EHV-1 and EHV-4 infection in vaccinated mares and their foals.

A silent cycle of equine herpesvirus 1 infection was described following epidemiological studies of unvaccinated mares and foals on a Hunter Valley stud farm. Following the introduction of routine vaccination with an inactivated whole virus equine herpesvirus 1 (EHV-1) and equine herpesvirus 4 (EHV-4) vaccine in 1997, a subsequent study identified excretion of EHV-1 and EHV-4 in nasal swab samples tested by PCR from vaccinated mares and their unweaned, unvaccinated foals. The current sero-epidemiological investigation of vaccinated mares and their young foals found serological evidence of EHV-1 and EHV-4 infection in mares and foals in the first 5 weeks of life. The results further support that EHV-1 and EHV-4 circulate in vaccinated populations of mares and their unweaned foals and confirms the continuation of the cycle of EHV-1 and EHV-4 infection.

Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, pregnant mares and young foals.

The envelope glycoprotein D of equine herpesvirus 1 (EHV-1 gD) has been shown in laboratory animal models to elicit protective immune responses against EHV-1 challenge, and hence is a potential vaccine antigen. Here we report that intramuscular inoculation of EHV-1 gD produced by a recombinant baculovirus and formulated with the adjuvant Iscomatrix elicited virus-neutralizing antibody and gD-specific ELISA antibody in the serum of over 90% of adult mixed breed horses. The virus-neutralizing antibody responses to EHV-1 gD were similar to those observed after inoculation with a commercially available killed EHV-1/4 whole virus vaccine. Intramuscular inoculation of EHV-1 gD DNA encoded in a mammalian expression vector was less effective in inducing antibody responses when administered as the sole immunogen, but inoculation with EHV-1 gD DNA followed by recombinant EHV-1 gD induced increased gD ELISA and virus-neutralizing antibody titres in six out of seven horses. However, these titres were not higher than those induced by either EHV-1 gD or the whole virus vaccine. Isotype analysis revealed elevated gD-specific equine IgGa and IgGb relative to IgGc, IgG(T) and IgA in horses inoculated with EHV-1 gD or with the whole virus vaccine. Following inoculation of pregnant mares with EHV-1 gD, their foals had significantly higher levels of colostrally derived anti-gD antibody than foals out of uninoculated mares. The EHV-1 gD preparation did not induce a significant mean antibody response in neonatal foals following inoculation at 12 h post-partum and at 30 days of age, irrespective of the antibody status of the mare. The ability of EHV-1 gD to evoke comparable neutralizing antibody responses in horses to those of a whole virus vaccine confirms EHV-1 gD as a promising candidate for inclusion in subunit vaccines against EHV-1.

Primary and challenge infection of mice with equine herpesvirus 1, strain HSV25A.

Clinical signs, haematology, lymphocyte subset analysis, viral clearance, lung histopathology and humoral and cell-mediated (CMI) immune responses were monitored throughout the acute and convalescent phases of infection in groups of BALB/c mice infected intranasally with equine herpesvirus 1 (EHV-1), strain HSV25A. Primary infection caused a leucocytosis due to a neutrophilia during days 1 and 2 post-infection (pi) and a B lymphocytosis at day 1 pi. Serum ELISA antibodies were detected by 7 days pi and neutralising antibodies by 2 weeks pi. Mice infected with EHV-1 were not protected against disease when challenged with EHV-1 12 weeks later. However, viral clearance from lungs was significantly faster and the antibody response was markedly enhanced within the first few days of challenge infection. A CMI response was detected by 5 days after primary infection, but the level of responsiveness was not increased by challenge infection, although the lungs of challenged mice had markedly increased numbers of mononuclear cells around blood vessels and bronchioles. Specific antibodies to glycoprotein (g) B were detected by 2 weeks pi, 4 weeks earlier than the detection of antibodies to gC and 10 weeks before those to gD. The primary response was relatively short-lived with neither ELISA antibody nor lymphocyte proliferation was evident by 6 months pi.

Equine herpesvirus 1 glycoprotein D expressed in Pichia pastoris is hyperglycosylated and elicits a protective immune response in the mouse model of EHV-1 disease.

Equine herpesvirus 1 glycoprotein D (EHV-1 gD) has been shown in mouse models and in the natural host to have potential as a subunit vaccine, using various expression systems that included Escherichia coli, baculovirus and plasmid DNA. With the aim of producing secreted recombinant protein, we have cloned and expressed EHV-1 gD, lacking its native signal sequence and C-terminal transmembrane region, into the methylotrophic yeast Pichia pastoris. The truncated glycoprotein D (gD) gene was placed under the control of the methanol inducible alcohol oxidase 1 promoter and directed for secretion with the Saccharomyces cerevisiae alpha-factor prepro secretion signal. SDS-PAGE and Western blot analysis of culture supernatant fluid 24 h after induction revealed gD-specific protein products between 40 and 200 kDa. After treatment with PNGase F and Endo H, three predominant bands of 34, 45 and 48 kDa were detected, confirming high mannose N-linked glycosylation of Pichia-expressed gD (Pic-gD). N-terminal sequence analysis of PNGase F-treated affinity-purified protein showed that the native signal cleavage site of gD was being recognised by P. pastoris and the 34 kDa band could be explained by internal proteolytic cleavage effected by a putative Kex2-like protease. Pic-gD, when used in a DNA prime/protein boost inoculation schedule, induced high EHV-1 ELISA and virus neutralizing antibodies and provided protection from challenge infection in BALB/c mice.

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).

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.

Chromosomal DNA probes for the identification of Bacteroides tectum and Bacteroides fragilis from the oral cavity of cats.

A dot-blot hybridisation assay using high molecular weight DNA as whole chromosomal probes was used to differentiate Bacteroides tectum from Bacteroides fragilis. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes. The whole chromosomal probes were specific--differentiating B. tectum from B. fragilis and both from a variety of other species (including other members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. However, even at very high stringencies, B. tectum homology groups I, II and III were not distinguishable from one another using either 32P-labelled or DIG-labelled probes. Thus, DIG-labelled whole chromosome probes directed against cellular DNA released directly onto nitrocellulose membranes is considered a useful method for diagnostic veterinary laboratories wishing to identify B. tectum and distinguish it from B. fragilis and other oral anaerobic flora of cats.

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 association of two recombinant proteinases of a feline strain of Porphyromonas gingivalis with periodontal disease in cats.

Serum from 40 domestic cats with various grades of periodontal disease was used to probe two recombinant functional proteinases from feline strain VPB 3457 of Porphyromonas gingivalis expressed in E. coli. One recombinant proteinase (VPB 2856) was constructed using polymerase chain reaction and had 91% DNA identity with the prtC collagenase gene of the human type strain of P. gingivalis, while the other proteinase (VPB 2814) was isolated from a size selected genomic library and had an amino-terminal sequence with no significant identity with deposited sequences. Thirteen of 40 cats showed a serum antibody response to VPB 2856 using Western immunoblot detection. All the 13 cats had an overall periodontal grade of 3 or greater and greater than 1.68x10(5) cfu P. gingivalis at the canine and premolar periodontium sample sites. Fourteen of 40 cats showed a serum antibody response to VPB 2814. Thirteen of these 14 cats had an overall periodontal grade of 3 or greater. Regression analysis of overall periodontal grade against the serum antibody response showed significant positive relationships for both VPB 2856 (r2 = 0.351; p<0.001) and VPB 2814 (r2 = 0.247; p<0.001). Regression analysis of the total colony forming units of feline strain P. gingivalis against the grade of serum antibody response showed a positive relationship for both VPB 2856 (r2 = 0.662; p<0.001) and VPB 2814 (r2 = 0.531; p<0.001). These data provide strong evidence that the recombinant proteinases of feline P. gingivalis expressed in E. coli clones VPB 2856 and VPB 2814 are associated with periodontal disease in cats.

Serum antibody responses of cats to soluble whole cell antigens of feline Porphyromonas gingivalis.

The whole cell soluble antigens of two strains (VPB 3457 and VPB 3492) of feline Porphyromonas gingivalis were analysed by Western blotting using serum taken from 40 domestic cats with various grades of periodontal disease. Five strongly immunogenic protein bands (70, 34, 27, 24 and 19kDa) from VPB 3457 and seven from VPB 3492 (58, 44, 34, 27, 25, 24 and 21kDa) were selected for further study. A significant positive correlation was found between the serum antibody response to the 70, 34, 27, 24 and 19kDa bands of VPB 3457 and the 58, 44, 25, 24 and 21kDa bands of VPB 3492 and the overall periodontal grade. A significant positive correlation was also found between the serum antibody response to the 24kDa band of VPB 3457 and the total colony forming units of P. gingivalis. N-terminal sequencing of the 44kDa band of VPB 3492 showed 75% identity with the translated amino acids from the hag A (haemagglutinin) gene of a human strain of P. gingivalis and N-terminal amino acid sequence of the 27kDa band of VPB 3457 showed 88% identity with the amino acid sequences translated from DNA of purported genes coding for variously named proteinases of human strains of P. gingivalis.

The isolation and enumeration of three feline oral Porphyromonas species from subcutaneous abscesses in cats.

Samples were examined from 15 subcutaneous fight wound abscesses from 15 cats. All abscesses were closed at the time of sampling and cats had received no prior treatment. Samples were processed within 20 min and quantitative assessment made of total facultative and obligately anaerobic flora isolated. 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 members of this genus and quantification of these species was made from each cat using colony lifts and southern hybridisation from nitrocellulose membranes taken from replicate plates from each abscess sample. Twelve of the 15 abscesses yielded a variety of facultative and obligately anaerobic (FOA) bacterial species and members of the genus Porphyromounas were enumerated from each of these 12 abscesses. Of the 12 abscesses in which Porphyromonas species were detected, seven contained one species only (five contained only P. gingivalis and two contained only P. salivosa) three abscesses contained two species (both P. gingivalis and P. circumdentaria) and two abscesses contained all three species of Porphyromonas. These results show that members of the genus Porphyromonas are likely to be significant contributors to the purulent disease process in subcutaneous abscesses in cats.

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.

Serum antibody responses of cats to soluble whole cell antigens and isolated fimbriae of feline Porphyromonas salivosa (macacae) and associations with periodontal disease.

The whole cell soluble antigens of two strains (NCTC 11632 and VPB 3313) of feline Porphyromonas salivosa (macacae) were analyzed by Western blotting using serum taken from 40 domestic cats with various grades of periodontal disease. Nine strongly immunogenic protein bands (66, 52, 42, 29, 27, 23, 22, 21 and 19kDa) were selected from both strains for further study. Both strains showed a significant association between overall periodontal grade and serum responses to the 66 and 21kDa bands with significant responses across both strains to all other bands except the 52kDa band. Similarly, both strains showed a significant association between the total colony forming units and serum responses to the 66 and 42kDa bands with significant responses across both strains to all other bands except the 19kDa band. When sera from 25 of these cats were tested by Western blotting against the isolated fimbriae of VPB 3313, there was a significant association between the grade of response of cats to the 42kDa fimbrial preparation and (1) the total reactivity of the mouth (the sum of the responses to all individual whole cell antigens), (2) the total colony forming units of P. salivosa (macacae) at the premolar site, and (3) to their responsiveness to the 42kDa band in the soluble whole cell antigen preparations. These findings suggest that P. salivosa (macacae) is a strong immunogen in the mouths of cats and those cats with more severe periodontal disease have a greater serum antibody reactivity to various soluble whole cell antigens, specifically including the fimbriae of this organism, than those with less severe periodontal disease. Overall, the findings suggest that this organism may be a contributor to periodontal disease in cats.

Chromosomal DNA probes for the identification of asaccharolytic anaerobic pigmented bacterial rods from the oral cavity of cats.

A dot-blot hybridisation assay using isolated high molecular weight DNA as whole chromosomal probes of the cat pigmented asaccharolytic Bacteroides/Porphyromonas species was used against both purified high molecular weight DNA and DNA released on membranes from whole cells for the identification of B. salivosus and for its differentiation from the other anaerobic species isolated from normal and diseased mouths of cats and horses. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes (Boehringer-Mannheim). The whole chromosomal probes were specific--differentiating B. salivosus from a variety of species (including members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. Likewise, asaccharolytic black pigmented Group 2 strains were distinguishable from all strains tested. However, cat strains of P. gingivalis which show 68-76% DNA-DNA homology with human strain P. gingivalis ATCC 33277T, were not distinguishable from each other using either 32P-labelled or DIG-labelled probes. The minimum amount of pure Bacteroides DNA which could be detected by the 32P-labelled probe was 100-300 pg, while the amount of pure DNA detected by the DIG system was 1-3 mg after room temperature colour development for 1 h and 100-300 pg after 6 h colour development.

High molecular weight polypeptide bands specific for equine herpesvirus 4.

Serum neutralisation (SN) and immunoblotting were used in attempts to distinguish between natural infections with the closely related viruses equine herpesvirus 1 (EHV-1) and equine herpes-virus 4 (EHV-4). Horse sera (n = 323) collected in 1990 from studs with no experience of EHV-1 abortions as well as 197 sera collected in 1992 from studs with a history of EHV-1 abortions were tested by SN. The two groups differed in the proportion with measurable EHV-1 antibody, the 1992 group being significantly higher. Both groups had high proportions with EHV-4 antibody and no serum had antibody to EHV-1 alone. Pools of positive sera were prepared as probes in immunoblots. High molecular weight (> 200 kDa) polypeptide bands specific for EHV-4 were detected. No bands specific for EHV-1 only were found. The specific EHV-4 polypeptides shared some properties with gp2 of EHV-1.

Bacteriological warfare amongst cats: what have we learned about cat bite infections?

Cat bite infections are one of the most common infectious diseases presenting to veterinary practices and to emergency rooms at human hospitals. This review describes the disease in humans and cats, the origin of organisms involved in cat bite abscesses and the importance of selected organisms such as members of the genus Porphyromonas in the disease. It also discusses future directions, the importance of identifying significant organisms and why an understanding of antimicrobial susceptibility patterns is of consequence to the outcome of the disease in humans and cats.

Comparison of the pathogenesis of acute equine herpesvirus 1 (EHV-1) infection in the horse and the mouse model: a review.

The mouse models of the respiratory and abortion forms of equine herpesvirus 1 (EHV-1) infection have been used to investigate the vaccine potential of various EHV-1 immunogens, the effect of antiviral agents on EHV-1 infection and the pathogenicity of EHV-1 strain variants and deletion or insertional mutants. This review examines the similarities and differences in the pathogenesis of primary EHV-1 infection in the natural host, the horse, and in the mouse by comparing tissue tropism, clinical signs of infection, the effects of EHV-1 on pregnancy, haematological changes following infection, viral clearance, histopathology and latency. The evidence suggests that the mouse model provides a valid method for investigation of virological and histopathological aspects of EHV-1-induced disease in the horse. However, the extent to which useful and valid comparisons and extrapolations can be made of immunological parameters from mouse to horse is yet to be determined.

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.

Immunization of BALB/c mice with DNA encoding equine herpesvirus 1 (EHV-1) glycoprotein D affords partial protection in a model of EHV-1-induced abortion.

DNA-mediated immunization was assessed in a murine model of equine herpesvirus 1 (EHV-1) abortion. Whilst there are differences between the model and natural infection in the horse, literature suggests that EHV-1 infection of pregnant mice can be used to assess the potential ability of vaccine candidates to protect against abortion. Female BALB/c mice were inoculated twice, 4 weeks apart, with an expression vector encoding EHV-1 glycoprotein D (gD DNA). They were mated 15 days after the second inoculation, challenged at day 15 of pregnancy and killed 3 days later. The gD DNA-inoculated mice had fewer foetuses which were damaged or had died in utero (6% in gD DNA, 21% vector DNA and 28% in nil inoculated groups challenged with EHV-1), a reduction in the stunting effect of EHV-1 infection on foetuses (gD DNA: 0.40g+/-0.06, vector DNA: 0.34g+/-0.10), reduced placental and herpesvirus-specific lung histopathology and a lower titre of virus (TCID(50)+/-SEM/lung) in maternal lung than control groups (gD DNA 4.7+/-0.3, vector 5.3+/-0.2, nil 5.6+/-0.2). Maternal antibody to EHV-1 gD was demonstrated in pups born to a dam inoculated 123 days earlier with gD DNA. Although protection from abortion was incomplete, immunization of mice with gD DNA demonstrated encouragingly the potential of this vaccine strategy.

Characterization of the glycoprotein D gene products of equine herpesvirus 1 using a prokaryotic cell expression vector.

The gene encoding equine herpesvirus 1 (equine abortion virus; EHV-1) glycoprotein D was engineered into the prokaryotic vector pEX, and expressed as a beta-galactosidase fusion product, which was recognized by pooled equine sera and anti-EHV-1 rabbit sera. Antibodies raised against the EHV-1 gD fusion product identified strong bands in infected cells at 66 and 68 K and at 138 K in purified virus, thus characterizing the several forms of this major envelope glycoprotein which is an important candidate for inclusion in subunit vaccines.