Investigation of a herpesvirus outbreak in mixed breeds of adult domestic ducks using next generation sequencing.

This report characterizes the first lethal outbreak of Marek's disease on a large farm of mixed-breed adult ducks (>18,000) and identifies the pathogen that resulted in high mortality (35%). Clinical signs included inappetence, respiratory distress, depression, muscle weakness, and ataxia. Post mortem revealed enlarged fragile liver mottled with miliary whitish spots and an enlarged spleen. Histopathology revealed hepatocellular necrosis with eosinophilic intra-nuclear inclusion bodies, necrosis of splenic follicles and degeneration/necrosis of renal tubules. The disease was tentatively diagnosed as a herpesvirus infection, confirmed by virus isolation from the liver. DNA was isolated from 15-year-old archival formalin-fixed tissues from infected ducks and subjected to next generation sequencing (NGS). Despite highly degraded DNA, short stretches of G- and C-rich repeats (TTAGGG and TAACCC) were identified as telomeric repeats frequently found in herpesviruses. Megablast and further investigative bioinformatics identified presence of Marek's disease virus (MDV), a Gallid alphaherpesvirus type 2 (GAHV-2), as the cause of the acute fatal infection. The source of infection may be attributed to a dead migratory flamingo found close to the duck enclosures three days prior to the outbreak; hence, GAHV-2 may also be responsible for the fatal infection of the flamingo accentuated by heat stress. Considering the possible spread of this highly contagious and lethal virus from a flamingo to the ducks, and the increasing zoonosis of animal viruses into humans, such as monkey B alphaherpesvirus transmission from macaques to humans with ~80% fatality, this observation has important ramifications for human health and safety of the poultry industry.

Camelids and Cattle Are Dead-End Hosts for Peste-des-Petits-Ruminants Virus.

Peste-des-petits-ruminants virus (PPRV) causes a severe respiratory disease in small ruminants. The possible impact of different atypical host species in the spread and planed worldwide eradication of PPRV remains to be clarified. Recent transmission trials with the virulent PPRV lineage IV (LIV)-strain Kurdistan/2011 revealed that pigs and wild boar are possible sources of PPRV-infection. We therefore investigated the role of cattle, llamas, alpacas, and dromedary camels in transmission trials using the Kurdistan/2011 strain for intranasal infection and integrated a literature review for a proper evaluation of their host traits and role in PPRV-transmission. Cattle and camelids developed no clinical signs, no viremia, shed no or only low PPRV-RNA loads in swab samples and did not transmit any PPRV to the contact animals. The distribution of PPRV-RNA or antigen in lymphoid organs was similar in cattle and camelids although generally lower compared to suids and small ruminants. In the typical small ruminant hosts, the tissue tropism, pathogenesis and disease expression after PPRV-infection is associated with infection of immune and epithelial cells via SLAM and nectin-4 receptors, respectively. We therefore suggest a different pathogenesis in cattle and camelids and both as dead-end hosts for PPRV.

Ignatzschineria cameli sp. nov., isolated from necrotic foot tissue of dromedaries (Camelus dromedarius) and associated maggots (Wohlfahrtia species) in Dubai.

Five bacterial strains, UAE-HKU57T, UAE-HKU58, UAE-HKU59, UAE-HKU60 and UAE-HKU61, were isolated in Dubai, UAE, from necrotic foot tissue samples of four dromedaries (Camelus dromedarius) and associated maggots (Wohrlfartia species). They were non-sporulating, Gram-negative, non-motile bacilli. They grew well under aerobic conditions at 37 °C, but not anaerobically. The pH range for growth was pH 7.0-9.0 (optimum, pH 7.5-8.0) and the strains could tolerate NaCl concentrations (w/v) up to 2 % (optimum, 0.5 %). They were catalase- and cytochrome oxidase-positive, but caseinase-, gelatinase- and urease-negative. Their phenotypic characters were distinguishable from other closely related species. Phylogenetic analyses of the almost-complete 16S rRNA gene and partial 23S rRNA gene, gyrB, groEL and recA sequences revealed that the five isolates were most closely related to undescribed Ignatzschineria strain F8392 and Ignatzschineria indica, but in most phylogenies clustered separately from these close relatives. Average nucleotide identity analysis showed that genomes of the five isolates (2.47-2.52 Mb, G+C content 41.71-41.86 mol%) were 98.00-99.97% similar to each other, but ≤87.18 % similar to other Ignatzschineriaspecies/strains. Low DNA relatedness between the five isolates to other Ignatzschineriaspecies/strains was also supported by Genome-to-Genome Distance Calculator analysis. The chemotaxonomic traits of the five strains were highly similar. They were non-susceptible (intermediate or resistant) to tetracycline and resistant to trimethoprim/sulphamethoxazole. The name Ignatzschineria cameli sp. nov. is proposed to accommodate these five strains, with strain UAE-HKU57T (=CCOS1165T=NBRC 113042T) as the type strain.

Serodiagnosis of aspergillosis in falcons (Falco spp.) by an Afmp1p-based enzyme-linked immunosorbent assay.

Aspergillosis in falcons may be associated with high mortality and difficulties in clinical and laboratory diagnosis. We previously cloned an immunogenic protein, Afmp1p, in Aspergillus fumigatus and showed that anti-Afmp1p antibodies were present in human patients with A. fumigatus infections. In this study, we hypothesise that a similar Afmp1p-based enzyme-linked immunosorbent assay (ELISA) could be applied to serodiagnose falcon aspergillosis. A specific polyclonal antibody was first generated to detect falcon serum IgY. Horseradish peroxidase-conjugate of this antibody was then used to measure anti-Afmp1p antibodies in sera collected from falcons experimentally infected with A. fumigatus, and the performance of the Afmp1p-based ELISA was evaluated using sera from healthy falcons and falcons with documented A. fumigatus infections. All four experimentally infected falcons developed culture- and histology-proven invasive aspergillosis. Anti-Afmp1p antibodies were detected in their sera. For the Afmp1p-based ELISA, the mean ± SD OD450 nm using sera from 129 healthy falcons was 0.186 ± 0.073. Receiver operating characteristics curve analysis showed an absorbance cut-off value of 0.407. One negative serum gave an absorbance outside the normal range, giving a specificity of 99.2%. For the 12 sera from falcons with confirmed aspergillosis, nine gave absorbance values ≥ cut-off, giving a sensitivity of 75%. The Afmp1p-based ELISA is useful for serodiagnosis of falcons with aspergillosis.

Disseminated Cryptococcus deuterogattii (AFLP6/VGII) infection in an Arabian horse from Dubai, United Arab Emirates / Infección diseminada por Cryptococcus deuterogattii (AFLP6/VGII) en un caballo árabe en Dubái, Emiratos Árabes Unidos

Background. During the past decades there has been an increase in cryptococcal infections caused by the basidiomycetous yeast species Cryptococcus gattii sensu lato, among humans and animals that live in endemic regions in Australia, Europe and the Americas. Unlike human cryptococcosis, little epidemiological data are available about C. gattii sensu lato infections in horses. Case report. A fatal case of a disseminated C. gattii sensu lato infection in an 11-year-old Arabian gelding imported from South Africa into the United Arab Emitares is reported. Tissue samples were studied by conventional mycology procedures and the obtained cryptococcal isolate was molecularly characterized by mating-type determination, amplified fragment length polymorphism (AFLP) fingerprinting, and multi-locus sequence typing (MLST). Phylogenetic analysis was performed to investigate the geographic origin of the cryptococcal isolate. The isolate was identified as Cryptococcus deuterogattii (AFLP6/VGII), mating-type α. Phylogenetic analysis showed that it was closely related to another C. deuterogattii isolate from the Middle East. Conclusions. A second case of a C. deuterogattii infection in the Middle East is described. It is likely that the horse acquired the infection in the Middle East, as the isolate is closely related to that of a recent human case from that region (AU)

Antecedentes. Durante las dos últimas décadas, las infecciones criptocócicas causadas por el hongo levaduriforme basidiomiceto Cryptococcus gattii sensu lato se han incrementado entre los seres humanos y los animales que viven en regiones endémicas de Australia, Europa y América. A diferencia de la criptococosis humana, existen muy pocos datos epidemiológicos disponibles sobre las infecciones por C. gattii sensu lato en los caballos. Caso clínico. Se expone el caso de una criptococosis diseminada fatal por C. gattii sensu lato en un caballo árabe castrado de 11 años de edad, importado desde Sudáfrica a los Emiratos Árabes Unidos. Las muestras de tejido analizadas por métodos microbiológicos convencionales permitieron el aislamiento de un criptococo que fue posteriormente caracterizado por técnicas moleculares para la determinación del tipo sexual, la obtención del perfil AFLP (amplified fragment length polymorphism) o polimorfismo de tamaño de fragmentos amplificados, y la tipificación por secuenciación multilocus (multi-locus sequence typing [MLST]). Se llevó a cabo un análisis filogenético para investigar el origen geográfico del criptococo aislado. Mediante PCR y AFLP el aislamiento fue identificado como Cryptococcus deuterogattii (AFLP6/VGII) y tipo sexual α. El análisis filogenético mostró que el aislamiento se encuentra muy próximo a otro único aislamiento de C. deuterogattii de Oriente Medio. Conclusiones. Este es el segundo caso descrito de infección por C. deuterogattii en Oriente Medio. Parece que el caballo adquirió la infección en aquella región, ya que el aislamiento muestra una relación muy próxima con otro de un caso reciente en un ser humano de esa región (AU)

Disseminated Cryptococcus deuterogattii (AFLP6/VGII) infection in an Arabian horse from Dubai, United Arab Emirates.

BACKGROUND: During the past decades there has been an increase in cryptococcal infections caused by the basidiomycetous yeast species Cryptococcus gattii sensu lato, among humans and animals that live in endemic regions in Australia, Europe and the Americas. Unlike human cryptococcosis, little epidemiological data are available about C. gattii sensu lato infections in horses. CASE REPORT: A fatal case of a disseminated C. gattii sensu lato infection in an 11-year-old Arabian gelding imported from South Africa into the United Arab Emitares is reported. Tissue samples were studied by conventional mycology procedures and the obtained cryptococcal isolate was molecularly characterized by mating-type determination, amplified fragment length polymorphism (AFLP) fingerprinting, and multi-locus sequence typing (MLST). Phylogenetic analysis was performed to investigate the geographic origin of the cryptococcal isolate. The isolate was identified as Cryptococcus deuterogattii (AFLP6/VGII), mating-type α. Phylogenetic analysis showed that it was closely related to another C. deuterogattii isolate from the Middle East. CONCLUSIONS: A second case of a C. deuterogattii infection in the Middle East is described. It is likely that the horse acquired the infection in the Middle East, as the isolate is closely related to that of a recent human case from that region.

Uniparental chicken offsprings derived from oogenesis of chicken primordial germ cells (ZZ).

Cloning (somatic cell nuclear transfer) in avian species has proven unachievable due to the physical structure of the avian oocyte. Here, the sexual differentiation of primordial germ cells with genetic sex ZZ (ZZ PGCs) was investigated in female germline chimeric chicken hosts with the aim to produce uniparental offspring. ZZ PGCs were expanded in culture and transplanted into the same and opposite sex chicken embryos which were partially sterilized using irradiation. All tested chimeric roosters (ZZ/ZZ) showed germline transmission with transmission rates of 3.2%-91.4%. Unexpectedly, functional oogenesis of chicken ZZ PGCs was found in three chimeric hens, resulting in a transmission rate of 2.3%-27.8%. Matings were conducted between the germline chimeras (ZZ/ZZ and ZZ/ZW) which derived from the same ZZ PGCs line. Paternal uniparental chicken offspring were obtained with a transmission rate up to 28.4% and as expected, all uniparental offspring were phenotypic male (ZZ). Genotype analysis of uniparental offsprings was performed using 13 microsatellite markers. The genotype profile showed that uniparental offspring were 100% genetically identical to the donor ZZ PGC line, shared 69.2%-88.5% identity with the donor bird. Homozygosity of the tested birds varied from 61.5% to 84.6%, which was higher than the donor bird (38.5%). These results demonstrate that male avian ZZ PGCs can differentiate into functional ova in an ovary, and uniparental avian clones are possible. This technology suggests novel approaches for generating genetically similar flocks of birds and for the conservation of avian genetic resources.

Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein:protein interaction and modulates its downstream signaling.

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the "junctional epitope" nature of VHH6, a camelid single domain antibody recognizing the IL-6-gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions.

Chlamydotis macqueenii and C. undulata (Aves: Otididae) are new hosts for Caryospora megafalconis (Apicomplexa: Eimeriidae) and proposal of the genus Avispora gen. nov.

Oocysts of a coccidian morphologically matching features of Caryospora megafalconis Klüh, 1994 were found in fecal samples and contents of the large intestines in five wild caught Clamydotis macqueenii (Gray) and 19 captive bred C. undulata (Jaquin). Scrapings of the intestinal mucosa of necropsied birds revealed macrogamonts and unsporulated oocysts. Sporulation in a potassium dichromate solution at 26 °C was completed in 48 h. Intestinal contents and sporulated oocysts obtained from feces of infected bustards as well as sporulated oocysts of C. megafalconis and C. neofalconis Böer, 1982 from two Falco rusticolis Linnaeus and one F. peregrinus Tunstall were used for DNA sequencing of the cox1, 18S ribosomal ribonucleic acid (rRNA), and 28S rRNA genes. The phylogenetic trees for all three genes showed that sequences of the material from bustards were identical with C. megafalconis from falcons. C. neofalconis and C. daceloe Yang et al., 2014 were situated in the neighboring clades. Contrary to this, subsequent sequences of C. bigenetica Wacha and Christiansen, 1982 from rattlesakes are at a distinct distance suggesting that despite morphological similarities of the oocysts, there are differences between Caryospora species of birds and reptiles. For this reason, it might be reasonable to transfer avian Caryospora species into a new genus Avispora.

VITAMIN B6 (PYRIDOXINE HYDROCHLORIDE) TOXICOSIS IN FALCONS.

This manuscript reports three independent accidental cases of vitamin (Vit) B6 toxicosis in gyrfalcons (Falco rusticolus) and peregrine falcons (Falco peregrinus) and a toxicology study that was conducted to characterize the clinical responses of gyrfalcons and gyrfalcon × peregrine falcons to a range of single intramuscular (IM) and oral (PO) doses of Vit B6. Both lethal and nonlethal doses were determined. Twelve female gyrfalcons died following IM injection of 1 ml of a vitamin B preparation. Within 30 min of injection, the birds passed pistachio green-colored urates and progressed to vomiting, anorexia, cessation of normal activity, ptosis, collapse, and death, occurring 24-36 hr post injections. Three individuals vomited frothy, partially digested blood and had clonic spasms and convulsions. Postmortem and histopathology revealed multifocal severe hepatic necrosis, splenic lymphoid tissue depletion and hemorrhages with arterial necrosis, and acute renal tubular necrosis. Following administration of a different, oral, mineral-vitamin supplement, a total of 21 peregrine falcons in two separate European facilities died suddenly. Histology of the liver showed diffuse congestion and multifocal coagulative necrosis with mild infiltration of heterophils. The particular nutritional supplement, used by both breeders, was analyzed and found to contain 5-9.7% Vit B6. Other randomly selected lots of the product contained 0.007-0.27% Vit B6. According to the product label, Vit B6 should have been present at 0.004%. To confirm the hypothesis that Vit B6 was responsible for the deaths of the falcons in Abu Dhabi, Vit B6 (British Pharmacopoeia [BP] grade) in powder form was diluted in water for injection and administered IM to four groups of falcons. Groups of four gyrfalcon × peregrine hybrid falcons or gyrfalcons (or both) were given a single IM dose of 5, 10, 15, or 20 mg/kg of Vit B6 or received an oral dose of 25, 50, or 75 mg of Vit B6. Only birds in the lowest-dose groups survived. The maximum nonlethal single doses of Vit B6 in falcons were 5 mg/kg i.m. and 25 mg/kg p.o.

First isolation of West Nile virus from a dromedary camel.

Although antibodies against West Nile virus (WNV) have been detected in the sera of dromedaries in the Middle East, North Africa and Spain, no WNV has been isolated or amplified from dromedary or Bactrian camels. In this study, WNV was isolated from Vero cells inoculated with both nasal swab and pooled trachea/lung samples from a dromedary calf in Dubai. Complete-genome sequencing and phylogenetic analysis using the near-whole-genome polyprotein revealed that the virus belonged to lineage 1a. There was no clustering of the present WNV with other WNVs isolated in other parts of the Middle East. Within lineage 1a, the dromedary WNV occupied a unique position, although it was most closely related to other WNVs of cluster 2. Comparative analysis revealed that the putative E protein encoded by the genome possessed the original WNV E protein glycosylation motif NYS at E154-156, which contained the N-linked glycosylation site at N-154 associated with increased WNV pathogenicity and neuroinvasiveness. In the putative NS1 protein, the A70S substitution observed in other cluster 2 WNVs and P250, which has been implicated in neuroinvasiveness, were present. In addition, the foo motif in the putative NS2A protein, which has been implicated in neuroinvasiveness, was detected. Notably, the amino-acid residues at 14 positions in the present dromedary WNV genome differed from those in most of the closely related WNV strains in cluster 2 of lineage 1a, with the majority of these differences observed in the putative E and NS5 proteins. The present study is the first to demonstrate the isolation of WNV from dromedaries. This finding expands the possible reservoirs of WNV and sources of WNV infection.

Outbreaks of highly pathogenic avian influenza H5N1 clade 2.3.2.1c in hunting falcons and kept wild birds in Dubai implicate intercontinental virus spread.

Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 have continued to perpetuate with divergent genetic variants in poultry within Asia since 2003. Further dissemination of Asian-derived H5 HPAIVs to Europe, Africa and, most recently, to the North American continent has occurred. We report an outbreak of HPAIV H5N1 among falcons kept for hunting and other wild bird species bred as falcon prey in Dubai, United Arab Emirates, during the autumn of 2014. The causative agent was identified as avian influenza virus subtype H5N1, clade 2.3.2.1c, by genetic and phylogenetic analyses. High mortality in infected birds was in accordance with systemic pathomorphological and histological alterations in affected falcons. Genetic analysis showed the HPAIV H5N1 of clade 2.3.2.1c is a reassortant in which the PB2 segment was derived from an Asian-origin H9N2 virus lineage. The Dubai H5N1 viruses were closely related to contemporary H5N1 HPAIVs from Nigeria, Burkina-Faso, Romania and Bulgaria. Median-joining network analysis of 2.3.2.1c viruses revealed that the Dubai outbreak was an episode of a westward spread of these viruses on a larger scale from unidentified Asian sources. The incursion into Dubai, possibly via infected captive hunting falcons returning from hunting trips to central Asian countries, preceded outbreaks in Nigeria and other West African countries. The alarmingly enhanced geographical mobility of clade 2.3.2.1.c and clade 2.3.4.4 viruses may represent another wave of transcontinental dissemination of Asian-origin HPAIV H5 viruses, such as the outbreak at Qinghai Lake caused by clade 2.2 ('Qinghai' lineage) in 2005.

On the life cycle and morphology of development stages of Paraspiralatus sakeri Gibbons et al., 2004 (Nematoda: Spiroidea, Spirocercidae), a heteroxenic stomach parasite of falcons.

Pitted darkling beetles (Adesmia cancellata) were infected with nematode eggs found in the alimentary tract of a gyrfalcon (Falco rusticolus) naturally infected with Paraspiralatus sakeri. Third-stage larvae in numbers between 1 and 84 were removed from the beetles 5 weeks postinfection and were used for morphological studies as well as to infect domestic chicken, yellow-bellied geckos (Hemidactylus flaviviridis) and fringe-toed lizards (Acanthodactylus schmidti). All experimental animals, necropsied 4-38 weeks later, were positive for spirally coiled nematode larvae located under the skin and in the interstitium of skeletal muscles. Despite similarities in general morphology, larvae from beetles and reptiles and chicken differed strikingly in the total body length and body width. Differences in length of the muscular oesophagus and distances of cervical papillae, nerve ring and excretory pore from the anterior end were less distinct. Morphology of these larvae matched with larvae found in subcutaneous cysts in naturally infected houbara bustards (Chlamydotis macqueeni) from Pakistan and UAE as well as with those detected in the muscles of an ocellated skink (Chalcides ocellatus).

Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species.

Camel antibodies have been widely investigated, but work has focused upon the unique heavy chain antibodies found across camelid species. These are homodimers, devoid of light chains and the first constant heavy chain domain. Camelid species also display conventional hetero-tetrameric antibodies with identical pairs of heavy and light chains; in Camelus dromedarius these constitute 25% of circulating antibodies. Few investigations have been made on this subset of antibodies and complete conventional camel IgG sequences have not been reported. Here we study the sequence diversity of functional variable and constant regions observed in 57 conventional heavy, 18 kappa and 35 lambda light chains of C. dromedarius and Camelus bactrianus. We detail sequences of the full kappa and lambda light chain, variable and CH1 region for IgG1a and IgG1b and the CH2 and CH3 region for IgG1a. The majority (60%) of IgG1 variable region sequences aligned with the human IgHV3 family (clan III) and had leader sequences beginning with MELG whereas the remaining sequences aligned with the IgHV4 (clan II) and had leader sequences beginning with MRLL. Distinct differences in CDR length were observed between the two; where CDR1 was typically 5 and 7 residues and CDR2 at 17 and 16 residues, respectively. CDR3 length of IgHV4 (range 11 to 20) was closer to that typical of VHH antibodies than that of IgHV3 (range 3 to 18 residues). Designed oligonucleotide primers have enabled identification of paired heavy and light chains of conventional camel antibodies from individual B cell clones.

¹5N, ¹³C and ¹H resonance assignments and secondary structure determination of a variable heavy domain of a heavy chain antibody.

Heavy chain antibodies differ in structure to conventional antibodies lacking both the light chain and the first heavy chain constant domain (CH1). Characteristics of the antigen-binding variable heavy domain of the heavy chain antibody (VHH) including the smaller size, high solubility and stability make them an attractive alternative to more traditional antibody fragments for detailed NMR-based structural analysis. Here we report essentially complete backbone and side chain (15)N, (13)C and (1)H assignments for a free VHH. Analysis of the backbone chemical shift data obtained indicates that the VHH is comprised predominantly of ß-sheets corresponding to nearly 60% of the protein backbone.

Comparing the presence of different genes in Salmonella subspecies I-IV and development of a diagnostic multiplex PCR method for identification of Salmonella subspecies.

Reptile-associated salmonellosis in humans has become a growing problem worldwide. Reptiles are frequently asymptomatic carriers of Salmonella and therefore, they are considered as an important reservoir for these bacteria. The classical biochemical method for Salmonella subspecies detection is time consuming, especially in samples from reptiles since they frequently carry more than one Salmonella subspecies. The aim of this study was therefore to develop a multiplex PCR assay for a rapid and accurate differentiation of Salmonella subspecies I, II, IIa, IIIb and IV. In the present study, the occurrence of the genes invA, ttrCA, iroB, STM4075, sciA, STM3690, sadA, gatD, foxA, pagN, fljB, iucD, spvB, lacZ, iutA, mdcA and irp2 was examined in 41 Salmonella strains from Middle Eastern animals (mainly reptiles) by monoplex PCR. According to the results a multiplex PCR assay was developed based on the genes ttrCA, sciA, foxA, iutA. Compared to biochemical analysis this method allowed a fast identification of the subspecies from all the Middle Eastern Salmonella strains (n = 41), as well as 79 strains from German children (n = 18) with reptile associated salmonellosis and other humans and animals (n = 61) with salmonellosis.These results revealed the multiplex PCR as a fast assay for a specific identification of Salmonella subspecies I, II, IIIa, and IIIb.

Generation and characterization of non-competitive furin-inhibiting nanobodies.

The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFß (transforming growth factor ß) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.

Prevalence, serovars, phage types, and antibiotic susceptibilities of Salmonella strains isolated from animals in the United Arab Emirates from 1996 to 2009.

The aim of this study was to give some insights into the prevalence, serovars, phage types, and antibiotic resistances of Salmonella from animal origin in the United Arab Emirates. Data on diagnostic samples from animals (n = 20,871) examined for Salmonella between 1996 and 2009 were extracted from the databases of the Central Veterinary Research Laboratory in Dubai and from typed strains (n = 1052) from the Robert Koch Institute, Wernigerode Branch in Germany and analyzed for general and animal-specific trends. Salmonella was isolated from 1,928 (9 %) of the 20,871 samples examined. Among the 1,052 typed strains, most were from camels (n = 232), falcons (n = 166), bustards (n = 101), antelopes (n = 66), and horses (n = 63). The predominant serovars were Salmonella Typhimurium (25 %), Salmonella Kentucky (8 %), followed by Salmonella Frintrop (7 %), and Salmonella Hindmarsh (5 %). When analyzed by animal species, the most frequent serovars in camels were Salmonella Frintrop (28 %) and Salmonella Hindmarsh (21 %), in falcons Salmonella Typhimurium (32 %), in bustards Salmonella Kentucky (19 %), in antelopes Salmonella Typhimurium (9 %), and in horses Salmonella Typhimurium (17 %) and S. Kentucky (16 %). Resistance of all typed Salmonella strains (n = 1052) was most often seen to tetracycline (23 %), streptomycin (22 %), nalidixic acid (18 %), and ampicillin (15 %). These data show trends in the epidemiology of Salmonella in different animal species which can be used as a base for future prevention, control, and therapy strategies.

Cutaneous and pulmonal habronemosis transmitted by Musca domestica in a stable in the United Arab Emirates.

Nematode larvae found in histological cuts of lung tissue of a horse from a farm in Al Dhaid (UAE) were determined to belong to the Habronematidae family. The clinical examination of the other 18 horses present in the farm revealed summer sores (cutaneous habronemosis) in two stallions. Nematode larvae were found in 147 (=26.2%) out of 561 male but only in 64 (=8.7%) out of 739 female Musca domestica caught at the farm in November and December 2008. Conversely, all 15 Stomoxys calcitrans specimens caught in the farm resulted negative for nematode larvae. The housefly population caught in the barn showed a prevalence of 20.9% with nematode larvae, while flies trapped outside the building on the territory of the farm had a much lower prevalence of 1.1%. The intensity of infection varied between one and 29 larvae per head. Larvae retrieved at the fly dissection were subjected to a ribosomal DNA-targeting semi-nested PCR protocol able to discriminate among the three nematode species Habronema muscae, Habronema microstoma, and Draschia megastoma. The larvae were identified to be H. muscae.

Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species.

BACKGROUND: The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring. METHODOLOGY/PRINCIPAL FINDINGS: Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster. CONCLUSION: This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity.