17 Chlamydiosis

Chlamydial pathogens cause a wide-range of infections and disease, known as chlamydioses, in humans, other mammals and birds. The causative organisms are Gram-negative obligate intracellular bacteria that undergo a unique biphasic developmental cycle involving the infectious elementary body and the metabolically-active, non-infectious reticulate body. At least two species, Chlamydophila psittaci and Chlamydophila abortus, are recognized as causes of zoonotic infections in humans worldwide, mainly affecting persons exposed to infected psittacine and other birds, especially ducks, turkeys, and pigeons, and less commonly to animals, particularly sheep. Outbreaks occur amongst aviary workers, poultry processing workers, and veterinarians. Infection is transmitted through inhalation of infected aerosols contaminated by avian droppings, nasal discharges, or products of ovine gestation or abortion. Person to person transmission is rare. Control strategies have met with variable success depending on the degree of compliance or enforcement of legislation. In the UK control is secondary, resulting from protection of national poultry flocks by preventing the importation of Newcastle disease virus using quarantine measures. Improved standards of husbandry, transport conditions, and chemoprophylaxis are useful for controlling reactivation of latent avian chlamydial infection. Vaccination has had limited effect in controlling ovine infection. Improved education of persons in occupational risk groups and the requirement for notification may encourage a more energetic approach to its control.

The disease, psittacosis, was first recognized by Ritter in 1879 (Harris and Williams 1985) who described several cases of unusual pneumonia or ‘pneumotyphus’ associated with exposure to tropical pet birds in Switzerland. Morange named the disease after the Greek word for parrot, psittakos, having established parrots to be the source of infection in a similar outbreak in Paris in 1894. The term ‘ornithosis’ was suggested for infection in non-psittacine birds. Currently the term ‘chlamydiosis’ is used to describe chlamydial infection in all avians and mammals.

The original description of chlamydial elementary bodies is attributed to Halberstaedter and von Prowazek in 1907 who observed intracytoplasmic inclusions containing large numbers of minute particles in conjunctival epithelial cells from humans and apes with trachoma. They classified these ‘Chlamydozoa’ (Greek, meaning ‘a mantle’) between bacteria and protozoa incorrectly, but they rightly inferred that these inclusions were the aetiological agents of trachoma.

The causative organism of psittacosis was described initially in 1930 independently by three researchers (Coles 1930; Levinthal 1930; Lillie 1930). Each reported minute spherical bodies within reticuloendothelial cells in infected parrots. In 1930, Bedson and co-workers described the agent as ‘an obligate intracellular parasite with bacterial affinities’ (Bedson et al. 1930)—a concept not generally accepted for another 30 years. Thereafter, the generic name Bedsoniae was used to describe the agent. In due course the agent was cultivated in fertile hens’ eggs.

In 1932, Bedson and Bland demonstrated the complex replication cycle from initial infection to the release of progeny of Chlamydia psittaci (Bedson and Bland 1932) (now Chlamydophila psittaci) (Fig. 17.1). They found two cell-type populations: the small, infectious elementary body (EB) and the larger, metabolically-active, non-infectious reticulate body (RB), subsequently recognized to be unique to all members of the family Chlamydiaceae (Fig. 17.2). It is now clear that chlamydiae are small prokaryotes that have evolved to a highly parasitic existence and do not constitute the missing link between bacteria and viruses as once thought.

The causal relationship between elementary bodies and psittacosis was demonstrated in 1932 by Bedson and co-workers. They demonstrated pathogenicity for experimental budgerigars, viable chick embryos, and mouse tissue cultures. Burnet (1935), investigating the ecology of psittacosis, showed that fledglings acquired infection from asymptomatic parent birds. Human infections were most commonly acquired via the aerosol route from inhalation of infected avian excreta or fomites. Infection in domestic mammals was first reported in 1936, following abortions in sheep (Greig 1936), although this was not confirmed as being an organism of the psittacosis group (ovine C. psittaci strains; now known as C. abortus) until 1950 (Stamp et al. 1950). The disease was named enzootic abortion of ewes (EAE), or ovine enzootic abortion (OEA).

Chlamydia psittaci has considerable pneumopathogenic potential and is known to cause human disease ranging from asymptomatic infection to severe pneumonia and death. Haematogenous spread of the organism from the respiratory tract results in a systemic illness affecting multiple organ systems. The detailed clinical and pathological observations of 18 patients in a chlamydial outbreak in Louisiana (Treuting and Olson 1944) are noteworthy.

Human infection in pregnancy (Giroud et al. 1956) with EAE agent was recognized in 1967 as a zoonoses. Although human infection with the ovine strains is relatively rare in comparison to the avian strains, the risk for pregnant women and the developing fetus is considerable (Longbottom and Coulter 2003). Infection can result in spontaneous abortion and stillbirths.

Cases of conjunctivitis and atypical pneumonia in humans in close contact with infected cats have also been reported, suggesting a probable zoonotic role for the agent responsible (now known as Chlamydophila felis) (Longbottom and Coulter 2003).

The disease, psittacosis, arises from human contact with psittacines, from which many of the early outbreaks were derived. As ownership of psittacines became fashionable, large-scale importation of South American birds into the USA and Europe occurred. This resulted in the 1929–30 pandemic with its associated high mortality and serious recognition of the disease. In 1935 chlamydiosis was also found to be prevalent in wild psittacines in Australia. Further sporadic cases of chlamydiosis occurred in the USA and Germany, incriminating domestically bred budgerigars. Non-psittacine birds have also been implicated in the transmission of this infection. Pneumonitis infections, due to chlamydiosis, in women in the Faroe Islands (Bedson 1940) in 1938, were probably contracted while preparing fulmar petrels for consumption. In the 1950s outbreaks occurred in turkey-processing plant employees in the USA (Irons et al. 1951) and later domestic ducks were implicated in outbreaks in Czechoslovakia (Strauss 1967). Poultry-associated cases were first reported in Western Europe in 1975 (in Denmark). More recently outbreaks have occurred in a veterinary teaching hospital (Heddema et al. 2006) and in poultry processing plants in the UK, especially among workers on eviscerating lines or who plucked birds (Newman et al. 1992; Gaede et al. 2008; Williams et al. 2008). Serological surveys showed that asymptomatic infections were common in persons at risk. An association between sheep contact and human abortion was noted by Giroud and co-workers, many years after the agent causing EAE was first described. Since that time a number of human C. psittaci infections (now known to be due to C. abortus) have been ascribed to contact with lambing or aborting ewes and to respiratory illness in laboratory staff, as well as workers in vaccine plants and abattoirs (Palmer and Salmon 1990; Longbottom and Coulter 2003).

Recognition of the zoonotic potential of C. psittaci resulted in adoption of various strategies in an attempt to control the spread of chlamydial infection to humans and to protect the domestic poultry industries from the velogenic, viscerotropic Newcastle disease. National embargoes on psittacine importation were first recorded in the 1930s in the USA, UK, and Federal Republic of Germany after the pandemic in 1929–30. This resulted in an increase in bird smuggling and import bans were revoked and replaced in the 1960s with import permits, health certification, quarantine measures, and prophylactic antibiotics.

In the UK, the Psittacosis or Ornithosis Order MAFF 1953 provided statutory powers to detain and isolate affected birds and to disinfect premises. In 1976, import restrictions were re-imposed through the MAFF Importation of Captive Birds Order, because of an increase in human cases. These strategies met with limited success in some countries and have encouraged psittacines to be bred domestically. However, chlamydiosis still occurs globally and is more frequently associated with poultry industries. Commercial importation of psittacines ceased in the US in 1993 with the implementation of the Wild Bird Conservation Act. However, legal importation of pet birds, interstate quarantine of psittacines and prophylaxis with chlorotetracycline remains under USDA supervision; but there is no federal control after quarantined birds are released. Some countries made chlamydiosis a statutory notifiable disease to encourage more energetic control measures.

The disease was made notifiable in 1972 in Australia and in 1978 in Norway. Avian chlamydiosis in humans is not notifiable in the UK, except in Cambridgeshire, but in 1989 it was added to the list of prescribed industrial diseases under the Social Security Act (1975) (Industrial Injuries Advisory Council 1989). More recently the International Animal Health Code and an EC Council Directive have stipulated animal health requirements covering international trade and importation.

Despite vaccines being available to control ovine chlamydiosis, EAE remains the most common infectious cause of abortion in sheep in the UK and many countries worldwide, responsible for over 40% of all diagnosed cases of abortion in the UK. Although there is no specific legislation relating to infection control, many farms participate in voluntary accredited health schemes, specifically the Sheep and Goat Health Scheme in England and Wales, and the Premium Health Scheme for Sheep in Scotland, as a way of controlling infection (Longbottom and Coulter 2003). However, these schemes are reliant on serological testing using the complement fixation test (CFT) which cross reacts with other chlamydial species infecting these animals and so is not 100% effective.

Chlamydiae are small, coccoid, obligate, intracellular Gram-negative bacteria. Their developmental cycle is unique, involving alternation between elementary bodies (EBs) and reticulate bodies (RBs). The small (0.3 μm) spore-like infectious EB (Fig. 17.2) binds and enters the eukaryotic host cell, where it differentiates into a metabolically active RB (0.5–1.6 μm) within a cytoplasmic membrane-bound vacuole. The RB undergoes division by binary fission, followed by re-differentiation back into EBs and release from the cell to complete the cycle (Fig. 17.2) (Treharne 1991). Their metabolic repertoire is distinct from that of free-living bacteria, being partially reliant on host-derived intermediates, which is consistent with an intracellular lifestyle, a small genome size (1.0–1.3 megabase pairs) and the probability that they are undergoing a process of reductive evolution (Thomson et al. 2005). Structurally, the cell wall is comprised of protein and lipopolysaccharide (LPS) but lacks peptidoglycan, although genomic sequencing of chlamydiae has shown they have the full set of genes necessary for peptidoglycan biosynthesis.

In 1999 the order of Chlamydiales was reclassified based on sequence analysis of the 16S and 23S ribosomal RNA genes (Everett et al. 1999). Analysis identified three new distinct families (Parachlamydiaceae, Simkaniaceae and Waddliaceae), in addition to the original single family member, the Chlamydiaceae. The Chlamydiaceae, which previously comprised the single genus Chlamydia, was divided into two genera, Chlamydia and Chlamydophila. The emended genus Chlamydia included the original species C. trachomatis as well as two new species, C. muridarum and C. suis, whereas the new genus Chlamydophila comprised the original species C. psittaci, C. pecorum and C. pneumoniae, and three new species, C. abortus, C. felis and C. caviae. More recent changes have been proposed reflecting the increase in diversity of chlamydial species found in the environment, including the new families Rhabdochlamydiaceae and Criblamydiaceae, and new genera of the Parachlamydiaceae (Protochlamydia), Simkaniaceae (Fritschea) and Chlamydiaceae (Clavochlamydia) (Table 17.1). Although there is little evidence on the epidemiology, prevalence or zoonotic potential of these agents, there have been reports supporting a role for Waddlia and Parachlamydia in bovine abortion, human miscarriage, and pneumonia (Borel et al. 2007; Baud et al. 2007, 2009). Rhabdochlamydia has also been associated with community-acquired pneumonia in humans (Casson et al. 2008).

Molecular research of chlamydial structure has been largely focused on the outer membrane of the elementary body (EB) because of its central role in the infection process. The main constituents of the outer membrane are proteins of molecular mass 40, 57, and 12 kDa together with lipopolysaccharide (LPS). The major outer membrane protein (MOMP), composed of the 40 kDa protein, is the dominant surface-exposed protein, comprising 60% of the total membrane protein content. MOMPs from different chlamydial species exhibit high sequence homology but still contain species- (biovar-) and serovar-specific epitopes located in surface-accessible variable domains. The protein functions as a porin and structurally is composed of three protein monomers in its native form, with each protein molecule forming a separate channel through which nutrients may pass from the host cell (Wyllie et al. 1998).

Other major protein components are the 60 kDa and 12 kDa cysteine-rich proteins. These EB-associated proteins, unlike MOMP which is also present in the RB, are developmentally regulated and probably involved in the condensation of the RB to EB late in the developmental cycle. The 60 kDa protein is thought to be located in the periplasm, where it forms disulphide bond cross-links with itself, as well as with the 12 kDa lipoprotein anchored on the periplasmic surface of the outer membrane and with MOMP forming a supramolecular structure responsible for structural rigidity of the EB outer membrane. The final major component of the membrane complex is the deeply truncated genus-specific LPS, which forms the basis of laboratory diagnosis. LPS is overproduced during replication and can be incorporated into the host cell surface.

There are many other proteins of interest present in the chlamydial outer membrane complex, including components of the type III secretion system, the 27 kDa macrophage infectivity potentiator-like protein, which is similar to an important virulence factor of Legionella pneumophila and the 57 kDa heat-shock protein which may also have an important pathogenic role. This genus-specific antigen, closely related to the groEL protein of E. coli, can elicit delayed conjunctival hypersensitivity in guinea pigs (Watkins et al. 1986) and monkeys (Schachter 1978) sensitized by prior chlamydial infection. This would explain the exacerbation of disease observed in animals immunized with whole organism based vaccines following reinfection, and is an important consideration for vaccine development strategies (Longbottom and Livingstone 2006). In addition, a group of antigens and corresponding genes have been identified as minor but immunodominant components of the protective chlamydial outer membrane complex (Longbottom et al. 1998).

Subsequently, genome sequencing of multiple chlamydial strains has revealed a large family of these polymorphic membrane proteins or pmps, while bioinformatic analysis has suggested them to be autotransporter proteins of the type V secretion system, an important virulence system of Gram-negative bacteria (Henderson and Lam 2001). These proteins may play a role in adhesion and immune evasion. Another important group of proteins that directly interact with the host are those secreted through the type III secretion system. These include the large number of candidate inclusion membrane proteins (Inc proteins), the chlamydial protease-like activity factor (CPAF antigen), CopN and the actin-recruiting phosphoprotein Tarp.

Chlamydiae cause a broad spectrum of clinically distinct diseases. It is unclear whether these diverse features are mediated by differences in pathogenetic characteristics or are due to variation of the host’s immune response. Disease mechanisms may, in part, involve direct activity on the host cell as shown by the dose-related immediate toxic response when injected into experimental animals. This toxicity, originally thought to be associated with the endotoxic LPS, now appears to be due to the genus-specific heat-shock protein (57 kDa). Circulating chlamydial LPS immune complexes have been incriminated in the pathogenesis of some chronic disease.

Chlamydophila psittaci does not exhibit host cell-type specificity and can cause productive infection in various cell types, including mononuclear phagocytes which contribute to systemic spread. It is possible that monocytes may degrade internalized immune complexes prior to acquisition by dendritic cells, which in turn are uniquely capable of eliciting primary T-cell responses (Stagg et al. 1992). In vivo, the pivotal role of dendritic cells shapes the immunological outcome, either by T-cell stimulation or by generation of nonspecific killing mechanisms. This is observed in post-chlamydial reactive arthritis where dendritic cells drive inflammatory processes.

Protective immunity to chlamydial reinfection is associated with mucosal antibody, is serovar specific and short lived. Surface-exposed outer membrane constituents of the chlamydial EBs (MOMP, Pmps and LPS), are immunoaccessible and are likely targets of the immune response. However, LPS neutralizing antibody is not stimulated. MOMP is antigenically complex and exhibits unique and common moieties conferring species serovariation. While Pmps have been shown to be minor but immunodominant components of the chlamydial outer membrane complex of chlamydiae that has been shown to protect from infection (Longbottom et al. 1998).

Chlamydiae primarily infect the mucosal epithelium and resolution of infection occurs without adverse sequelae (Schachter 1992), although severe chronic inflammation may occur. Repeated exposure to chlamydial antigens may contribute to the immuno-pathology. Evidence from experimental animal work implies that multiple episodes of infection provoke hypersensitivity responses, causing irreversible tissue damage. Application of non-infectious detergent-extracted chlamydial antigens to the conjunctivae of immune guinea-pigs results in delayed hypersensitivity (Watkins et al. 1986). The offending protein was the genus-specific heat-shock 57 kDa antigen. Clarification of the role of the host response to this protein and its invocation of autoimmunity is required.

In humans, C. psittaci infects via the respiratory tract and is transported to, and replicates in, the reticuloendothelial cells of the liver and spleen. Spread via the haematogenous route to the lungs produces EB-rich fibrinous exudates. This process accounts for the long incubation period (7–20 days). Man is an incidental, dead-end host.

Maternal infection with ovine C. psittaci results in a predilection for, and replication in, placental trophoblasts. EBs are released into intervillous spaces and infect other chorionic villi, inducing intense inflammation. Placental insufficiency and fetal anoxic death follow. Trophoblast destruction releases large amounts of thromboplastin, and possibly chlamydial toxins, into the circulation resulting in disseminated intravascular coagulation and shock.

Avian chlamydiosis is a generalized infection affecting all major organs (Meyer 1965). Oedema, haemorrhage, and extensive lymphocytic infiltration are common. Inapparent infection is more common than overt disease, which is precipitated by stress following capture, transport, re-housing or food shortage. EBs are shed during overt and inapparent infection. Subclinical intestinal infections are common both in abortion-affected and healthy ruminants, although their pathogeneticity remains unclear (Storz 1988).

Chlamydial adhesion consists of two factors acting in unison where specific binding to cell receptors and/or non-specific physical interactions occur. Adhesion is trypsin resistant, heat- and periodate oxidation-sensitive and inhibited by heparin and heparan sulphate. Non-specific attachment of chlamydial MOMP by electrostatic and hydrophobic interactions is also likely to be important.

Endocytosis is essential for chlamydial development, although conceivably more than one route is used. This may depend on the mode of presentation, the chlamydial strain or the host cell. Endocytosis is ‘parasite determined’, blocked at low temperatures (4–8°C) involving local segmental responses of the host cell membrane similar to clathrin-dependent receptor-mediated endocytosis. Intracellular lysosomal fusion with the chlamydia-containing endocytic vesicle is probably inhibited, by undefined surface properties of the EBs, and occurs prior to replication. Reduction of the disulphide bonds, which cross-link the MOMP, causes loss of EB envelope rigidity and initiates the conversion of EBs to RBs. Increase in porin activity facilitates nutrient exchanges between the developing RB and host cell. Complete differentiation into metabolically active RBs occurs within 9 or 10 hours. Macromolecular synthesis of proteins and nucleic acids utilizes host cell ATP and nucleotides. RBs divide by binary fission and by 30 hours reorganize into a new generation of EBs, stimulated probably by reduction of ATP within the endocytic vesicle. Extensive cross-linking of MOMP increases the rigidity of the outer membrane. The endocytic vacuole enlarges due to EB accumulation, followed by release into the extracelluar environment.

Non-productive and persistent infections are common in chlamydial disease but the mechanism is unknown. Chlamydiae may survive in cells for long periods in a non-replicative form or, alternatively, multiply at a low level. Tissue culture studies indicate that tryptophan concentrations are critical for persistent infection, although interferon and depletion of other cell nutrients may be involved.

Drugs inhibiting C. psittaci replication interfere with protein synthesis or cell wall synthesis (Collier and Ridgway 1984). Rifampicin has the highest antichlamydial activity, followed by tetracyclines and macrolides, e.g. erythromycin and clarithromycin. The newer quinolones, e.g. ofloxacin, also show high antichlamydial activity. Penicillins and some cephalosporins possess moderate antichlamydial activity by their action on RBs. Inhibition of binary fission and development of abnormal forms occurs and can be reversed by removal of antibiotics. Such drugs are not recommended for therapy.

EB suspensions are thermolabile, lose infectivity within hours at 35–37°C and within days at 4°C. Survival can be improved by buffering at pH 7.2 containing 0.4 M sucrose and can be maintained for months/years at −70°C or in liquid nitrogen. Freeze-drying is variably successful.

Infectivity is destroyed or greatly reduced, within 1 min at room temperature, by chemical agents at concentrations routinely used for disinfection, e.g. alcohols, iodine, and hypochlorite, although 1% phenol was less rapid. Chlamydophila psittaci is stable in dust, feathers, faeces, and products of abortion at ambient temperatures, an ecologically important factor in transmission. Infectivity has been documented in canary feed for 2 months, in poultry litter for up to 8 months, straw and hard surfaces for 2–3 weeks, and in diseased turkey carcasses for more than 1 year.

Globally, chlamydioses occur throughout the animal kingdom affecting ectothermic vertebrates, avians, mammals, and man. Common reservoirs of zoonotic avian chlamydiae include psittacine birds, pigeons, pheasants, and seabirds. Bird species of the economically important poultry industries, for example turkeys, geese, and ducks, are also natural hosts. Chlamydophila abortus infections are also of economic importance in farm animals, e.g. sheep, goats, cattle, and pigs. Close domestic association between symptomatic cats and humans provides ample opportunity for zoonotic C. felis spread.

Humans: usually 4–15 days, may be as long as 1 month. Avians: unknown in natural infection but experimental infection in turkeys produced symptoms in 5–10 days. Mammals: varies but in non-pregnant ruminants C. abortus rapidly enters a latent phase, upon becoming pregnant the organism recrudesces resulting in infection of the placenta.

The onset of disease may be insidious or rapid, with fever, headache and generalized malaise. After a few days an irritating non-productive cough develops followed by sputum production. Chest signs are often limited to râles with little evidence of consolidation, which is at variance with the radiological findings. Epistaxis and mucocutaneous manifestations frequently occur. Complications include hepato-splenomegaly, meningitis or meningoencephalitis, myocarditis, or pericarditis (Crosse 1990). During acute illness the white cell count is often normal with leucopenia developing in about 25% of cases.

Infection with C. abortus is particularly significant in pregnancy as several cases of abortion, critical puerperal sepsis, and shock, including renal failure, hepatic dysfunction, disseminated intravascular coagulation, with significant mortality, have occurred in women in contact with sheep (Bloodworth et al. 1987). Uncommonly, neural involvement, flu-like illness, respiratory symptoms, and conjunctivitis have been reported in children and adults following sheep exposure.

Chlamydiosis is often asymptomatic but a generalized infection affecting all major organs may occur. Loss of condition with yellow-green gelatinous diarrhoea, anorexia, respiratory distress and nasal discharge occurs. Conjunctivitis may be the only symptom. Ducks typically have serous or purulent nasal and ocular discharges, whereby feathers around the eyes and nostrils become encrusted.

Feline pneumonitis manifests febrile, depressive, and anorexic illness with mucopurulent discharge from eyes and nostrils (Storz 1988). Recovery after 2–4 weeks frequently results in a subclinical carrier state which may relapse. In very young or elderly cats pneumonitis may be fatal.

In ruminants chlamydiae can cause respiratory, intestinal, placental, and arthropathogenic manifestations. Intestinal infections cause diarrhoea in young animals, initiate pathology in other parts of the body, thus representing important transmission mechanisms. Many ruminants harbour chlamydiae in the alimentary tract without clinical symptoms. Observations in dogs and pigs are similar, and intestinal chlamydial infection in mammals is comparable to acute/persistent avian infection.

Placental and fetal infections of ruminants, with ensuing abortion, are economically important causes of reproductive failure. In experimentally infected animals, clinical observations show a clear sequence of events. Fever and marked leucopenia can occur 1–2 days after inoculation and continue for 3–5 days. The placental junction is breached and thereafter events in utero proceed independently of those in the dam. Abortion occurs late in gestation, usually in the last trimester at around 2–3 weeks preterm, but may be as early as day 100 of gestation. Ovine enzootic abortion spreads by contact at lambing, and infection acquired at this time remains latent until subsequent pregnancy when recrudescence results in abortion. In affected flocks, loss can be up to 30% during an abortion storm, thereafter the annual incidence falls to 5–10% (Aitken and Longbottom 2007). Infertility problems may occur after chlamydial abortion.

Bovine mastitis (Storz 1988), caused by naturally occurring infection in milking herds, causes severe reduction, or transitory cessation, of milk production. Chlamydiae can be recovered from the milk but its epidemiological significance is unknown.

Polyarthritis of lambs (stiff lamb disease) occurs in epizootic proportions in the USA and is economically important. The age of affected lambs ranges from 4 days to several weeks. Varying degrees of mobility, anorexia, and conjunctivitis are observed and, with disease progression, lambs are reluctant to bear weight on their limbs. In Germany, chlamydiae have been isolated from synovial specimens of pigs with chronic, non-purulent synovitis.

Infection may be generalized but major changes occur in the lungs which appear congested. Typically, areas of normal alveoli containing air, interspersed with areas of affected alveoli with an EB-rich cellular fibrinous or serous exudate are seen. Interstitial infiltration and mucosal oedema is rare. Necrotic areas and Kupffer cell vacuolation occur in the liver, and the spleen is typically congested. Cardiac muscle may be oedematous with interstitial infiltration and vegetations may occur on heart valves. Occasionally, adrenals may be haemorrhagic. When the central nervous system is affected, congestion and oedema of the brain and cord is observed, sometimes with chromatolysis of nerve cells or intracytoplasmic inclusions in meningeal cells.

Pathological features of placentitis caused by chlamydiae from ovine infection reveal a focal acute microinfarction due to patchy infiltration of inflammatory cells and fibrin deposits in the intervillous spaces (Wong et al. 1985). Destruction and desquamation of trophoblasts in the absence of chorio-amnionitis occurs with an associated deciduitis.

Lung and air sac congestion results from focal inflammatory cell infiltration, oedema, and haemorrhage. Histiocytic and lymphatic cells accumulate in interalveolar septa and propria of the large bronchioli. Other mucous membranes are infiltrated diffusely or focally with lymphocytes, mononuclear cells, and heterophils. Similar changes occur in other major organs. Eosinophilic necrosis occurs in the liver.

Intestinal infection can be cytocidal, resulting in atrophied, irregularly shaped, vesiculated microvilli, and ultimately cell degeneration. Oedema and cellular infiltration of the lamina propria occurs. Diarrhoea results from loss of enterocyte function.

Placental and fetal infections are complex pathological phenomena. The major pathology is localized placentitis with necrotic cotyledons and thickened, opaque periplacentomes. Margins of placental lesions consist of zones of hyperaemia and haemorrhage. Cytoplasmic inclusions are present in chorionic cells of the intercotyledonary region and endometrial cells. Exudate is present in the inter- and peri-placentome and the chorio-allantois is oedematous.

Fetal infection occurs through haematogenous spread from the placenta to the fetal circulation. Oral, conjunctival, and respiratory lesions are reported, but fetal death is associated with terminal anoxia due to placental insufficiency.

Diagnosis is often delayed or mixed and many patients recover without specific therapy. Severely ill patients require supportive therapy, fluid level maintenance, oxygen therapy, and measures to combat shock. Tetracyclines and macrolides, particularly erthyromycin and clarithromycin, are the drugs of choice, although some quinolones, ofloxacin and ciprofloxacin, are effective. Tetracyclines have better bioavailability in the central nervous system. They are administered either orally or parenterally, dependent on clinical severity. Early therapeutic intervention results in excellent response and is particularly important in pregnancy, but inadequate regimens may lead to relapse.

Chemotherapy is identical for treatment and prophylaxis (Grimes 1985). Treatment can be affected by feed-administered chlorotetracycline or by parenteral administration. Antibiotic concentration in feed varies according to species, e.g. 2,500–5,000 p.p.m. for parrots and 500 p.p.m. for budgerigars. The prescribed time for treatment is 45 days for oral administration in parrots and 30 days for budgerigars. For injection, 75 mg/kg body weight every 5 days is recommended; however, repeated injections may result in muscle damage. Blood concentrations greater than 1 μg/ml are considered adequate. Vibramycin-calcium syrup is effective and is the treatment of choice in Europe. The efficacy of some quinolones for treating avian psittacosis is being evaluated.

Injection of a long-acting oxytetracycline preparation will maintain ovine pregnancy until nearer the expected parturition date (Aitken et al. 1990). It is recommended as a ‘one-off’ procedure to minimize the development of resistance. Tetracyclines do not eliminate placental infectivity, and it is advisable to institute treatment of the whole flock. Ewes not close to lambing may require a further dose of tetracycline after 2 weeks. Despite treatment, some ewes still abort and remain potential sources of infection for other naïve ewes.

Mortality rates from avian sources were as high as 40% in the pre-antibiotic era. In the Louisiana outbreak, 8 of 19 cases died and during the 1929 pandemic 20–30% of infected cases died. Nowadays fatalities are rare, accounting for less than 5% of affected cases, but significant morbidity is common. Ovine chlamydial infection in pregnant women may result in an 80% fatality rate. Fetal or neonatal death is common.

Infection is usually inapparent with overt disease being precipitated by stress, e.g. overcrowding, inadequate nutrition, or transportation. Epizootics accompanied by high mortality rates occur among flocks of birds. Sporadic deaths occur in older birds that escaped infection as nestlings. Unchecked, mortality in domestic poultry flocks may reach 30%.

Ovine and bovine adult infection is usually asymptomatic and rarely results in death. Infection during pregnancy can result in up to 30% of aborting ewes in flocks.

There are two main approaches to diagnosing chlamydial infections in mammals and birds. One involves direct detection of the organism in swab and tissue samples by cytochemical staining of smears, histochemical detection in tissue sections, immunofluorescent detection of antigen, by the detection of nucleic acid by PCR or DNA microarray, or following isolation in tissue culture. The other involves serological methods to identify anti-chlamydial antibodies in blood samples (Sachse et al. 2009). With minor modification, many of the laboratory tests used for chlamydial diagnosis are applicable in all hosts. The merits of the commonly used tests are outlined in Tables 17.2 and 17.3.

The mainstay of diagnosis is the complement fixation (CF) test, but immunofluorescence tests are also used. Antigen and genome detection have been evaluated and appear to be both reliable and rapid, although not widely used (Sillis et al. 1992). Culture is not routinely attempted. Recently a new MOMP based nested PCR/enzyme immunoassay (EIA) has been developed that detects positive cases of human psittacosis (Vanrompay et al. 2007).

Available diagnostic tests are not reliable enough to allow screening or culling programmes to be 100% effective. The CF test and microimmunofluorescence test are currently used for routine diagnosis, as well as a latex agglutination test for detecting IgM (Andersen 2008). Veterinarians only accept a positive culture result as a basis for complying with notification regulations and there is a great need for a test which is accurate, rapid and economical (Spencer 1989). Immunological and genomic techniques on ante-mortem specimens are becoming popular, whereas conventional histopathology is declining. Most recently, a C. psittaci genotyping method based on DNA microarray technology has been developed (Sachse et al. 2008).

Laboratory diagnosis is generally performed by CF test on paired blood samples collected at the time of abortion and then at least three weeks later. Diagnosis can also be made by detection of chlamydiae in placentae, aborted fetal tissue or on vaginal swabs using histochemical stains such as Giemsa, Gimenez or modified Ziehl-Nielsen, or using immunological methods (Longbottom 2008). Other more sensitive and specific molecular methods of detection can be carried out in specialist laboratories.

Confirmation of human infection may only be sought in moderate to severe cases, resulting in many unrecognized mild and asymptomatic infections. In many countries, human disease is not notifiable whereas in Norway and Sweden notification has occurred for nearly 40 years. In the USA, CDC report fewer than 50 confirmed cases a year. In Britain fewer than 40 cases were reported annually before 1966 but this has now increased to more than 400 cases per year, with periodic peaks. Bird exposure was reported in only 20% of cases. In Japan, where pet bird keeping is very popular, the annual incidence is estimated at 250–300 cases. Most countries detected an infection peak in 1981 linked to spread by migratory geese. However, the epidemiology of chlamydial respiratory infection needs re-evaluation following the recognition of C. pneumoniae. The CF test remains the most routinely used laboratory investigation for human chlamydiosis, although it cannot differentiate between the chlamydial species. Epidemics of C. pneumoniae occur and the contribution of this species to the reported human cases requires definition.

The incidence data of avian and ovine infections are subject to similar distortions of underdiagnosis and under-reporting. Differences in ecology, surveillance, reporting procedures and market value of animals over time affect this data. In the UK 84–89% of reported ovine cases occur during January to March each year, hence the increased risk to pregnant women at this time.

Ovine C. psittaci is enzootic and large numbers of EBs are shed in fetal fluids and placentae. Ewes do not abort twice as a result of chlamydial infection, but chlamydial excretion can occur in the faeces and in fetal and placental products in subsequent pregnancies. Hence healthy sheep are a source of infection. Human infection with ovine C. psittaci is sporadic and no outbreaks have occurred. Bovine abortion is epizootic and poses minimal risk for humans as infection is relatively chronic with low-level excretion in aborted material. Serological evidence that stockmen and veterinarians face significant exposure to ruminant chlamydiae is available, although this may require further evaluation using more specific serology.

Seasonality has not been observed in most countries, but peaks are usual in July and August in Czechoslovakia and early in the year in Japan. This may provide information on the source of some infections. In the duck industry human infection relates to ducklings hatching in the spring and reaching a summer peak when birds are processed. Japanese observations may relate to continuous contact with pet birds indoors during the winter.

The first European chlamydiosis outbreak was related to a sick bird in a Swiss household, resulting in 50% mortality. Similar outbreaks were observed worldwide until the 1930 pandemic involving at least 1,000 cases with 20% mortality. The pandemic was due to large-scale importation of infected Amazon parrots to satisfy fashionable demand. Since then, over 70 types of psittacine birds have been found to harbour chlamydiae. In Britain infection among domestically bred budgerigars is uncommon, although a recent Slovenian outbreak did implicate budgerigars (Dovč et al. 2007).

Infection is not confined to psittacines, as exemplified by the Louisiana outbreak where egrets were implicated (Treuting and Olson 1944). Eight of 19 affected cases died, including nurses involved in caring for primary cases. Many wild game and garden birds are also known to be infected and remain an important source of infection to humans and other birds. Periodically, epizootics of infection occur in aviaries, resulting in a high avian mortality.

Feral pigeons in towns and cities worldwide are commonly infected. However, zoonotic spread of infection from asymptomatic pigeons appears to be low. Handling sick pigeons and killing or dressing wild pigeons is a major risk. Sporadic infections acquired from racing pigeons are frequent. Stress of flying long distances, housing in insanitary lofts, competing for food, and transportation are all factors which may trigger avian disease.

Since the introduction of intensive poultry rearing, several major outbreaks of chlamydiosis affecting man have been reported, mainly associated with ducks and turkeys. These were evident in Czechoslovakia when the formation of large poultry plants and increased production was associated with an increase in pneumonia. Between 1949 and 1960, 1,072 cases were diagnosed, with a further 500 during the next 5 years. The mortality rate in humans was 0.7%.

An outbreak of psittacosis occurred in the Minnesota turkey industry in 1986 (Hedberg et al. 1989). The risk of acquiring infection varied by work area, with employees in the evisceration area and those in the live hang area many more times more likely to acquire infection than employees in other areas.

Ducks have been associated with a number of outbreaks of human chlamydiosis (Table 17.4). The first outbreak in the British duck industry occurred in 1980 when a cluster of cases led to an epidemiological survey (Andrews et al. 1981). Seventy-two per cent of plant workers were seropositive compared with 37% of the duck farm workers. Attack rates were highest in workers on the eviscerating line. In another British duck outbreak in 1985 (Newman et al. 1992) new employees were three times more likely to become infected cases than established employees. An explosive outbreak of duck-associated C. psittaci infection among British veterinarians in 1980 (Palmer et al. 1981) was associated with a visit to one of the duck plants involved in the previous outbreak. The highest attack rates were associated with contact with feathers. No illness was observed in workers at the plant. The exposure of a susceptible group to a heavily contaminated environment may account for these findings. Similar findings were reported in the UK (Williams et al. 2008).

Transmission to man occurs by the respiratory route from healthy and sick birds through faecal droppings and conjunctival/nasal secretions. Organisms survive well on feathers or in dust and cases have occurred in workers processing duck feathers for pillows. Ruminants and cats transmit C. psittaci to man via aerosols of infected body fluids. Outbreaks in poultry plant workers have implicated parenteral transmission via cuts and abrasions (Hedberg et al. 1989), although this is contentious.

In birds, exposure of nestlings to infection is the major mode of transmission. Vertical transmission through the egg or from parent to young through feeding by regurgitation occurs in some species. Contamination of the nesting site with infected exudates may be important in gulls and shore birds. Wild birds are important in transmission to commercial poultry. In ruminants (Stamp et al. 1950), infection is spread by contact at lambing and infection remains latent until the following pregnancy when recrudescence results in abortion which contaminates pastures. The theory that birds feeding on this material may act as a vector to other flocks remains to be proved.

Lice and mites have been shown to carry chlamydiae but their role in the epidemiology of psittacosis is unknown. Survival of the agent of epizootic bovine abortion on the surface of ectoparasites has been demonstrated.

Regulations aimed at preventing importation of Newcastle disease virus into countries to protect the domestic poultry flocks, also prevent the importation of avian chlamydiae. Currently there are no effective vaccines available.

After the 1930 pandemic, numerous countries instituted a complete import ban of psittacine birds. However, due to inadequately controlled prohibitions, illegal smuggling became rife, resulting in the introduction in many countries of import permits with quarantine. The USA no longer imposes import restrictions as chlamydiosis is not felt to be a serious public health threat (Grimes 1985). The restriction on import permits to only a few birds per permit or to a small number of licensed premises may be more effective.

The International Animal Health Code (Office International des Epizootics) and the European Union Council Directive EC318/2007 lays down specific trade and import conditions. Psittacines must have appropriate health certification and birds must originate from one of the approved registered breeding establishments complying with appropriate transit legislation. In the US, state regulatory agencies may impose quarantine on interstate movement of diseased poultry. The US National Association of State Public Health Veterinarians published a compendium of measures to control chlamydioses in humans and pet birds in 2009.

Recrudescence frequently occurs in carrier birds due to transit stress, and overt symptoms or death may occur early in quarantine, thus eliminating infective birds. Not all carriers will be identified in the quarantine period, but healthy carriers constitute less of a human or avian health hazard. Quarantine periods vary in different countries, e.g. 35 days in the UK and Europe. In the US uninterrupted treatment of psittacines with chlortetracycline for 45 days, but only 30 days for budgerigars, is recommended by the National Association of State Public Health Veterinarians (2009); a measure designed mainly to protect the quarantine station employees. Ideally, following treatment, quarantined birds should be tested for chlamydiosis. More stringent measures apply in Germany (Gerbermann 1989).

Preventative strategies in poultry plants include the use of masks, hermetic domes over plucking machines, and installation of good ventilation to reduce spread by inhalation. Defeathering carcasses previously immersed in scalding water and heat processing of feathers/down have been used. The most effective measures focus on identifying and treating infected flocks before processing, while remembering that apparently healthy birds are infectious.

Education of exposed occupational groups is important to raise awareness and to improve husbandry. Pregnant women should avoid sheep exposure and laboratory personnel should apply good laboratory practice with strict containment measures.

Preventing domestic birds acquiring infection is difficult because of the high carriage rate in wild birds. However, poultry or pet birds kept indoors can be given prolonged antibiotic therapy. In sheep, transmission to other ewes can be reduced by isolating aborted ewes, destroying placentae, and disinfecting the area. Prophylactic tetracycline treatment of other ewes should be considered. Protective vaccines are available but they tend to give only short-lived, modest protection against disease. Formalized vaccines are of value, but the live, attenuated, vaccines have better efficacy against disease, although they do not prevent infection or shedding.