Disseminated Mycobacterium Genavense Infection in a FIV-Positive Cat

Abstract

An 8-year-old FIV-positive Australian cat was presented with coughing, periocular alopecia, pyrexia and inappetence. Skin scrapings demonstrated Demodex cati mites. Antibiotics were administered and it was treated successfully for periocular demodectic mange, but the cat continued to exhibit respiratory signs and lose weight. Further investigation revealed an ascarid infection and active chronic inflammation of undetected cause affecting the lower airways. Repetitive treatment with pyrantel failed to eradicate the ascarid infection. The cat became cachectic and developed moist ulcerative dermatitis of the neck, severe non-regenerative anaemia, leucopenia and thrombocytopenia. Necropsy and histopathology revealed mycobacteriosis affecting skin, lungs, spleen, lymph nodes, liver and kidney. Attempted culture of frozen tissues at a mycobacteria reference laboratory was unsuccessful. Paraffin-embedded, formalin-fixed tissue was retrieved and examined using PCR to amplify part of the 16S rRNA gene. A diagnosis of disseminated Mycobacterium genavense infection was made based on the presence of acid fast bacteria in many tissues and partial sequence of the 16S rRNA gene. Although M genavense has been identified previously as a cause of disseminated disease in AIDS patients, this is the first report of infection in a cat. It was suspected that the demodecosis, recurrent ascarid infections and disseminated M genavense infection resulted from an immune deficiency syndrome consequent to longstanding FIV infection.

Generalized infections with mycobacteria, particularly members of the Mycobacterium avium complex (MAC), are well-known complications of infection with the human immunodeficiency virus (HIV). Recently, a putative new species of mycobacteria, provisionally named M genavense has been identified as a cause of disseminated disease in acquired immune deficiency syndrome (AIDS) patients (Hirschel et al 1990, Böttger 1990, 1994, Böttger et al 1992, Coyle et al 1992, Wald et al 1992, Nadal et al 1993). Histopathologically, M genavense infections in HIV-positive patients are characterized by masses of foamy macrophages and, depending on the immunological reactivity of the host, by ill-formed granulomas in various tissues (Maschek et al 1994). Interestingly, mycobacteriosis due to M genavense has also been well described as a fatal condition in various avian species (Hoop et al 1993, 1996, Kiehn et al 1996, Ramis et al 1996, Ferrer et al 1997) and in a single canine patient (Kiehn et al 1996).

Initially, identification of M genavense was based on amplification and direct sequence determination of polymerase chain reaction (PCR) amplified 16S rRNA gene fragments (Rogall et al 1990), as the organism failed to grow on solid medium. More recently, however, M genavense strains have been isolated using enriched liquid media, and subcultured onto solid Middlebrook 7H11 agar supplemented with mycobactin J, although growth was observed to be poor, extremely slow and variable (Jackson et al 1992, Wald et al 1992, Coyle et al 1992, Böttger et al 1993, Tortoli et al 1994). This preliminary work was extended in various investigations, which are summarized in Böttger and collaborators' (1993) taxonomic description: M genavense can be differentiated from other slow growing mycobacteria by its fastidious growth, in particular its preference for broth media (such as BACTEC 13A medium, Middlebrook 7H12 or 7H9 media), inability to grow on standard (unsupplemented) solid media used for isolation of mycobacteria and its characteristic morphological appearance (tiny acid-fast coccobacilli). Enzymatic and biochemical characteristics of this organism have been published, and have enabled it to be distinguished from M avium, M malmoense, M ulcerans and M haemophilum (Böttger et al 1993).

The present report concerns a feline immunodeficiency virus (FIV)-positive cat that developed a systemic disease which resulted in poor appetite, weight loss, lower respiratory signs, skin lesions and eventually bone marrow failure. Disseminated mycobacteriosis was diagnosed at necropsy, but attempted culture in 1988 was unsuccessful. Paraffin-embedded formalin fixed tissue from this patient was retrieved 10 years later and examined using the PCR to amplify and direct sequence part of the 16S rRNA gene as part of a study of uncharacterized mycobacterial infections of cats and dogs. A diagnosis of disseminated M genavense infection was made retrospectively based on the histology, presence of acid fast organisms in many organs and partial sequence of the 16S rRNA gene.

Case report

An 8-year-old castrated male Siamese cross cat was presented to the University of Sydney Veterinary Centre in June 1988 for mucoid ocular discharge, periocular alopecia, coughing, inappetence, pica (eating newspaper) and weight loss. The cat had been seen as a kitten for signs of upper respiratory tract disease, and had experienced mild recurrent signs such as sneezing and nasal discharge throughout its life. The patient belonged to an inner-city, multi-cat household, was an avid pigeon hunter and had been vaccinated annually against feline herpesvirus, feline calicivirus and feline parvovirus. The cat had a low grade fever (38.9 to 39.1°C). Numerous mites (Demodex cati) were observed in skin scrapings of the periocular skin. The cat weighed 4.8 kg, compared with its normal 5.0 kg weight. Vitamin E and cythionate (Proban; Boehringer Ingelheim, doses unrecorded) were prescribed for demodecosis, while amoxicillin-clavulanic acid (50 mg every 12 h; Clavulox, Pfizer) was prescribed empirically for its respiratory signs.

Although the periocular skin lesions resolved, coughing and poor appetite persisted and the cat weighed 4.2 kg at the end of July. Physical findings then included a poor dry hair coat and crackles on auscultation of the ventral thorax. A large number of ascarid eggs were visualized following concentration of faeces by zinc sulphate flotation. The haematocrit was 0·30 l/l. Abdominal radiographs were unremarkable, while thoracic radiographs demonstrated changes consistent with bronchial disease, most evident in the cranial lung lobes. Cytology of material collected via transtracheal aspiration revealed much mucus, some of which was inspissated, scattered erythrocytes, scattered epithelial cells and non-degenerate neutrophils. Some squamous cells were also present, suggesting oropharyngeal contamination of the sample. No bacteria were seen in Gram-stained smears, and sparse numbers of mixed bacterial species were cultured aerobically on blood agar. This was compatible with contamination by oral flora. A Baermann test for lungworm was negative. The cat was considered to have an active chronic inflammation of the airways but no specific diagnosis was made, and the cat was treated empirically with abomectin (200 μg/kg; Avomec, Merk and Co) and chloramphenicol (125 mg every 12 h). Coughing improved subsequent to this treatment, and the cat gained 0.2 kg in weight.

The cat was represented in August for a moist ulcerative dermatitis of the dorsal cervical region considered to be an eosinophilic plaque. The cat had lost condition and was becoming cachectic. A depot corticosteroid injection was administered and a topical corticosteroid cream was prescribed. Seven days later the skin lesion had improved and a second depot corticosteroid injection was given. The cat continued to have a reduced appetite and to lose weight, and was represented the day after having a seizure. Physical findings included cachexia and pale gums. Mites could not be found in repeated skin scrapings, and the hair had regrown around the eyes. Despite regular anthelmintic treatment with pyrantel, ascarid eggs were still present in large numbers in the faeces. Haematology demonstrated a non-regenerative anaemia (haematocrit of 0.17 l/l, haemoglobin of 59 g/l, uncorrected aggregate reticulocyte count of 1%, total reticulocyte count of 28×10⁹/l), leucopenia (corrected value of 1.1×10⁹/l) due to lymphocytopenia (0.35×10⁹/l), absolute eosinopenia and neutropenia with a left shift (segmented neutrophils 0.29×10⁹/l, bands 0·12×10⁹/l, metamyelocytes 0.05×10⁹/l, thrombocytopenia (100×10⁹/l) and normoblastaemia (95 nucleated erythroid cells per 100 leukocytes; consisting of 11 pronormoblasts, 11 early, five intermediate and 68 late normoblasts). Erythrocytes showed moderate poikilocytosis and many contained Howell-Jolly bodies. The non-regenerative anaemia with inappropriate normoblastaemia, profound leucopenia and thrombocytopenia strongly suggested the cat had bone marrow disease. Splenic disease may also have been contributing to the normoblastaemia and increased numbers of Howell-Jolly bodies. Serology was negative for feline leukaemia virus antigen (ViraCHEK; Synbiotics) but positive for antibody to FIV (Cite/Agritech Systems). The owner elected to have the cat euthanased.

Necropsy examination and histopathology

A necropsy was performed 2 h after death. The cat was in poor condition, dental calculus was present in association with mild gingival hyperaemia, and an oval 2×4 cm ulcerated, crusting skin lesion was evident on the dorsal cervical area. The lungs were congested and contained numerous, scattered, 0.5–1.0 cm, discrete, soft, grey nodules. The mandibular, retropharyngeal, superficial cervical and mediastinal lymph nodes were enlarged, firm and homogeneous grey-white on cut section. The kidneys had pale cortices, the spleen was slightly enlarged with rounded edges while the liver had a pronounced lobular pattern. Tissue samples of the skin lesion, various lymph nodes, lung, spleen, liver, adrenal, small intestine, kidney and brain were collected and stored at −20°C while others were fixed in 10% phosphate buffered formalin, processed routinely and embedded in paraffin. Sections (6 μm) were cut and stained with haematoxylin and eosin (H&E), Brown and Brenn's modification of the Gram, and Putt's modification of the Ziehl-Neelsen acid fast stain.

Aggregates of large, round macrophages with ample, grey, finely granulated cytoplasm were detected in the deep dermis and subcutis (underlying the dorsal cervical skin lesion), associated with portal triads in the liver, in both red and white pulp of the spleen, in both cortical and medullary regions of the lymph nodes and associated with large airways and in alveolar spaces in the lungs. Giant, multinucleated macrophages were associated with the aggregates, particularly in the lungs, spleen and lymph nodes (Fig 1). Occasional plasma cells and lymphocytes were associated with the aggregates, particularly in the lungs where there was also bronchial gland hyperplasia. The lymph nodes were almost completely replaced by granulomatous inflammation. The liver had enlarged, finely granulated Kupffer cells, while the spleen had prominent extramedullary haematopoiesis and reduced white pulp. The kidney had prominent pink, amorphous cast material in the lumens of the medullary tubules and cortical distal tubules. The glomeruli contained scattered large macrophages and increased amounts of mesangial matrix and cell nuclei suggesting a mesangio-proliferative glomerulonephritis. Aggregates of macrophages and multinucleated cells, Kupffer cells and glomerular macrophages were packed with acid fast, beaded Gram-variable, short (1.5–4 μm), straight bacilli (Figs 2 and 3). In some macrophages the bacilli appeared to be aligned roughly in parallel while in others the arrangement was more haphazard. There was no evidence of involvement of the gastrointestinal tract or of the brain in any disease process and acid-fast organisms were not seen in sections of these tissues. Unfortunately, samples of bone marrow were not obtained at necropsy for microscopic examination.

Fig 1.

There are numerous large, round macrophages with grey, finely granulated cytoplasm and giant, multinucleated macrophages which have replaced the normal architecture of the lymph node. H & E; ×151.

Fig 2.

Accumulated macrophages around a bronchial airway show numerous intracellular, beaded Gram-variable, short straight bacilli. Brown and Brenn's modification of the Gram stain; ×754.

Fig 3.

Accumulates of giant, multinucleated and single macrophages within the spleen are packed with acid fast bacilli. Putt's modification of the Ziehl-Neelsen acid fast stain; ×302.

Microbiology

Isolation of the acid-fast organisms was attempted at the Tuberculosis Section of the State Health Laboratory, Queensland from frozen portions of lung, liver, spleen, kidney, lymph nodes and adrenal glands. Small samples of each tissue were macerated individually in sterile normal saline, with and without decontamination with 2% sodium hydroxide. The resultant suspensions were centrifuged and the sediments used to make smears for acid-fast staining and for inoculation onto Lowenstein-Jensen (LJ) Medium, LJ Medium with sodium pyruvate, LJ Medium with ferric ammonium citrate, LJ Medium plus various antimicrobials, and Herrold's Medium containing mycobactin J. Cultures were incubated in a sloped position for 24 h, and then incubated upright at both 32° and 36°C but no visible growth of mycobacterial colonies was obtained after 12 weeks. Numerous small acid fast coccobacilli were found in smears prepared from each sediment.

Paraffin-embedded formalin fixed tissue was retrieved 10 years later and examined using the PCR to amplify the 16S rRNA gene for direct sequence analysis. To extract DNA, a small section of paraffin-embedded tissue was cut using a sterile blade and suspended in 500 μl extraction buffer (100 mM Tris-HCl, 150 mM NaCl, 50 mM EDTA pH 7.4). The sample was dewaxed by heat treatment at 100°C for 10 min and transferred to a 2 ml screw cap tube (Sarstedt Ltd, Germany) containing 0.5 ml sterile 0.1 mm diameter zirconia beads (Biopsec Products, USA). The tissue was agitated for 2 min in a Mini-Bead Beater (Biospec Products), 50 μl of proteinase K (20 mg/ml) was added prior to incubation overnight at 50°C. The digested tissue was agitated for a further 2 min. Sequence capture was performed on the treated tissue as described (Mangiapan et al 1996). Briefly, 16S rRNA gene sequences were extracted from the treated tissue specimen by hybridization with biotinylated capture oligonucleotides Cap pA (5′-AAAAAAGAGTTTGATCCTGGCTCAG) and Cap pH (5′-AAAAAAAGGAGGTGATCCAGCCGCA) (Gibco BRL, Life Technologies Ltd, UK) at 42°C for 3 h and captured onto streptavidin coated magnetic beads (Dynal, Norway). The magnetic beads were washed, as described, and resuspended in 25 μl of sterile deionised water.

PCR amplification of extracted DNA (5 μl) was performed using 16S rRNA gene primers pA (5+-AGAGTTTGATCCTGGCTCAG) (Edwards et al 1989) and MSHE (5′-GCGACAAACCCCACCTACGAG) as described (Hughes et al 1997). Amplification mixes were subjected to 4 min denaturation at 94°C, 30 cycles of 95°C for 30 s, 60°C for 1 min and 72°C for 1 min followed by a final extension period of 72°C for 7 min and refrigeration. Reamplification of 5 μl aliquots of PCR product was performed using the same PCR conditions and cycling parameters. Controls for PCR and extraction were included and precautionary measures to prevent contamination were taken as described previously (Hughes et al 1994). PCR products (5 μl) were visualized by electrophoresis on 1% (w.v) agarose gel stained with ethidium bromide (0.5 μg/ml) (Sigma Chemical Company, USA). The PCR product from the paraffin embedded tissue specimen was purified using the QIAquick PCR purification kit (QIAGEN Ltd, UK) according to the manufacturer's instructions. Sequence analysis of the purified PCR product was performed by The Advanced Biotechnology Centre (UK) using primers pA, MSHE and MSHB (5′-GGGATAAGCCTGGGAAACT). Sequences were analysed with DNASIS version 7, software (Hitachi; supplied by Hitachi software Engineering America Ltd, USA), and sequence comparisons were made with EMBL and GenBank databank sequences.

A PCR product of expected size (∼600 bp) was obtained for the paraffin-embedded tissue specimen. Nucleotide sequence identity was determined for a 464 bp region of this 16S rRNA gene product including variable regions V2 and V3 (Kempsell et al 1992). DNA similarity searches with EMBL and GenBank databank sequences revealed 100% nucleotide identity of this sequence with M genavense (EMBL sequence accession number X60070) over 464 bp (mismatch due to the presence of one unidentified nucleotide in the M genavense database sequence was not considered).

Discussion

The ante-mortem investigation of this case was incomplete due to financial considerations and lack of commitment on the part of the owner to pursue a definitive diagnosis. Thus, procedures that may have demonstrated the presence of mycobacteria in cytological specimens or tissue sections, such as skin biopsy, bone marrow aspiration, abdominal ultrasonography with aspiration biopsy, bronchoalveolar lavage and lung biopsy, were not performed. Terminally, haematology suggested disruption of bone marrow and splenic functions resulting in non-regenerative anaemia with inappropriate normoblastaemia, leukopenia and thrombocytopenia. With the benefit of hindsight, skin biopsy, lymph node aspiration or bone marrow biopsy would have been most informative. Not only did the laboratory data strongly suggest bone marrow aspiration as the diagnostic procedure most likely to provide a definitive diagnosis, but bone marrow involvement is observed commonly in disseminated mycobacterial infections including those caused by M genavense (Farhi et al 1986, Maschek et al 1994). A definitive diagnosis of a disseminated mycobacterial infection was made at necropsy, but even though appropriate tissue specimens were collected, mycobacterial culture was unsuccessful. In retrospect the unsuccessful attempts at culture were due to the very fastidious nature of the causal organism, which had at that time not yet been identified as a human or avian pathogen.

The FIV and FeLV status of the cat was determined as bone marrow involvement and wasting syndrome were considered compatible with an AIDS-like syndrome. As serology demonstrated the cat was FIV-positive, it is tempting to draw parallels between the behaviour of M genavense in human AIDS patients and the present case. The cat had two consecutive infections with organisms considered to be associated with immunodeficiency, namely demodecosis and disseminated mycobacteriosis, and recurrent infection with a common gastrointestinal parasite despite appropriate anthelmintic treatment. It is therefore conceivable that in this particular patient, FIV infection had progressed to the stage where the cat had overt immunodeficiency. In our experience this is unusual sequela to FIV infection (Walker et al, 1995, Malik et al 1997), as we do not commonly observe sequential opportunistic infections in cats with longstanding FIV infection. The replacement of much of the lymph node and splenic tissue by mycobacterial granulomas would likely have contributed also to the immunodeficient state. The mesangioproliferative glomerulonephritis could be referable to longstanding FIV infection (Reinacher & Frese 1991) and/or, chronic antigenic stimulation associated with the disseminated mycobacterial infection.

Although there is already a body of evidence that M genavense is an important avian pathogen, it has been reported only once previously in a companion mammal. This patient was a young Pug dog with generalized lymphadenomegaly, hepatosplenomegaly and fever, presumably reflecting disseminated mycobacteriosis. This case was diagnosed following lymph node biopsy and treated with apparent success using clarithromycin, ethambutol and enrofloxacin; long-term follow-up was, however, lacking (Kiehn et al 1996). A cause of immunodeficiency was not identified in this canine patient, unlike the vast majority of human patients that develop disseminated M genavense infections as a consequence of HIV infection. Strong circumstantial evidence pointed to the cat of this report having generalized immunodeficiency, possibly referable to its FIV status. It would have been interesting to determine whether the CD4 count in our patient (Walker et al 1995) was as low as in human AIDS patients with disseminated M genavense infections.

The histopathological findings in the present case are similar but not identical to those observed in three AIDS patients with M genavense infection (Maschek et al 1994). Aggregates of large, granular macrophages packed with acid fast bacilli, but not surrounded by lymphoid elements or fibroplasia, was the key feature in our case and in AIDS patients. However, the additional feature of large multinucleated macrophages, a prominent feature in some of the cat's tissues, was not reported in human patients. Although the inflammatory response would be considered granulomatous according to a strict definition of the term, there was a virtual absence of an ordered lymphoid component suggesting strongly that the cat was immunologically incompetent. The corticosteroids given to the patient late in its clinical course would no doubt have contributed to its immunodeficient status, and to the dramatic gross and microscopic lesions observed at necropsy. The substantial involvement of the cat's lung tissue, and absence of gastrointestinal lesions are in contrast to AIDS patients, where gut but not pulmonary involvement is typical (Maschek et al 1994). While species variation might account for the varying patterns of tissue involvement, it is also possible that the cat's history of chronic respiratory disease may have predisposed to establishment of the organism in the lungs. Certainly the extent and severity of the pulmonary pathology suggests that the lower respiratory tract was the portal of entry and from where bacteraemia was established. In birds, disseminated infections involving many organs and tissues including the respiratory tract have been reported. However, primary involvement of the gastrointestinal tract would appear to be most common (Hoop et al 1993, Ramis et al 1996, Portaels et al 1996, Ferrer et al 1997), although cases with isolated involvement of skin have also been documented (Ferrer et al 1997).

Although it is likely that the cat of the present report would not have responded to therapy because of underlying immunodeficiency, limited available information suggests drugs including clarithromycin, rifampicin, clofazamine, amongst others, have efficacy against M genavense, as witnessed by human patients and one affected dog that improved substantially or were cured using multi-drug regimens (Bessesen et al 1993, Matsiota-Bernard et al 1995, Albrecht et al 1995). Based on limited published information, clarithromycin would appear to be the most effective agent.

This report serves to highlight the usefulness of PCR methodology in mycobacteriology, which permitted a retrospective diagnosis of disseminated M genavense infection based on formalin fixed, paraffin-embedded material collected 10 years earlier. PCR and direct sequencing of species-specific variable regions of the 16S rRNA gene is a powerful technique for detecting and classifying mycobacterial disease in human and veterinary patients, providing a rapid diagnosis even when causal organisms are fastidious, slow-growing or impossible to culture in vitro.