Challenging diagnosis and successful treatment of localised Mycobacterium avium subsp. hominissuis glossitis in a dog on long-term immunomodulatory therapy.

CASE HISTORY: A 3-year-old, intact female mixed-breed dog, weighing 7 kg, was presented with generalised swelling of the tongue, leading to impaired deglutition and episodes of dyspnoea. From the age of 2 years, the dog had been under immunosuppressive therapy due to atopic dermatitis. CLINICAL FINDINGS AND TREATMENT: Multiple nodular lesions at the apex of the tongue were noted as well as mandibular and retropharyngeal lymph node enlargement. Serum biochemistry results showed inflammatory changes. The results of several biopsies taken over 7 months indicated persistent pyogranulomatous and necrotising glossitis despite ongoing antimicrobial treatment, first with amoxicillin/clavulanic acid and then pradofloxacin. No foreign material, acid-fast bacteria or fungal hyphae were detected throughout. The final diagnosis of Mycobacterium avium subsp. hominissuis (Mah) was reached after PCR and bacterial culture were carried out on the third biopsy sample. Therapy was initiated with rifampicin, clarithromycin and doxycycline, leading to complete remission of the lesions. DIAGNOSIS: Severe chronic pyogranulomatous and necrotising glossitis associated with infection by Mah. CLINICAL RELEVANCE: This report describes challenges in the diagnosis and therapy of a localised Mah infection in an iatrogenically immunocompromised dog. Successful treatment was only achieved with a specific combination of antibiotics administered long-term. ABBREVIATIONS: AF: Acid-fast; ALP: Alkaline phosphatase; CT: Computed tomography; MAC: Mycobacterium avium complex; Mah: Mycobacterium avium subsp. hominissuis.

Phenotypic and genotypic approach to characterize a Trueperella pecoris strain isolated from necrotic vestibulitis of a camel (Camelus dromedarius).

The present study was designed to characterize phenotypically and genotypically a Trueperella (T.) pecoris strain isolated from necrotic vestibulitis of a 10-year-old camel (Camelus dromedarius). The species identity of T. pecoris 203/7 investigated in the present study could be confirmed by phenotypic properties and by phylogenetic analyses based on partial sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, elongation factor Tu encoding gene tuf and the target gene rpoB encoding the ß-subunit of bacterial RNA polymerase. T. pecoris strain 203/7 was grouped within the genus Trueperella in the family Arcanobacteriaceae. The 16S rRNA gene analysis showed a sequence identity of 99·9% to reference strain T. pecoris DSM 111392T . The present isolate was clearly identified as T. pecoris, the most recently described species of the genus Trueperella. Strain T. pecoris 203/7 was isolated in moderate numbers from necrotic vestibulitis of the camel and could be of some importance for the infectious process. However, the investigated strain represents the first isolation of T. pecoris from a camel.

Health assessment of harbour porpoises (PHOCOENA PHOCOENA) from Baltic area of Denmark, Germany, Poland and Latvia.

Harbour porpoise (Phocoena phocoena), the only resident cetacean species of the Baltic Sea is formed of two subpopulations populations, occurring in the western Baltic, Belt Seas and Kattegat and the Baltic Proper, respectively. Harbour porpoises throughout these areas are exposed to a large number of human activities causing direct and indirect effects on individuals, that might also harm this species on a population level. From Latvia, Poland, Germany and Denmark 385 out of 1769 collected dead harbour porpoises were suitable for extensive necropsy. The animals were collected between 1990 and 2015 and were either by-caught or found dead on the coastline. Following necropsies, histopathological, microbiological, virological and parasitological investigations were conducted. Females and males were equally distributed among the 385 animals. Most animals from the different countries were juveniles between 3 months and 3 years old (varying between 46.5 and 100% of 385 animals per country). The respiratory tract had the highest number of morphological lesions, including lungworms in 25 to 58% and pneumonia in 21 to 58% of the investigated animals. Of those with pneumonia 8 to 33% were moderate or severe. The alimentary, hearing, and haematopoietic systems had inflammatory lesions and parasitic infections with limited health impact. 45.5 to 100% of the animals from the different countries were known by-caught individuals, of which 20 to 100% varying between countries had netmarks. Inflammatory lesions, especially in the respiratory tract were found in higher numbers when compared to control populations in areas with less human activities such as arctic waters. The high number of morphological changes in the respiratory tract and of bycatches especially among immature animals before reaching sexual maturity is of serious concern, as well as the low number of adult animals among the material. Data on health status and the causes of death are valuable for management. A next step in this regard will combine data from health and genetic investigations in order to detect differences between the two populations of the Baltic.

Intestinal displacements in older harbour and grey seals.

Intestinal displacements including volvulus, torsion and invagination have been reported in various terrestrial and marine mammals. We conducted pathological investigations on 157 seals that had either stranded on the coasts of the North or Baltic Sea between 1996 and 2015 (115 harbour seals, >19 mo old; 21 grey seals, >13 mo old) or died while in human care (18 harbour seals, >19 mo old; 3 grey seals, >13 mo old). Intestinal displacements were found in 23% of the examined free-living harbour seals, in 5% of the stranded grey seals and in 17% of the harbour seals in human care. Intestinal volvulus, found in 24 cases, was characterized by twisting of the intestine along the mesenteric axis (180-540°) resulting in vascular obstruction and haemorrhagic infarction. In harbour seals, the sex ratio of individuals suffering from volvulus tended to be biased towards females during April to June, suggesting an elevated risk for pregnant females around birth time. Invagination was detected in 11 cases, 5 of which suffered from additional volvulus. Pathological findings associated with intestinal volvulus and invagination were sero-haemorrhagic effusions in the abdominal cavity. Enteritis, parasitic infection with gastric nematodes and intestinal acanthocephalans and bacterial infection with predominantly Clostridium perfringens and Escherichia coli were observed in most of the affected animals. In total, 30 investigated harbour and grey seals suffered from intestinal displacements. Pregnant females seemed to be more vulnerable around birth time. Causes of intestinal displacements remain undetermined, but are likely multifactorial.

First report on the isolation of Trueperella abortisuis from companion animals.

The present study gives a detailed phenotypic and genotypic characterization of three Trueperella abortisuis strains isolated from a ten year old male Hovawart dog with an abscess of anal sac, from urine of an eight year old European shorthair cat with urolithiasis and nephrolithiasis and from a 14year old Maine Coon cat with a perianal abscess, respectively. All three strains could be identified phenotypically, by MALDI-TOF MS analysis and genotypically by sequencing the 16S rDNA and the molecular target genes gap and tuf. The present study gives a first description of T. abortisuis of this origin.

Occurrence of anthropozoonotic parasitic infections and faecal microbes in free-ranging sperm whales (Physeter macrocephalus) from the Mediterranean Sea.

Sperm whales (Physeter macrocephalus) are the largest toothed whales and only living member of family Physeteridae. Present survey represents first report on cultivable faecal microbes and gastrointestinal helminths and protozoans infecting free-ranging sperm whales inhabiting Mediterranean Sea waters surrounding Balearic Archipelago, Spain. Twenty-five individual sperm whale scat samples, including one calf, were collected without disturbance of animals during the summer of 2016. Parasitological diagnostic methods, such as sodium acetate acetic formalin (SAF) method, carbol fuchsin-stained faecal smears, Giardia/Cryptosporidium coproantigen ELISAs and an Anisakis-specific PCR were applied for further identification. Five bacterial genera, i.e. Acinetobacter, Clostridium, Enterococcus, Staphylococcus and Streptococcus, and one fungus namely Cladosporium were identified. Parasitological infections included seven different parasite species with some of them bearing anthropozoonotic potential. Thus, four of these parasites were zoonotic, i.e. Anisakis, Balantidium, Diphyllobothriidae gen. sp. and Giardia. Additionally, Zalophotrema curilensis eggs, spirurid-like eggs and Cystoisospora-like oocysts were identified. Molecular characterization identified Anisakis physeteris as the species infecting these whales. This survey provides first records on occurrence of two zoonotic enteropathogenic protozoan parasites (Giardia and Balantidium) and of facultative pathogenic bacteria (Clostridium and Enterococcus) in sperm whales. Presented data should be considered as a baseline study for future monitoring surveys on anthropozoonotic pathogens affecting free-living sperm whale populations and enhance investigations on possible impact on public health as well as on isolated Mediterranean sperm whale subpopulation.

Lactococcus lactis, causative agent of an endocarditis valvularis and parietalis thromboticans in the allis shad, Alosa alosa (L.).

Since the 1940s, the anadromous allis shad, Alosa alosa (L.), has suffered population declines throughout its distribution range in Europe. In context of EU-LIFE projects for the reintroduction of the allis shad in the Rhine system, a comprehensive study was started in 2012 to investigate infectious diseases occurring in allis shad. In course of the study, 217 mature and young-of-the-year allis shad originating from the wild population from the Gironde-Garonne-Dordogne system (GGD-system) and the Rhine system as well as 38 allis shad from the breeding population were examined by use of bacteriological and histological methods. In 2012 and 2014, an endocarditis valvularis thromboticans caused by a coccoid bacterium was detected in 16% and 25% of mature allis shad originating from the GGD-system. Results of microbiologic examinations, including biochemical characteristics, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence analysis, revealed Lactococcus lactis as causative agent of this infection. This is the first report of an endocarditis valvularis and parietalis thromboticans caused by Lactococcus lactis in fish. Possible sources of infection as well as the impact for the reintroduction programme are discussed.

Bacteriological findings in the canine uterus during Caesarean section performed due to dystocia and their correlation to puppy mortality at the time of parturition.

Canine intrauterine bacteriological flora during dystocia is unknown. Thus, frequency (bacterial growth (not) detected), quality (species and number of different bacterial isolates) and quantity (colony-forming units) of intrauterine bacteria in relation to in utero foetal death in 50 bitches undergoing emergency Caesarean section were investigated. Bacterial growth was quantified from single colonies, (+) (0.5), to strong growth, +++ (3) and was observed in 34 bitches (68%), with Staph. epidermidis (n = 12), Staph. intermedius-group (n = 7), ß-haemolytic streptococci (n = 6), Staph. aureus, α- and γ-haemolytic streptococci (n = 4 each) being most common and one to four bacteria per sample. Regarding the quantity, most often (n = 46) low growth was identified. In bitches with living pups only (group I), mean number of isolates was 0.78 ± 0.83 compared to 1.60 ± 1.10 (living + stillborn pups, group II) and 1.0 ± 1.15 (stillborn pups only, group III) and mean bacterial growth in groups I/II/III was + (1.0, quantity), + (1.4) and ++ (1.6). Taking just positive samples into consideration, mean number of bacterial isolates was significantly higher in group II compared to I (p = .0088). We concluded that the canine uterus cannot be considered free of bacteria during dystocia. Mean numbers of different bacterial isolates and quantity of bacterial growth are higher in bitches with in utero foetal death.

Identification of Arcanobacterium hippocoleae by MALDI-TOF MS analysis and by various genotypical properties.

In the present study an Arcanobacterium hippocoleae strain isolated from a uterus swab of an apparently healthy mare could be identified by phenotypic properties, by MALDI-TOF MS analysis and genotypically by investigating the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region and the genes encoding the ß subunit of bacterial RNA polymerase (rpoB), elongation factor tu (tuf) and glyceraldehyde 3-phosphate dehydrogenase (gap). The presented data are one of the few reports about the species A. hippocoleae and might help to elucidate the role this species plays in infections of horses.

Application of a loop-mediated isothermal amplification (LAMP) assay for molecular identification of Trueperella pyogenes isolated from various origins.

In the present study 28 Trueperella pyogenes strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene cpn60 encoding chaperonin. No cross reaction could be observed with control strains representing four species of genus Trueperella and seven species of closely related genus Arcanobacterium. The present cpn60 LAMP assay might allow a reliable and low cost identification of T. pyogenes also in laboratories with less specified equipment.

Development of a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of Arcanobacterium pluranimalium.

In the present study 28 Arcanobacterium pluranimalium strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene pla encoding pluranimaliumlysin. No comparable reaction could be observed with control strains representing five species of genus Arcanobacterium and five species of genus Trueperella. The presented pla LAMP assay might allow a reliable and low cost identification of this bacterial pathogen also in laboratories with less specified equipment.

Identification of Arcanobacterium pluranimalium by matrix-assisted laser desorption ionization-time of flight mass spectrometry and, as novel target, by sequencing pluranimaliumlysin encoding gene pla.

In the present study 13 Arcanobacterium pluranimalium strains isolated from various animal origin could successfully be identified phenotypically by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and the pluranimaliumlysin encoding gene pla. The detection of mass spectra by MALDI-TOF MS and the novel genotypic approach using gene pla might help to identify A. pluranimalium in future and might elucidate the role this species plays in infections of animals.

Further characteristics of Arcanobacterium canis, a novel species of genus Arcanobacterium.

Comparable to previously conducted phenotypical and genotypical investigations characterizing Arcanobacterium canis, a newly described species with the type strain A. canis DSM 25104 isolated from an otitis externa of a dog, four additional A. canis strains isolated from infections of three dogs and one cat could reliably be identified by phenotypic properties, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and by sequencing the genomic targets 16S rDNA, 16S-23S rDNA intergenic spacer region, 23S rDNA, and the genes rpoB and gap. All four A. canis investigated in the present study were isolated from the infected animals together with several other bacterial species indicating that the pathogenic importance of A. canis remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approaches might help to identify A. canis in future and might elucidate the role this species plays in infections of dogs and cats.

[Bacterial susceptibility testings of the lower airways of diseased dogs]. / Bakterienisolate aus dem unteren Respirationstrakt von erkrankten Hunden und deren aktuelle Resistenzsituation.

OBJECTIVE: In this retrospective study over 5 years the bacterial flora of the deep airways of diseased dogs was sampled using bronchioalveolar lavage and its in-vitro susceptibility to numerous antimicrobial agents was analysed. MATERIAL AND METHODS: Susceptibility testing of bacterial isolates from 84 dogs performed from 2004 to 2009 were evaluated and compared to data collected in 1999/2000. RESULTS: The 99 bacterial isolates comprised Pasteurella spp. (27.3%), Bordetella bronchiseptica (20.2%), Staphylococcus spp. (18.2%), Escherichia coli (15.2%), Klebsiella spp. (8.1%), Pseudomonas spp. (7.0%), and Streptococcus spp. (4.0%). Lavage samples of eight dogs revealed simultaneous occurrence of more than one species. The majority of the tested Bordetella bronchiseptica-isolates were susceptible to fluoroquinolones as well as tetracycline, doxycycline and polymyxin B. The number of amoxicillin/clavulanic acid- and chloramphenicol-resistant isolates decreased compared to 1999/2000. The increase of chloramphenicol-susceptible isolates was statistically significant. The tested Staphylococcus-isolates revealed a susceptibility to fluoroquinolones, amoxicillin, cephalexin, doxycycline, and polymyxin B. Compared to the earlier study, a major portion exhibited susceptibility to chloramphenicol and tetracycline. None of the Klebsiella spp. tested in 1999/2000 was resistant to enrofloxacin, whereas only 62.5% of the isolates examined during 2004-2009 were susceptible. A susceptibility of all tested Klebsiella -isolates was detectable with respect to polymyxin B. Regarding E.coli , none of the tested antibiotics revealed an in-vitro activity against all of the tested isolates. The increase of amoxicillin/clavulanic acid-resistant isolates was statistically significant. CONCLUSION AND CLINICAL RELEVANCE: The study demonstrates the benefits of bronchioalveolar lavage as a diagnostic tool to ensure a responsible utilisation of antibiotics. Most of the tested bacterial isolates were susceptible to fluoroquinolones.

First description of Trueperella (Arcanobacterium) bernardiae of animal origin.

In the present study a Trueperella (Arcanobacterium) bernardiae strain isolated from an anal swab of a three-day-old piglet could be identified phenotypically, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing the 16S rDNA, the 16S-23S rDNA intergenic spacer region (ISR) and by sequencing the superoxide dismutase A encoding gene sodA. The present study gives the first information about the presence of T. (A.) bernardiae in specimen of animals.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for species identification of bacteria of genera Arcanobacterium and Trueperella.

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.

Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins.

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.

Characterization of Arcanobacterium abortisuis by phenotypic properties and by sequencing the 16S-23S rDNA intergenic spacer region.

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 ß-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.