wurde unter der Speziesbezeichnung Arcanobacterium bialowiezense sp. nov. und das Isolat mit der Stammnummer W106/04 unter der Speziesbezeichnung Arcanobacterium bonasi sp. nov. bei der Deutschen Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig) hinterlegt. A. bialowiezense oder A. bonasi wurden bei 87% der kranken Bullen nachgewiesen, aber weder bei gesunden Bullen noch bei den weiblichen Tieren gefunden. Die phylogenetische Verwandtschaft zu A. pyogenes unterstreicht eine mögliche Bedeutung in der Ätiologie und Pathogenese der Balanoposthitis beim Wisent. Es ist denkbar, dass die beiden Isolate auch ähnliche Virulenzfaktoren besitzen und somit am Krankheitsgeschehen der Balanoposthitis beteiligt sein können. Momentan ist es nur möglich eine Hypothese über den Verlauf der Krankheit aufzustellen und diese als Basis für weitere Untersuchungen zu verwenden. Demnach kommt es möglicherweise durch prädisponierende Faktoren zu einer Vorschädigung des Gewebes mit der anschließenden Ausbildung einer fokalen Hyperkeratose. Eventuell liegt auch ein genetischer Defekt zugrunde, der in der geringen genetischen Variabilität der Wisente begründet ist und zu einer Störung der lokalen Immunabwehr führt (Lünser et al., 2005). Ein solcher Defekt hätte zur Folge, dass Krankheitserreger leichter in die Haut einwandern und sich dort vermehren können. In vorangegangenen Untersuchungen wurde die Beteiligung von Fusobakterien an der Balanoposthitis des Wisents nachgewiesen (Jakob et al., 2000). Durch einen Synergismus von Fusobakterien mit den beiden neuen Spezies A. bonasi und A. bialowiezense könnte die Infektion noch verstärkt werden wie es für A. pyogenes und F. necrophorum beschrieben ist (Kaczmarowski, 2003). Aufgrund der hohen Verwandtschaft der beiden Arcanobacterium spp. zu A. pyogenes kann man vermuten, dass sie ähnliche Virulenzfaktoren besitzen. Neuraminidasen unterstützen die Anhaftung der Arcanobakterien an Hautzellen, wodurch sie in der Lage sind die Haut zu besiedeln (Esmay et al., 2003). PLO, ein haemolytisches Exotoxin (Ding, 1996; Billington, 1997), wirkt zytolytisch indem es an Membranen tierischer Zellen bindet und dort oligomere Poren bildet (Billington, 2000). Es ist außerdem beschrieben, dass PLO die Komplementkaskade aktiviert (Paton, 1984). Bei einer überschiessenden Reaktion kann dies zu Entzündungsreaktionen und Nekrosen führen (www.mucos.cz). Da es möglich war einen der Erreger mittels PCR bei einem Bullen in Stadium I der Erkrankung nachzuweisen ist nicht auszuschließen, dass die Arcanobakterien primär an der Balanoposthitis beteiligt sind.The Białowieża National Park is situated on both sides of the border between Poland and Belarussia. After the second world war it was separated into the Polish and the Belarussian part. Today the Polish part comprises 594 km2 and the Belarussian part 874 km2. The largest of the few remaining free-ranging populations of European bison exists in the Białowieża Primeval Forest. Since 1980 a chronic necrotising disease of the prepuce and penis (Balanoposthitis) has been observed in this population. The European (E.) bison belongs to the family Bovidae (Gray, 1872), the subfamily Bovinae (Gray, 1821) and the genus bison. There are two species classified within the genus bison: the European bison (Bison bonasus) and the North American bison (Bison bison). The European bison is a threatened species (Appendix III (protected fauna species) of the Bern Convention on the conservation of European wildlife and natural habitats, endangered species on the 2003 IUCN Red List of Threatened Species) and the Balanoposthitis is a serious problem for the present Białowieża population (Anonymous, 2002). A chronic necrotizing inflammation of the external genital organs of male E. bison was first described in 1980 (Kita et al., 1994). Advanced disease is characterized by crushing of the hairs around the preputial orifice, edema of the surrounding skin, and accumulation of thick exudate and necrotic tissue within the preputial cavity. In addition, paraphimosis, constriction of the distal penis, and in some cases necrosis and auto-amputation of the glans penis, have been observed. Histopathologically, the necrotic tissue is demarcated from the underlying connective tissue by inflammatory cells and granulation tissue. Numerous lymphoid follicles in the surrounding connective tissue are also present (Jakob et al., 2000). Due to the inflammatory and necrotic changes of the prepuce and penis in late stages of Balanoposthitis affected bulls are not able to take part in reproduction. This regular loss of bulls (between 5 and 10% per year) obviously leads to a further reduction of the genetic potential of this threatened species. For this reason the objective of this study was to investigate whether certain bacteria are primarily or secondarily involved in the disease process of the Balanoposthitis. Additionally, a survey of the physiological bacteriological flora of the male and female E. bison genital tract was conducted. For this purpose genital samples of healthy and diseased bulls as well as female E. bison were examined bacteriologically. From genital swabs of bulls showing Balanoposthitis two new bacteria were isolated which were morphologically similar to Arcanobacterium (A.) pyogenes. However strain W3/01 and strain W106/04 showed no serolysis on Löffler serum Agar and they also differed in enzymatic reactions from A. pyogenes. 16S rDNA sequence analysis showed that the two isolates W3/01 and W106/04 shared 97.2% sequence similarity. Highest sequence similarities (95.5-96.1%) to A. pyogenes DSM 20630 and A. benardiae DSM 9152 were found. The isolates with strain numbers W3/01 and W106/04 were deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig) under the species name of Arcanobacterium bialowiezense sp. nov. and Arcanobacterium bonasi sp. nov., respectively. A. bialowiezense or A. bonasi were isolated from 87% of the diseased bulls, but not from healthy bulls or female E. bison. The phylogenetic relationship to A. pyogenes underlines a possible role in the etiology and pathogenesis of the Balanoposthitis. It is possible that both isolates have virulence factors similar to A. pyogenes, and for this reason they might be involved in the disease process. At this stage it is only possible to hypothesize about the process of the disease and use this as a basis for further studies: Predisposing factors might scarify the tissue resulting in a hyperkeratosis. Possibly a genetic defect due to the low genetic variability leads to a failure of the local immune response (Lünser et al., 2005). This would make it easier for pathogenic microorganisms to invade the skin and proliferate. In preliminary studies involvement of Fusobacteria was demonstrated (Jakob et al., 2000). A synergism between Fusobacterium (F.) spp. and A. bonasi and A. bialowiezense could further enhance the infection as it has been described for A. pyogenes und F. necrophorum (Kaczmarowski, 2003). The phylogenetic relationship of these two Arcanobacterium spp. to A. pyogenes leads to the assumption that they might have similar virulence factors: Neuraminidase promotes the adherence of Arcanobacterium spp. to skin cells, which enables them to colonize the skin (Esmay et al., 2003). PLO, a hemolytic Exotoxin (Ding, 1996; Billington, 1997), has a cytolytic effect through binding to the membranes of animal cells and building large oligomere pores (Billington, 2000). PLO also activates the complement cascade (Paton, 1984). By overreacting this can lead to inflammation and necrosis (www.mucos.cz). As DNA of one of the new Arcanobacterium spp. was isolated by PCR in a bull at Stage I of the disease it is not possible to exclude a primary etiological role in the disease process