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.

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.

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)).

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.

Resistencia a penicilina G y oxacilina, de cepas de Staphylococcus aureus aisladas de mastitis bovina subclínica / Penicillin G and oxacillin resistance in Staphylococcus aureus strains isolated from bovine subclinical mastitis

Sixty-eigth out of 530 different S. aureus field strains isolated from subclinical cases of bovine mastitis from Germany (n = 26), Indonesia (n = 16), Mexico (n = 16) and Brazil (n = 10), respectively, were selected to be studied in the present work. The strains were tested phenotypically as well as genotypically for the presence of penicillin G- and oxacillin-resistance. For the primary phenotypical species identification of the 530 S. aureus strains, plasmacoagulase-test and Api 32 Staph system was applied. This was confirmed by molecular detection of the S. aureus specific genes encoding 23 S rRNA, thermostable nuclease (nuc), clumping factor (clfA), coagulase (coa) and protein A region Xr (spa). The selection of the 68 strains was carried out by the random selection of one strain per herd; additionally, only strains with different macrorestriction profiles were included here. Genotypic resistance to semisynthetic penicillins (methicillin/oxacillin) and penicillin G was studied through the identification of mecA- and blaZ-genes, respectively. The mecA gene was detected in only one S. aureus isolate from Brazil, which was not phenotypically resistant against methcillin, as shown by the use of standard disc diffusion method, BBL-Chromoagar and MIC-determination by Vitek II. In contrast penicillin-resistance of strains based on the presence of the blaZ-gene could be observed in 50 (73.5%) of the investigated strains. However, only 40 (58.8%) of these 50 blaZ-positive strains were phenotypically penicillin-resistant. According to the presented data, resistance to semisynthetic penicillins in S. aureus field strains seems to be not a major problem in dairy herds of the investigated countries despite the long-term use of these antibiotics in the field.

De 530 diferentes cepas de S. aureus aisladas de casos de mastitis subclínica bovina, se seleccionaron 68 cepas de S. aureus procedentes de Alemania (n = 26), Indonesia (n = 16), México (n = 16) y Brasil (n = 10), para estudiarlas en la presente investigación. Las cepas fueron analizadas fenotípica y genotípicamente para observar su resistencia a penicilina G y oxacilina. Para una identificación inicial se utilizó el sistema Api 32 Staph y la prueba de coagulasa. El resultado se confirmó por la detección molecular de los genes específicos de S. aureus 23S rRNA, nucleasa termoestable (nuc), factor aglutinante (clfA), coagulasa (coa) y la proteína A (spa) región Xr. La selección primaria de las cepas sospechosas se hizo al azar, seleccionando una cepa por hato; además sólo se incluyeron en el estudio cepas con diferentes perfiles de macrorrestricción. La resistencia genotípica a meticilina y penicilina se estudió mediante la identificación de los genes mecA y blaZ, respectivamente. El gen mecA fue detectado sólo en un aislamiento de S. aureus de Brasil y no fue resistente fenotípicamente a la meticilina, lo cual se demostró mediante los métodos de difusión estándar en discos, el uso del Chromoagar-BBL y la determinación de la concentración mínima inhibitoria (MIC, por sus siglas en inglés) por Vitek II. La detección genotípica de la resistencia de las cepas a la penicilina se basó en la detección del gen blaZ; y se observó en 50 cepas investigadas (73.5%). Sin embargo, sólo 40 cepas (58.8%) fueron fenotípicamente resistentes a la penicilina. Los resultados obtenidos muestran que la resistencia de las cepas aisladas de campo S. aureus a las penicilinas semisintéticas, actualmente no es un problema importante en las vacas lecheras, a pesar del uso extensivo de esas sustancias antibióticas en el campo en los países investigados.

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.