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Tuberculosis of pet birds

Bacterial diseases of pet birds

Tuberculosis (Mycobacteriosis) is an ancient or ‘heritage’ disease which was observed even before the Neolithic age and in Egyptian mummies. In 1882, Robert Koch first isolated Mycobacterium, stained with alkaline methylene blue and vesuvin and established its etiologic relationship with tuberculosis. Koch described that the same human or bovine type Mycobacterium may cause avian tuberculosis.


 Strauss and Gamaleia (1891) and Maffucci (1892) cited in Cobbett (1917) illustrated that the etiological Mycobacterium of avian tuberculosis was different from human or bovine type. Hinshaw (1933) and Ackermann et al. (1974) reported occurrence of tuberculosis in Amazon parrot (Amazona farinosa). Coyle et al. (1992) proposed a new Mycobacterial species (Mycobacterium genavense) isolated from human patients with AIDS. Simultaneously in 1993, Mycobacterium genavense
was reported from pet birds in Europe (Hoop et al. 1993).

Etiology:

Mycobacterium avium subsp. avium and M. genavense are the most common cause of tuberculosis in pet birds. M. genavense is common in psittacine birds, whereas, M. avium subsp. avium mostly infects the birds kept in aviaries. M. tuberculosis, M. bovis, M. intracellularae, M. gordonae, M. simiae, M. intermedium, M. peregrinum, M. terrae, M. avium subsp. paratuberculosis, M. trivial, M. fortuitum, M. diernhoferi, M. chelonae, M. smegmatis, M. flavescens, M. scrofulaceum, M. celatum, M. nonchromogenicum and M. marinum are also associated etiology. M. avium subsp. hominissuis was detected in a female blue-fronted Amazon parrot (Amazona aestiva).
 Mycobacterium is gram positive, straight or slightly curved rod with occasional coccobacillary, club and branched form. In the tissues, it measures 1–4 μm in length and 0.2–0.3 μm in width. It occurs singly, in pair or in bundle. It is difficult to demonstrate their gram positive nature due to high lipid content of the cell wall.
 The stains are relatively impermeable to the bacterial cell. They can be easily stained by Ziehl-Neelsen (ZN) or acid fast staining technique.
 The genus Mycobacterium (Actinomycetes family) contains more than 100 species. Some are pathogenic for man and animals which grow slowly in artificial media in laboratory (slow growers) than the fast growers (M. smegmatis). Previously, slow growing Mycobacterium was sub divided into three types based on their host specificity i.e. human type, bovine type and avian type. Recent classification reveals that slow growing Mycobacterium is composed of several species. The most
common species are Mycobacterium tuberculosis complex and M. avium-intracellulare complex (MAC). Mycobacterium tuberculosis complex is comprised of M. tuberculosis, M. bovis, M. caprae, M. microrti, M. africanum and M. canettii.
MAC is composed of two major species—M. avium and M. intracellulare.
M. avium is subdivided into four subspecies (ssp.) i.e. ssp. avium (Maa), ssp. paratuberculosis (Map), ssp. silvaticum and recently added ssp. hominissuis.
MAC is considered as ‘atypical mycobacteria’ and members of this group are highly resistant against environmental changes such as high and low temperatures, dryness, extreme pH and common disinfectants. There are total 28 serotypes of MAC and the serotypes 1–6, 8–11 and 21 belonged to M. avium ssp. avium. Mycobacterium genavense forms a deep branch of Mycobacterial phylogentic tree with other members such as M. interjectum and M. simiae. This group is characterized by slow growth albeit contains the signature molecule of fast growers (short helix 18). M. genavense can be distinguished from other slow growers by their fastidious growth and preference for liquid medium. M. genavense was first reported from human AIDS patients with disseminated infections (Hirschel et al. 1990).

Host Susceptibility:

Among psittacine birds, grey-cheeked parakeets (Brotogeris pyrrophterus), amazons (Amazona spp.), budgerigars (Melopsittacus undulatus) and pinous parrots (Pionus spp.) are the most commonly affected species with tuberculosis. Other psittacines such as green-winged macaws (Ara chloroptera), cockatoos (Cacatua spp.), conures (Aratinga auricapillus, Cyanoliseus patagonus) and red-crowned parakeet (Cyanoramphus novaezelandieae) are also detected to be infected with M. genavense and M. tuberculosis. M. avium subsp. avium is reported to cause infection in cockatiels (Nymphicus hollandicus). The non-cultivable Mycobacterium is detected in blue and yellow macaw (Ara ararauna) and grey-cheeked parakeet (Brotogeris pyrrhopterus).
The common parrots (African grey parrot, Senegal parrot) are not considered as natural host of M. tuberculosis although infection in African grey parrot is reported which was transmitted from human. M. bovis can produce natural infection in parrots and the budgerigars are experimentally infected with M. bovis producing clinical syndrome within 70 days.
Among non-psittacine group of birds, canary (Serinus canaria), gouldian finch (Chloebia gouldiae) and zebra finch (Poephila guttata castanotis) are commonly infected with M. genavense. Synergistic infection of M. genavense and avian polyoma virus was detected in European goldfinch (Carduelis carduelis). Occasionally canaries are also infected with M. tuberculosis.
Avian tuberculosis is a disease of adult birds although occasionally detected in young (<1 year old) canaries.

Transmission:

MAC is transmitted in birds chiefly by ingestion, inhalation and rarely through arthropods. M. avium ssp. avium is excreated through faeces of infected birds and survive in soil (up to 4 years), sawdust (8 months at 37 °C) and water for a long period. Bird to bird transmission in aviaries may occur through infected faeces or rarely by cannibalism. Occasionally skin abrasion acts as a route of mycobacterial
infection in pet birds.
Ingestion is considered as a potential route of transmission of M. genavense infection in pet birds. The environment specially drinking water is an identified source of M. genavense infection. Lung involvement in pet birds suggests inhalation as an additional route of transmission. Birds may act as reservoir of M. genavense.
Bird to bird transmission of M. genavense is rare although the possibility could not be excluded entirely. Immunosuppression plays a role in transmission of M. genavense in human but whether the same condition facilitates the transmission in pet birds is obscure.
Transmission of M. tuberculosis in pet birds (green-winged macaws, blue-fronted amazon) from human is observed due to close contact with owners suffering from tuberculosis and feeding the birds with pre-chewed food.

Pathogenesis:

M. avium subsp. avium.
MAC enters the host chiefly through ingestion route of transmission and become present in the intestine. The waxy cell wall of the bacteria protects it from gastric acids and enzymes. Several pathogen associated molecular patterns (PAMPs) are expressed by virulent Mycobacterium which can recruit ‘microbicidal’ macrophages through toll like receptor (TLR) mediated signaling. During M. tuberculosis infection in human, these PAMPs in the bacterial surface are masked with a lipid, namely phthiocerol dimycoceroserate (PDIM). The PAMPs are not recognized by the host immune system and the bacteria can avoid the reactive nitrogen species (RNS) generated by ‘microbicidal’ macrophages.
MAC (M. avium subsp. avium) does not contain PDIM in their surface but use a different strategy (still unexplored) to resist RNS. MAC is benefited with these RNS as commensal present in the gut are sensitive to it. Commensal mediated competitive inhibition is thus excluded and probably MAC enters M-cells like host adopted Salmonella to invade the underlying blood monocytes. The M-cells are specialized cells of the follicle associated epithelium and the region is relatively free from commensal mediated competitive inhibition. The invasion of monocytes is followed by bacterimia and subsequent haematogenous spread to liver, spleen and other organs.
MAC enters the macrophages (histiocytes) of periarteriolar lymphoid sheath (PALS) zone in spleen within 10 days post infection in birds. Mycobacterium has several virulence factors which promote their survival within macrophages using different strategies such as acid resistance, avoidance of acidification etc.
MAC (M. avium subsp. avium) specifically restricts vacuole maturation and prevents the fusion of phagosome and lysosome for their survival within macrophages. Haematogenous spread of the organism leads to infection of bone marrow, lungs, air sacs, gonads and rarely, kidney and pancreas. The organs become enlarged due to accumulation of macrophages within organ parenchyma.
 Granuloma formation is an attempt of host tissue to localize the infection, although Mycobacterium exploits it for their multiplication and further dissemination. The growth of Mycobacterium occurs in the macrophages present in a granuloma. The infected macrophages undergo apoptosis and leave the encased bacteria which are engulfed by newly recruited macrophages. This process of apoptosis and re-phagocytosis within a granuloma is regulated by a mycobacterial secreation system (ESX-1/ESAT6) detected in M. tuberculosis but absent in M. avium subsp. avium. Typical tuberculous granulomas in different organs are not frequent in M. avium subsp. avium infection, although observed in lungs and periocular region of parrots. Granulomas in different organs (liver, kidney, intestine, muscle and subcutaneous tissues) are observed in red-crowned parakeet (Cyanoramphus novaezelandiae) and green-winged macaw (Ara chloroptera) infected with M. tuberculosis.
Mycobacterium genevense M. genevense infection in pet birds mostly occurs through the oral route like MAC.
There is every possibility that they follow the same pattern of pathogenesis although still unexplored. M. genevense causes non-tuberculoid form of mycobacteriosis in pet birds albeit occurrence of granulomas are observed in glottis of amazon parrot (Amazona ochracephala), aorta of cockatiel (Nymphicus hollandicus), small intestine of canary-winged parakeet (Brotogeris versicolurus) and brain of spectacled amazon parrot (Amazona albifrons).

Clinical Symptoms:

Incubation period of mycobacteriosis in pet birds is 6 months to 4 years. Clinical syndrome in psittacine birds varies widely. In acute form, sudden death without any symptom is common. In chronic form, constant loss of weight along with diarrhoea, frequent micturition with excessively large quantity and low specific gravity of urine, depression, laboured breathing, distension of abdomen and poor feathering primarily suggests about mycobacteriosis. The condition fails to respond to common antibiotics.
 Cutaneous masses are sometimes observed in skin and conjunctiva. Inflammation of feather follicles (folliculitis) is occasionally observed which includes perifollicular swelling, erythema, pruritus and pain, restlessness, shivering and feather damaging behaviour.

Lesion:

The liver and spleen are enlarged, mottled and whitish. Miliary abscess in liver are observed in budgerigars experimentally inoculated with M. avium subsp. avium. The intestine becomes tubular, thickened and tan coloured. Typical tuberculous granulomas in different organs are not frequent in M. avium subsp. avium infection, albeit observed in lungs and periocular region of parrots, pericardium of gang gang cockatoo (Callocephalon fimbriatum) and cervical esophagus of blue-fronted parrots
(Amazona aestiva) occluding the lumen. Involvement of lung is rare in pet birds affected with mycobacteriosis. Some post mortem findings in canary (Serinus canarius), eurasian goldfinch (Carduelis carduelis) and the red siskin (Spinus cucullatus) reported the occurrence of lung lesions. In a rare case of M. genevense infection in an amazon parrot (Amazona albifrons), perivascular cuffs of macrophages in the grey and white matter of the brain and spinal cord, gliosis and mild
vacuolation of white matter were observed.

Diagnosis:

Clinical Specimens
Blood/sera, cloacal swabs, tracheal swabs, biopsies from organs can be collected as ante-mortem samples for diagnosis of mycobacteriosis in the laboratory.
Post-mortem samples include vital organs such as liver, spleen, intestine, heart, lung and bone marrow. All the specimens should be immediately sent to the laboratory following the standard regulations for sending biohazardous substances. If there is delay in sending, refrigeration of the samples should be done to prevent the growth of contaminants. Addition of 0.5% boric acid may preserve the samples for 1 week.

Diagnostic Techniques:

 - (a) Clinical signs and history of direct contact with owner and other birds suffering from tuberculosis give a tentative diagnosis.
 - (b) Haematological parameters: Following haematological changes can be correlated with tuberculosis in pet birds although these changes are non-specific and are observed in other inflammatory and chronic infections also.
* Moderate to marked increases in white blood cell numbers (heterophilia, monocytosis, lymphocytosis)
* Decreased packed cell volume (PCV) (except during early stage of infection)
* Increased enzyme concentration (alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase)
 - (c) Demonstration of acid-fast organisms: Demonstration of the organisms can be done in the smears prepared from ante-mortem samples by acid-fast (or ZN) staining. Mycobacterium appears as red coloured slender rods singly or in bundle. Fluorescent acid fast stain may be used for better detection.
In post mortem specimens, cytoplasm of infected cells is laden with acid-fast organisms.
 - (d) Gross and histopathology: Enlarged liver, thickened intestinal loop, increased opacity in endosteal bone in the humerus, tibia, ulna, femur in advanced cases is suggestive for tuberculosis. Presence of visible granuloma is not a constant feature although may be detected in lungs. In decomposed carcasses bone marrow is the best specimen. Histopathogical findings such as presence of acid-fast bacilli in inflammatory cells can also tentatively diagnose mycobacteriosis.
 - (e) Isolation of organism: This is considered as gold standard method for confirmatory diagnosis of Mycobacterium. To isolate the organism, the tissue sample must be processed in proper way. Tissue samples are homogenized in pestle and mortar after keeping in the solution of hypochlorite (1:1000) for 4–16 h. It is decontaminated by the addition of acid (5% oxalic acid), alkali (2–4% sodium hydroxide) or detergent (0.375–0.75% hexadecylpyridiniumchloride, HPC). The acid or alkali mixture is neutralized, centrifuged and the sediment is used for culture. M. avium subsp. avium can be isolated in Dorset egg medium, Lowenstein-Jensen (LJ), Herrold’s and Middlebrook’s (7H10,7H11) medium containing pyruvate. Glycerol and ‘mycobactin’ is also added as growth enhancer. Mycobactin extracted from the environmental Mycobacteria, acts as siderophore helping in acquisition of iron. Mycobactin is produced by all cultivable Mycobacteria except M. avium subsp. avium and M. avium subsp. paratuberculosis. Incubation period is 8 week at 40 °C. Even it can grow at 42–43 °C. It produces smooth or rough type of colonies. In liquid culture a radiometric method using 14C labelled substrate can be used for rapid detection (BACTEC system).
M. genavense isolation is difficult although can be done on special media with prolonged incubation for 2–9 months. Conventionally, after decontamination of the samples with 2(N) NaOH, the samples can be inoculated in Herrold’s egg yolk medium with and without mycobactin J and Sula’s liquid medium and incubated at 37 °C. The growth is periodically checked in every two weeks. The successful isolation of M. genavense from blue headed parrot (Pionus menstruus) was observed in Herrold’s egg yolk medium without mycobactin J after 270 days of incubation. In specialized laboratories, newly developed liquid culture systems [manual mycobacteria growth indicator tube
(M-MGIT), BACTEC system) are used for confirmatory isolation of M. genavense.
 - (f) Immunological/serological tests: Use of tuberculin test with purified protein derivative of M. avium subsp. avium correlates poorly with clinical disease in psittacine birds. ELISA was experimentally developed with M. fortuitum, M. vaccae, and M. avium antigens for detection of antibodies against M. avium subsp. avium in birds. However ELISA produces variable results in different species of birds. Immunological or serological tests for detection of mycobacteriosis in pet birds are not routinely followed.
 - (g) Molecular biology: Polymerase chain reaction (PCR) can detect Mycobacterium from fresh samples, faeces and paraffin-blocked tissues. The species-specific PCR targets IS901 and hsp65 genes for detection of M. avium subsp. avium and M. genavense, respectively. Differentiation of both the species can be done by sequencing of the 16SrRNA gene. A nested polymerase chain reaction (PCR) from the consensus sequences of the hsp65 gene, followed by analysis with restriction enzymes can also differentiate M. avium and M. genavense. Real-time TaqMan PCR assay is developed to detect hsp65 gene of M. genavense and MAC subsp. Other recent technologies such as GenoType assay and DNA microarrays can be used for diagnosis of avian tuberculosis. For detection of genetic diversity among the strains of M. avium subsp. avium, mycobacterial interspersed repetitive units—variable-number tandem-repeat markers (MIRU-VNTR) typing can be successfully used.

Zoonosis:

M. avium subsp. avium is considered as a potential zoonotic risk in the immunocompromised persons albeit majority of human infection is caused by another member of M. avium group (M. avium subsp. hominissuis). M. genavense is associated with gastrointestinal or pulmonary mycobacteriosis in immunosuppressed patients associated with AIDS. Other species of mycobacteria associated
with pet bird causes opportunistic and sporadic infections in human. The cases of reverse zoonosis (anthroponosis) are also reported where M. tuberculosis was transmitted from the infected owners to their pet birds (green-winged macaws, blue-fronted amazon).

Treatment and Control Strategy:

Successful treatment of pet birds is reported with various combinations of anti-tuberculous drugs at highest tolerable dose for a prolonged period (9 months or more). Single anti-tuberculous drug is not preferred due to possibility of resistance development. Emergence of multidrug-resistant tuberculosis (MDR-TB; resistant to isoniazid and rifampicin) and extremely drug-resistant tuberculosis (XDR-TB; in addition to being multidrug-resistant the bacteria are resistant to fluoroquinolone and 1 of 3 antibiotics such as capreomycin, kanamycin and amikacin) is a global problem now a days. In most of the cases, dose is fixed on the basis of human paediatric studies because pharmacokinetic properties of anti-tuberculous drugs in pet birds are still unknown. The anti-tuberculous drugs and antibiotics used against M. avium infections in pet birds are isoniazid, rifampin, rifabutin, ethambutol, clofazimine, ciprofloxacin, enrofloxacin, streptomycin and amikacin. Successful therapy of M. genavense infections with combinations of moxifloxacin, clarythromycin, ethambutol and amikacin in humans has been reported but there is no specific drug recommended for M. genavense infection. Treatment with clarithromycin, rifampin, and ethambutolm against M. marinum infection in a blue-fronted Amazon parrot was not successful.
 Experimentally, different combinations of anti-tuberculous drugs and antibiotics such as isoniazid (30 mg/kg) + ethambutol (30 mg/kg) + rifampin (45 mg/kg) for 12–18 months, clofazimine (6 mg/kg) + ethambutol (30 mg/kg) + rifampin (45 mg/kg) for 9–18 months, ciprofloxacin (80 mg/kg) + ethambutol (30 mg/kg) + rifampin (45 mg/kg) for 9–12 months was used successfully against confirmed cases of tuberculosis in different pet birds (grey-cheeked parakeet, double yellow-headed Amazon, lilac-crowned Amazon). Although combination of azithromycin (43 mg/kg), rifampin (45 mg/kg), and ethambutol (30 mg/kg) administered orally once daily for 180 days in ring-neck doves (Streptopelia risoria) naturally infected with M. avium subsp. avium failed to eradicate the infection. Further, treatment with combination of clarithromycin (61 mg/kg bw), moxifloxacin (25 mg/kg bw) and ethambutol (60 mg/kg bw) administered in budgerigars experimentally infected with M. avium subsp. avium by crop gavage every 12 h for 18 weeks significantly improved the situation but failed to recover completely. Combination of minocycline (10 mg/kg p.o. b.i.d.) and clarithromycin (10 mg/kg p.o. s.i.d.) significantly reduced oral plaques in blue penguins (Eudyptula minor) naturally infected with M. intracellulare. Due to zoonotic potential specially for children and elderly persons and immunocompromised patients, prolonged treatment and poor success rate, difficulty of drug administration to avian patients maintaining proper doses, natural and acquired antimicrobial resistance, poor owner compliance and moreover, lack of a proper treatment schedule, the debate exists regarding advice of treating pet birds against tuberculosis. Nevertheless, euthanasia is the preferred measure in the prevention of tuberculosis in pet birds in relation to human health. For prevention of further transmission, removal of all organic matter and debris from cages, washing the cages and surroundings properly with disinfectants and maintenance of biosecurity measures are required. Chlorohexidine and quaternary ammonium compounds can act as mycobacteriostatic disinfectants.

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