Impact of antibiotics on spermatozoa quality and bacterial load of chilled-stored camels (Camelus dromedarius) semen.

This experiment was conducted to assess the effect of different antibiotics in tris-fructose egg yolk-based diluent on bacterial load and sperm quality of dromedary camels during processing and cold storage. Ten semen ejaculates were collected from five male dromedary camels. Each sample was fractioned into four equally divided aliquots and diluted in one of four tris-fructose egg yolk. The first extender contained no antibiotic (NC). The second extender included streptomycin sulphate (1000 µg/ml) and benzyl penicillin (1000 IU/ml) (SP). The third extender was supplied with 250 µg/ml gentamicin sulphate (Gent). The fourth extender contained 500 µg/ml gentamicin sulphate,100 µg/ml tylosin tartrate, 300 µg/ml lincomycin hydrochloride and 600 µg/ml spectinomycin hydrochloride (GTLS). After dilution, the extended semen samples were cooled to 5 °C within 2 h and finally stored at 5 °C for 72 h. Microbial concentration, motility of spermatozoa, live spermatozoa, plasma membrane and acrosome integrity percentages were evaluated just after dilution at 35 °C, 0, 24, 48 and 72 h from the start of cooling to 5 °C. The results revealed that the diluent containing gentamicin had significantly (P<0.05) maximum motility percentage at the different examination intervals. The pattern of live spermatozoa percentage was varied between the different treatments at different examination intervals. The diluent supplied with Gent was distinguished with a significant peak percentage (P<0.05) of swelled spermatozoa among the other antibiotics supplied diluents. The number of colony-forming units isolated from the semen samples kept in diluent containing no antibiotics was significantly (P<0.05) higher than that isolated from the diluents supplemented with antibiotics. In conclusion, the semen diluents fortified with gentamicin generally keep the motility, acrosomal and plasma membrane integrity and live spermatozoa for 72-h preservation of dromedary semen.

Normal radiographic and ultrasonographic appearance of the adult dromedary camel tarsus (one humped camel).

Six cadaver pelvic limbs were obtained from clinically sound dromedary camels and examined radiographically and ultrasonographically using a 7.5 MHz convex transducer. Radiographic examination was performed in dorsoplantar, lateromedial, dorsolateral-plantaromedial oblique and plantarolateral-dorsomedial oblique projections, and the bony structures and articulations of the tarsal joint were outlined. The tarsus was ultrasonographically investigated in four planes (dorsal, medial, lateral and plantar), and each plane was scrutinized in four levels (calcaneal tuber, tibial malleoli, base of calcaneus and proximal end of metatarsus) in both transverse and longitudinal views. Limbs were examined grossly, frozen at -20°C and sectioned. Radiographic and ultrasonographic findings correlated well with the gross anatomy and frozen sections. The normal appearance of bony and soft structures of the tarsus described in this study provided basic reference data for ultrasonographic and radiographic investigations of tarsal disorders in the dromedary camel.

Computed tomography and cross-sectional anatomy of the normal dromedary camel tarsus (one humped camel).

The purpose of this study was to provide a detailed computed tomographic (CT) anatomic reference for the dromedary camel tarsus. Six cadaver pelvic limbs, obtained from three clinically and radiographically sound dromedary camels, were scanned in both soft tissue and bone windows starting from the calcaneal tuber towards the proximal metatarsus. Limbs were frozen at -20°C and sectioned transversely via an electric bone saw. The CT images were evaluated and correlated with their corresponding cryosections. The resulting images provided detailed anatomic features for bones, joints and soft tissue components of the tarsus and are intended to serve as a basic reference for the CT scanning of the dromedary camel tarsal pathology.

Glycoprotein E (gE) specified by bovine herpesvirus type 5 (BHV-5) enables trans-neuronal virus spread and neurovirulence without being a structural component of enveloped virions.

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alpha-herpesvirus that causes fatal encephalitis in calves. We previously demonstrated that deletion of a glycine-rich epitope in the gE ectodomain dramatically reduced BHV-5 neurovirulence. To investigate the role of gE cytoplasmic tail sequences in the neuropathogenesis of BHV-5 in rabbits, we constructed a BHV-5gE recombinant virus with a short residual cytoplasmic domain lacking the YXXL motifs and the acidic (BHV-5gEAm480). In vitro, BHV-5gEAm480 produced on the average smaller plaques, compared with wild-type BHV-5, but it produced on the average substantially larger plaques than the gE ORF-deleted BHV-5. The truncated gE was not phosphorylated, and was not endocytosed from the cell surface. Importantly, the truncated gE was not incorporated into enveloped infectious virions, but its glycosylation and interaction with gI were not affected. In a rabbit model of infection, the BHV-5gEAm480 remained highly virulent, while the gE-null virus was avirulent. The gEAm480 mutant virus invaded most of the central nervous system (CNS) structures that are invaded by the wild-type BHV-5. The number of neurons infected by BHV-5gEAm480 was very similar to the number infected by BHV-5 wild-type and gEAm480-rescued viruses. Collectively, the results suggest that gE functions in transsynaptic transmission of BHV-5 and neurovirulence without being a structural component of the virion particle.

The Us9 gene of bovine herpesvirus 1 (BHV-1) effectively complements a Us9-null strain of BHV-5 for anterograde transport, neurovirulence, and neuroinvasiveness in a rabbit model.

The alphaherpesvirus envelope protein Us9 is a type II viral membrane protein that is required for anterograde spread of bovine herpesvirus 5 (BHV-5) infection from the olfactory receptor neurons to the brain. In a rabbit seizure model, Us9-deleted BHV-5 failed to invade the central nervous system (CNS) following intranasal infection. However, when injected directly into the olfactory bulb, retrograde-spread infection from the olfactory bulb (OB) to the piriform cortex and other areas connected to the OB was not affected. In contrast to BHV-5, wild-type BHV-1 failed to invade the CNS following intranasal infection. In this study, we show that mature BHV-1 Us9 is a 30- to 32-kDa protein, whereas mature BHV-5 Us9 is an 18- to 20-kDa protein. In vitro, BHV-1 Us9 is expressed at 3 h postinfection (hpi), whereas BHV-5 Us9 is expressed at 6 hpi. Despite these differences, BHV-1 Us9 not only complemented for BHV-5 Us9 and rescued the anterograde-spread defect of the BHV-5 Us9-deleted virus but conferred increased neurovirulence and neuroinvasiveness in our rabbit seizure model. Rabbits infected with BHV-5 expressing BHV-1 Us9 showed severe neurological signs at 5 days postinfection, which was 1 to 2 days earlier than BHV-5 wild-type or Us9-reverted BHV-5 virus. The data underscore the importance of both Us9 genes for virion anterograde transport and neuroinvasiveness. However, Us9 is not the determinant of the differential neuropathogenesis of BHV-1 and BHV-5.

In the absence of glycoprotein I (gI), gE determines bovine herpesvirus type 5 neuroinvasiveness and neurovirulence.

Bovine herpesvirus type 5 (BHV-5) is an alphaherpesvirus that causes fatal encephalitis in calves. Envelope glycoproteins E (gE) and gI of alphaherpesviruses are important for the pathogenesis in vivo. Previously the authors determined that BHV-5 gE is important for BHV-5 neurovirulence. To determine the role of gI in BHV-5 neurovirulence, the authors have constructed gI-deleted and gI-revertant BHV-5 and analyzed their neuropathogenic properties in a rabbit seizure model. Following intranasal infection, 40% of the rabbits infected with the gI-deleted virus showed severe neurological signs. gI-deleted BHV-5 invaded all the central nervous system (CNS) structures invaded by the gI-revertant BHV-5; however, the number of neurons infected by the gI-deleted virus was similar or slightly reduced (two to four fold). Thus, the gI-deleted virus retained significant neurovirulence and/or neuroinvasive properties when compared with the gE-deleted BHV-5. Pulse-chase analysis revealed that the gE of gI-deleted virus was processed to a larger and a diffused 94- to 100-kDa protein (instead of 94 kDa). The 94- to 100-kDa protein was processed in the Golgi with delayed kinetics but it was endoglycosidase H (EndoH) resistant. In cells infected with gI-deleted virus, there was a reduction in cell-surface gE expression compared to wild-type, which correlated to reduced amount of gE processed in the Golgi. The authors believe that in the absence of gI, BHV-5 gE is sufficient for BHV-5 neurovirulence.

A glycine-rich bovine herpesvirus 5 (BHV-5) gE-specific epitope within the ectodomain is important for BHV-5 neurovirulence.

The bovine herpesvirus 5 (BHV-5) gE ectodomain contains a glycine-rich epitope coding region (gE5 epitope), residues 204 to 218, that is significantly different from the corresponding gE region of BHV-1. Deletion of the gE epitope significantly reduced the neurovirulence of BHV-5 in rabbits. Pulse-chase analyses revealed that the epitope-deleted and wild-type gE were synthesized as N-glycosylated endoglycosidase H-sensitive precursors with approximate molecular masses of 85 kDa and 86 kDa, respectively. Like the wild-type gE, epitope-deleted gE complexed with gI and was readily transported from the endoplasmic reticulum. Concomitantly, the epitope-deleted and wild-type gE acquired posttranslational modifications in the Golgi leading to an increased apparent molecular mass of 93-kDa (epitope-deleted gE) and 94-kDa (wild-type gE). The kinetics of mutant and wild-type gE processing were similar, and both mature proteins were resistant to endoglycosidase H but sensitive to glycopeptidase F. The gE epitope-deleted BHV-5 formed wild-type-sized plaques in MDBK cells, and the epitope-deleted gE was expressed on the cell surface. However, rabbits infected intranasally with gE epitope-deleted BHV-5 did not develop seizures, and only 20% of the infected rabbits showed mild neurological signs. The epitope-deleted virus replicated efficiently in the olfactory epithelium. However, within the brains of these rabbits there was a 10- to 20-fold reduction in infected neurons compared with the number of infected neurons within the brains of rabbits infected with the gE5 epitope-reverted and wild-type BHV-5. In comparison, 70 to 80% of the rabbits exhibited severe neurological signs when infected with the gE5 epitope-reverted and wild-type BHV-5. These results indicated that anterograde transport of the gE epitope-deleted virus from the olfactory receptor neurons to the olfactory bulb is defective and that, within the central nervous system, the gE5 epitope-coding region was required for expression of the full virulence potential of BHV-5.

Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis.

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves. In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17- and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb.