Investigation of Genetic Defects in Severe Combined Immunodeficiency Patients from Turkey by Targeted Sequencing.

Primary immunodeficiencies (PIDs) represent a large group of disorders with an increased susceptibility to infections. Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiencies (PIDs) with marked T-cell lymphopenia. Investigation of the genetic aetiology using classical Sanger sequencing is associated with considerable diagnostic delay. We here established a custom-designed, next-generation sequencing (NGS)-based panel to efficiently identify disease-causing genetic defects in PID patients and applied this method in SCID patients of Turkish origin with previously undefined genetic aetiology. We used HaloPlex enrichment technology, a targeted, NGS-based method which was designed to diagnose patients with SCID and other PIDs. Our HaloPlex panel included a total of 356 PID-related genes, and we searched disease-causing mutations in 19 Turkish SCID patients without a genetic diagnosis. The coverage of targeted regions ranged from 97.47% to 99.62% with an average of 98.31% for all patients. All known SCID genes were covered with a percentage of at least 97.3%. We made a genetic diagnosis in six of 19 (33%) patients, including four novel disease-causing mutations identified in RAG1, JAK3 and IL2RG, respectively. We showed that this NGS-based method can provide rapid genetic diagnosis for patients suffering from SCID, potentially facilitating clinical treatment decisions.

An in vitro attenuated strain of Histomonas meleagridis provides cross-protective immunity in turkeys against heterologous virulent isolates.

In the current study, cross-protective immunity induced by a well-defined clonal strain of Histomonas meleagridis, attenuated by prolonged in vitro cultivation against different clonal heterologous isolates of the same parasite was investigated. For this purpose, 86 turkey poults were assigned to groups consisting of 9-10 birds. Birds of four groups were vaccinated on their 1st day of life followed by re-vaccination on their 14th day of life when the remaining turkeys were left untreated. The challenge was performed using four strains of H. meleagridis that were isolated from chickens or turkeys from different outbreaks of histomonosis in Europe and three of them showed diversities in their genome. Hence, every strain used for the challenge was applied to a group of vaccinated and a group of non-vaccinated birds while birds of the negative control group were sham inoculated. Non-vaccinated birds suffered from severe histomonosis due to the challenge with fatalities reaching from 5 to 10 turkeys per group. Vaccinated birds did not contract clinical signs of the disease following challenge and the increase in weight was unaffected compared to birds of the negative control group. A significant difference in lesion scores was recorded between vaccinated and non-vaccinated groups, with very few instances of liver involvement in the former groups. Livers of vaccinated birds that were without recordable macroscopic lesions were also found negative by immunohistochemical investigation. According to the data obtained, the present study demonstrates, for the first time, the cross-protective capability of a tentative vaccine strain of H. meleagridis attenuated in vitro against heterologous virulent isolates of different origin.

A single strain of Tetratrichomonas gallinarum causes fatal typhlohepatitis in red-legged partridges (Alectoris rufa) to be distinguished from histomonosis.

Typhlohepatitis was observed in a flock of 2500 red-legged partridges in Great Britain, characterized by the sudden deaths of 15 birds within 2 days. Necropsy of five dead birds revealed severe lesions in the caeca with thickened caecal walls, a reddened lining and bloody contents. The livers contained multiple miliary lesions and similar pathological changes were found in the spleens of some birds. Microscopic examination of intestinal contents showed the occurrence of coccidial oocysts in two partridges. Different methods for the detection of bacteria from liver and intestine samples were conducted without positive results. Histopathological examination revealed the presence of protozoan parasites in the caecum, liver and spleen of the affected birds. In situ hybridization (ISH) for the detection of trichomonads resulted in positive findings and polymerase chain reaction (PCR) confirmed the presence of Tetratrichomonas gallinarum in the lesions. Additionally, archived tissues of red-legged partridges from different flocks suffering from severe typhlohepatitis in Great Britain in 2008 and 2009 were re-investigated by ISH and PCR. Beside the sporadic occurrence of histomonosis, in most of the cases trichomonads were detected by ISH in the caecum and liver of affected birds. Furthermore, dissemination of the flagellate into the lung and bursa of Fabricius could be demonstrated. Analyses of T. gallinarum DNA obtained from the different cases resulted in homologous nucleotide sequences. Altogether, the results demonstrate the circulation of a virulent strain of T. gallinarum in reared red-legged partridges.

Riemerella anatipestifer outbreaks in commercial goose flocks and identification of isolates by MALDI-TOF mass spectrometry.

Several outbreaks of Riemerella anatipestifer in commercial geese occurred within a short time period. A serious disease was recognized in the affected birds, mainly characterized by depression and severe neurologic disturbances. The morbidity ranged from 20 to 30% and the mortality from 5 to 20%. Generally, the clinical signs started at the age of 8 to 10 days. Post-mortem examination revealed fibrinous pericarditis, perihepatitis and airsacculitis in all birds. Some of the birds also had synovitis of the tibio-tarsal joints and oedematous swelling of the subcutaneous tissues around these joints and metatarsus. Histology revealed a characteristic severe inflammation with heterophilic granulocytes in different organs. Bacteriological investigation was made from several organs and R. anatipestifer could be isolated from all birds investigated. The identification of these clinical isolates, done for the first time by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, confirmed the aetiology. Sequence analysis showed 100% similarity between the clinical isolates, indicating a common source of infection.

Outbreak of cutaneous form of poxvirus on a commercial turkey farm caused by the species fowlpox.

The present report documents the occurrence of a poxvirus infection in commercial meat turkeys. The affected farm had six flocks, with a total of 11,680 birds at different ages; birds from two of these flocks were affected. The clinical picture was characterized by severe epithelial lesions and proliferations on the head and neck regions as reported for the cutaneous form of poxvirus infection. Except for these lesions, no adverse clinical signs or gross pathologic lesions were observed. Only a low number of birds was affected (n = 20) and no increase of mortality could be seen. Bacteriologic investigations from the lesions revealed multiresistant Staphylococcus aureus. Eosinophilic inclusions (Bollinger bodies) in histologic examinations in the cytoplasm of keratinocytes were noticeable. Typical pox virions were demonstrated by electron microscopy, and poxvirus was isolated on the chorioallantoic membrane of specific-pathogen-free chicken eggs. Further identification of the poxvirus species was carried out by PCR and sequencing, revealing an infection with the species fowlpox. Layers in vicinity of the turkey farm that also were affected by fowlpox were considered as potential source of infection. Although it is assumed that avian poxviruses are strongly species specific, the present case report reinforces the changing picture of poxvirus infections in turkeys. Furthermore, it supports the assumption of previous data that fowlpox virus has to be seen as recently emerging pathogen in turkeys.

Outbreaks of Streptococcus gallolyticus subsp. pasteurianus in Goslings Characterized by Central Nervous Symptoms.

The present report describes outbreaks of Streptococcus gallolyticus subsp. pasteurianus in young geese flocks in Austria. The flocks, comprising 160-1450 goslings of 2-3 wk of age, experienced increased mortalities The clinical signs were characterized by severe central nervous symptoms, namely leg paddling and torticollis. The postmortem investigation revealed hepatitis, splenitis, and a low amount of liquid fluid in the coelomic cavity. Livers were of fragile texture, with white necrotic areas. The latter were also found in spleens. No macroscopic lesions were seen in brains. Bacteriologic investigation followed by bacterial identification by matrix-assisted laser desorption time-of-flight mass spectrometry and phylogenetic analysis of the partial 16S rRNA region revealed the presence in heart, liver, spleen, and brain of S. gallolyticus subsp. pasteurianus. Histologic investigation revealed multifocal necrosis in liver and spleen samples together with infiltration of mononuclear cells and heterophilic granulocytes. Furthermore, in the lesions, coccoid bacteria could be identified. No histopathologic changes were observed in brain samples from goslings, except in one bird in which accumulation of coccoid bacteria in blood vessels of the brain samples was present. Antibiotic sensitivity tests revealed identical profiles for all strains, which were susceptible to penicillins, cephalosporins, chloramphenicol, imipenem, and tylosin. However, resistance was found against quinolones, aminoglycosides, tetracycline, and trimethoprim-sulfamethoxazole, which are commonly used to treat infections with gram-positive bacteria.

Reporte de caso­Brotes de Streptococcus gallolyticus subsp. pasteurianus en gansitos caracterizados por síntomas nerviosos centrales. El presente informe describe brotes de Streptococcus gallolyticus subsp. pasteurianus en parvadas de gansos jóvenes en Austria. Las bandadas, que comprendían entre 160 a 1450 gansos de 2 a 3 semanas de edad, experimentaron aumento de la mortalidad. Los signos clínicos se caracterizaron por síntomas severos del sistema nervioso central, incluyendo, movimientos de pataleo y tortícolis. La investigación post mórtem reveló hepatitis, esplenitis y la presencia de líquido en la cavidad celómica en poca cantidad. Los hígados presentaron textura frágil, con áreas necróticas blancas. Estos últimos también se encontraron en bazos. No se observaron lesiones macroscópicas en el cerebro. La investigación bacteriológica seguida de la identificación bacteriana mediante espectrometría de masas MALDI-TOF y el análisis filogenético de la región parcial de ARNr 16S revelaron la presencia en el corazón, el hígado, el bazo y en el cerebro de S. gallolyticus subsp. pasteurianus. La investigación histológica reveló necrosis multifocal en muestras de hígado y bazo junto con infiltración de células mononucleares y granulocitos heterófilos. Además, en las lesiones se pudieron identificar bacterias de morfología cocoide. No se observaron cambios histopatológicos en muestras de cerebro de los gansitos, excepto en un ave en la que se observó acumulación de bacterias cocoides en los vasos sanguíneos de las muestras de cerebro. Las pruebas de sensibilidad a los antibióticos revelaron perfiles idénticos para todas las cepas, que eran susceptibles a penicilinas, cefalosporinas, cloranfenicol, imipenem y tilosina. Sin embargo, se encontró resistencia contra quinolonas, aminoglucósidos, tetraciclina y trimetoprim-sulfametoxazol, que se usan comúnmente para tratar infecciones con bacterias grampositivas.

Characterization of Austrian koi herpesvirus samples based on the ORF40 region.

Using a PCR that amplifies a region of the thymidine kinase (TK) gene, an epidemic spread of koi herpesvirus (KHV) was determined in koi carps in Austria in 2007. A total of 15 virus samples from different locations in Austria were analyzed to determine their genetic relatedness following PCR and nucleic acid sequencing of the open reading frame 40 (ORF40) region of the KHV genome. ORF40-specific PCR amplification products that were obtained from tissue samples shared 100% nucleotide sequence identity with the published sequence of the Japanese strain of KHV. The ORF40 sequence of one isolate from the UK that was included in the present study was 100% identical with the published sequence of an Israeli strain of KHV. This is the first study that used a larger number of samples and a PCR method, which allowed distinguishing all 3 strains of KHV. The present investigation provides information on the epidemiology of KHV infections in Europe and describes a useful molecular tool for epidemiological studies.

Identification and molecular characterization of numerous Histomonas meleagridis proteins using a cDNA library.

SUMMARYHistomonas meleagridis is a protozoan parasite of various galliform birds causing a type of enterohepatitis termed histomonosis or 'blackhead disease'. Due to the ban of chemotherapeutic substances and an increase in free-range poultry production, histomonosis is currently a re-emerging disease. So far limited molecular knowledge is available. In the present work, mRNAs coding for antigenic proteins of H. meleagridis were identified. For this purpose, a cDNA expression library was constructed from a mono-eukaryotic culture of H. meleagridis. The library was screened with polyclonal rabbit serum raised against purified H. meleagridis trophozoites. Polyclonal rabbit serum specifically recognized the same major H. meleagridis antigens as chicken and turkey sera originating from animal trials, but displayed a significantly lower bacteria-dependent background signal. After 2 rounds of screening, a total of 95 positive clones were sequenced. Bioinformatics analyses were performed on nucleotide and deduced amino acid sequences, identifying 37 unique clones. Based on the homology to other protozoan parasites, mostly Trichomonas vaginalis, the clones were grouped according to functional aspects: structural proteins, possible surface proteins, oxygen reducing proteins, ribosomal proteins, protein kinases and various other intracellular proteins.

Phylogenetic analysis of spring viraemia of carp virus isolates from Austria indicates the existence of at least two subgroups within genogroup Id.

Genetic relationships between 22 spring viraemia of carp virus (SVCV) isolates from Austria collected between 1994 and 2007 were determined based on the partial nucleotide sequence of the glycoprotein gene (G gene). Phylogenetic analyses located all Austrian isolates except one in genogroup Id. One isolate collected in 2007 was placed within the SVCV Ia genogroup. More importantly, the study also revealed 3 distinct clusters within genogroup Id, designated Id1, Id2 and Id3. Existence of subgroups Id2 and Id3 within the genogroup Id was supported by high bootstrap values. The genetic clustering could neither be linked to host species nor to geographic localization of fish farms. Furthermore, no clear link could be established between the pathological lesions and phylogenetic relationship. However, time-dependent division of the isolates was observed. Viruses from the Id1 cluster were mainly sampled in Austria in the 1990s and up until 2003, whereas all viruses from the Id2 subgroup were isolated after 2003.

Is C-reactive protein a better predictor of recurrent carotid disease following carotid endarterectomy than established risk factors for atherosclerosis?

BACKGROUND: The study was undertaken to identify possible relations between established and novel risk factors for atherosclerosis (gender, age, diabetes mellitus, hypertension, smoking and C-reactive protein) and the possibility of carotid restenosis following carotid endarterectomy (CEA). PATIENTS AND METHODS: A prospective study of 193 consecutive patients, admitted electively for carotid endarterectomy during 68 months, was conducted. 131 patients had symptomatic and 62 asymptomatic carotid disease. An attempt was made to follow-up on all operated arteries with duplex sonography at 2 months, 6 months, 12 months, and 24 months postoperatively. The correlation of previously mentioned variables (gender, age, diabetes mellitus, hypertension, smoking and average values of C-reactive protein) with progressive or recurrent disease was determined by chi-square analysis and analysis of variance. RESULTS: Of all 193 examined patients 29 demonstrated increasing degree of artery stenosis, while recurrent artery stenosis of > 69% was diagnosed in 11 patients. Age, gender, hypertension, and diabetes did not play a significant role in the presence of progressive (or recurrent) disease, while active smokers and patients with preoperative and average C-reactive protein (CRP) levels over 3.0 mg/L had a greater propensity to develop progression (or recurrence) of carotid disease. CONCLUSIONS: Increased levels of CRP in serum may be a better predictor of carotid restenosis after CEA than other established risk factors for vascular disease--except active smoking.

Effects of hyperbaric oxygen therapy on experimental burn wound healing in rats: a randomized controlled study.

A body of data supports the efficacy of hyperbaric oxygen (HBO2) therapy in the treatment of thermal burns, but the role of HBO2 in the treatment of burn injury remains a subject of controversy. The aim of this study was to evaluate possible positive effects of HBO2 on the experimental burn wound healing. Deep second degree burns were produced on the depilated backs of 70 male Wistar rats using a validated burn protocol. The animals were assigned randomly to one of two groups: 35 to the control group, which was treated with silver sulphadiazine and placebo gas, and 35 to the experimental group, which was treated with silver sulphadiazine and HBO2. The main outcome measure was wound healing, characterized by formation of post-burn edema, neoangiogenesis, number of regeneratory active follicles, necrosis staging, margination of leukocytes, and time of epithelization. A significant reduction of the post-burn edema after treatment with HBO2 (p = 0.009) was found. HBO2 had a beneficial effect on neoangiogenesis (p = 0.009). The number of preserved regeneratory active follicles was significantly higher (p = 0.009) and epithelial regeneration was more rapid in the experimental group (p = 0.048). There were no significant differences for margination of leukocytes (p = 0.55) or necrosis staging (p = 1.00). These data further support earlier conclusions that HBO2 is beneficial in the healing of burn wounds.

Haloperidol-stomach lesions attenuation by pentadecapeptide BPC 157, omeprazole, bromocriptine, but not atropine, lansoprazole, pantoprazole, ranitidine, cimetidine and misoprostol in mice.

The focus was on haloperidol (central dopamine antagonist)-stomach lesion, a longly described suitable counterpart of dopamine blocker cysteamine-duodenal lesion. In this, the contribution of blockade of central/peripheral dopamine receptors and prostaglandins synthesis, along with influence of antiulcer agents was evaluated in mice. Male NMRI Hannnover mice were sacrificed 24 h after haloperidol (25 mg/kg b.w. i.p., given alone or with saline (haloperidol+saline) (i) or in combination (ii,iii)). Supporting central dopamine predominance for haloperidol stomach lesion induction, co-administration of peripheral dopamine receptor antagonist domperidone (5 mg/kg i.p.) (haloperidol+ domperidone) (ii), or prostaglandin synthesis inhibitor indomethacin (10 mg/kg s.c.) (haloperidol+ indomethacin) (iii) did not aggravate this lesion. (i) In haloperidol+saline challenged mice the lesions were inhibited by co-administration (/kg i.p.) of a gastric pentadecapeptide BPC 157, GlyGluProProProGlyLysProAlaAspAspAlaGlyLeuVal, M.W. 1419 (10 microg, 10 ng, 10 pg, but not 1 pg, 100 fg, 10 fg), bromocriptine (10 mg), omeprazole (10 mg, 100 mg, but not 1 mg). Atropine (10, 100, 200 mg), pirenzepine (10, 100, 200 mg), misoprostol (10, 100, 200 microg), pantoprazole (1, 10, 100 mg), lansoprazole (0.1, 1, 10 mg), cimetidine (10, 100, 200 mg) and ranitidine (10, 100, 200 mg) were not effective. (ii) Dopamine peripheral blockade influence: in haloperidol+domperidone mice, previously effective bromocriptine, pentadecapeptide BPC 157 (10 microg) or omeprazole (10 mg) did not attenuate stomach lesions. (iii) Prostaglandins synthesis blockade effect: in haloperidol+indomethacin mice, previously effective agents, bromocriptine or omeprazole were not active, while BPC 157 effect was only lessened.

Identification of a new reovirus causing substantial losses in broiler production in France, despite routine vaccination of breeders.

Numerous cases of tenosynovitis appeared in France causing high morbidity in free-range and standard broilers. The main clinical findings were lameness, stunting and non-uniform bodyweights. Although the natural mortality was low, the economic losses due to birds that had to be removed from the flock prematurely, downgrading of carcases and lower average weights at slaughter were substantial. Postmortem examinations, bacteriological, virological and serological examination confirmed the aetiology of avian orthoreovirus (ARV)-induced tenosynovitis. The isolated ARVs were analysed serologically and genetically. Sequencing of σC RT-PCR products and phylogenetic analysis revealed a new type of ARV. The virus was not neutralised in serum neutralisation test using monovalent sera from vaccinated chickens. Together with the flock data, epidemiology of these recent reovirus outbreaks in France was reconstructed. It is concluded that these reovirus isolates differ serologically and genetically from the well described reovirus isolates used in commercial vaccines which were not capable of preventing the disease. The outbreaks resulted in substantial losses in broilers from vaccinated breeders.

Phylogenetic analysis of avian hepatitis E virus samples from European and Australian chicken flocks supports the existence of a different genus within the Hepeviridae comprising at least three different genotypes.

Using PCRs that amplify regions of helicase and capsid genes, the presence of avian hepatitis E virus (avian HEV) was determined in samples from European and Australian chicken flocks (collected from 2005 to 2007 and 1986 to 1995, respectively). A total of 27 virus samples from 9 countries were analysed to determine the phylogenetic relationship following PCRs and nucleic acid sequencing of the helicase and capsid regions of 18 avian HEV samples. For comparison, helicase and capsid sequences of completely sequenced avian HEVs from Europe, Australia and the USA were used. In addition, available helicase and capsid sequences of other avian HEVs and four mammalian HEVs were included. At least three genotypes within the avian HEV were revealed. These genotypes tend to be differentiated geographically. Altogether, the present investigation is of importance to understand the epidemiology of avian HEV infections in chickens and gives new insight into the phylogenetic relationships between isolates. Furthermore, we would like to propose that avian HEV represent a separate genus within the Hepeviridae consisting of different genotypes.

The Tpv2 family of retrotransposons of Phaseolus vulgaris: structure, integration characteristics, and use for genotype classification.

The Tpv2 family of transposable elements of common bean, Phaseolus vulgaris, belongs to the Ty1/copia group of long terminal repeat (LTR) containing retrotransposons. By reverse transcriptase (RT)-PCR and by analysis of genomic clones, we characterized four of the approximately 40 copies present in the Phaseolus genome, and the genomic environment of their integration sites. Tpv2 integrated preferentially into actively transcribed regions. While none of the isolated elements had all the functional domains necessary for transposition, analysis of bean cultivars suggested that some members of the Tpv2 family transposed in recent breeding history. Probes derived from Tpv2, as well as flanking genomic sequences, may be useful for classifying Phaseolus cultivars.