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Chlamydiosis of pet birds

Chlamydiosis of pet birds

Halberstaedter and von Prowazek (1907), a Czech zoologist, first described Chlamydia, although it was actively infecting people for centuries before its discovery.
 First description of Chlamydia associated trachoma in human was found in the Ebers papyrus dated around 1550 B.C. In modern times, Ritter (1879) first described Chlamydia psittaci infection in human acquired from parrots. During 1890–1930, numerous outbreaks of human psittacosis occurred in Europe, North and South America, associated with parrots and other pet birds. In 1929–30, pandemic psittacosis outbreaks in human were reported due to import of infected psittacine birds from South America to Europe and North America.

 In 1930, Levinthal, Coles, and Lillie, independently described the properties of the pathogen, and accordingly Chlamydia was known as Levinthal-Coles-Lillie (LCL) agent. Moulder (1962) first revealed the structural and chemical composition of C. psittaci. Hatch (1975) demonstrated the requirement of adenosine-triphosphate (ATP) supplementation for growth of Chlamydia.
 In literatures, first description of chlamydiosis in parakeets was reported from Germany (Strauch and Rott 1958). Further study after few years also revealed the presence of Chlamydia in parrots and other parakeets in Germany (Schmittdiel 1966).
 Human psittacosis outbreaks specially in persons associated with poultry, turkey
and duck industry were reported from United States and European countries during
1980–90.

Etiology:

All the Chlamydiae are placed under the order Chlamydiales, and family Chlamydiaceae. Based on cluster analysis of 16S and 23S rRNA genes, the family Chlamydiaceae was divided into two genera i.e. Chlamydia and Chlamydophila.
 Recent genome comparison study of the two genera proposed to unite the Chlamydia in a single genus. The latest edition of the Bergey’s manual of systematic bacteriology also described the single genus of Chlamydia. Pathogenic species under the genus Chlamydia are C. psittaci, C. trachomatis, C. suis, C. muridarum, C. abortus, C. caviae, C. felis, C. pecorum and C. pneumoniae. Among the pathogenic species, C. psittaci is mostly associated with avian chlamydiosis (psittacosis) in pet birds, ornithosis in poultry, and zoonotic infection in human. The 16S rRNA gene based phylogenetic study indicates the presence of a distinct cluster of C. psittaci strains which are associated with chlamydiosis in Psittaciformes (cockatoos, parrots, parakeets, lories etc.) and Columbiformes (pigeons) birds.
 Although, other Chlamydial species such as C. abortus, C. trachomatis and C. pecorum are occasionally detected in brown skua, parrots, parakeets, and pigeons. Three new avian species of Chlamydia, namely C. ibidis sp. nov., C. avium sp. nov., and C. gallinacea sp. nov. are proposed. Among them, C. ibidis and C. avium are isolated from feral sacred ibis (Threskiornis aethiopicus), psittacines, and pigeons.
 Earlier, avian isolates of C. psittaci was divided into six serovars (serotypes) which can infect different species of birds (A–F, Table 2.1). Based on major outer membrane protein (ompA) sequence, C. psittaci is currently divided into 15 genotypes. Among them, nine genotypes (A–F, E/B, M56, WC) are associated with different species of birds and mammals (Table 2.1).

Host Susceptibility:

 Avian strains of C. psittaci are detected in more than 460 species of birds under 30 orders. The pet birds belonged to the order Psittaciformes (cockatoos, parrots, parakeets, lories etc.) and Columbiformes (pigeons) are most susceptible to C. psittaci infection. In parrots, the worldwide prevalence of C. psittaci varies from 16–81%. The infection in pet birds is reported from Europe, Brazil, Africa, USA, Iran and India. Free ranging Galapagos doves (Zenaida galapagoensis) and rock doves (Columba livia) in Spain; monk parakeets (Myiopsitta monachus), Amazon parrots, red-tailed Amazon (Amazona brasiliensis) in Brazil; ring necked parakeet (Psittacula krameri), Alexandrine parakeet (Psittacula eupatria), African grey parrot (Psittacus erithacus), Timneh grey parrot (Psittacus erithacus timneh) in Iran were reported to be infected with C. psittaci. In India, C. psittaci was isolated from pigeons (Columba livh), parrots (Psittacula krameri) and crows (Corvus splendens).   Infection of Passeriformes birds is not common, although, canaries were detected to be infected with C. psittaci in Croatia.
C. psittaci was also detected in healthy asymptomatic birds such as in Ara macao, and Amazona ochrocephala in Costarica, and free-living Hyacinth macaw (Anodorhynchus hyacinthinus) and blue-fronted parrot (Amazona aestiva) in Brazil.
 The syndrome was not expressed either due to infection with low virulent strain or resistance of some bird species.
 The seroprervalence studies revealed the presence of C. psittaci antibodies in macaws (Ara macao, Ara ambigua), hyacinth macaws (Anodorhynchus hyacinthinus), budgerigars (Melopsittacus undulatus), lovebirds (Agapornis sp.), cockatiels (Nymphicus hollandicus), Alexandrine parakeets (Psittacula eupatria), Eurasian siskins (Carduelis spinus), oriental skylarks (Alauda arvensis), and black-tailed grosbeaks (Coccothraustes migratorius) in different countries. The presence of C. psittaci antibodies indicates the exposure of the birds to the organism. In a study in China, highest seroprevalence was observed in cockatiel which was followed by Alexandrine parakeets, lovebirds, and budgerigars. It seems that lovebirds and budgerigars among the psittacine birds are relatively resistant against C. psittaci infection, although, the reason is unexplored.
 Among the wild predator birds, white-tailed sea eagle (Haliaeetus albicilla) and the peregrine falcon (Falco peregrinus) are detected to be infected with C. psittaci.

Transmission:

 Inhalation of contaminated dust, airborne particles from the feathers and ingestion are major ways of C. psittaci transmission in the birds. Direct contact during close proximity with the infected birds also helps in transmission. Throughout the breeding season, specially during incubation of eggs, male psittacine birds prefer to feed the females by regurgitation. In this process the feeds are often mixed with secreations of the crop, pharynx and nasal cavity. Transmission of C. psittaci is observed from parent birds to their nestlings during feeding.
 Asymptomatic carrier birds infected with C. psittaci, excreate the organisms through faeces, nasal and lacrimal discharge, oropharyngeal mucus, crop milk and other secreations. Shedding is increased during coexisting infections and stress conditions such as shipment, breeding, crowding, chilling and nutritional deficiencies.
 When the excreated faecal material dries, the organisms are aerosolized. Elementary bodies (infections form) of C. psittaci survive in the dried faeces for several months, in the contaminated feed for up to two months, on glass for 15 days, and in straw for 20 days.
 Mechanical transmission of C. psittaci by biting arthropods such as flies, mites and lice are observed. Vertical transmission is infrequently observed in parakeets, seagulls, snow geese and poultry.
Zoonotic transmission of C. psittaci in human occurs mostly through inhalation of contaminated dust, feathers and aerosolized excreations. Direct contact with infected pet birds or their cages, utensils, beddings contaminated with discharges can transmit the bacteria. Sometimes, biting of the infected birds also helps in transmission. Person to person spread is rarely reported although possible through inhalation.

Pathogenesis:

Chlamydia follows a unique life cycle with tri-phasic developmental stages. The infectious form (elementary body, EB) is extremely small (250–350 nm in diameter), pear to spherical shaped particle with electron dense irregular nucleoid. It has rigid cell wall with disulphide cross linkage among the cysteine rich amino acids of outer membrane proteins (OMP). This form can survive for a prolonged period in the environment. After transmission, infection starts with the attachment of elementary
bodies to the host cell membrane. In birds, apical surface of columnar epithelial cells in intestine acts as preferred site for attachment of C. psittaci.
 Primary attachment of EB takes place by electrostatic interactions, most likely with glycosaminoglycan (GAG) moieties on the host cell surface. This reversible binding is followed by receptor mediated irreversible attachment. Protein disulfide isomerase (PDI), present in the host cell membranes and causing disulphide reduction, helps in attachment of EBs. Chlamydial major outer membrane protein (MOMP) mostly acts as adhesin to bind with the host cellular receptor. Following receptor mediated attachment, the EB enters the cell via endocytosis [microfilament dependent/independent process (clathrin mediated)]. Some Chlamydial strains enter the host cells through cholesterol-rich lipid raft domains. Among different pathways, C. psittaci strains prefer to use clathrin-coated vesicles for cellular entry. C. psittaci elementary bodies contain rosette like long projections (Matsumoto’s projection) on their surface which acts as type three secreation system (T3SS). C. psittaci-T3SS helps in introduction of Chlamydial proteins into the host cell cytoplasm. These T3SS-injected proteins interact with host cellular proteins and cause modulation of host cell function. After cellular entry, vesicles containing EBs escape the lysosomes in the host cell cytoplasm and reach near the nucleus within 8–12 h after entry. In C. psittaci infection, IncB proteins (T3SS-effector protein) interact with host cell proteins (dynein motor proteins) for intracellular transport of vesicles with EBs into the nuclear zone. The EB is converted into reticulate bodies (RB) in this nuclear zone.
 The EB loses its electron dense DNA core and its cell wall loses its rigidity due to break of disulphide bridges. The reticulate bodies are non-infectious form, larger in size (500–2000 nm diameter) and metabolically active. RBs multiply by binary fission and start genus specific protein synthesis. The structural reorientation started and RBs are transformed into intermediate bodies (IB, 300–1000 nm in diameter).
 A central electron-dense core with radially arranged nucleoid fibres surrounding the core is observed in the IBs. The IBs are converted into progeny EBs within a vesicle after 30 h of the entry of initial EBs. Chlamydial micro colony with 100–500 EBs within the vesicle is called ‘inclusion body’ and it is generated after 48–50 h. The inclusion bodies move to the golgi apparatus region with the help of host dynein proteins. EB is released to attack new cells by rupturing the vesicles and the cycle is repeated. The signal for release of EBs is yet unknown but it is associated with host cellular apoptosis. Suppression of host cellular apoptosis can induce persistent Chlamydial infection.
 Intracellular survival of EBs depends on escape from the lysosomal breakdown process. The EBs can induce delayed maturation of lysosomes as an escape mechanism. The intracellular inclusion bodies are covered with a mesh of host cytoskeletal filaments which prevent the exit of the content and consequent activation of the host immune system. Close attachment of C. psittaci inclusions with
the mitochondria helps in acquisition of ATP because they cannot synthesize it.
 Moreover, intracellular survival of the inclusions depends on acquisition of lipids such as sphingomyelin, phosphatidylinositol and phosphatidylcholine. Golgi apparatus of the host cells act as major source of lipids for the inclusions and often the golgi apparatus are fragmented to provide the lipid. Some non-replicating reticulate bodies persist within the host cytoplasm and produce latent infection. The growth cycle of Chlamydia within the body of the host is disrupted due to nutritional deprivation, treatment with antibiotic and activated immune system. In disrupted growth cycle, reticulate bodies are converted into enlarged pleiotrophic ‘aberrant’ RBs. The aberrant RB contains chromosome but the genes associated with growth (genes encoding membrane proteins, transcription regulators, cell division factors, EB-RB differentiation factors) are not expressed. Further, the genes encoding chlamydia protein associated with death domains (CADD) are down regulated which causes suppression of host cell apoptosis and persistence of infection. Interaction of host cellular protein (G3BP1) and chlamydial IncA (T3SS-effector protein) also suppress host cellular apoptosis.   When the inducers of the disrupted growth cycle (antibiotic, immune system products) are removed, the aberrant RB is again converted into normal RB and they can complete the growth cycle.

Clinical Symptoms:

 In birds, chlamydiosis has an incubation period of 3–10 days. Clinical symptoms are not specific. General syndrome such as loss of condition, anorexia, fever, diarrhoea, respiratory problems, nasal and ocular discharges are observed. Expression of syndrome and associated mortality (up to 80%) depends on virulence of C. psittaci strains, age, species, nutritional and immune status of the pet birds. Occasionally, sub-clinical C. psittaci infection without visible syndrome is observed in birds. During stress conditions, the sub-clinical infection is activated with increased shedding of C. psittaci.

Lesion:

The pet birds with avian chlamydiosis do not show any pathognomonic gross lesion. Conjunctivitis, lateral nasal adenitis, sinusitis, fibrinous airsacculitis, lung congestion, fibrinous pneumonia, pericarditis with presence of fibrinous cover, peritonitis, hepatitis with multifocal necrosis and splenitis are observed. In pigeons, conjunctivitis, swollen eyelids, rhinitis, presence of fibrinous exudates over peritoneum, air sac and pericardium, enlarged, soft and dark coloured liver and spleen are observed. The budgerigars, infected with C. psittaci and Reovirus, showed distinct cachexia, hepatomegaly and spleenomegaly. The livers become enlarged, mottled and tan-brown in colour.   Other lesions include uric acid deposit in kidney, conjunctivitis and air-sacculitis. Carrier birds with asymptomatic infection do not show any gross lesion.
 Atherosclerosis is considered as a well defined ailment specially in aged pet birds. African gray parrots, macaws and Amazon parrots are most susceptible to this condition. Sudden death without prior symptom is the cardinal sign of atherosclerosis. Like human, the risk factors for atherosclerosis include high cholesterol and triglyceride concentrations, sex, age, species, obesity and inactivity, and moreover, C. psittaci infection. Arteriosclerotic plaques are observed between the intima and internal elastic lamina of the blood vessels in many species of birds (Fig. 2.7). The plaques are composed of fibrous tissues and are observed as pale yellowish areas at the thickened portion of intima. In severe cases, the plaques become circumferential lesion which cause narrowing of the lumen and reduced
blood flow.

Diagnosis:

Clinical Specimens.
From live birds, pharyngeal/choanal slit swabs, conjunctival swabs and nasal swabs can be collected aseptically as ante-mortem samples. Faeces or cloacal swabs are less preferred because shedding of Chlamydia is not consistent. Post mortem samples collected from the dead birds include lungs, spleen, liver and air sacs.
 Chlamydiae are relatively labile organisms and special precautions are required for their detection. Samples should be maintained in cold chamber and processed immediately after collection. The tissue samples can be preserved at −80 °C for prolonged period. DNA extracted from the tissue samples can be stored in stabilization buffer. For successful isolation of Chlamydia, the clinical samples should be collected in special Chlamydia transport medium such as 2SP (0.2 M sucrose
phosphate medium containing 10 lg/ml of gentamicin, 25 U of nystatin and 25 lg/ml of vancomycin) and SPG (75 g of sucrose, 0.52 g of KH2PO4, 1.22 g of Na2HPO4, 0.72 g of glutamic acid and water in 1litre, pH 7.4–7.6) supplemented with bovine serum albumin, streptomycin, vancomycin and nystatin. Broad spectrum antibiotics like tetracycline, chloramphenicol, macrolides, sulphonamides,
penicillin should not be added as they have anti-chlamydial effect.

Diagnostic Techniques:

(a) Direct examination: Smears prepared from collected faecal samples, conjunctiva or impression smears of tissue samples can be stained with Macchiavello, Castaneda, Giemsa, Giménez, modified Gimenez (PVK stain), Stamp, modified Z-N, and methylene blue for demonstration of Chlamydial
inclusion bodies. Giemsa stain is more useful in the smears prepared from conjunctival scrapings. The inclusion bodies appear purple/blue with Giemsa, Castaneda and methylene blue stain and red with Macchiavello, Giménez, Stamp, and modified Z-N stains.

 (b) Isolation of Chlamydia from clinical samples: Isolation of Chlamydia can be done in the yolk sacs of embryonated hen eggs, laboratory animals and cell culture. Fertile chicken eggs (6–8 days old) are inoculated through the yolk sac route. The embryo dying three or more days after incubation is examined for chlamydial inclusions. Mice are ideal laboratory animal for isolation of Chlamydia. The mice usually die within ten days of intranasal, intracerebral or intraperitoneal inoculation and the EBs can be isolated from viscera and peritoneal exudates. Cell lines treated with a metabolic inhibitor (cycloheximide at 2 lg/ml) can be used for isolation of Chlamydiae. McCoy, HeLa, monkey kidney cells, L-929, Buffalo Green Monkey (BGM) cells, mouse fibroblast cells, fish and lizard cells are used. The inoculated cells should be incubated at 35–37 °C for 48–72 h and the intracytoplasmic inclusion bodies are detected by staining (Giemsa) or fluorescein conjugated monoclonal antibody. Isolation by cell culture is still considered as gold standard method for detection of Chlamydia. However, it requires biosafety level-3 (BSL3) laboratory with expertise.
 The clinical samples should be decontaminated by antibiotics like gentamicin (50 lg/ml), vancomycin (75 lg/ml) and nystatin (500 unit/ml) before inoculation into eggs, animals or cell lines. Transport medium (2SP) can be used as buffer.

 (c) Detection of C. psittaci antigen: ELISA based antigen detection kits are available for detection of C. trachomatis infection in human. The same kit can be used for detection of C. psittaci because the two species share common antigen. However, minimum 600 elementary bodies are needed in the samples for detection.

 (d) Serological tests: Serological tests can be used as supplementary diagnostic tests along with detection of antigen or isolation. Presence of antibodies in the host cannot confirm active infection. Sometimes, false negative results are produced if the samples are collected before development of antibody or during treatment with antibiotics. The serological methods such as micro immunofluorescence (MIF) test, ELISA, CFT, elementary body agglutination (EBA) tests are used for detection of anti-Chlamydia antibodies. MIF is more sensitive and can detect all types of immunoglobulins in the sera. ELISA based tests using whole organism, LPS, lipoglycoprotein of Chlamydia as antigen are sensitive but less specific for detection of C. psittaci. Whereas, ELISA with recombinant major outer membrane protein (MOMP) of C. psittaci as antigen, can more specifically detect C. psittaci. CFT can detect anti-Chlamydia Ig G only, not Ig M. Further, CFT is tedious, time consuming, less sensitive test and the antigens (complement fixing) are not commercially available. The EBA test can detect anti-Chlamydia Ig M only, and as a consequence, infection in early stage can only be diagnosed.

 (e) Molecular biology: PCR is a specific, sensitive, and rapid technique to detect C. psittaci. Successful application of PCR depends on quality of extracted DNA from the clinical samples. Guanidine-detergent lysing solution should be used for lysis of eukaryotic host cells and Chlamydia for extraction of DNA. The 16S rRNA gene is conserved in the genus Chlamydia and is a suitable target gene for detection of Chlamydia up to species level. Major outer membrane protein (ompA) is used as a target gene in nested PCR, although, variations exist in the MOMP gene sequence among C. psittaci strains. SYBR green-based real time PCR targeting ompA, 23S rRNA gene, inclusion membrane protein A gene (incA), molecular cysteine-rich protein gene (envB) of C. psittaci and microarray-based detection assays are also developed for detection of Chlamydia.

Zoonosis:

 Human psittacosis cases are reported in Europe, USA, South America, Japan and Australia. Other than the persons who rear the birds in their home, occupational risk groups such as veterinarians, pet shop workers, avian quarantine workers, poultry processing plant workers, bird breeders, and farm workers are most susceptible. Even psittacosis outbreak was detected among custom officers in some countries due to their exposure to imported parakeets in the airport. Incubation period in human is 5–14 days. Clinical syndrome in human includes fever, chills, headache, pneumonia, renal disorders, and miscarriages in pregnant women. All the vital organs are affected with the progression of infection and endocarditis, hepatitis, myocarditis, arthritis and encephalitis are reported. Ocular infection with follicular kerato-conjunctivitis is also observed.

Treatment and Control Strategy:

 Doxycycline, tetracycline and enrofloxacin were successfully used in budgerigars and psittacine birds to cure avian chlamydiosis. Doxycycline is the drug of choice for the birds and the treatment should be continued for 45 days. It may induce toxicity in some bird species and produce signs of depression, inactivity, anorexia, greenish or yellowish urine. Use of the drug in those birds should be stopped immediately and supportive symptomatic treatment should be started. Recommended dose of doxycycline in feed is 300 mg/kg feed for 45 days. In drinking water, 400 mg of doxycycline hyclate/litre of water will maintain therapeutic concentration in psittacine birds. Administration of the drug through the feed or drinking water is suitable for aviaries. For pet bird owners, oral dministration of the capsule in individual bird is appropriate. Recommended oral dose of the drug is
40–50 mg/kg body weight in every 24 h for cockatiels, Senegal parrots, blue-fronted, orange-winged Amazon parrots; 25 mg/kg body weight in every 24 h for African gray parrots, blue and gold macaws, green-winged macaws; and 25– 50 mg/kg body weight in every 24 h for other psittacine birds. Injectable doxycycline is administered at doses of 75–100 mg/kg body weight, intramuscularly (pectoral muscle), in every 5–7 days for the first 30 days and subsequently in every 5 days for the rest of the treatment period. Long acting oxytetracycline can be injected sub-cutaneously at the dose of 75 mg/kg body weight in every 3 days in cockatoos, blue-fronted and orange-winged Amazon parrots, and blue and gold macaws. The oxytetracycline injection causes irritation at the site. If tetracycline is
orally administered or used in feed, dietary calcium sources (mineral block, oyster shell, supplemented pellets) should be reduced. To control the psittacosis infection in aviaries, general precautionary measures, such as quarantine of newly introduced birds for 30 days and periodical testing for C. psittaci infection, separation of birds after return from bird shows or fairs, rodent control, control of exposure to wild birds, regular disinfection of the cages and utensils, proper ventilation to reduce aerosol load within the unit should be followed. Use of prophylactic antibiotic is not recommended as it may produce resistant bacteria. Recommended disinfectants for C. psittaci infection are 1:1,000 dilution of quaternary ammonium compounds, 70% isopropyl alcohol, 1% lysol,
and chlorophenols. Use of vacuum cleaner in the aviaries is not preferred as it will aerosolize infectious particles. No vaccine is commercially available for the pet birds against C. psittaci infection. Experimental DNA vaccination in budgerigars with plasmid DNA expressing MOMP of C. psittaci was found effective.

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