Daria N. Love

Rapid, single-step differentiation of equid herpesviruses 1 and 4 from clinical material using the polymerase chain reaction and virus-specific primers

Sets of primers were designed which enabled specific amplification of homologous regions of the glycoprotein C and gene 76 genetic loci of equine herpesviruses 1 and 4 (EHV-1 and EHV-4). The resultant virus-specific polymerase chain reaction (PCR) products arising from each loci could be discriminated easily on the basis of size on an agarose gel, allowing rapid differentiation of the two equine herpesviruses. Specificity of the amplifications were confirmed by Southern hybridization and restriction endonuclease digestion. The PCR test was applied to nasal swab samples from weanling foals and to archival aborted fetal tissue samples and the results compared to those obtained by virus isolation. A strong correlation was found between this PCR assay and virus isolation methods of EHV-1 and EHV-4 detection and discrimination.

Oral associated bacterial infection in horses: studies on the normal anaerobic flora from the pharyngeal tonsillar surface and its association with lower respiratory tract and paraoral infections

Two hundred and seventy bacterial isolates were obtained from the pharyngeal tonsillar surface of 12 normal horses and 98 obligatory anaerobic bacteria were characterised. Of these, 57 isolates belonging to 7 genera (Peptostreptococcus (1); Eubacterium (9); Clostridium (6); Veillonella (6); Megasphera (1); Bacteroides (28); Fusobacterium (6)) were identified, and 16 of these were identified to species level (P. anaerobius (1); E. fossor (9); C. villosum (1); B. fragilis (1); B. tectum (2); B. heparinolyticus (2)). Three hundred and twenty isolates were obtained from 23 samples from horses with lower respiratory tract (LRT) or paraoral (PO) bacterial infections. Of the 143 bacteria selected for detailed characterisation, obligate anaerobes accounted for 100 isolates, facultative anaerobes for 42 isolates and obligate aerobes for one isolate. Phenotypic characterisation separated 99 of the isolates into 14 genera. Among the obligately anaerobic species, Gram-positive cocci including P. anaerobius comprised 25% of isolates, E. fossor 11% and other Gram-positive rods (excluding Clostridium sp.) 18% of isolates. The Gram-negative rods comprised B. fragilis 5%, B. heparinolyticus 5%, asaccharolytic pigmented Bacteroides 3% and other Bacteroides 13%, while a so-far unnamed species of Fusobacterium (7%), and Gram-negative corroding rods (3%) were isolated. Among the facultatively anaerobic isolates, S. equi subsp. zooepidemicus accounted for 31% of isolates, followed by Pasteurella spp. 19%, Escherichia coli 17%, Actinomyces spp. 9%, Streptococcus spp. 9%. Incidental facultative isolates were Enterococcus spp. 2%, Enterobacter cloaceae 2%, Actinobacillus spp. 2% and Gram-negative corroding rods 5%. On the basis of the similarities (as determined by DNA hybridization data and/or phenotypic characteristics) of some of the bacterial species (e.g. E. fossor and B. heparinolyticus) isolated from both the normal pharyngeal tonsillar surfaces and LRT and PO diseases of horses, it is considered that the most likely source of bacteria involved in these disease processes is flora from the oral cavity.

The obligate and facultatively anaerobic bacterial flora of the normal feline gingival margin

Samples from the gingival margins of 14 cats considered normal on clinical examination were cultured for facultative and obligate anaerobic bacteria. All mouths were free from any gingival marginal inflammation and tartar build-up; all cats were between 6 and 12 months of age. A mixed growth was obtained from all samples. The mean number of bacterial species per sample was 10.7 with a range of 7-16 isolates. Of the 150 isolates processed, 109 (72.66%) were obligate anaerobes. Of the facultatively anaerobic species, Actinomyces (including A. viscosus, A. hordeovulneris and A. denticolens) comprised 12%, Pasteurella multocida 9.33% of isolates and Propionibacterium species 6% of all isolates. Gram-negative bacilli belonging to the genera Bacteroides and Fusobacterium were isolated from 12 of the 14 samples, and comprised 77% of the obligate anaerobes isolated. Clostridium villosum comprised 10.1% of obligately anaerobic isolates, Wolinella species made up 6.42%, while 4.58% were Peptostreptococcus anaerobius. The most commonly isolated obligately anaerobic species was C. villosum and the most commonly isolated facultatively anaerobic species was P. multocida. These findings show a bacterial flora of the normal feline mouth which is very similar in composition to that of cat fight abscesses and feline pyothorax.

Genetic structure of populations of β-haemolytic Lancefield group C streptococci from horses and their association with disease

The genetic structure of beta-haemolytic Lancefield group C streptococci isolated from horses in Australia was examined by multilocus enzyme electrophoresis. The 249 isolates comprised 70 classified phenotypically as Streptococcus equi subspecies equi, 177 classified as S equi subspecies zooepidemicus and two which were unclassifiable. Forty-one electrophoretic types were identified which could be classified into three major clusters, A, B and C. Of the isolates, 178 fell into cluster B (types 4 to 22) and lay within a genetic distance of 0.36. Sixty-nine of the 70 S equi subspecies equi isolates fell into type 12, which suggests that they were members of a single clone, and the isolates from abscesses were significantly more likely to belong to type 12 than those from horses with no clinical signs (P < 0.001). There were no other significant associations between electrophoretic types or clusters and the isolation of the organism from particular sites. These data suggested that S zooepidemicus may be the archetypal species from which the clone designated subspecies equi has been derived. If isolates of the subspecies equi from other geographical regions also prove to be members of electrophoretic type 12, this hypothesis would be strengthened.

Isolation and characterisation of bacteria from pyothorax (empyaemia) in cats

Abstract Samples from 19 cases of feline empyaemia were examined. All cases had no prior treatment and specimens were collected by thoracocentesis after preparation of the skin as for aseptic surgery. Of the 87 bacterial strains isolated, 70 (80.5%) were anaerobes and 17 (19.5%) were facultative anaerobes. Fourteen cases contained mixtures of anaerobes and facultative anaerobes, and five contained anaerobes only. Bacteroides was the most commonly isolated genus (42.5% of all isolates). Clostridium villosum (16.1%) was the most frequently isolated bacterial species followed by Peptostreptococcus anaerobius and Pasteurella multocida (each 12.6%). The most commonly isolated anaerobic species was Clostridium villosum sp.nov. (20% of anaerobic isolates and 16.1% of all isolates) and Pasteurella multocida was the most commonly isolated facultatively anaerobic species (64.7% of facultative isolates and 12.6% of all isolates).

Isolation and characterisation of bacteria from abscesses in the subcutis of cats.

Thirty-six closed abcesses in the subcutis of cats were examined. Of 168 bacterial strains isolated, 121(72%) were anaerobes and 47 (28%) were facultative anaerobes. Twenty-six abscesses contained mixtures of facultative anaerobes and anaerobes, six contained anaerobes only and four contained facultative anaerobes only. Bacteriodes was the genus most commonly isolated (28.6% of all isolates) followed by Fusobacterium (19.0%) and Pasteurella (multocida) (13.1%). Peptostreptococcus anaerobius was the most commonly isolated anaerobic species (13.2% of anaerobic isolates and 9.5% of all isolates)and Past, multocida was the most commonly isolated facultative anaerobe (46.8%; 13.1%of all isolates).

Associations amongst three feline Porphyromonas species from the gingival margin of cats during periodontal health and disease.

Digoxigenin labelled whole chromosomal DNA probes directed against three feline members of the genus Porphyromonas (P. gingivalis VPB 3492, P. circumdentaria NCTC 12469T and P. salivosa VPB 3313) were used to identify and quantify organisms in samples taken from the gingival margins of 40 domestic cats with different grades of periodontal disease. At the right upper canine tooth, the grade of periodontal disease ranged from 0 to 5 and the cfu of facultative/obligate anaerobes ranged from 5.5 x 10(4) to 2.0 x 10(6)). In 38 of the 40 cats, at least one of the three Porphyromonas species was isolated and regression analysis showed that the cfu of total Porphyromonas sp. was a highly significant indicator of the grade of periodontal disease (p < 0.001, R2 0.510). Feline P. gingivalis was isolated from 37 of the 40 cats and regression analysis showed that it was a highly significant predictor of the grade of periodontal disease (p < 0.001, R2 0.561). The cfu of P. salivosa was a significant predictor of the grade of periodontal disease (p < 0.001, R2 0.286) and regression analysis showed that there was a significant positive relationship between cfu of P. circumdentaria and grade of periodontal disease (p = 0.018, R2 0.116). The periodontal grades at the right upper third premolar tooth ranged from 0 to 6. The cfu of facultative/obligate anaerobes isolated ranged from 1.2 x 10(5) to 7.9 x 10(6), and regression analysis showed that cfu was a significant predictor of periodontal grade (p < 0.001, R2 0.378). The cfu of total Porphyromonas species ranged from 1.2 x 10(4) to 1.7 x 10(6) and regression analysis of the cfu against the grade of periodontal disease showed a highly significant association (p < 0.001, R2 0.633). The cfu of P. gingivalis ranged from 0 to 1.1 x 10(6) and regression analysis of the cfu of P. gingivalis against the grade of periodontal disease showed a highly significant association (p < 0.001, R2 0.439). The cfu of P. salivosa was a significant predictor of the grade of periodontal disease (p < 0.001, R2 0.479) and the same association was found between cfu of P. circumdentaria and grade of periodontal disease (p = 0.002, R2 0.204). This study has established Porphyromonas as anumerically significant and highly prevalent genus in feline periodontal disease.

Immune responses and protective efficacy of recombinant baculovirus-expressed glycoproteins of equine herpesvirus 1 (EHV-1) gB, gC and gD alone or in combinations in BALB/c mice

Baculovirus-expressed glycoproteins of EHV-1 gB, gC and gD alone or in combination evoked antibody responses and protected vaccinated mice against a challenge with EHV-1. gB, gD, gB + gC, gB + gD and gC + gD elicited very high levels of ELISA antibodies while gC and gC + gD elicited high levels of virus neutralising antibodies. Western blotting demonstrated that the antibodies produced were not only specific for the baculovirus-expressed glycoproteins gB, gC and gD, but also highly specific for each EHV-1 glycoprotein. Vaccination of mice with gB or gD prevented clinical signs of infection in mice challenged with EHV-1 and all vaccinated groups of mice except controls showed a rapid clearance of virus from the lungs and a reduction in lesions characteristic of herpesviruses in the lungs post-challenge. Notably, the lungs of mice vaccinated with gB, gD or gB + gD and challenged with EHV-1 showed prominent peribronchiolar and perivascular aggregations of mononuclear cells, predominantly lymphocytes. Immunocytochemical staining of these sections showed large numbers of T cells, suggesting an active role for these cells at the site of virus replication post-challenge.

Phylogenetic analysis of members of the genus Porphyromonas and description of Porphyromonas cangingivalis sp. nov. and Porphyromonas cansulci sp. nov.

The partial 16S rRNA gene sequences of representative strains of two groups of anaerobic, gram-negative, pigmented, asaccharolytic, rod-shaped bacteria isolated from subgingival plaque of dogs with naturally occurring periodontal disease were determined. A comparative analysis of the rRNA sequence data revealed that the two groups of organisms represent previously unknown lines of descent within the genus Porphyromonas. On the basis of our phylogenetic findings and the phenotypic distinctiveness of the organisms, two new species, Porphyromonas cangingivalis and Porphyromonas cansulci, are proposed.

Bacteroides salivosus sp. nov., an asaccharolytic, black-pigmented species from cats

A new species, Bacteroides salivosus sp. nov., is proposed for black-pigmented, asaccharolytic Bacteroides strains from cats, isolated from subcutaneous abscesses and empyemas, as well as from gingival margins of normal mouths. The bacterium is an obligately anaerobic, gram-negative, brown- or black-pigmented, asaccharolytic, nonmotile, nonsporeforming rod that does not grow in 20% bile and has a guanine-plus-cytosine content of 42 to 44 mol%. It has 12% deoxyribonucleic acid homology with the human type strain of Bacteroides gingivalis (ATCC 33277T) and 1% deoxyribonucleic acid homology with the human type strain of Bacteroides asaccharolyticus (ATCC 25260T). Strain VPB 157 (NCTC 11632T) is the type strain. Unlike B. gingivalis, B. salivosus produces catalase, and the colonies of the type strain fluoresce at 24 and 48 h, although some other strains do not fluoresce. It does not agglutinate sheep erythrocytes. Unlike either B. asaccharolyticus or Bacteroides endodontalis, it has trypsinlike activity and produces large quantities of phenylacetic acid.