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A new hepatitis E virus (HEV-7) was recently found in dromedaries and 1 human from the United Arab Emirates. We screened 2,438 dromedary samples from Pakistan, the United Arab Emirates, and 4 African countries. HEV-7 is long established, diversified and geographically widespread. Dromedaries may constitute a neglected source of zoonotic HEV infections.

Background Among all known picornaviruses, only two species, equine rhinitis A virus and equine rhinitis B virus (ERBV) are known to infect horses, causing respiratory infections. No reports have described the detection of ERBV in fecal samples of horses and no complete genome sequences of ERBV3 are available. Methods We performed a molecular epidemiology study to detect ERBVs in horses from Dubai and Hong Kong. Complete genome sequencing of the ERBVs as well as viral loads and genome, phylogenetic and evolutionary analysis were performed on the positive samples. Results ERBV was detected in four (13.8 %) of the 29 fecal samples in horses from Dubai, with viral loads 8.28 × 103 to 5.83 × 104 copies per ml, but none of the 47 fecal samples in horses from Hong Kong by RT-PCR. Complete genome sequencing and phylogenetic analysis showed that three of the four strains were ERBV3 and one was ERBV2. The major difference between the genomes of ERBV3 and those of ERBV1 and ERBV2 lied in the amino acid sequences of their VP1 proteins. The Ka/Ks ratios of all the coding regions in the ERBV3 genomes were all

Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23) from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5′-UCUAAAC-3′ as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 0.0001). Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1.

We report the complete genome sequence of influenza virus H9N2 associated with a fatal outbreak among chickens in Dubai. All segments are clustered with avian H9N2 viruses circulating in the Middle East but distinct from those in southeast Asia. It is not a reassortant virus or transmitted from other regions.

The activity of alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), lactoperoxidase (LPO), lipase (LIP) and leucine arylamidase (LAP) in raw and pasteurised camel milk was studied, in order, to find a heat treatment indicator suitable to verify an effective pasteurisation. The studies have shown that ALP, GGT and LIP are not suitable as marker because residual activities were detected in pasteurised milk. LAP activity in raw camel milk is too low and the data variation is too high for serving as a marker. The LPO results look promising. The enzyme activity in raw camel milk is high and the respective value in pasteurised milk is predominantly below the detection limit of the method.

Rhodococcus (R). equi, a recognized pathogen in horses, is emerging as a human opportunistic pathogen, especially in immunocompromized people. It affects also New World camelids, but there are no reports of R. equi infection in Old World camelids yet. Four cases of disseminated R. equi infection in adult breeding dromedaries occurred at one camel farm near Dubai within 16 months of each other. At necropsy the lungs were diffusely consolidated with large caseous areas. Histology revealed severe suppurative to necrotising pneumonia with multiple encapsulated abscesses. Immunohistochemistry enabled the detection of 15- to 17-kDa antigens (VapA) of R. equi in the lung sections. High numbers of R. equi were isolated from the lung lesions as well as from liver, spleen and mediastinal lymph nodes, indicative of septicaemia. The isolated strains were PCR-positive for the specific virulence plasmid (VapA-Gen) of R. equi, indicating virulent strains and containing an 85-kb type I plasmid. This is the first report of disseminated R. equi infection in Old World camelids. Since adult camels in general do not suffer from bacterial caused pneumonia (except tuberculosis), this is a new emerging disease for camels.

Three strains of Gram-negative, rod-shaped, non-spore-forming bacteria (M 2040T, M 1973 and M 1878-SK2), isolated from milk of camels at a camel-milk production farm in the United Arab Emirates, were investigated for their taxonomic allocation. On the basis of 16S rRNA gene sequence similarities, all three strains were shown to belong to the Alphaproteobacteria and were most closely related to Chelatococcus asaccharovorans and Chelatococcus daeguensis (95.1 and 95.2 % sequence similarity to the respective type strains). meso-Diaminopimelic acid was detected as the characteristic peptidoglycan diamino acid. The predominant compound in the polyamine pattern was spermidine, and sym-homospermidine was not detectable. The quinone system was ubiquinone Q-10. The polar lipid profile included the major compounds phosphatidylcholine and diphosphatidylglycerol and moderate amounts of phosphatidylethanolamine, phosphatidylglycerol, an unidentified glycolipid and two unidentified aminolipids. Minor lipids were also detected. The major fatty acid profile, consisting of C19 : 0 cyclo ω8c and C18 : 1 ω7c, with C18 : 0 3-OH as the major hydroxylated fatty acid, was similar to those of the genus Chelatococcus. The results of DNA–DNA hybridization experiments and physiological and biochemical tests allowed both genotypic and phenotypic differentiation of the isolates from described Chelatococcus species. Isolates M 2040T, M 1973 and M 1878-SK2 were closely related on the basis of DNA–DNA reassociation and therefore represent a single novel species. In summary, low 16S rRNA gene sequence similarities of 95 % with Chelatococcus asaccharovorans and marked differences in polar lipid profiles as well as in polyamine patterns support the description of a novel genus and species to accommodate these strains, for which the name Camelimonas lactis gen. nov., sp. nov. is proposed. The type strain of Camelimonas lactis is M 2040T (=CCUG 58638T =CCM 7696T).

Camelid IgG has been reported to be less immunogenic, less able to activate the complement cascade and more thermostable than IgG from other mammals, and has the ability to bind antigens that are unreactive with other mammalian IgGs. We are investigating whether these attributes of camelid IgG translate into antivenom with immunological and venom-neutralising efficacy advantages over conventional equine and ovine antivenoms. The objective of this study was to determine the preclinical venom-neutralising effectiveness of IgG from camels immunised with venoms, individually or in combination, of the saw-scaled viper, Echis ocellatus, the puff adder, Bitis arietans and the spitting cobra, Naja nigricollis - the most medically-important snake species in West Africa. Neutralisation of the pathological effects of venoms from E. ocellatus, B. arietans and N. nigricollis by IgG from the venom-immunised camels, or commercial antivenom, was compared using assays of venom lethality (ED(50)), haemorrhage (MHD) and coagulopathy (MCD). The E. ocellatus venom ED(50), MHD and MCD results of the E. ocellatus monospecific camel IgG antivenom were broadly equivalent to comparable ovine (EchiTAbG, MicroPharm Ltd, Wales) and equine (SAIMR Echis, South African Vaccine Producer, South Africa) antivenoms, although the equine antivenom required half the amount of IgG. The B. arietans monospecific camel IgG neutralised the lethal effects of B. arietans venom at one fourth the concentration of the SAIMR polyspecific antivenom (a monospecific B. arietans antivenom is not available). The N. nigricollis camel IgG antivenom was ineffective (at the maximum permitted dose, 100 mul) against the lethal effects of N. nigricollis venom. All the equine polyspecific antivenoms required more than 100 microl to be effective against this venom. The polyspecific camel IgG antivenom, prepared from five camels, was effective against the venom-induced effects of E. ocellatus but not against that of B. arietans and N. nigricollis venoms. No direct correlation was evident between either camel IgG relative avidity or titre and the effectiveness of venom neutralisation in preclinical assays.

Antivenom is the most effective treatment of snake envenoming and is manufactured from the IgG of venom-immunised horses and sheep. Camelids have a unique IgG structure which may account for the report that camel IgG is less immunogenic and less likely to activate complement than equine or ovine IgG. Camelid IgG therefore offers potential safety advantages over conventional IgGs used for antivenom manufacture. The reported thermostability of camelid IgG also holds promise in the inclusion of a relatively inexpensive anti-microbial heat step in antivenom manufacture. However, these potential benefits of camelid IgG would be much reduced if any one of the three camel IgG subclasses dominated, or under-performed, the serological response of camels to venom immunisation because of the prohibitive manufacturing costs of having to purify, or exclude, one or more IgG subclasses. This study compared the titre, antigen-specificity, relative avidity and ability to neutralise the haemorrhagic and coagulopathic effects of Echis ocellatus venom of each IgG subclass from the venom-immunised camels. The results demonstrated that no one IgG subclass consistently out-performed or under-performed the others in their immunoreactivity to venom proteins and ability to neutralise venom-induced pathologies. We concluded therefore that IgG taken from a pool of immunised camels could be processed into antivenom without requiring the implementation of expensive chromatographic separations to select, or indeed to exclude, a specific IgG subclass. The immunoreactivity of the heavy and light chain, IgG1 subclass, was markedly more vulnerable to extreme heat treatment than the heavy chain-only IgG2 and IgG3 subclasses.

VHHs or Nanobodies are single-domain antigen-binding fragments derived from heavy chain antibodies found in camelids. It has already been shown that complex protein mixtures and even whole organisms elicit good immune responses in camelids; therefore we hypothesized that VHHs selected from a dromedary immunized with poliovirus type 1 might inhibit the in vitro replication of poliovirus through binding to essential biological sites on the viral capsid. In this study, we aimed to determine whether VHHs inhibit wild-type and vaccine strains of poliovirus type 1. Interestingly, VHHs showed a potent antipolio activity with EC50 values in the low nanomolar range. Moreover, these antibody fragments completely blocked viral multiplication at higher concentrations. Remarkably, no (immune) escape variants against some of these VHHs could be generated. In conclusion, VHHs fulfil several in vitro requirements to be assigned as potential antiviral compounds for further development of an anti-poliovirus drugs.

Fifteen non pregnant female dromedaries of different age were intra-tracheally and intra-nasally infected with a Brucella melitensis strain belonging to the genetic group East Mediterranean (former African group). The development of Brucella antibodies in the infected dromedaries was investigated over a period of 12 months by comparing 15 different serological tests. Three uninfected control dromedaries remained negative during the entire experiment. Our investigations revealed that only 2 serological tests were characterised by a high degree of sensitivity for the diagnosis of brucellosis in dromedaries. These two tests are a Rose Bengal Test (RBT) from Vircell, Spain and a competitive ELISA (cELISA) from Ingenasa, also from Spain. The blood culture revealed that all tested sodium citrate blood samples were negative. Results of the enriched citrate blood samples of the infected 15 dromedaries fluctuated and became positive for a short time, but at the end of the experiment only one remained positive. Direct examination by PCR did not show any evidence of the pathogen in the EDTA blood of the 15 infected dromedaries. Also, all nasal swabs taken up to a period of 3 months post infection remained negative in both PCR and microbial culture.

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

There is accumulating evidence that birds of prey are susceptible to fatal infection with highly pathogenic avian influenza (HPAI) virus. We studied the antigenic, molecular, phylogenetic, and pathogenic properties of 2 HPAI H5N1 viruses isolated from dead falcons in Saudi Arabia and Kuwait in 2005 and 2007, respectively. Phylogenetic and antigenic analyses grouped both isolates in clade 2.2 (Qinghai-like viruses). However, the viruses appeared to have spread westward via different flyways. It remains unknown how these viruses spread so rapidly from Qinghai after the 2005 outbreak and how they were introduced into falcons in these two countries. The H5N1 outbreaks in the Middle East are believed by some to be mediated by wild migratory birds. However, sporting falcons may be at additional risk from the illegal import of live quail to feed them.

A virus was isolated from aborted dromedary (Camelus dromedarius) fetuses during an abortion storm in Dubai, United Arab Emirates. Laboratory investigations showed the causative agent to be indistinguishable from equine rhinitis A virus (ERAV), a picornavirus. Two pregnant dromedaries experimentally infected with the camel virus isolate both aborted and an identical virus was reisolated from both fetuses, thus confirming the diagnosis. The extremely high prevalence of antibody (>90 %) and the high titres recorded against ERAV in the dromedary herd clearly showed that ERAV does infect dromedaries. Unlike horses, where ERAV targets the upper respiratory tract, in dromedaries the target organ appears to be the genital tract.

Close relatedness and genomic plasticity characterizing the high-threat pathogens Burkholderia pseudomallei and Burkholderia mallei render the molecular diagnosis of these species hard to guarantee with a maximal confidence level. This article describes fast molecular assays derived from compiled sequences of housekeeping genes determined in more than 1,000 strains. The assays proved to be robust and appropriate for general detection as well as species identification purposes.

A PCR assay targeting the metalloprotease gene (mprA) of Burkholderia pseudomallei was developed for the specific detection of this organism in pure cultures and clinical samples. All other closely related organisms including B. mallei the causative agent of glanders, and B. thailandensis tested negative. Burkholderia pseudomallei DNA was successfully amplified from paraffin-embedded lung tissue of a camel with a generalized B. pseudomallei infection. The developed PCR assay can be used as a simple tool for the specific and sensitive detection of B. pseudomallei.

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.

The recent emergence of Middle East respiratory syndrome coronavirus from the Middle East and its discovery from dromedary camels has boosted interest in the search for novel viruses in dromedaries. The existence of astroviruses (AstVs) in dromedaries was previously unknown. We describe the discovery of a novel dromedary camel AstV (DcAstV) from dromedaries in Dubai. Among 215 dromedaries, DcAstV was detected in faecal samples of four [three (1.5 %) adult dromedaries and one (8.3 %) dromedary calf] by reverse transcription-PCR. Sequencing of the four DcAstV genomes and phylogenetic analysis showed that the DcAstVs formed a distinct cluster. Although DcAstV was most closely related to a recently characterized porcine AstV 2, their capsid proteins only shared 60-66 % amino acid identity, with a mean amino acid genetic distance of 0.372. Notably, the N-terminal halves of the capsid proteins of DcAstV shared ≤ 85 % amino acid identity, but the C-terminal halves only shared ≤ 49 % amino acid identity compared with the corresponding proteins in other AstVs. A high variation of the genome sequences of DcAstV was also observed, with a mean amino acid genetic distance of 0.214 for ORF2 of the four strains. Recombination analysis revealed a possible recombination event in ORF2 of strain DcAstV-274. The low Ka/Ks ratios (number of non-synonymous substitutions per non-synonymous site to number of synonymous substitutions per synonymous site) of the four ORFs in the DcAstV genomes supported the suggestion that dromedaries are the natural reservoir where AstV is stably evolving. These results suggest that AstV is a novel species of the genus Mamastrovirus in the family Astroviridae. Further studies are important to understand the pathogenic potential of DcAstV.

The recent emergence of Middle East respiratory syndrome coronavirus from the Middle East and the discovery of the virus from dromedary camels have boosted interest in the search for novel viruses in dromedaries. Whilst picornaviruses are known to infect various animals, their existence in dromedaries was unknown. We describe the discovery of a novel picornavirus, dromedary camel enterovirus (DcEV), from dromedaries in Dubai. Among 215 dromedaries, DcEV was detected in faecal samples of four (1.9 %) dromedaries [one (0.5 %) adult dromedary and three (25 %) dromedary calves] by reverse transcription PCR. Analysis of two DcEV genomes showed that DcEV was clustered with other species of the genus Enterovirus and was most closely related to and possessed highest amino acid identities to the species Enterovirus E and Enterovirus F found in cattle. The G+C content of DcEV was 45 mol%, which differed from that of Enterovirus E and Enterovirus F (49–50 mol%) by 4–5 %. Similar to other members of the genus Enterovirus, the 5′ UTR of DcEV possessed a putative type I internal ribosome entry site. The low ratios of the number of nonsynonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site (K a/K s) of various coding regions suggested that dromedaries are the natural reservoir in which DcEV has been stably evolving. These results suggest that DcEV is a novel species of the genus Enterovirus in the family Picornaviridae. Western blot analysis using recombinant DcEV VP1 polypeptide showed a high seroprevalence of 52 % among serum samples from 172 dromedaries for IgG, concurring with its much higher infection rates in dromedary calves than in adults. Further studies are important to understand the pathogenicity, epidemiology and genetic evolution of DcEV in this unique group of animals.

An adult male dromedary bull was diagnosed with pulmonary tuberculosis (Tb). The dromedary was severely emaciated and died 2 months after the onset of the disease. It exhibited typical Tb lesions in both lungs and lung lymph nodes. A guinea pig inoculated with lung tissue from the Tb camel died after 3 weeks from typical Tb. Mycobacteria were isolated from the dromedary's lung and lung lymph nodes and also from different organs of the guinea pig. The microorganism was identified as member of the antelope clade of the Mycobacterium tuberculosis complex.

A polymerase chain reaction (PCR) assay targeting the flagellin P ( fliP )-I S 407 A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP -IS 407 A sequence of B. mallei American Type Culture Collection (ATCC) 23344 T allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of B. mallei ATCC 23344 T . B . mallei DNA was also amplified from various tissues of horses with a generalized B . mallei infection. The developed PCR assay can be used as a simple and rapid tool for the specific and sensitive detection of B. mallei in clinical samples.

The human population is rising and will soon reach 9 billion people. In parallel, the demand for animal protein is increasing and with it is the threat of zoonotic diseases. We must therefore be on our guard. The close association of people with animals promotes the opportunity for zoonotic infections and real danger may arise when animals are imported with no health background. Therefore, it is essential to implement strict import controls, and establish efficient quarantine facilities. Many viral, bacterial, and zoonotic diseases have been diagnosed on the Arabian Peninsula, either by isolating the pathogens or through serological surveys. Most of them are briefly discussed in this paper.

The occurrence of spontaneous ovulation in dromedaries was examined in two separate studies including 20 non-lactating, barren and 12 lactating dromedaries, respectively. Lactating camels were milked twice a day with an automatic bucket milking machine. Ovarian activity was monitored by repeated ultrasonography. Blood samples for progesterone were collected daily or two to three times a week. To compare CL development after spontaneous and induced ovulations, ovulation was induced by a GnRH analogue in eight lactating dromedaries. Spontaneous ovulation was observed in one non-lactating camel (1 of 20 camels, 5%; 1 of 70 follicular waves, 1.4%), whereas, spontaneous ovulation was detected more frequently in lactating dromedaries (5 of 12 camels, 41.7%; 13 of 91 follicular waves, 14.3%). In one lactating camel, spontaneous ovulation occurred repeatedly for nine times. There was a significant effect of type of ovulation (spontaneous versus induced, P0.05) and day (P0.001) on serum progesterone concentration. Mean serum progesterone levels and total progesterone production (AUC) were higher after induced ovulation. Luteal diameter and serum progesterone concentration were positively correlated (r = 0.71, P0.001), but there was a significant difference between morphological and functional development of the CL. In dromedaries, morphological development starts earlier, morphological regression starts later and last longer than functional development and regression of the CL. Compared to induced ovulation, functional development of the CL after spontaneous ovulation might be altered but the morphological development is not affected.

Twenty days after an open castration, a 5-year-old dromedary was presented to the Dubai Camel Hospital with severe central nervous symptoms. The dromedary showed the following signs: off feed, stiff gait with extended neck, external swelling of the preputial sheath and groin region, and foamy saliva drooling from the mouth. The dromedary was unable to swallow. Three days after admission, the camel developed lockjaw, and on the fifth day it was unable to stand owing to paralysis of the hindquarters. Because of the severity of the disease and because it did not respond to treatment, the camel was euthanized 26 days after the operation and submitted to the Central Veterinary Research Laboratory for further investigation. Both castration wounds were closed and spermiducts were filled with necrotic masses from which Clostridium tetani was isolated. Two mice, which were injected with the filtrate of the thioglycolate broth, developed typical signs of tetanic spasm of the hind leg. Faecal samples from camel and horse paddocks that were only 50 metres apart were negative for C. tetani. However, C. tetani was isolated from two soil samples of the horse paddock. It is recommended that camels should be vaccinated against tetanus prior to castration.

There is no current comprehensive assessment of the molecular phylogeny of the coccidia, as all recently published papers either deal with subsets of the taxa or sequence data, or provide non-robust analyses. Here, we present a comprehensive and consistent phylogenetic analysis of the available data for the small-subunit ribosomal RNA gene sequence, including a number of taxa not previously studied, based on a Bayesian tree-building analysis and the covariotide model of evolution. The assumptions of the analysis have been rigorously tested, and the benefits and limitations highlighted. Our results provide support for a number of prior conclusions, including the monophyly of the families Sarcocystidae (cyst-forming coccidia) and Eimeriidae (oocyst-forming coccidia), but with bird-host Isospora species in the Eimeriidae and mammal-host species in the Sarcocystidae. However, it is clear that a number of previously reported relationships are dependent on the evolutionary model chosen, such as the placements of Goussia janae, Lankesterella minimia and Caryospora bigenetica. Our results also confirm the monophyly of the subfamilies Toxoplasmatinae and Sarcocystinae, but only some of the previously reported groups within these subfamilies are supported by our analysis. Similarly, only some of the previously reported groups within the Eimeriidae are supported by our analysis, and the genus Eimeria is clearly paraphyletic. There are unambiguous patterns of host-parasite relationship within the coccidia, as most of the well-supported groups have a consistent and restricted range of hosts, with the exception of the Toxoplasmatinae. Furthermore, the previously reported groups for which we found no support all have a diverse range of unrelated hosts, confirming that these are unlikely to be natural groups. The most interesting unaddressed questions may relate to Isospora, which has the fewest available sequences and host-parasite relationships apparently not as straightforward as elsewhere within the suborder.

The present study describes the laboratory investigations after a single right eye drop (3.1 × 109 CFU live bacteria) immunisation of 6 dromedary camels (Camelus dromedarius) with live attenuated B. melitensis Rev 1 vaccine. The experiment was conducted over a period of 5 months. The vaccine strain was isolated for 16 days from only the right eye of the vaccinated dromedaries, but not from the left eye and both nostrils. Similar pattern of results was obtained by polymerase chain reaction. It was negative for the left eye, both nostrils (except for one dromedary) and for EDTA blood and serum. All vaccinated dromedaries seroconverted from day 16 after vaccination until 4 months shown by Rose-Bengal test and slide-agglutination test. No serological reactions were found after 5 months. The complement fixation test remained negative throughout the experiment. Information about the vaccination against brucellosis in camels, the within host disperse of the vaccine strain and the serological response are scarce. The experiment provided basic data about the feasibility of Brucevac conjunctival vaccine in camels. However, to prove if the immunised dromedaries acquired a lifelong immunity against brucellosis, pregnant vaccinated dromedaries need to be challenged with a field B. melitensis strain. We also recommend changing the conjunctival vaccination route to subcutaneous or intramuscular to prevent accidental infection due to B. melitensis vaccine strain excreted by lacrimation.

Embryonic gonadal tissue was dissected individually from Houbara bustard embryos at eight days post-incubation. Houbara bustard gonadal cells containing gonadal primordial germ cells (gPGCs) were injected into the blood stream of 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 bustard PGCs in chimeric chicken testis were assessed by PCR with Houbara bustard species-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara bustard cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germ line chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara bustard eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara bustard and the other was female but died before hatching.

We confirm a natural infection of dromedaries with glanders. Multilocus variable number tandem repeat analysis of a Burkholderia mallei strain isolated from a diseased dromedary in Bahrain revealed close genetic proximity to strain Dubai 7, which caused an outbreak of glanders in horses in the United Arab Emirates in 2004.

Summary Foot-and-mouth disease viruses are often restricted to specific geographical regions and spread to new areas may lead to significant epidemics. Phylogenetic analysis of sequences of the VP1 genome region of recent outbreak viruses from Libya and Saudi Arabia has revealed a lineage, O-Ind-2001, normally found in the Indian subcontinent. This paper describes the characterization of field viruses collected from these cases and provides information about a new real-time RT-PCR assay that can be used to detect viruses from this lineage and discriminate them from other endemic FMD viruses that are co-circulating in North Africa and western Eurasia.

There is a large illegal trade in trapped houbara bustards Chlamydotis undulata macqueenii, which are smuggled into the Middle East for falconry. Mortality is high and is associated with poor transport conditions, malnutrition, overcrowding and exposure to multiple infectious diseases, in particular aspergillosis, avian pox and paramyxovirus type 1 virus. Other pathogens include Salmonella sp., Pseudomoims sp., Trichomonas sp., intestinal endoparasites, avian leucosis, reovirus, adeno-virus, paramyxovirus type 2 and Chlamydia sp. Various regional initiatives have been initiated to confiscate and rehabilitate illegally trapped birds. This paper reviews the causes of morbidity and mortality seen in illegally traded houbara bustards and provides health recommendations for those involved in rehabilitation.

To the Editor: In 2012, a novel coronavirus termed Middle East respiratory syndrome coronavirus (MERS-CoV) emerged on the Arabian Peninsula; the virus has been responsible for >800 human cases. Recently, evidence of MERS-CoV infection in dromedaries was obtained from the Canary Islands, the Arabian Peninsula, and Africa (1–3). Viral sequences from dromedaries and from humans infected with MERS-CoV were highly similar, suggesting a prominent role of dromedaries as an animal reservoir of the virus (4). However, the serologic assessment of other animal species has been incomplete. Investigations of domestic animal species have been restricted to goats, sheep, and cattle (3) and a limited study of horses (n = 3) (5). No evidence of recent infection was found in either study.

Summary During the spring of 1995, 1996 and 1997 following tests on six peregrine falcons (Falco peregrinus) and two gyr falcons (Falco rusticolus), Salmonella typhimurium was isolated from liver, spleen and small intestines. Four of the falcons (two peregrines and two gyrs) had also contracted Chlamydia infection, three peregrines a pox infection and one peregrine a Herpesvirus infection. It is believed that this dual infection was fatal for these birds. The disease was marked by anorexia, dehydration and green-coloured droppings. Necropsy of all falcons revealed discolouration of the liver and enlargement of liver and spleen. Miliary necrosis was detected in all livers. A total of 12 salmonella serovars, including S. typhimurium, were cultured from faeces of 48 falcons which showed no clinical signs.

Blood samples were obtained from adult (> 1.5 years) and juvenile (2-8 weeks, 9-16 weeks and 17-24 weeks) captive buff-crested bustards (Eupodotis ruficrista gindiana) to study age-related changes. A total of twelve different tests were conducted using a Hitachi 90011 wet chemistry analyzer. A comparison of the values obtained was made between adult and juvenile buff-crested bustards and from the literature with other bustard species. Significant differences between adult and juvenile buff-crested bustards were found for glucose, uric acid, total protein, alkaline phosphatase, asparatate amino transferase and calcium. The results obtained from this study provide blood chemistry values for this species and demonstrate age-related differences between adult and juvenile birds.

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.

MβL (metallo-β-lactamase) enzymes are usually produced by multi-resistant Gram-negative bacterial strains and have spread worldwide. An approach on the basis of phage display was used to select single-domain antibody fragments (VHHs, also called nanobodies) that would inhibit the clinically relevant VIM (Verona integron-encoded MβL)-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of the NbVIM_38 nanobody were then characterized. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the micromolar range for all β-lactams tested. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the α2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor.

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 10(7) camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

This study was aimed at evaluating reliable serological tests for diagnosing brucella infection in dromedaries. All four types of serological tests used in this study were based on detection of brucella antibodies, not Brucella antigen. Three different serological tests were compared with the routine OIE recommended Rose Bengal Test (RBT) performed at the Central Veterinary Research Laboratory (CVRL), Dubai. Our comparative results showed no significant differences on 353 dromedary sera between complement fixation test (CFT-Animal and Plant Health Agency) and MSA™ Camel Brucella Rapid Ab test. The CFT-APHA sensitivity was determined to be 98.98% and MSA™ Rapid Camel Brucella Ab test was 97.96%. FPA performed the worst among the three with sensitivity at 84.69%. MSA™ Camel Brucella Rapid Ab test rank the highest in specificity at 99.92%, followed by CFT at 98.43%. FPA again performed inferior in specificity compared to the other two tests. In conclusion, CFT and MSA™ Camel Brucella Rapid Ab tests are reliable tests comparable to RBT test used in this study. Since CFT test cannot be performed in the field, hence the new MSA™ Camel Brucella Rapid Ab test is also suitable for field testing with its high sensitivity and specificity, which is comparable to Rose Bengal Test (RBT) for the diagnosis of dromedary Brucellosis.

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 β-sheets corresponding to nearly 60% of the protein backbone.

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.