Introduction

Chlamydia are the only animal pathogenic members of the PVC group in the phylum Chlamydiota. Coxiella burnetii is included with most gram-negative bacteria in Gammaproteobacteria and is related to Legionella, a bacterium causing pneumonia in immune-compromised people. Both are obligate intracellular pathogens with biphasic lifecycles. They produce an environment resistant non-replicating elementary body/small cell variant (spore-like) and an environment susceptible, intracellular replicating reticulate body/large cell variant. Most members have a wide host range, including people (ZOONOSIS). Transmission of these parasites are usually through the oral, respiratory and venereal routes. They cause multisystemic infections in animals. Both can result in persistent infections in apparently healthy hosts.

Life Cycle of the obligate intracellular bacteria Chlamydia and Coxiella burnetti

Chlamydia life cycle

Chlamydia species being members of the PVC group, are gram-negative, obligate intracellular pathogens. Unlike viruses, Chlamydia synthesise genetic material and protein.  Since they cannot manufacture their own ATP, they are dependent on the host cells for all energy dependent processes.

They have a biphasic life cycle: the smaller dense, non-replicating forms that are the infective particles known as elementary bodies and will after phagocytosis block phagosome-lysosome fusion and move to close to the Golgi apparatus.  They then re-organise into a larger less dense form known as reticulate bodies and divide by binary fission and are metabolically active. The cycle is usually completed within 24 to 48 hours. Tryptophan acts as a promoter of cell division. Thus, the bacteria become persistent when the tryptophan is, for example, broken down by Interferon-gamma produced by T-cells.

Coxiella burnetii life cycle

The intracellular parasite, Coxiella burnetii,  uses host cell NAD. Thus it can be cultured in cell-free reduced oxygen in complex media containing NAD that is moderately acidic.

Monocytes and macrophages phagocytose the infectious small-cell variant (SCV) by an actin-dependent process. It does this without stimulating inflammation. The acidic products of the phagolysosome stimulate conversion to the metabolically active large cell variant (LCV). The LCV divides by binary fission, expanding the cell. The bacteria persist in these cells by preventing the cell from dying (preventing apoptosis). The bacteria will eventually produce SCVs and allow the cell to die releasing the SCVs to infect other cells.

Diseases caused by Chlamydia and Coxiella burnetii

A number of multisystemic diseases are caused by Chlamydia species with the most important being C. psittaci. Most animals tend to be subclinically affected by both Chlamydia species and Coxiella burnetti and these bacteria are known to affect people (ZOONOSIS). Occasionally stressed and non-immune animals will exhibit clinical disease. The Table below is a list of the better known Chlamydia species and C. burnetti and the diseases they cause.

Chlamydiosis

Transmission

Chlamydia are associated with epithelial cells and are usually shed in faeces, respiratory secretions, venereally and in utero. Most of the animal chlamydiae can infect people (zoonosis) and other animal species.

General Pathogenesis

Chlamydia elementary bodies can survive for up to 30 days in the environment and are very light. Thus, they are often found in faecal dust and feather dander and are easily inhaled. They also enter the host sexually, by ingestion and through direct contact. Localised infections occur in the eye causing conjunctivitis. The elementary body then adheres to epithelial cells where they are internalised. In the phagosome, they reorganise to the reticulate body and divide. During the early stage of cell infection, Chlamydia will directly inhibit natural cell apoptosis to allow it to replicate. During the later stages of infection when a mature vesicle is present, it will then encourage cell apoptosis and also rupture the cell. Moderate amounts of gamma interferon will stop the apoptosis by reducing tryptophan in the cell allowing the bacteria to persist in the host cell. Although cell rupture is responsible for some of the cell damage, most is associated with cytokine induced inflammation and a type IV hypersensitivity response. Rupture of the cells with the release of elementary bodies allows the organism access to new cells and also to circulating antibodies.

These bacteria can also enter phagocytes and be transported throughout the body localising in for example the placenta, brain, lungs and joints.

Chlamydia pecorum

Chlamydia pecorum affects a wide host range. It is however, in the koala, where the disease takes on its most severe form.  There is a concern that chlamydiosis is contributing to the decline of koala population in especially Queensland. Infected koalas transmit it sexually, by contact and by the joeys eating of “pap” a nutritious faecal fluid from the mother’s cloaca that provides the joeys with the microorganisms necessary to digest eucalyptus leaves. In utero transmission may also occur. In koalas, C. pecorum causes conjunctivitis, infertility, renal disease and cystitis. Up to 40% of infected female koalas can become permanently infertile. Koala retrovirus type B, a gamma retrovirus is considered to predispose koalas to chlamydiosis. Note that Koala retrovirus A has incorporated itself into the genome of almost all koalas in Queensland. It does not seem to play a role in predisposing koalas to chlamydiosis.  Koalas can also be infected with Chlamydia pneumoniae, however, the disease is usually mild.

In other animals, C. pecorum has been associated with abortion, conjunctivitis, encephalomyelitis, enteritis, pneumonia and polyarthritis in other animals.

Control of this endemic disease in koalas is difficult. An experimental recombinant vaccine has been developed that decreases Chlamydia shedding and disease, but not infection. Other control measures include antibiotic treatment of diseased animals, desexing of animals with genital infections and euthanasia of diseased animals.

Psittacosis or Ornithosis

Chlamydia psittaci has been isolated from at least 465 avian species.  The prevalence of chlamydiosis in water birds such as ducks is also high. Bacteria are spread by close contact with the inhalation, or less commonly, the ingestion of contaminated faecal dust or respiratory secretions. Birds are often infected in the nest by the close contact and crop feeding by the mother. Percutaneous infections either via skin injury or by biting arthropods are rare. The infection is usually clinically silent in most birds. However, when stressed birds, especially parrots, can develop a multisystemic disease with the liver being the primary target organ. Affected birds have a fever, ruffled feathers, green diarrhoea and closed eyes. Some will develop respiratory signs.

Conjunctivitis in cats

Chlamydia felis is a bacterium endemic in domesticated cats worldwide, primarily causing conjunctivitis, rhinitis and respiratory problems. Another cause of bacterial conjunctivitis in cats is Mycoplasmopsis felis. Viral causes of “snuffles”, namely feline herpesvirus type 1 and feline calicivirus, can also cause conjunctivitis. Zoonotic of humans with C. felis is rare. Attenuated FP Baker 905 strains are used as live vaccines for cats. They are non-core vaccines.

Enzootic abortion in sheep

Chlamydia abortus is the cause of enzootic abortion in sheep and infertility in other animals including humans and cattle. The disease is absent in Australia and is a Notifiable exotic disease. Uterine secretions are a rich source of this bacterium.  Oral and sometimes venereal transmission occurs where the bacteria will invade the epithelial cells of the placenta causing a placentitis. Late term abortions, stillbirths and weak lambs are born, some with evidence of a multisystemic disease. C. abortus infection generally remains inapparent until an animal aborts late in gestation or gives birth to a weak or dead foetus. Since it can circulate throughout the body, it can also be found in the udder, where it causes a subclinical mastitis. Human infections are rare and generally mild with people developing influenza-like symptoms and a dry cough. Pregnant women can abort. A commercial vaccine is available in Great Britain.

Q-fever

Coxiella burnetii infections

Q-fever is a highly contagious disease of animals and humans caused by Coxiella burnetii and is characterised in humans by fever, muscle pain and malaise and in animals by abortion. It is a PC3 bacterium. Query Fever (“Q” fever) was first recognised in abattoir workers in Brisbane in 1937 by Edward Holbrook Derrick. With the exception of New Zealand, it has a global distribution. Coxiella burnetii, a member of the gamma subdivision of the Proteobacteriaceae (related to Legionella), is an obligate intracellular gram-negative bacterium.

Coxiella burnetii is a zoonotic agent and is considered a Category B biowarfare agent as it is highly contagious: as little as 1 infective particle (ID50 – infectious dose needed to infect 50% of individuals) is required to cause disease in people.  It is also very stable in aerosols at different temperatures and can survive on surfaces for up to 60 days.

Coxiella burnetii is found in most animal species with most infections being sub-clinical. In Australia, the agent is usually maintained in wild animals such as kangaroos, bandicoots and rodents where it can be transmitted to domestic animals by infected ectoparasites, such as ticks, or by faecal dust.  In infected (carrier) animals, the bacteria are shed in the faeces, urine, milk, blood, foetal material and uterine secretions. The placenta is considered especially infectious as it contains billions of bacteria. The disease is highly contagious in domestic ruminants, with a prevalence rate of up to 80% in infected herds.

Small cell variants which are highly resistant to heat, desiccation and acids being small are mostly inhaled from faecal dust or aborted material. Although the lungs are the most common initial site of infection the bacteria are transported throughout the body. They are present in high numbers in the reticuloendothelial system (i.e. bone marrow, lymph nodes).  Inflammation tends to be non-necrotising with the so-called “doughnut” granulomata being described. C. burnetti is also able to alter its antigenicity by varying the composition of the O-polysaccharide chain length of the LPS in its outer membrane from the full length (smooth) to a truncated length (rough). Smooth form variants are more virulent than rough form variants.

Of the domestic ruminants, goats are most likely to exhibit reproductive disease i.e. infertility, abortions, still births, weak offspring and retained placentae. These clinical signs are predominantly attributable to placentitis.

Coxiella cheraxi, isolated from Redclaw, first described by Tan and Owens (2000), can cause up to 80% mortality in these animals.

Q-fever in people

It has been estimated by the WHO that if 50kg of C. burnettii were aerolised in an urban area with a population of 500 000, 125 000 will have acute illness, 9 will have chronic disease and 150 will die.

Domestic ruminants (goats, sheep and cattle) are considered to be the primary reservoirs for human infections. In humans, the disease is usually occupational with farm workers, veterinary personnel, abattoir workers, tanners and laboratory workers constituting the greatest risk. The bacterium can survive for up to 8 months in wool. However, consumers of raw milk products and those living in areas with high livestock density where they breathe in faecally-laden dust may also be at risk. In Europe, the effective range of spread is less than 5 km. Due to climatic conditions, this distance will be a lot less in Australia, about 400m. (A recent outbreak of Q-fever in people in Germany was sourced to infected sheep foetal cells that were used as an antiaging cell therapy. In a recent survey 60% of workers in a Geelong cattle sales years had antibodies to C. burnettii, in 2023 there were 86 notifications of Q-fever in people in the Darling Downs and SW Queensland, There have been 89 notifications of Q-fever in Queensland during Jan-Feb 2024 – a 90% increase compared to last year the same time).

The disease is notifiable in people in most States of Australia. Affected people show a flu-like illness with high fever and “sweats”, 7 to 40 days after exposure. The fever lasts for 7-14 days when most patients will recover. A small number of patients may progress to interstitial pneumonia, granulomatous hepatitis or nervous disease. Rarely, pregnant women may miscarry or go into early labour. Up to 50% of infected people never show any signs of illness. 2% of infected persons can develop endocarditis or myocarditis which may be fatal in 10% of cases. Immunosuppressed, and those with concurrent valvular disease are at high risk of endocarditis.  A long-term effect in 20% of patients is the so-called “chronic fatigue syndrome” which can last between 10 to 15 years.

The diagnosis is made by qPCR on the serum in the acute phase of the disease and shows a 4-fold rise of C. burnettii specific IgM in the chronic phase. A serum IgG antibody titre of 1:80 is considered diagnostic in patients suffering from endocarditis. Treatment is by the use of intracellular antibiotics such as doxycycline, fluoroquinolones, chloramphenicol and hydroxycholorquine. Treatment is most effective in the first few days of a fever (early acute disease). Chronically affected persons may have to be treated for up to 4 years.

Clinical manifestations in people can prevented by the use of a formalin-inactivated bacterin intradermal vaccine – Q-vax. Pre-vaccine testing of individuals to detect an immune response (antibody or intradermal testing) is necessary to avoid adverse effects of the vaccine. Note the antibodies to infection with C. burnettii are very long-lived, sometimes life-long. As the vaccine is manufactured in embryonating hen’s eggs, caution is taken when vaccinating persons with a known hypersensitivity to egg proteins. Due to its high expense and poor availability, the vaccine is only used for high risk groups i.e. veterinarians, abattoir workers, graziers etc. To protect the general public: animals in petting zoos should be tested free of the agent; animals in their last trimester of gestation should not be transported or displayed; unpasteurised (high temperature) milk should not be drunk; and manure from infected farms should not be used in gardens. Immune-suppressed individuals and pregnant women should avoid working with pregnant or animals recently given birth.  Laboratory workers can only work on this agent in PC3 facilities.

Diagnosis and control of Chlamydiosis and Q-fever

Laboratory Diagnosis

The samples collected are dependent on clinical signs but may include conjunctival swabs, respiratory, vaginal and placental epithelial scrapings. Laboratory diagnosis is made by one or more of the following:

1. Characteristic cytoplasmic inclusion bodies of infected cells in cytological and histological specimens using a modified acid-fast stain. Both Chlamydia and Coxiella stain red (acid-fast) with this stain.  Brucella also stains acid-fast with themodified acid-fast stain.
2. RealTimePCR on genus or species specific chromosomal DNA – now the test of choice
3. Antibody tests on clotted blood using the ELISA. In many cases, an acute and convalescent phase sera are required to demonstrate a 4-fold rise in antibody titer. In humans, in the case of Q-fever diagnosis, antibodies to Phase II antigen (lipopolysaccharide type) is indicative of acute disease, whereas detection of predominantly Phase 1 proteins is indicative of chronic disease.
4. (Isolation of the organism from infected tissue.  The tissue is inoculated into the yolk sac of seven-day chick embryos for both Chlamydia and Coxiella or in McCoy human cells for Chlamydia. Coxiella can also be cultured in a complex media containing NAD. Note that nowadays these bacteria are only cultured for research purposes or when vaccines are manufactured).

Treatment and Control

1. Animals infected with Coxiella burnetii are generally not treated as they rarely show clinical signs and goats will only abort once and remain fertile. Tetracyclines, especially doxycycline, are the preferred therapeutic agents in humans and animals. Macrolides are effective, but take longer to eliminate the bacteria, making treatment with them more expensive.
2. Chlamydia can survive for up to 30 days and Coxiella 60 days in the environment; therefore it is important the animal pens or cages used by infected animals are depopulated and thoroughly cleaned and disinfected prior to putting in new animals.
3. All milk for human consumption should be pasteurised. Coxiella burnetii being heat-resistant, is the target bacteria for pasteurisation.
4. Attenuated FP Baker 905 strains are used as live vaccines against Chlamydia felis in cats. They are non-core vaccines.(C. abortus bacterins are available in some countries and are effective in reducing abortions. In parts of Europe, a modified live vaccine is available that is administered once to ewes just before their first breeding season). There is an inactivated vaccine that has been used to prevent Coxiella burnetii abortions in goats in Europe. It is not registered in Australia.
5. People working with livestock should be vaccinated against Coxiella burnetii.

END OF CHAPTER