avium subspecies hominissuis’ infection in a domestic

cat

Mycobacterium avium subspecies hominissuis (MAH), a species

of nontuberculous Mycobacterium (NTM), is a slow-growing bacteria that is

widely detected in the environment. In human medicine, NTM produce pulmonary infiltrates and

disseminated diseases, mainly in immunocompromised hosts [5, 7]. Incidence of NTM infections is

reportedly increasing worldwide, and the prevalence of NTM species varies between countries.

In Japan, MAH is the most common pathogen of NTM diseases [14, 24, 25]. A previous study has revealed that combination antibiotics following guidelines

for the treatment of pulmonary NTM disease achieves positive clinical results.

In dogs and cats, MAH infection occurs via ingestion of infected meat or water, or contact

with polluted soils. Despite the widespread

distribution of MAH, infection in dogs and cats is uncommon due to their innate immunity

[13]. Little is therefore known about the clinical

implications, treatment, and prognosis of MAH infection in cats. We report here a case of

disseminated MAH infection in a young male Somali cat who recovered due to combination

antibiotics and describe a side effect observed during treatment.

A 4-year-old neutered male Somali cat was kept completely indoors, and received a medical

check-up every year. Diffuse parenchymal lung disease had been detected without any symptoms

or blood test abnormalities in a medical check-up 3 months previously. Antibiotics and

prednisolone (0.55–1.1 mg/kg every 24 hr) had been administered for the previous 2 months due

to symptoms such as a poor appetite, moderate tachypnea, and a mild fever. The antibiotics

clindamycin (5.5 mg/kg every 12 hr), doxycycline (11 mg/kg every 12 hr), and enrofloxacin (5.6

mg/kg every 24 hr) were administered separately. Despite the drug administration, his symptoms

and lung disease were not improved. He was therefore referred to the Veterinary Medical Center

of Osaka Prefecture University.

On physical examination, a body weight of 4.4 kg, body temperature of 39.0°C, heart rate of

180/min, and rhonchi lung sounds (respiratory rate: 30/min) were observed. A complete blood

cell count revealed neutropenia (Table

1). The general chemical profile of serum showed no abnormalities (Table 1). The chest X-ray revealed an interstitial

lung pattern (Fig. 1A). Ultrasound imaging indicated a swollen lymph node in the right abdomen (Fig. 1B). When fine needle aspiration (FNA) cytology of

the swollen lymph node was performed, small lymphocytes and large macrophages were observed

without neutrophils and eosinophils, which indicated granulomatous inflammation of the lymph

node (Fig. 2). According to additional examinations, such as feline coronavirus titer, alpha 1-acid

glycoprotein, serum amyloid A, and anti-filarial antibody, feline infectious peritonitis and

filariasis were ruled out and aggressive inflammation was suspected (Table 1).

Due to worsening of lung disease and lymph node swelling, dynamic computed tomography (CT)

and bronchoscopy were performed on day 38 of illness. The dynamic CT was performed in the

arterial phase (20 sec after the injection of contrast medium), in the portal vein phase (60

sec) and in the equilibrium phase (180 sec) using 2 ml/kg nonionic contrast

medium (300 mg/ml iohexol, Daiichi Sankyo Co., Tokyo, Japan). CT scan

revealed bilateral peribronchial consolidation, swollen jejunum lymph node with uniform

distribution of contrast medium, and multiple prominent nodules of the liver (Fig. 3A). These nodules exhibited lower CT values than that of liver parenchyma in plain image

and were not enhanced with dynamic CT (Fig. 3B).

Contrast enhancement of peripheral areas of the liver nodules was observed in the arterial

phase; however, it disappeared in the portal vein phase. During bronchoscopy, intratracheal

foreign bodies, increased mucus production, and redness of bronchial mucosa were not found.

Cytology of bronchoalveolar lavage (BAL) showed a small number of neutrophils and macrophages

without any bacteria. When FNA cytology of the liver was performed, neutrophils, small

lymphocytes, and large macrophages were observed. The specimens of bronchoscopy and liver FNA

were submitted to research laboratory (Japan Clinical Laboratories, Inc., Kyoto, Japan) for

culture of general bacteria, fungus, and Mycobacterium species. These

examinations revealed that general bacteria and fungus were culture-negative, and

Mycobacterium species were smear-negative with Ziehl-Neelsen staining but

culture-positive in Mycobacteria Growth Indicator Tube systems (Becton, Dickinson and Co.,

Franklin Lakes, NJ, U.S.A.). The pathogen was confirmed as M. avium through

DNA-DNA hybridization techniques with DDH Mycobacteria “Kyokuto” (Kyokuto Pharmaceutical

Industrial Co., Ltd., Tokyo, Japan).

Antitubercular drug susceptibility testing determined by the proportion test method on

egg-based ogawa media (Vite Spectrum SR, Kyokuto Pharmaceutical Industrial Co., Ltd.) revealed

that the bacterial isolate was resistant to isoniazid, rifampicin, streptomycin, ethambutol,

kanamycin, enviomycin, ethionamide, para-aminosalicylic acid, and levofloxacin; however, it

was sensitive to cycloserine (Table 2).

In order to identify the subspecies of M. avium isolate recovered from the

clinical sample, we determined the nucleotide sequence of hsp65 (GenBank

accession number LC497502) and compared single nucleotide polymorphisms (SNPs) among the

M. avium strains, which were categorized into “codes” 1 to 9, and 15 to 17

depending on their sequence (Table 3) as described previously. The

sequence from our sample, however, was not a complete match with any hsp65

sequences from code 1 to 9, and 15 to 17. We then created the phylogenetic tree of

hsp65 using the sequences deposited in GenBank belonging to codes 1 to 9,

and 15 to 17 [1, 12] (Fig. 4). Codes 1 to 3, 7 to 9, and 15 to 17 are found in MAH. Code 4 is from M.

avium subsp. avium, and codes 5 and 6 are from M.

avium subsp. paratuberculosis. The hsp65 sequence

from our specimen, shown as strain MY332, was closest to that in code 9 and located in the

group composed of codes 1, 2, 8, 9, 15, 16, and 17. These results indicated that the isolate

from the clinical sample was MAH.

Due to the positive culture results for the Mycobacterium species, we

started orally combined administration of antibiotics (isoniazid 10 mg/kg every 24 hr,

rifampicin 10 mg/kg every 24 hr, and enrofloxacin 5 mg/kg every 24 hr) on day 66 of illness.

However, atonic seizures occurred in increasing frequency from day 93 of illness. The chest

X-ray and ultrasound imaging revealed the improvement of lung disease and jejunum lymph node

swelling, which indicated the therapeutic response of MAH infection. We administered pyridoxal

phosphate (1 mg/kg every 24 hr) and clarithromycin (10 mg/kg every 12 hr) instead of

isoniazid. Atonic seizures disappeared within a week. On day 107 of illness, lung disease was

in remission and the jejunum lymph node had begun to shrink. On day 246 of illness, CT scan

revealed that the peribronchial consolidation and the multiple nodules of the liver had

disappeared (Fig. 3C and 3D). Furthermore, bronchus

and liver FNA specimens were culture-negative in Mycobacteria Growth Indicator Tube systems.

We stopped antibiotic administration on day 429 of illness, and there were no clinical signs,

evidence of lung disease in chest X-Ray, or obvious lymph node swelling in ultrasound images

on day 771 of illness.

Here, we diagnosed MAH infection in a domestic cat who was referred because of lung disease

of an unknown origin. During clinical investigation, we detected an interstitial lung pattern,

a swollen abdominal lymph node, and multiple nodules of the liver. Cytology of the lymph node

and the liver indicated signs of granulomatous inflammation without any bacteria. However,

cultures of bronchus and liver FNA specimens were positive for NTM, which was confirmed as MAH

through SNP analysis and analysis of the phylogenetic tree of hsp65. After

the administration of combination antibiotics for 6 months, NTM culture of bronchial and liver

FNA specimens results turned negative. We stopped administration of antibiotics after an

additional 6 months, and there were no signs of relapse on day 771 of illness.

Infections of Mycobacterium species in domestic cats have been reported

sporadically. In Great Britain, previous reports have revealed that 1.16% of 11,782 feline

tissue samples submitted to diagnostic laboratories were confirmed to have

Mycobacterium infections, and

15% of cultured Mycobacterium species were M. avium. In

Japan, Mycobacterium infections in domestic cats have been historically

uncommon, with only two cases reported: unclassified Mycobacterium species

(MFM001 strain) and MAH in the Kanto region. This is the third reported case of

Mycobacterium species infection in a domestic cat in Japan. In Japan,

pulmonary NTM diseases are commonly diagnosed in human medicine with an incidence rate of 14.7

cases per 100,000 person-years in 2016, which is 2.6 times higher than the incidence rate

reported in 2007. The most common pathogens of

pulmonary NTM diseases were M. avium in the northern and eastern parts of

Japan and M. intracellulare in the southern and western parts of Japan.

Mycobacterium infection should also be considered a potential disease for

domestic cats. In a previous study in Australia, it was observed that certain lines of

Abyssinian and Somali cats likely suffer from a familial immunodeficiency that predisposes

them to infection with slow-growing mycobacteria, including M. avium. Whilst immunological predisposition of those breeds has

not been well proven, it will be beneficial to pay attention to the breed of cats with

suspected Mycobacterium infections.

Species of M. avium are divided into four subspecies: M.

avium. subsp. avium, M. avium. subsp.

silvaticum, M. avium subsp.

paretuberculosis, and M. avium. subsp.

hominissuis. These subspecies are genetically very close. However, their

host range and pathogenicity differs. Furthermore, recent studies have suggested that genomic

differences affect the virulence and antibiotic resistance of MAH [4, 14]. In domestic cats, there have

been a few reports that have identified the pathogen of NTM infection as MAH [3, 15, 20]. In this report, SNP analysis and analysis of the

phylogenetic tree of hsp65 enabled the correct evaluation of the clinical

findings, pathology and therapeutic response depending on the bacterial species.

M. avium is ubiquitous worldwide in soil and water under certain conditions

and remains viable in the environment for at least two years. Despite the widespread distribution and survivability of M.

avium, infections in human and veterinary medicine are quite rare. Therefore,

individual innate immunity is the most important for the prevention of M.

avium infection, and no evidence has been found for the spread of M.

avium among humans or animals. However, due to uncommon diseases, it is difficult

to conclude that the epidemiology of each Mycobacterium species, subspecies,

and strain has been established. Thus, we should prevent immunocompromised humans or animals

(f.e., resulting from chemotherapy) to come into contact with infected animals.

M. avium infection in cats is usually caused by ingestion of the organism

via the environment or contaminated food. Our case was completely kept indoors; therefore, we

suspected inapparent infection in youth or infection from polluted water or food. After

ingestion, M. avium is phagocytosed by intestinal macrophages and eventually

causes diseases due to stress or acquired or inborn immunosuppression. In human and veterinary medicine, some cases of M.

avium infection have been diagnosed with blood culture; therefore, hematogenous

dissemination of M. avium has been considered [1, 5, 19]. Previously reported clinical findings of MAH infection in cats are

lymphadenopathy, skin abscesses, skin granulomas, meningoencephalitis, and lung nodules [3, 15, 20]. In the present study, diffuse parenchymal lung

diseases and granulomatous inflammation of the lymph node and liver were observed. We

suspected that our case was infected via ingestion of polluted water or food and MAH

disseminated to the jejunum lymph nodes, liver, and lungs. MAH infection should therefore be

considered a differential diagnosis for lung diseases or granulomatous inflammation of

uncertain cause. A previous study identified the same clinical findings and splenomegaly in

cats with M. avium infection, though potential subspecies were not analyzed

[2]. In the present study, no spleen abnormalities

were found with ultrasound imaging or CT scan. However, as we did not perform cytology of the

spleen, we should consider the possibility that minute spleen lesions were formed. Further

studies will be required to elucidate the relationship between clinical findings and

M. avium subspecies.

In some reports, NTM phagocytosed by macrophages were detected in cytology of granuloma or

BAL, with or without acid-fast staining [15, 21, 22]. However, we

did not detect MAH in cytology of the swollen lymph node, liver or BAL with Ziehl-Neelsen

staining. Previous studies revealed that Mycobacterium species at different

stages of infection can sometimes be extremely difficult to find. Thus, it is recommended that

Mycobacterium infection is not ruled out even in cases of negative results

of cytological preparations, and that histological examination or polymerase chain reaction

should nevertheless be performed [22, 23]. In the present study, lung disease was detected

without any symptoms or blood test abnormalities in a medical check-up three months earlier;

therefore, we considered our case to be at the early stage of MAH infection. In such cases, it

has been reported that specific culture for NTM species of granuloma, BAL and blood has proved

useful in human medicine. In our case, bronchus

and liver FNA specimens were submitted for culture of Mycobacterium species

and shown to be positive. This result indicated that culture for specific NTM species, as well

as cytology, should be considered in cases with lung diseases or granulomatous inflammation of

uncertain cause.

During combined administration of antibiotics, this case presented atonic seizures. As the

possible causes of seizures, we considered MAH infiltration into central nervous systems, side

effects of isoniazid, or other intracranial diseases. Chest X-ray and ultrasound imaging

revealed the improvement of lung disease and jejunum lymph node swelling, which indicated the

therapeutic response of MAH infection. Therefore, a side effect of isoniazid, whose

recommended dosage range was 10 − 20 mg/kg every 24 hr, was suspected as the cause of seizures. In human medicine, isoniazid

administration for patients with concurrent diseases, such as chronic kidney disease and

diabetes, sometimes causes atonic seizures, and these side effects can be prevented and

treated with pyridoxal phosphate. In the present

case, there were no clinical findings to indicate concurrent diseases. However, we should

consider the possibility that the multiple nodules in the liver potentially affected the

metabolism of isoniazid even though isoniazid was administered at the minimum recommended

dosage.

Three cats were previously reported with MAH infection. Two were diagnosed at necropsy [3, 20]. One, which

presented with chronic vomiting, diarrhea, weight loss, anorexia and an abdominal mass, was

treated with combined antibiotics, including azithromycin, rifampicin and enrofloxacin. However, after a short period of improvement of

clinical symptoms, the cat was euthanized because of severe protein-losing enteropathy.

Although treatment and prognosis of M. avium infection in domestic cats in

Australia has been reported, Mycobacterium subspecies were not identified in

these cases. This is therefore the first case of MAH

infection to be confirmed by gene analysis and to show recovery with combination antibiotic

treatment. The poor prognosis in previous cases could be attributed to difficulties in the

antemortem diagnosis and delayed start time for treatment of MAH infection after clinical

symptoms were observed. This case demonstrates that combined antibiotic administration based

on reports from human medicine is effective for MAH infection in cats, and early examinations,

diagnosis and treatment will lead to a good result. Susceptibility testing revealed that the

MAH isolate was resistant to most antitubercular drugs. However, we administered combined

antibiotics including isoniazid and rifampicin, which the MAH isolate was resistant to, and

clinical symptoms improved. Previous studies revealed that in vitro

susceptibility testing for Mycobacterium species was of little or no value

for predicting clinical efficacy [4, 11]. The present study indicated that it is difficult to

predict therapeutic effects of antitubercular drugs for MAH depending on in

vitro susceptibility testing in domestic cats.

In human medicine, combination antibiotics are administered for 12–24 months or until NTM

culture results are negative. However, there is no convincing scientific evidence [4, 9, 16, 28]. In domestic

cats, it was recommended that treatment of feline leprosy syndrome, which was caused by

Mycobacterium species, should ideally be continued for a further 3 months

after lesions have regressed in order to reduce the risk of recurrence. However, the optimal length of drug therapy for MAH has not yet been

established. Here, we administered combination antibiotics for 6 months until NTM culture

yielded negative results, and no symptoms or abnormal findings were detected in X-Ray or

ultrasound images. Further studies might be needed to identify the optimal treatment duration

of MAH infection.