Appraisal of the Australian Veterinary Prescribing Guidelines for antimicrobial prophylaxis for surgery in dogs and cats.

The Australian Veterinary Prescribing Guidelines for antimicrobial prophylaxis for surgery on dogs and cats are evidence-based guidelines for veterinary practitioners. Validation of these guidelines is necessary to ensure quality and implementability. Two validated tools, used for medical guideline appraisal, were chosen to assess the guidelines. The terminology from the GuideLine Implementability Appraisal (GLIA) and the Appraisal of Guidelines for Research and Evaluation version 2 (AGREE II) were adapted for use by veterinarians. A two-phase evaluation approach was conducted. In the first phase of the evaluation, the GLIA tool was used by two specialist veterinary surgeons in clinical practice. The results of this phase were then used to modify the guidelines. In the second phase, the AGREE II tool was used by 6 general practitioners and 6 specialists to appraise the guidelines. In phase 1, the specialist surgeons either agreed or strongly agreed that the guidelines were executable, decidable, valid and novel, and that the guidelines would fit within the process of care. The surgeons were neutral on flexibility and measurability. Additional clarity around one common surgical procedure was added to the guidelines, after which the surgeons agreed that the guidelines were sufficiently flexible. In phase 2, 12 veterinarians completed the assessment using the AGREE II tool. In all sections the scaled domain score was greater than 70%. The overall quality of the guidelines was given a global scaled score of 76%. This assessment has demonstrated that the guidelines for antimicrobial prophylaxis for companion animal surgery are valid and appear implementable.

Antimicrobials used for surgical prophylaxis by equine veterinary practitioners in Australia.

BACKGROUND: Antimicrobials are widely used in Australian veterinary practices, but no investigation into the classes of antimicrobials used, or the appropriateness of use in horses, has been conducted. OBJECTIVES: The aim of the study was to describe antimicrobial use for surgical prophylaxis in equine practice in Australia. STUDY DESIGN: Cross-sectional questionnaire survey. METHODS: An online questionnaire was used to document antimicrobial usage patterns. Information solicited in the questionnaire included demographic details of the respondents, the frequency with which antimicrobials were used for specific surgical conditions (including the dose, timing and duration of therapy) and practice antimicrobial use policies and sources of information about antimicrobials and their uses. RESULTS: A total of 337 members of the Australian veterinary profession completed the survey. Generally, the choice of antimicrobial was appropriate for the specified equine surgical condition, but the dose and duration of therapy varied greatly. While there was poor optimal compliance with British Equine Veterinary Association guidelines in all scenarios (range 1-15%), except removal of a nonulcerated dermal mass (42%), suboptimal compliance (compliant antimicrobial drug selection but inappropriate timing, dose or duration of therapy) was moderate for all scenarios (range 48-68%), except for an uninfected contaminated wound over the thorax, where both optimal and suboptimal compliance was very poor (1%). Veterinarians practicing at a university hospital had higher odds of compliance than general practice veterinarians (Odds ratio 3.2, 95% CI, 1.1-8.9, P = 0.03). MAIN LIMITATIONS: Many survey responses were collected at conferences which may introduce selection bias, as veterinarians attending conferences may be more likely to have been exposed to contemporary antimicrobial prescribing recommendations. CONCLUSIONS: Antimicrobial use guidelines need to be developed and promoted to improve the responsible use of antimicrobials in equine practice in Australia. An emphasis should be placed on antimicrobial therapy for wounds and appropriate dosing for procaine penicillin.

The role of Type 1, P and S fimbriae in binding of Escherichia coli to the canine endometrium.

Escherichia coli (E. coli) is the most commonly isolated infectious agent causing pyometra in bitches. Many E. coli strains isolated from the uteri of infected dogs carry several adhesin genes (fimH, papGIII and sfa). The objective of this study was to investigate the role of each adhesin gene product, acting alone or expressed in combination, in the bacterial binding to canine endometrium. E. coli strain P3, which was isolated from a uterus of a bitch naturally affected with pyometra, was shown by PCR to carry all three known fimbrial adhesin genes fimH, papGIII and sfa. Knockout (KO) mutants of this wildtype (P3-wt) strain were generated using insertional inactivation. Adhesion assays on anoestrous uteri of three post-pubertal bitches were undertaken. Overall, the number of bacteria adhering to canine endometrial biopsies was comparable between strains and no significant difference in the number of bound bacteria was found between the P3-wt strain and the single or double KO-strains. However, the triple knockout strain displayed less binding to the canine endometrium compared with the P3-wt strain. This study shows that a pathogenic E. coli strain (P3) isolated from the uterus of a bitch with pyometra was able to fully compensate for the loss of two of its three known adhesin genes. It was necessary to inactivate all three known adhesin genes in order to see a significant decrease in binding to canine endometrium.

Uropathogenic virulence factor FimH facilitates binding of uteropathogenic Escherichia coli to canine endometrium.

Pyometra is a potentially life-threatening condition in bitches and is often caused by Escherichia coli infection. Both pathogenic and non-pathogenic E. coli strains commonly carry the genes for type 1 fimbriae that mediate bacterial adhesion onto host epithelium. To investigate whether the type 1 fimbrial adhesin, FimH, facilitates the binding of uropathogenic E. coli to canine endometrium, the fimH gene was insertionally inactivated in a pathogenic E. coli strain. The ability of E. coli to bind to canine endometrial epithelial cells was determined in vitro using canine uterine biopsies. Binding of the fimH mutant was only 0.3% of that of the wild type. Complementation of the mutation restored the phenotype to that of the parent. This study has developed an in vitro model that allows quantitative and qualitative assessment of bacterial binding to canine endometrium and has demonstrated that the fimH gene plays a role in adherence of pathogenic E. coli to canine endometrium.

Effect of simulated stages of the canine oestrous cycle on Escherichia coli binding to canine endometrium.

Pyometra, a prevalent infectious uterine disease that affects intact middle-aged bitches, is typically associated with Escherichia coli. Our hypotheses were (i) that bacterial adhesion to canine endometrium differs between different stages of the oestrous cycle and (ii) that the adhesin FimH facilitates this adhesion. Twelve post-pubertal, ovariectomized greyhound bitches were treated with exogenous hormones to simulate different stages of the oestrous cycle. Tissue samples from each uterus were incubated with a pathogenic E. coli strain carrying the fimH gene, but no other adhesin genes (P4-wt)--or an E. coli strain in which fimH was insertionally inactivated (P4-∆fimH::kan)--or with phosphate-buffered saline as a negative control. After washing, tissue samples were homogenized for quantification of adherent bacteria. The differences in binding to canine endometrium at different stages of the oestrous cycle were not significant. However, the mean difference in binding of the P4-wt and the P4-∆fimH::kan across all stages of the simulated oestrous cycle was significant (p < 0.001 by paired t-test on geometric means). Individual differences in numbers of P4-wt bacteria bound between dogs might suggest genetic variations or epigenetic differences in FimH receptor expression by the endometrium, unrelated to the stage of the oestrous cycle.

Detection of a novel gammaherpesvirus in koalas (Phascolarctos cinereus).

A novel gammaherpesvirus was detected in wild koalas (Phascolarctos cinereus) captured at different locations during 2010. Sequence analysis of the DNA polymerase gene revealed that the virus was genetically distinct from all known gammaherpesviruses. This is the first herpesvirus to be definitively identified in the Vombatiforme suborder (koalas and wombats).

Glycoprotein G is a virulence factor in infectious laryngotracheitis virus.

Infectious laryngotracheitis virus (ILTV; Gallid herpesvirus 1) is an alphaherpesvirus that causes acute respiratory disease in chickens. The role of glycoprotein G (gG) in vitro has been investigated in a number of alphaherpesviruses, but the relevance of gG in vivo in the pathogenicity of ILTV or in other alphaherpesviruses is unknown. In this study, gG-deficient mutants of ILTV were generated and inoculated into specific-pathogen-free chickens to assess the role of gG in pathogenicity. In chickens, gG-deficient ILTV reached a similar titre to wild-type (wt) ILTV but was significantly attenuated with respect to induction of clinical signs, effect on weight gain and bird mortality. In addition, an increased tracheal mucosal thickness, reflecting increased inflammatory cell infiltration at the site of infection, was detected in birds inoculated with gG-deficient ILTV compared with birds inoculated with wt ILTV. The reinsertion of gG into gG-deficient ILTV restored the in vivo phenotype of the mutant to that of wt ILTV. Quantitative PCR analysis of the expression of the genes adjacent to gG demonstrated that they were not affected by the deletion of gG and investigations in vitro confirmed that the phenotype of gG-deficient ILTV was consistent with unaltered expression of these adjacent genes. This is the first reported study to demonstrate definitively that gG is a virulence factor in ILTV and that deletion of gG from this alphaherpesvirus genome causes marked attenuation of the virus in its natural host.

A model for cystic endometrial hyperplasia/pyometra complex in the bitch.

The objective of this study was to develop a reliable model for the study of the cystic endometrial hyperplasia and pyometra complex (CEH/P) in the bitch. Greyhound bitches (n = 15) were ovariectomised and allocated into three groups (Group 1, n = 5; Group 2, n = 5; Group 3, n = 10, including 5 used from Group 1). Simulated proestrus, estrus and diestrus were induced by treatment with estradiol benzoate and megestrol acetate. The duration of cervical opening during estrus was determined by the intra-vaginal infusion of radio-opaque medium and subsequent radiography of the uterus (Group 1). One milliliter of a culture of Escherichia coli (with five uro-pathogenic virulence factors as identified by PCR: pap, sfa, hlyA, cnf1 and fim) was inoculated intra-vaginally daily throughout the simulated estrus (Group 2). One milliliter of the culture (n = 6) or sterile Luria-Bertani broth (n = 4) was introduced directly into the uterus on simulated diestrus Days 8 or 12 (Group 3). Necropsies were performed 12 and 7-14 days after the inoculation (Groups 2 and 3). The cervix remained open throughout the duration of simulated estrus (5-6 days) in four out of five bitches, and for a shorter duration (3 days of a 6-day estrus period) in one bitch (Group 1). CEH/P was induced by inoculation of bacteria into the uterus (10/10 bitches) but not into the vagina (0/5 bitches), (P = 0.003). A model for the study of CEH/P has been validated.

A glycoprotein I- and glycoprotein E-deficient mutant of infectious laryngotracheitis virus exhibits impaired cell-to-cell spread in cultured cells.

In alphaherpesviruses, glycoprotein I (gI) and glycoprotein E (gE) form a heterodimer that functions in cell-to-cell spread of the virus. Generally, alphaherpesvirus mutants that lack these glycoproteins are replication competent in cell culture but show a reduced capacity for cell-to-cell spread and hence smaller plaque sizes. Infectious laryngotracheitis virus (ILTV), or Gallid herpesvirus 1, is an alphaherpesvirus that causes respiratory disease in chickens. The roles of gI and gE in ILTV have not been investigated previously. In this study, a glycoprotein I and glycoprotein E deletion mutant of ILTV (gI/gE-ve ILTV) was generated by replacing the region of the ILTV genome coding for the adjacent gI and gE genes with the gene for enhanced green fluorescent protein (eGFP). This gI/E-ve ILTV was readily propagated in cell culture in the presence of wildtype ILTV (wt ILTV). However, in the absence of wt ILTV the propagation of gI/gE-ve ILTV was severely impaired. Infection of permissive cell cultures with gI/gE-ve ILTV failed to produce plaques but single infected cells could be identified by fluorescence microscopy. This suggests that gI/gE has a more significant role in the cell-to-cell spread of ILTV in vitro than in many other alphaherpesviruses.

The cellular immune response in the tracheal mucosa to Mycoplasma gallisepticum in vaccinated and unvaccinated chickens in the acute and chronic stages of disease.

Mycoplasma gallisepticum causes a lymphoproliferative response in the tracheal mucosa of infected birds. The studies reported here aimed to determine, using immunohistochemical and immunofluorescent staining, which lymphocyte subsets were infiltrating the mucosa during the acute and chronic phases of disease and to determine whether these subsets differed in birds that had been vaccinated with strain ts-11. In vaccinates there was no detectable infiltration of T or B lymphocytes between 1 and 6 weeks after infection with a virulent strain. Unvaccinated birds had an initial influx of CD8+TCR- lymphocytes at 1 week, with the numbers decreasing over the next 5 weeks. CD8+TCR+ cells increased over the 6 weeks. The proportion of CD4+TCRalphabeta2+ cells also increased, whilst there was an increase in CD4+TCRalphabeta1+ cells at 3 weeks and then a decrease by 6 weeks. B lymphocytes were not detected until 3 weeks after infection, and their appearance coincided with a decrease in the concentration of mycoplasma DNA detectable in the trachea. The formation of clusters of CD8+TCR- lymphocytes was a prominent feature of the early response, while at 3 and 6 weeks after infection clusters of B cells became prominent, although in some cases they surrounded a cluster of CD8+ cells. These observations suggest a primary role for local antibody mediated responses in controlling M. gallisepticum infection, but also show that there are probably significant natural killer and cytotoxic T cell responses to infection, although the efficacy of these in controlling infection was not able to be determined.

Lymphocytic infiltration in the chicken trachea in response to Mycoplasma gallisepticum infection.

A prominent feature of disease induced by Mycoplasma gallisepticum is a lymphoproliferative response in the respiratory tract. Although this is also seen in other mycoplasma infections, including Mycoplasma pneumoniae, the phenotype of the lymphocytes infiltrating the respiratory tract has not been determined. In this study, the numbers and distribution of lymphocytes in the tracheas of chickens infected with a virulent strain of M. gallisepticum were examined. Three groups of chickens were experimentally infected with M. gallisepticum and three unchallenged groups were used as controls. One infected and one control group were culled at 1, 2 and 3 weeks post infection. Tracheas were removed and examined for the presence and number of T cells carrying CD4, CD8, TCRgamma7, TCRalphabeta1 or TCRalphabeta2 markers. There was no significant difference in the number of CD8+ cells in the upper, middle and lower trachea. High numbers of both CD4+ and CD8+ cells were found with variable numbers of TCRalphabeta1+ and TCRalphabeta2+, but no TCRgammadelta+, cells throughout the time course. The distribution of CD4 cells was dispersed, while the CD8+ cells were clustered in follicular-like arrangements. No difference was detected in the distribution of TCRalphabeta1+ and TCRalphabeta2+ cells. The titre of mycoplasma genomes in the trachea decreased significantly from 1 to 2 weeks, while the mucosal thickness of the trachea increased significantly from 1 to 2 weeks then decreased from 2 to 3 weeks, indicating resolution of the lesions following control of infection. This study is the first to examine the phenotypes of T lymphocytes infiltrating the respiratory tract during mycoplasma infections. The findings suggest involvement of specific stimulation of CD8+ cells, particularly in the acute phase of disease.

Multigene families encoding the major hemagglutinins in phylogenetically distinct mycoplasmas.

Mycoplasma synoviae has two major membrane antigens, MSPA and MSPB, both of which are phase variable and which may be coordinately involved in adhesion of the organism to erythrocytes. A single gene (vlhA) from M. synoviae was characterized, and polypeptides were expressed from nonoverlapping 5' and 3' regions in Escherichia coli. The expression product of the vlhA 5' region reacted with specific reagents against MSPB, while that of the 3' region reacted with specific reagents against MSPA. Analysis of the predicted amino acid sequence showed a characteristic signal peptidase II cleavage site, and the presence of the acylation site was confirmed by identification of a lipid-associated membrane protein, similar in molecular mass to MSPB, in [3H]palmitate-labelled membrane proteins. Further sequence analysis of the vlhA gene revealed a high identity with the Mycoplasma gallisepticum pMGA1.7 gene, a member of a large translated family. The vlhA gene was shown to hybridize to multiple restriction fragments of the M. synoviae genome, suggesting that it was also a member of a multigene family. These findings indicate that coordinate phase variation of the two major surface antigens of M. synoviae WVU may be due to their expression from the same gene and that homologous gene families encode the major hemagglutinins of two phylogenetically distinct mycoplasmas. The presence of homologous multigene families in such phylogenetically distinct species, but not in the genomes of more closely related species, suggests that the families may have been transferred horizontally.

Expression of two members of the pMGA gene family of Mycoplasma gallisepticum oscillates and is influenced by pMGA-specific antibodies.

Certain monoclonal antibodies and polyclonal antisera directed to pMGA, the major protein of Mycoplasma gallisepticum, were tested for the ability to influence the surface phenotype of the cell population which resulted from their inclusion in growth medium. The polyclonal antiserum and one monoclonal antibody (MAb 66) resulted in an alteration of surface phenotype; specifically, populations of cells grown either on plates or in broth cultures which contained these reagents ceased the expression of pMGA and instead expressed an antigenically unrelated new polypeptide (p82). Upon the removal of antibody, the progeny of these cells regained pMGA expression and produced antigenically sectored colonies. The basis of this switch between pMGA+ and pMGA- states was shown to be transcriptional. The p82 polypeptide, the expression of which resulted from growth of cells in antibodies, was another member of the pMGA gene family and was located just downstream from the pMGA gene normally expressed by the M. gallisepticum cells used. Collectively the results of this work suggest that this organism has evolved an unusual means of altering the antigenic composition of its surface in response to antibodies or to other environmental cues.

Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses.

Equine rhinovirus 1 (ERhV1) is a respiratory pathogen of horses which has an uncertain taxonomic status. We have determined the nucleotide sequence of the ERhV1 genome except for a small region at the 5' end. The predicted polyprotein was encoded by 6741 nucleotides and possessed a typical picornavirus proteolytic cleavage pattern, including a leader polypeptide. The genomic structure and predicted amino acid sequence of ERhV1 were more similar to those of foot-and-mouth disease viruses (FMDVs), the only members of the aphthovirus genus, than to those of other picornaviruses. Features which were most similar to FMDV included a 16-amino acid 2A protein which was 87.5% identical in sequence of FMDV 2A, a leader (L) protein similar in size to FMDV Lab and the possibility of a truncated L protein similar in size to FMDV Lb, and a 3C protease which recognizes different cleavage sites. However, unlike FMDV, ERhV1 had only one copy of the 3B (VPg) polypeptide. The phylogenetic relationships of the ERhV1 sequence and nucleotide sequences of representative species of the five genera of the family Picornaviridae were examined. Nucleotide sequences coding for the complete polyprotein, the RNA polymerase, and VP1 were analyzed separately. The phylogenetic trees confirmed that ERhV1 was more closely related to FMDV than to other picornaviruses and suggested that ERhV1 may be a member, albeit very distant, of the aphthovirus genus.

Expression studies on four members of the pMGA multigene family in Mycoplasma gallisepticum S6.

A large family of related genes known as pMGA exists in the avian pathogen Mycoplasma gallisepticum but only a single member of this family was previously found to be expressed in one strain of this bacterium. In this work two unrelated strains of M. gallisepticum were also shown by amino-terminal sequencing to express a unique pMGA polypeptide in both cases. To investigate pMGA gene selection in M. gallisepticum, mRNA expression was analysed in M. gallisepticum strain 56 using reverse transcription-PCR (RT-PCR) and Northern blot techniques with probes for several members of the pMGA multigene family. It was shown that the pMGA message is 2.2 kb in size and is monocistronic. RT-PCR detected four different pMGA mRNA molecules but their relative yields were significantly affected by magnesium concentration. By quantitative Northern analysis, the relative abundances of the four pMGA mRNAs in M. gallisepticum S6 total RNA was determined: the pMGA1.1 mRNA predominated [1.88 ng (micrograms total RNA)-1] but at least three other pMGA genes were found to be transcribed but at much lower levels (20 to 40-fold lower). The pMGA1.1 mRNA is expressed at a level five times higher than the tuf gene, known to be one of the most abundantly expressed proteins in the prokaryotic cell. The start point of transcription for pMGA1.1 was determined and probable promoter assigned. From these data it appears likely that transcriptional control plays a major role in the selection of pMGA gene expression in the M. gallisepticum cell.

A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1.

We describe a type-specific ELISA, which distinguishes antibody to equine herpesvirus 4 (EHV4; equine rhinopneumonitis) and EHV1 (equine abortion virus) thereby identifying horses that have been infected with either or both of these antigenically related viruses. The antigens used are parts of the EHV4 and EHV1 glycoprotein G (gG) homologues expressed in E. coli as fusion proteins [Crabb and Studdert, 1993: J Virol 67: 6332-6338). The expressed proteins comprise corresponding regions of the gG molecules that are highly divergent and encompass strong, typespecific epitopes. Plasma samples from 97 Thoroughbred and 174 Standardbred horses were tested, all of which were unvaccinated. All horses were strongly EHV4 ELISA positive while 30% were EHV1 ELISA positive. The type-specificity of the EHV1 gG antigen was tested in cross-absorption experiments and it was found that 96% (66 of 69) of EHV1 ELISA positive horses were true EHV1 antibody positives. It was also shown that 100% (26 of 26) horses known to have been exposed to EHV1, either by infection or immunisation with EHV1, had significant levels of antibody against the EHV1 gG antigen (i.e., all horses recognised the EHV1 epitope(s) contained within this molecule). Maintenance of EHV1 gG antibody was examined by testing sera obtained from mares four years after confirmed EHV1 abortion. Seven out of 10 of these mares remained EHV1 ELISA positive. In summary, the ELISA is highly specific and is sufficiently sensitive to detect all horses previously infected with EHV4 and most previously infected with EHV1.