Detection and quantitation of gallid herpesvirus 1 in avian samples by 5' Taq nuclease assay utilizing Minor Groove Binder technology.

A 5' Taq nuclease assay utilizing Minor Groove Binder technology and targeting the thymidine kinase gene of gallid herpesvirus 1 (GaHV-1) was designed and optimized for use in diagnosing avian infectious laryngotracheitis. The assay was specific for GaHV-1 in that it did not react with other avian viral or bacterial pathogens. The detection limit was 1.0x10(-2) median tissue culture infectious dose per reaction or 90 target copies per reaction. Fifteen out of 41 diagnostic samples from sick birds reacted in the assay, five of which produced a typical alphaherpesvirus cytopathic effect (CPE) on chicken kidney (CK) cells. Sequencing, using amplicons generated by a polymerase chain reaction with primers flanking the 5' Taq nuclease amplicon, confirmed the presence of GaHV-1 in six samples (two producing alphaherpesvirus CPE on CK cells, three not producing alphaherpesvirus CPE, and one that was not inoculated onto CK cells). Tracheal swabs taken from 18 healthy broilers did not react in the assay. The ability of the assay to determine viral load in samples was demonstrated. Overall the assay is suitable for the rapid diagnosis of infectious laryngotracheitis.

Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: a new tool for diagnosing infectious coryza.

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

Pasteurella multocida detection by 5' Taq nuclease assay: a new tool for use in diagnosing fowl cholera.

A 5' Taq nuclease assay utilising minor groove binder technology and targeting the 16S rRNA gene was designed to detect Pasteurella multocida (the causative agent of fowl cholera) in swabs collected from poultry. The assay was first evaluated using pure cultures. The assay correctly identified four P. multocida taxonomic type strains, 18 P. multocida serovar reference strains and 40 Australian field isolates (17 from poultry, 11 from pigs and 12 from cattle). Representatives of nine other Pasteurella species, 26 other bacterial species (18 being members of the family Pasteurellaceae) and four poultry virus isolates did not react in the assay. The assay detected a minimum of approximately 10 cfu of P. multocida per reaction. Of 79 poultry swabs submitted to the laboratory for routine bacteriological culture, 17 were positive in the 5' Taq nuclease assay, but only 10 were positive by culture. The other 62 swabs were negative for P. multocida by both 5' Taq nuclease assay and culture. The assay is suitable for use in diagnosing fowl cholera, is more rapid than bacteriological culture, and may also have application in diagnosing P. multocida infections in cattle and pigs.

Characteristics of a haemolytic extract from avian Pasteurella multocida.

In experimental fowl cholera, the intramuscular inoculation of Pasteurella multocida induces tissue damage that implies proteolytic or cytolytic activity of the bacteria. Such activity could not be demonstrated by conventional in vitro tests. The treatment of P. multocida strain VP21 with Tween-80 yielded an extract that lysed washed chicken red cells. Extracts were active to a maximum titre of 64. Haemolytic activity of the extract was neither affected by boiling nor by extremes of pH, indicating the active component was not a simple protein. Treatment with trypsin had no effect, but it was inactivated by Proteinase K. Yields were highest from bacteria grown in dextrose starch- or casein sucrose-yeast broths; were similar if cultured in air or anaerobically, but were reduced if the bacteria were grown in 5% CO(2). Haemolytic activity was eliminated on exposure to serum or serum albumen. The extract from strain VP21 haemolysed red cells from the chicken, rabbit, sheep, horse, bovine and human, with the highest titres observed on chicken cells. Six other avian strains and seven out of 10 strains of P. multocida from other species yielded an extract which haemolysed chicken red cells. The elaboration of this cytotoxic substance in vivo and its role in pathogenesis remains to be determined.

Survival of avian strains of Pasteurella multocida in chicken serum.

The ability of bacteria to survive in serum is considered a likely virulence determinant in diseases where the infective bacteria become septicaemic. Optimal conditions were established to test the survival of Pasteurella multocida in chicken serum. Serum was used at 90%, the inoculum was 10(3)-10(4)cfu in phosphate buffered saline pH 7.4. Survival was measured after incubation for 2-4 h; if survival was <50% the strain was considered serum susceptible. Susceptible strains were either killed or their growth was inhibited. Some resistant strains not only survived but grew rapidly in unheated serum. Thirty-five strains, all originally isolated from clinical fowl cholera, were tested; eight were susceptible, of which three were killed and five inhibited, and the remainder (27) were resistant. Ten serum-resistant P. multocida serogroup A strains were grown in hyaluronidase to remove the capsule and survival in chicken serum was re-tested. Three strains became susceptible, while seven strains remained resistant. Three serum susceptible strains were then tested in the presence of cytidine monophosphate-N-acetylneuraminic acid (CMP-NANA). This substance is present in the human serum, and is known to mask the effect of complement on Neisseria gonorrhoeae rendering susceptible strains resistant. Two of the three serum susceptible strains became resistant in the presence of CMP-NANA. Serum susceptibility/resistance was more complex than that of Escherichia coli, and the role of resistance to avian complement in the pathogenesis of fowl cholera remains to be determined.

Molecular studies on avian strains of Pasteurella multocida in Australia.

A collection of 45 strains of Pasteurella multocida was assembled. The strains had been isolated from cases of fowl cholera in eastern Australia over 8 years, and included mainly type A strains. All the strains were examined for plasmids and resistance to 10 antimicrobial agents and most of the strains were examined for restriction fragment length polymorphism. Nine strains were assayed for pathogenicity for mice. Twenty strains yielded no plasmid. Seven contained a single plasmid of 1.3 kbp and 18 contained 2 plasmids, of 2.4 and 7.5 kbp. All the strains were resistant to streptomycin, trimethoprim and lincomycin while one strain was resistant to tetracycline. There was no correlation between plasmid content and resistance to antimicrobial agents. Three strains that lacked plasmids were highly virulent for mice, 6 strains containing plasmids were not. Restriction fragment length polymorphism generated by HpaII allowed the 39 strains that were tested to be divided into 10 groups.

[Urologic complications of gynecologic surgery. Report of 16 cases]. / Les complications urologiques de la chirurgie gynécologique. A propos de 16 cas.

From 16 cases collected in eight years (January 1991-December 1999) in the I. Deen Teaching Hospital Department of Urology, the authors study the gynaecological and obstetric surgery urological complications. They noted that these complications interest in 80% of cases the 18-47 years old woman with a high parity in 70% of cases. Hysterectomy, whatever the indication is the main etiology: 62.50%, caeserean takes the second place with 25%. The anatomic lesions are ureteral in 68.75% of cases and bladder in 31.25%. In the 14 cases treated, in the department, uretero reimplantation has been done in 46.66%, bladder suture in 40% and nephrectomy in 6.66%. Thirteen women were healed and one died.

[Repair of a vesicovaginal fistula in the ventral supine position (34 cases)]. / Cure de fistule vésicovaginale par voie basse en décubitus ventral (34 cas).

The authors report the efficacy of a transvaginal approach in the ventral supine position for repair of vesicovaginal fistula based on a series of 34 consecutive patients. 34 patients, between the ages of 15 and 60 years, with vesicovaginal fistula (cervicotrigonal in one half of cases) of obstetric origin (73% of dystocia) in multiparous women (62% were treated by transvaginal closure of the fistula in the ventral supine position). 29 complete successes were obtained with 5 residual fistulae which were lost to follow-up. The use of the ventral supine position is valuable in the transvaginal repair of vesicovaginal fistulae.

[Epidemiologic and therapeutic features of urogenital fistulae in Guinea (Conakry)]. / Aspects épidémiologiques et thérapeutiques des fistules uro-génitales en Guinée (Conakry).

OBJECTIVE: The authors analyse the epidemiological and therapeutic aspects of 186 cases of urogenital fistulas and attempt to define a preventive approach to these lesions. METHODS: From January 1986 to December 31, 1993, 186 patients were admitted to the urology department of Ignace Deen hospital for urogenital fistulas. Each patient was submitted to the following assessment: complete clinical examination, laboratory examination, endoscopic examination, radiological examination. A therapeutic classification was established on the basis of this assessment: Group 1: complex fistulas. Group 2: difficult fistulas. Group 3: simple fistulas. RESULTS: Urogenital fistulas were predominantly observed in young primiparous women living in rural zones and the principal cause was a dystocic delivery: 179 cases (96.23%), while only 7 cases (3.7%) were due to gynaecological lesions. 246 primary and secondary repair operations were performed, corresponding to an average of 1.3 operations per patient. Cure was obtained in 131 patients (70.43%) including 37.63%) in Group 1, 8.61% in Group 2 and 21.19% in Group 3. In three cases of partial success, the fistulas were closed; two patients have persistent dysuria with reduced bladder capacity and one patient suffers from dyspareunia with impossibility of coital penetration. Finally, the 49 failures (26.34%) concerned 34 type 1 fistulas; 5 type 2 fistulas and 10 type 3 fistulas. CONCLUSIONS: In the light of our eight-year experience, urogenital fistula still appears to be a real problem in Guinea, where it represents a public health problem for which surgical cure still raises technical difficulties. In the fight for eradication of urogenital fistula in developing countries, emphasis must be placed on prevention with a just and equitable distribution of health care personnel in rural zones which are often underprivileged: constant improvement of the road network to allow rapid transfer of cases of foetomaternal dystocia to a reference centre; improvement of health structures; urological and obstetric surveillance of any woman operated for urogenital fistula.

Encephalomyocarditis virus infection in a splenectomised calf.

BACKGROUND: Encephalomyocarditis (EMC) caused by EMC virus (EMCV) was diagnosed in a 5-month-old splenectomised calf, which died suddenly on an experimental farm that had a high infestation of rodents. RESULTS: At postmortem examination, the lungs were dark purple and diffusely congested. On histological examination, the calf had severe necrotising myocarditis. EMCV was isolated from the heart. The polyprotein gene of the EMCV isolate was amplified by PCR and had 85-91% identity with published EMCV sequences, including 89% identity with isolates from Queensland. On phylogenetic analysis, the polyprotein gene had highest sequence identity with South Korean EMCV strain, CBNU. CONCLUSION: This is the first report of naturally occurring EMC in cattle in Australia and the first report of naturally occurring bovine EMC from which EMCV has been isolated.

Equine multinodular pulmonary fibrosis in three horses in Australia.

BACKGROUND: Equine multinodular pulmonary fibrosis (EMPF) is a recently described form of interstitial pneumonia associated with the presence of equine herpesvirus type 5 (EHV-5). Since 2007, several case reports from America, Europe and the United Kingdom have further characterised the clinical presentation and laboratory findings of this disease. CASE REPORTS: Three Thoroughbred broodmares were diagnosed with EMPF. Diagnosis was based on lung histopathology and positive identification of EHV-5 using PCR DNA amplification. There was multiple organ involvement in all three cases, including identification of EHV-5 in hepatic tissue in one case. Two of the three horses died. Treatment with acyclovir was unsuccessful in one horse and one horse survived without antiviral or corticosteroid treatment. CONCLUSION: This case series is, to the authors' knowledge, the first report of EMPF in Australia and adds to the clinical description of the disease.

Isolation of bovine herpesvirus type 5 from the semen of a healthy bull in Australia.

Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed.

Detection of Arcobacter spp. in piggery effluent and effluent-irrigated soils in southeast Queensland.

AIMS: To investigate the occurrence and levels of Arcobacter spp. in pig effluent ponds and effluent-treated soil. METHODS AND RESULTS: A Most Probable Number (MPN) method was developed to assess the levels of Arcobacter spp. in seven pig effluent ponds and six effluent-treated soils, immediately after effluent irrigation. Arcobacter spp. levels in the effluent ponds varied from 6.5 x 10(5) to 1.1 x 10(8) MPN 100 ml(-1) and in freshly irrigated soils from 9.5 x 10(2) to 2.8 x 10(4) MPN g(-1) in all piggery environments tested. Eighty-three Arcobacter isolates were subjected to an abbreviated phenotypic test scheme and examined using a multiplex polymerase chain reaction (PCR). The PCR identified 35% of these isolates as Arcobacter butzleri, 49% as Arcobacter cryaerophilus while 16% gave no band. All 13 nonreactive isolates were subjected to partial 16S rDNA sequencing and showed a high similarity (>99%) to Arcobacter cibarius. CONCLUSIONS: A. butzleri, A. cryaerophilus and A. cibarius were isolated from both piggery effluent and effluent-irrigated soil, at levels suggestive of good survival in the effluent pond. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide quantitative information on Arcobacter spp. levels in piggery effluent and to associate A. cibarius with pigs and piggery effluent environments.

Field isolates of fowlpox virus contaminated with reticuloendotheliosis virus.

The polymerase chain reaction (PCR) method was used to examine samples from field cases of fowlpox for the presence of reticuloendotheliosis virus (REV). The S-strain fowlpox vaccine, known to be contaminated with REV, served as a positive control. Fowlpox virus was grown from field samples and vaccines by inoculation of embryonated hen eggs by the chorioallantoic membrane (CAM) route. DNA was extracted from the CAM lesions and examined for REV proviral sequences using primers specific for the long terminal repeats of REV. Amplicons of the expected length were detected in all the 45 field samples from poultry and in the S strain vaccine. Two other vaccines and two isolates from wild birds contained no detectable REV sequences. The PCR products from the vaccine and one field isolate were sequenced and were identical. These products showed 81 to 87.5% homology with the published sequences for the long terminal repeats of REV. It was not determined whether the REV proviral DNA was integrated with cellular DNA, fowlpox DNA or both. Inoculation of day-old chickens with the S-strain vaccine resulted not only in the production of fowlpox lesions but also feathering defects and proventriculitis. This suggests that the REV present in the vaccine is replication competent. Problems being encountered with protection from fowlpox following vaccination in Australia might be attributed to simultaneous challenge with fowlpox virus and REV.