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.

PCR studies of feline leprosy cases.

16S rRNA gene sequence analysis provided evidence for two different mycobacterial species, Mycobacterium lepraemurium and a potentially novel species, as causative agents of 'feline leprosy'. Comparison of 16S rRNA gene sequence data obtained for M. lepraemurium and the potentially novel species indicated 12 nucleotide differences over a 446 bp region encompassing the V2 and V3 hypervariable regions. From available 16S rRNA gene sequence data, M. lepraemurium shared greatest nucleotide identity with M. avium subsp paratuberculosis and M. avium. The novel species had a long helix 18 in the V3 region and shared greatest nucleotide identity with M. leprae, M. haemophilum and M. malmoense. The novel species had an additional 'A' nucleotide at position 105 of the aligned 16S rRNA gene sequence, the only other mycobacterial database sequence having this same extra nucleotide being M. leprae. This nucleotide variation was exploited to develop specific PCR assays for the two species. These were found to be effective and specific when tested against a panel of mycobacteria including species found in feline leprosy lesions and closely related mycobacteria and also when applied directly to formalin-fixed, paraffin-embedded tissues from feline leprosy cases.

Detection of EHV-1 and EHV-4 DNA in unweaned Thoroughbred foals from vaccinated mares on a large stud farm.

REASONS FOR PERFORMING STUDY: A silent cycle of equine herpesvirus 1 infection has been described following epidemiological studies in unvaccinated mares and foals. In 1997, an inactivated whole virus EHV-1 and EHV-4 vaccine was released commercially in Australia and used on many stud farms. However, it was not known what effect vaccination might have on the cycle of infection of EHV-1. OBJECTIVE: To investigate whether EHV-1 and EHV-4 could be detected in young foals from vaccinated mares. METHODS: Nasal and blood samples were tested by PCR and ELISA after collection from 237 unvaccinated, unweaned foals and vaccinated and nonvaccinated mares during the breeding season of 2000. RESULTS: EHV-1 and EHV-4 DNA was detected in nasal swab samples from foals as young as age 11 days. CONCLUSIONS: These results confirm that EHV-1 and EHV-4 circulate in vaccinated populations of mares and their unweaned, unvaccinated foals. POTENTIAL RELEVANCE: The evidence that the cycle of EHV-1 and EHV-4 infection is continuing and that very young foals are becoming infected should assist stud farms in their management of the threat posed by these viruses.

Avian cryptococcosis.

Clinical and laboratory findings in 15 unreported cases of avian cryptococcosis from Australia were collated and contrasted with 11 cases recorded in the literature. Cryptococcus species produced localized invasive disease of the upper respiratory tract of captive parrots living in Australia. This resulted in signs referable to mycotic rhinitis or to involvement of structures contiguous with the nasal cavity, such as the beak, sinuses, choana, retrobulbar space and palate. Parrots of widely differing ages were affected and of the seven birds for which sex was determinable, six were male. Cryptococcus bacillisporus (formerly C. neoformans var. gattii) accounted for four of five infections in which the species or variety was determinable, suggesting that exposure to eucalyptus material may be a predisposing factor. In these cases, Cryptococcus appeared to behave as a primary pathogen of immunocompetent hosts. One tissue specimen was available from an Australian racing pigeon with minimally invasive subcutaneous disease; immunohistology demonstrated a C. neoformans var. grubii (formerly C. neoformans var. neoformans serotype A) infection, presumably subsequent to traumatic inoculation of yeast cells into the subcutis. Two similar cases had been reported previously in pigeons domiciled in America. Data for parrots, one pigeon and other birds studied principally in America and Europe (and likely infected with C. neoformans) suggested a different pattern of disease, more suggestive of opportunistic infection of immunodeficient hosts. In this cohort of patients, the organism was not restricted to cool superficial sites such as the upper respiratory tract or subcutis. Instead, infections typically penetrated the lower respiratory tract or disseminated widely to a variety of internal organs. Finally, three captive North Island brown kiwis, one residing in Australia, the other two in New Zealand, died as a result of severe diffuse cryptococcal pneumonia (two cases) or widely disseminated disease (one case). C. bacillisporus strains were isolated from all three cases, as reported previously for another kiwi with disseminated disease in New Zealand.

Inoculation with DNA encoding the glycoprotein gp2 reduces severity of equine herpesvirus 1 infection in a mouse respiratory model.

The envelope glycoprotein 2 (gp2) of equine herpesvirus 1 (EHV-1) has no known homologue in other herpesviruses with the exception of some equid alphaherpesviruses. In order to investigate the potential of gp2 as a vaccine antigen, expression vectors were constructed to encode full-length gp2, a truncated version lacking the membrane anchor, and the C-terminal region. Intramuscular inoculation of mice with these DNA constructs induced neutralizing antibody against EHV-1 and, following intranasal challenge with EHV-1, mice inoculated with any of the gp2 DNA constructs cleared virus more rapidly from their lungs than control mice. The rate of clearance was comparable to that for glycoprotein D DNA, indicating gp2 as a potential antigen for inclusion in a subunit vaccine.

Seroprevalence of equine herpesvirus 1 in mares and foals on a large Hunter Valley stud farm in years pre- and postvaccination.

OBJECTIVE: To examine the prevalence of equine herpesvirus 1 antibody in mares and foals on a large Hunter Valley Thoroughbred stud farm in New South Wales before and after the introduction of an inactivated whole virus vaccine. DESIGN: Cross-sectional serological surveys performed in February 1995 and 2000 to determine the prevalence of EHV-1 antibody-positive mares and foals. A further cross-sectional survey was carried out in 2001 to complement the 2000 data. STUDY POPULATION: Two hundred and twenty-nine mares and their foals were sampled in 1995 and 236 mares and their foals were sampled in 2000. The study population comprised all of the mares with foals at foot on this property at each sample period. Fifty mares were sampled in both studies. A further 264 mares and their foals were sampled in 2001. PROCEDURE: A blood sample was collected from each mare and foal at the beginning of February 1995, 2000 and 2001. Each sample was tested in triplicate using an antibody-detection ELISA that is type-specific for EHV-1 and EHV-4 antibodies. RESULTS: The prevalence of EHV-1 antibody-positive mares was not statistically different in 2000 compared to 1995. However, the prevalence of antibody-positive foals was significantly lower in 2000 than in 1995. In 2001, the prevalence of antibody-positive mares was higher than in 2000, but not different from that in 1995. The prevalence of antibody-positive foals in 2001 was not significantly different from the prevalence observed in 1995 or that observed in 2000. However, when the three studies were compared there was a significant variation in the prevalence of EHV-1 positive foals due to the variation between the 1995 and the 2000 data. CONCLUSIONS: Mares are the source of virus from which foals are infected early in life and following analysis of the 2001 data, the difference in the prevalence of EHV-1 antibody-positive foals between 1995 and 2000 was likely to be a reflection of seasonal, nutritional and management factors, rather than the result of vaccination.

Serological responses of mares and weanlings following vaccination with an inactivated whole virus equine herpesvirus 1 and equine herpesvirus 4 vaccine.

Equine herpesvirus 1 (EHV-1) is a major cause of respiratory disease and abortion in horses worldwide. Although some vaccines have been shown experimentally to reduce disease, there are few reports of the responses to vaccination in the field. This study measured antibody responses to vaccination of 159 mares (aged 4-17 years) and 101 foals (aged 3-6 months) on a large stud farm with a killed whole virus EHV-1/4 vaccine used as per the manufacturer's recommendations. Using an EHV glycoprotein D (gD)-specific ELISA and a type-specific glycoprotein G (gG) ELISA, respectively 13.8 and 28.9% of mares, and 42.6 and 46.6% of foals were classed as responding to vaccination. Additionally, 16.4 and 17.6% of mares were classified as persistently seropositive mares. Using both assays, responder mares and foals had lower week 0 mean ELISA absorbances than non-responder mares and foals. Responder mares were ten times more likely to have responder foals, and non-responder mares were six times more likely to have non-responder foals than other mares using the gG ELISA. Mares aged 7 years or less and foals aged 4 months or more were more likely to respond to vaccination than animals in other age groups. There was no association between response of mares and the number of previous vaccinations received and persistently seropositive mares did not respond to vaccination. This study documents the responses of mares and foals to vaccination in a large scale commercial environment in 2000, and suggests that knowledge of antibody status may allow a more selective vaccination strategy, representing considerable savings to industry.

The C-terminal regions of the envelope glycoprotein gp2 of equine herpesviruses 1 and 4 are antigenically distinct.

The unusual mucin-like high molecular mass (Mr) glycoprotein 2 (gp2) has only been described in the equid alphaherpesviruses, among which there is considerable antigenic cross-reactivity. Equine herpesvirus 1 (EHV-1) gp2 is cleaved into a highly glycosylated N-terminal subunit and a 42 kDa C-terminal cleavage product. In order to investigate their antigenic recognition by horses naturally infected with EHV-1 and/or equine herpesvirus 4 (EHV-4), the C-terminal cleavage product and high Mr gp2 were affinity purified. Cross-reactivity between EHV-1 and EHV-4 was observed for the high Mr gp2 using Western blotting. In contrast only horses with antibodies to EHV-1 detected the 42 kDa EHV-1 gp2 C-terminal cleavage product. This phenomenon was evident in pooled sera from adult horses and also in foals that had demonstrated seroconversion due to EHV-1 infection. The results indicate that the C-terminal region of EHV-1 gp2 is antigenically distinct from that of EHV-4 gp2 and can be detected only after an EHV-1-specific immune response.

Feline leprosy: two different clinical syndromes.

Feline leprosy refers to a condition in which cats develop granulomas of the subcutis and skin in association with intracellular acid-fast bacilli that do not grow on routine laboratory media. In this study, the definition was extended to include cases not cultured, but in which the polymerase chain reaction (PCR) identified amplicons characteristic of mycobacteria. Tissue specimens from 13 such cases from eastern Australia were obtained between 1988 and 2000. This cohort of cats could be divided into two groups on the basis of the patients' age, histology of lesions, clinical course and the sequence of 16S rRNA PCR amplicons. One group consisted of four young cats (less than 4 years) which initially developed localised nodular disease affecting the limbs. Lesions progressed rapidly and sometimes ulcerated. Sparse to moderate numbers of acid-fast bacilli were identified using cytology and/or histology, typically in areas of caseous necrosis and surrounded by pyogranulomatous inflammation. Organisms did not stain with haematoxylin and ranged from 2 to 6 microm (usually 2 to 4 microm). Mycobacterium lepraemurium was diagnosed in two cases based on the sequence of a 446 bp fragment encompassing the V2 and V3 hypervariable regions of the 16S rRNA gene a different sequence was obtained from one additional case, while no PCR product could be obtained from the remaining case. The clinical course was considered aggressive, with a tendency towards local spread, recurrence following surgery and development of widespread lesions over several weeks. The cats resided in suburban or rural environments. A second group consisted of nine old cats (greater than 9 years) with generalised skin involvement, multibacillary histology and a slowly progressive clinical course. Seven cats initially had localised disease which subsequently became widespread, while two cats allegedly had generalised disease from the outset. Disease progression was protracted (compared to the first group of cats), typically taking months to years, and skin nodules did not ulcerate. Microscopically, lesions consisted of sheets of epithelioid cells containing large to enormous numbers of acid-fast bacilli 2 to 8 microm (mostly 4 to 6 microm) which stained also with haematoxylin. A single unique sequence spanning a 557 bp fragment of the 16S rRNA gene was identified in six of seven cases in which it was attempted. Formalin-fixed paraffin-embedded material was utilised by one laboratory, while fresh tissue was used in another. The same unique sequence was identified despite the use of different primers and PCR methodologies in the two laboratories. A very slow, pure growth of a mycobacteria species was observed on Lowenstein-Jensen medium (supplemented with iron) and semi-solid agar in one of three cases in which culture was attempted at a reference laboratory. Affected cats were domicile in rural or semi-rural environments. These infections could generally be cured using two or three of rifampicin (10-15 mg/kg once a day), clofazimine (25 to 50 mg once a day or 50 mg every other day) and clarithromycin (62.5 mg per cat every 12 h). These findings suggest that feline leprosy comprises two different clinical syndromes, one tending to occur in young cats and caused typically by M lepraemurium and another in old cats caused by a single novel mycobacterial species.

Cryptococcosis in ferrets: a diverse spectrum of clinical disease.

Cryptococcosis was diagnosed in seven ferrets (five from Australia; two from western Canada) displaying a wide range of clinical signs. Two of the ferrets lived together. One (5-years-old) had cryptococcal rhinitis and presented when the infection spread to the nasal bridge. Its sibling developed cryptococcal abscessation of the right retropharyngeal lymph node 12 months later, soon after developing a severe skin condition. DNA fingerprinting and microsatellite analysis demonstrated that the two strains isolated from these siblings were indistinguishable. Two ferrets (2- to 3-years-old) developed generalised cryptococcosis: one had primary lower respiratory tract disease with pneumonia, pleurisy and mediastinal lymph node involvement, while in the other a segment of intestine was the primary focus of infection with subsequent spread to mesenteric lymph nodes, liver and lung. The remaining three ferrets (1.75 to 4-years-old) had localised disease of a distal limb, in one case with spread to the regional lymph node. Cryptococcus bacillisporus (formerly C. neoformans var gattii) accounted for three of the four infections in Australian ferrets where the biotype could be determined. The Australian ferret with intestinal involvement and the two ferrets from Vancouver had C. neoformans var grubii infections.

Feline immunodeficiency virus status of Australian cats with lymphosarcoma.

OBJECTIVE: To determine the FIV status of Australian cats with lymphosarcoma and relate this to patient characteristics, tumour characteristics (tissue involvement, histological grade and immunophenotype), haematological and serum biochemical values and FeLV status of affected cats. DESIGN: Prospective study of 101 client-owned cats with naturally-occurring lymphosarcoma. PROCEDURE: Western blot analysis, ELISA and immunochromatography were used to detect FIV antibodies in serum from cats with lymphosarcoma. RESULTS: On the basis of Western blot analysis (which was considered the most accurate method for determining FIV status), 50/101 (50%) of cats with naturally-occurring lymphosarcoma were positive for FIV antibodies. Of these 50 cats, 35 had tumours of B-cell phenotype, 13 had T-cell tumours and 2 had tumours classified as non-B/non-T. Tumours from eight of these FIV-positive cats contained FeLV gene sequences, including a 9-month-old cat with FeLV antigenaemia. Compared with FlV-negative cats with lymphosarcoma, FIV-positive cats were more likely to be domestic crossbreds (P = 0.004), male (P = 0.048) and have atypical (especially nasal) forms of lymphosarcoma (P = 0.09). Only 39 of 107 (36%) blood or sera tested using ELISA were positive for FIV antibodies (including 5 false-positives). CONCLUSIONS: The prevalence of FIV infection was considerably higher in our cohort of cats compared with series of lymphosarcoma cases from the Northern hemisphere. A positive FIV status was strongly associated with lymphosarcoma in Australian cats and it is possible that this infection may predispose to the development of lymphoid neoplasia. The presence of FIV infection would have been underestimated if commercial kits alone had been used for serology.

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.

Effects of training on resting peripheral blood and BAL-derived leucocyte function in horses.

In this study, the effects of prolonged, high intensity training on aspects of peripheral blood and bronchoalveolar lavage (BAL)-derived leucocyte function were evaluated in 8 horses. All horses undertook a 7 week endurance training programme, followed by 5 weeks of high intensity training (HIT). Thereafter, horses were divided into control (C) and overtraining (OT) groups. The frequency and intensity of training were increased more substantially for horses in the OT group. Training was terminated in week 32 when horses in the OT group demonstrated a significant performance reduction. Peripheral blood and BAL samples were collected from 4 horses in C and OT groups in training weeks 7, 11, 14, 18, 22, 28 and 32. Flow cytometric techniques were used to assess phagocytosis by peripheral blood neutrophils and pulmonary alveolar macrophages (PAM), and oxidative burst activity of neutrophils, PAM, peripheral blood and BAL-derived lymphocytes. Peripheral blood neutrophil phagocytosis (internalisation) increased during the initial HIT period and decreased from week 16 when the training workload was increased for both groups. The oxidative burst activity of peripheral blood neutrophils and lymphocytes similarly increased and then decreased in response to training. The oxidative burst activity of PAM was reduced towards the end of the overtraining phase of the programme. Pulmonary alveolar macrophage phagocytosis and oxidative burst activity of BAL-derived lymphocytes demonstrated no change throughout the course of the study. There was no difference in results obtained from C or OT group horses, suggesting that protracted HIT, rather than overtraining, was associated with impaired cell function. The detrimental effects observed in peripheral blood neitrophil and PAM function may indicate impaired nonspecific immunity which may adversely affect the health and performance of horses undergoing protracted periods of intense training.

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.

Treatment of canine leproid granuloma syndrome: preliminary findings in seven dogs.

OBJECTIVE: To determine effective treatment strategies for patients with refractory canine leproid granuloma syndrome. DESIGN: Multi-institutional retrospective/prospective case series using client-owned dogs. PROCEDURE: Seven dogs (four Boxers, one Dobermann, one Bullmastiff and one Bullmastiff cross-bred; ages 3 to 11 years) with leproid granulomas were treated successfully using a variety of treatment regimens. These cases were recruited because: lesions were either widely distributed over the dog; progressive, despite routine therapy, or were associated with particularly disfiguring lesions. The treatment regimen evolved during the course of the clinical study. RESULTS: Combination therapy using rifampicin (5 to 15 mg/kg p.o., every 24 h) and clarithromycin (8 to 24 mg/kg p.o. daily; dose divided every 8 or every 12 h) was used most frequently and proved to be effective and free from side effects. Total daily doses of clarithromycin in excess of 14 mg/kg were considered optimal and long treatment courses, in the order of 1 to 3 months, were used. Combination therapy using rifampicin (25 mg/kg; that is, higher than the recommended dose) and clofazimine was effective in one case, but resulted in hepatotoxicity. A topical formulation of clofazimine in petroleum jelly was used as an adjunct to oral rifampicin and doxycycline in another patient treated successfully. CONCLUSION: Based on our evolving clinical experience, a combination of rifampicin (10 to 15 mg/kg p.o., every 24 h) and clarithromycin (15 to 25 mg/kg p.o. total daily dose; given divided every 8 to 12 h) is currently recommended for treating severe or refractory cases of canine leproid granuloma syndrome. Treatment should be continued (typically for 4 to 8 weeks) until lesions are substantially reduced in size and ideally until lesions have resolved completely. A topical formulation, containing clofazimine in petroleum jelly may be used as an adjunct to systemic drug therapy. Further work is required to determine the most cost effective treatment regimen for this condition.

Mycobacterium genavense infection in two aged ferrets with conjunctival lesions.

Mycobacterium genavense infection was diagnosed in two adult ferrets. Disseminated mycobacteriosis was diagnosed in a castrated 5-year-old sable ferret with generalised peripheral lymph node enlargement and a proliferative lesion of the conjunctiva of the nictitating membrane. The diagnosis was based on characteristic cytology and sequence analysis of the 16S rRNA gene amplified using the polymerase chain reaction from fresh biopsy material. Therapy with rifampicin, clofazimine and clarithromycin probably cured the infection. An entire 4-year-old female ferret with conjunctival swelling, serous ocular discharge and swelling of the subcutaneous tissues of the nasal bridge was diagnosed as having M genavense infection on the basis of typical cytology, histopathology and sequence analysis of 16S rRNA amplicons from formalin-fixed paraffin-embedded tissue. This patient was treated successfully using rifampicin. Both ferrets subsequently died as a result of other disease conditions, 10 and 4 months following initiation of therapy, respectively. This is the first report documenting M genavense as a cause of disseminated mycobacterial disease in ferrets. Conjunctival involvement may be a feature of disseminated mycobacteriosis in the ferret. The possibility that these infections were the consequence of a ferret retrovirus infection should be considered further.

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.