The zoonotic potential of *Map* has been debated for almost a century because of similarities between Johne´s disease in cattle and Crohn´s disease in humans. A quarter century later since *Map* was first proposed as an etiologic agent in Crohn´s disease based on the isolation

gastroesophageal reflux disease [160,161].

bisexual men, when compared to other HIV risk categories.

Shin et al. designed a five-target multiplex PCR to discriminate MAC organisms isolated. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all *Myco‐ bacterium species*, a chromosomal target called DT1 that is unique to *Maa* serotypes 2 and 3, to *M. avium* subsp. *silvaticum*, and to *M. intracellulare*, and three insertion sequences, IS*900*, IS*901*, and IS*1311*. The results for the pattern of amplification allowed to determine whether isolates were mycobacteria, or members of MAC, and to classify them into one of the three major MAC subspecies, *Map*, *Maa*, *Mah* [18].

#### **5. Public health concerns**

Zoonotic aspects of mycobacteria transmitted by the environment and wildlife highlights a major health problem. MAC causes a variety of disorders including tuberculosis-like diseas‐ es in animals and in human immunocompetent or immunocompromised patients. Suscepti‐ bility to mycobacterial infections depends of risk factors since they are ubiquous in the soil and water [150]. Human exposure to mycobacterium present in wildlife and in nature can occur by a variety of routes. Humans are continuously exposed at a low level (50 to 5000 bacilli per day). Contact with water, municipal or natural are also important routes for my‐ cobacteria infection. Birds are major excretors of the agent in their faeces and the bacteria can persist in the soil and in water for long [1].

Healthy humans have a low susceptibility to MAC infection and only a very small percent‐ age of mycobacteria progress trough to infection, but in immunocompromised individuals infected with HIV or leukaemia patients, treated with steroid therapy, chemotherapy or oth‐ er immunosuppressive medication, should be carefully considered regarding their possibili‐ ty to come in contact with birds with mycobacterial infection [1,151]. Prior to the introduction of highly active antiretroviral treatment more than 40% of patients developed *M. avium* complex bacteriemia two years after the AIDS diagnosis [152] and a disseminated MAC infection was found in as much as 50% of autopsied AIDS patients [153,154]. This has predominantly been attributed to the impairment of the adaptative part of the immune sys‐ tems in HIV-1 infected individuals due to the loss of CD4+ T cells, as the susceptibility to opportunistic infections including *M. avium* infection is correlated with a decline in this cell type [155]. MAC usually produces clinical disease only when CD4+ are very low (< 50 cells/ ml), which is seen in 4 to 5 per cent of HIV infected patients [88]. A recent study showed that exposure of dendritic cells to HIV-1 promotes or facilitates the intracellular growth of *M. avium* [153]. Signs and symptoms associated with MAC disease in AIDS cases are persistent high grade fever, high sweats, anaemia and weight loss in addition to nonspecific symptoms of malaise, anorexia, diarrhea, myalgia and occasional painful adenophaty [88]. Epidemiolo‐ gy of MAC complex in patients without HIV infection remains somewhat difficult to deter‐ mine since the disease is relatively uncommon, it is not a reportable health event and environmental exposure varies greatly according to the geographic region [156]. In an epide‐ miological survey in USA from 2000 to 2003 performed in [156] in patients without HIV in‐ fection the rate of positive non-tuberculous cultures was 17.7 per 100,000. Surveys conducted in Europe estimated the rate of disease anywhere in the human body to be 0.8-3.1 per 100,000 [157,158]. In a recent study, Bodle et al. estimated the incidence of non-tubercu‐ lous mycobacteria in the respiratory tract disease in 2.0 per 100,000 and the disease in any‐ where in the human body in 2.7 per 100,000 [156]. Another recently published study showed that the patient´s lung disease was likely acquired by inhalation of aerosols while showering [159]. *M. avium* may reach the lungs by aspiration because a considerable percentage of pa‐ tients with non-tuberculous mycobacteria (NMT) disease have been found to experience gastroesophageal reflux disease [160,161].

agnosis [108]. They have developed a multiplex PCR based on IS*900*, IS*901*, IS*1245* and the *dnaJ* gene. This method allows the detection of *M. a. paratuberculosis*, *M. a. hominissuis* and *M. a. avium*/*M. a. silvaticum* in one PCR reaction (PCR multiplex) and theoretically enables the detection of mixed infections of *M. a. paratuberculosis* and *M. a. avium* or M*. a. paratuberculosis* and *M. a. hominissuis*. The sensitivity of this multiplex PCR is 103 CFU for each bacterial strain in one PCR reaction, which also enabled the use of this test directly for DNA isolated

Shin et al. designed a five-target multiplex PCR to discriminate MAC organisms isolated. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all *Myco‐ bacterium species*, a chromosomal target called DT1 that is unique to *Maa* serotypes 2 and 3, to *M. avium* subsp. *silvaticum*, and to *M. intracellulare*, and three insertion sequences, IS*900*, IS*901*, and IS*1311*. The results for the pattern of amplification allowed to determine whether isolates were mycobacteria, or members of MAC, and to classify them into one of the three

Zoonotic aspects of mycobacteria transmitted by the environment and wildlife highlights a major health problem. MAC causes a variety of disorders including tuberculosis-like diseas‐ es in animals and in human immunocompetent or immunocompromised patients. Suscepti‐ bility to mycobacterial infections depends of risk factors since they are ubiquous in the soil and water [150]. Human exposure to mycobacterium present in wildlife and in nature can occur by a variety of routes. Humans are continuously exposed at a low level (50 to 5000 bacilli per day). Contact with water, municipal or natural are also important routes for my‐ cobacteria infection. Birds are major excretors of the agent in their faeces and the bacteria

Healthy humans have a low susceptibility to MAC infection and only a very small percent‐ age of mycobacteria progress trough to infection, but in immunocompromised individuals infected with HIV or leukaemia patients, treated with steroid therapy, chemotherapy or oth‐ er immunosuppressive medication, should be carefully considered regarding their possibili‐ ty to come in contact with birds with mycobacterial infection [1,151]. Prior to the introduction of highly active antiretroviral treatment more than 40% of patients developed *M. avium* complex bacteriemia two years after the AIDS diagnosis [152] and a disseminated MAC infection was found in as much as 50% of autopsied AIDS patients [153,154]. This has predominantly been attributed to the impairment of the adaptative part of the immune sys‐ tems in HIV-1 infected individuals due to the loss of CD4+ T cells, as the susceptibility to opportunistic infections including *M. avium* infection is correlated with a decline in this cell type [155]. MAC usually produces clinical disease only when CD4+ are very low (< 50 cells/ ml), which is seen in 4 to 5 per cent of HIV infected patients [88]. A recent study showed that exposure of dendritic cells to HIV-1 promotes or facilitates the intracellular growth of *M. avium* [153]. Signs and symptoms associated with MAC disease in AIDS cases are persistent

from the tissue of the heavily infected sheep.

108 Insights from Veterinary Medicine

major MAC subspecies, *Map*, *Maa*, *Mah* [18].

can persist in the soil and in water for long [1].

**5. Public health concerns**

Disease patterns of MAC are different in immunocompromised patients. In adults, infection is mainly pulmonary [1,151]. MAC is the most common of the nontuberculous mycobacteria found in apparently healthy children [162] and it's infection is characterized by a chronic granulomatous lymphadenopathy in the neck region that preferably is treated by excision of the affected lymph node [162,163]. The main hypothesis of infection is that oral contact with *M. avium*-infected water courses causes lymphadenitis in the head and neck region in chil‐ dren [162,164]. Among the members of MAC, *Maa* is predominant (87-98%) in AIDS patients and induces disseminated mycobacteriemia rather than restrict the bacteria to the lungs [1]. Since the advent of AIDS, HIV has become the major risk factor for MAC infection. In AIDS patients the main route of infection is the gastrointestinal tract and *M. avium* is naturally tol‐ erant to the low pH that exists in the stomach [19,165]. Regarding therapeutics, *M. avium* is of special concern because drugs commonly used from treating tuberculosis in humans are not effective [166] and MAC strains are resistant to isoniazid, the most popular anti-tubercu‐ losis drug [19,167]. Preexisting pulmonary conditions, patients with current illness or immu‐ nosuppressive medication are the most important risk factors for MAC infection amongst patients without HIV infection. MAC was also reported as the most common pathogen caus‐ ing post transplant non-tuberculous mycobacteria disease [156]. Other factors are local trau‐ mas and surgical procedures injuries [168]. Chronic obstructive pulmonary disease, emphysema, pneumoconiosis, aspiration due to oesophageal disease, previous gastrectomy and chronic alcoholism are some of the conditions which have been linked to disease [88,154]. Disseminated MAC infection is more frequent in caucasians compared with His‐ panic or African-americans individuals. However, there are no differences related with age [169,170]. Infection is more frequent in men than in women, particularly in homosexual and bisexual men, when compared to other HIV risk categories.

The zoonotic potential of *Map* has been debated for almost a century because of similarities between Johne´s disease in cattle and Crohn´s disease in humans. A quarter century later since *Map* was first proposed as an etiologic agent in Crohn´s disease based on the isolation of the organism from several patients the association as a causative agent or in an incidental away remains unresolved. Milk and water are potential sources for acquiring *Map* infection [171]. However, only a few samples of milk, positive by PCR for the presence of *Map* have yielded positive results in culture, suggesting that *Map* remains undetectable probably be‐ cause the low number of viable *Map* in samples [172]. Published reports indicate that *Map* may not be completely inactivated by pasteurization of milk [173]. Serological response to *Map* in humans is also not conclusive [172]. Traditional methods of detecting bacteria, cul‐ ture and stain are largely ineffective in detecting *Map* in humans. Bacteria are very difficult to culture and *Map* is able to exist in a spheroplast (cell wall deficient form) in humans so it cannot be identified by Ziehl-Neelsen staining [174-176]. Polymerase chain reaction has identified *Map* in greater than 90% of biopsy specimens from Crohn´s patients [177] and via‐ ble *Map* was detected in peripheral blood and serum in a higher proportion of individuals with Crohn's disease [178,179]. However, culture of *Map* from human specimens is a contro‐ versial question since some authors pointed out the difficulty of culturing *Map* [180,181]. At this moment is not possible to know if *Map* is a primary etiological agent or secondary in‐ vader and further research is need to understand the possible links between this agent and Crohn disease [1].

The evidence for the zoonotic potential should not be neglected particularly in immunocom‐

Recent reports, suggesting an association between MAC and autoimmune and other chronic human diseases, alert to the importance of developing new studies on MAC biology, molec‐

Research to understand the impact of MAC in public health is needed as well as the deter‐ mination of transmission routes between humans and wildlife, which requires interdiscipli‐

The work was supported by the strategic research project PEst-OE/AGR/UI0772/2011 fi‐

1 CECAV- Animal and Veterinary Research Center, University of Trás-os-Montes and Alto-

3 Department of Genetics and Biotechnology, IBB-Institute for Biotechnology and Bioengin‐ eering, Centre of Genomic and Biotechnology, University of Trás-os-Montes and Alto-

[1] Biet F, Boschiroli ML, Thorel MF, Guilloteau LA. Zoonotic Aspects of Mycobacteri‐ um bovis and Mycobacterium avium-intracellulare Complex (MAC). Veterinary re‐

[3] Turenne CY, Wallace R, Behr MA. Mycobacterium avium in the Postgenomic Era.

[4] Smole SC, McAleese F, Ngampasutadol J, von Reyn CF, Arbeit RD. Clinical and Epi‐ demiological Correlates of Genotypes within the Mycobacterium avium Complex

[2] Prescott LM, Harley JP, Klein DA. Microbiology. New York: McGraw-Hill; 2002.

2 School of Agriculture, Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal

, Ana Matos2

, Manuela Matos3

*Mycobacterium avium* Complex in Domestic and Wild Animals

http://dx.doi.org/10.5772/54323

111

and

nary collaboration among medical, veterinary and other public health officials.

nanced by the Foundation for Science and Technology (FCT).

, Maria de Lurdes Pinto1

Douro, Department of Veterinary Sciences, Vila Real, Portugal

Clinical Microbiology Reviews. 2007;20(2) 205-229.

promised patients, both humans and animals.

ular diagnosis and epidemiology.

**Acknowledgements**

**Author details**

Ana Cláudia Coelho1

Maria dos Anjos Pires1

Douro, Vila Real, Portugal

search. 2005;36(3) 411-436.

**References**

There is a recent interest in *Map* as an immune trigger of several autoimmune diseases [182,183]. Environmental agents are postulated to trigger autism. Recently, a theory propos‐ ing a mechanism by which *Map* triggers autism through molecular mimicry to the heat shock protein HSP65, which stimulates antibodies that cross react with myelin basic protein, a common feature of autism [184]. Another recent study has associated the presence of *Map* with Blau syndrome, an autossomal dominant, and systemic inflammatory disease. The mu‐ tations of Blau syndrome are on the same gene on chromosome 16 (CARD 15) that confers susceptibility to Crohns´disease [185]. *Map* was also implicated in sarcoidosis [186,187], which is a multisystemic granulomatous disease with many features in common with myco‐ bacterial infection, and that, like Crohn´s disease, can be mimicked by slow bacterial infec‐ tions [188,189].

*Map* was also linked to ulcerative colitis, irritable bowel syndrome, autoimmune (type 1) diabetes, Hashimoto thyroiditis and multiple sclerosis [179,184]. Increasing evidence sug‐ gests a role for *Map* in autoimmune (type I) diabetes. It is postulated that this bacterium acts via molecular mimicry between its antigens (HSP65) and the pancreatic enzyme glutamic acid decarboxylase (GAD) [190-195].

## **6. Conclusion**

MAC comprises slow growing mycobacteria that are ubiquitous in the environment (soil and water), and have a wide source range, causing disease in various domestic and wild mammals and birds. MAC can affect a wide-range of wild animals, but little has been pub‐ lished up to the moment on the clinical signs, which are rarely exhibited or not documented. When present, the occurrence of clinical signs and lesions is highly variable in timing, though often similar to those of their domesticated counterparts.

The evidence for the zoonotic potential should not be neglected particularly in immunocom‐ promised patients, both humans and animals.

Recent reports, suggesting an association between MAC and autoimmune and other chronic human diseases, alert to the importance of developing new studies on MAC biology, molec‐ ular diagnosis and epidemiology.

Research to understand the impact of MAC in public health is needed as well as the deter‐ mination of transmission routes between humans and wildlife, which requires interdiscipli‐ nary collaboration among medical, veterinary and other public health officials.

## **Acknowledgements**

of the organism from several patients the association as a causative agent or in an incidental away remains unresolved. Milk and water are potential sources for acquiring *Map* infection [171]. However, only a few samples of milk, positive by PCR for the presence of *Map* have yielded positive results in culture, suggesting that *Map* remains undetectable probably be‐ cause the low number of viable *Map* in samples [172]. Published reports indicate that *Map* may not be completely inactivated by pasteurization of milk [173]. Serological response to *Map* in humans is also not conclusive [172]. Traditional methods of detecting bacteria, cul‐ ture and stain are largely ineffective in detecting *Map* in humans. Bacteria are very difficult to culture and *Map* is able to exist in a spheroplast (cell wall deficient form) in humans so it cannot be identified by Ziehl-Neelsen staining [174-176]. Polymerase chain reaction has identified *Map* in greater than 90% of biopsy specimens from Crohn´s patients [177] and via‐ ble *Map* was detected in peripheral blood and serum in a higher proportion of individuals with Crohn's disease [178,179]. However, culture of *Map* from human specimens is a contro‐ versial question since some authors pointed out the difficulty of culturing *Map* [180,181]. At this moment is not possible to know if *Map* is a primary etiological agent or secondary in‐ vader and further research is need to understand the possible links between this agent and

There is a recent interest in *Map* as an immune trigger of several autoimmune diseases [182,183]. Environmental agents are postulated to trigger autism. Recently, a theory propos‐ ing a mechanism by which *Map* triggers autism through molecular mimicry to the heat shock protein HSP65, which stimulates antibodies that cross react with myelin basic protein, a common feature of autism [184]. Another recent study has associated the presence of *Map* with Blau syndrome, an autossomal dominant, and systemic inflammatory disease. The mu‐ tations of Blau syndrome are on the same gene on chromosome 16 (CARD 15) that confers susceptibility to Crohns´disease [185]. *Map* was also implicated in sarcoidosis [186,187], which is a multisystemic granulomatous disease with many features in common with myco‐ bacterial infection, and that, like Crohn´s disease, can be mimicked by slow bacterial infec‐

*Map* was also linked to ulcerative colitis, irritable bowel syndrome, autoimmune (type 1) diabetes, Hashimoto thyroiditis and multiple sclerosis [179,184]. Increasing evidence sug‐ gests a role for *Map* in autoimmune (type I) diabetes. It is postulated that this bacterium acts via molecular mimicry between its antigens (HSP65) and the pancreatic enzyme glutamic

MAC comprises slow growing mycobacteria that are ubiquitous in the environment (soil and water), and have a wide source range, causing disease in various domestic and wild mammals and birds. MAC can affect a wide-range of wild animals, but little has been pub‐ lished up to the moment on the clinical signs, which are rarely exhibited or not documented. When present, the occurrence of clinical signs and lesions is highly variable in timing,

though often similar to those of their domesticated counterparts.

Crohn disease [1].

110 Insights from Veterinary Medicine

tions [188,189].

**6. Conclusion**

acid decarboxylase (GAD) [190-195].

The work was supported by the strategic research project PEst-OE/AGR/UI0772/2011 fi‐ nanced by the Foundation for Science and Technology (FCT).

## **Author details**

Ana Cláudia Coelho1 , Maria de Lurdes Pinto1 , Ana Matos2 , Manuela Matos3 and Maria dos Anjos Pires1

1 CECAV- Animal and Veterinary Research Center, University of Trás-os-Montes and Alto-Douro, Department of Veterinary Sciences, Vila Real, Portugal

2 School of Agriculture, Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal

3 Department of Genetics and Biotechnology, IBB-Institute for Biotechnology and Bioengin‐ eering, Centre of Genomic and Biotechnology, University of Trás-os-Montes and Alto-Douro, Vila Real, Portugal

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1129-1135.

and Type-1 Diabetes Mellitus. PLoS ONE. 2009; 4(9):e7109. http://dx.doi.org/ 10.1371%2Fjournal.pone.0007109.

**Chapter 5**

**Psittacosis**

Maria dos Anjos Pires

http://dx.doi.org/10.5772/53720

**1. Introduction**

ed [8].

disease [18,19].

João Morais, Ana Cláudia Coelho and

Additional information is available at the end of the chapter

emphasizes the scientific reports worldwide [12-15].

animals are showing clinical signs of disease [16,17].

There are three morphologically distinct forms of *Chlamydophila*:

*Chlamydophila psittaci* (Bacteria kingdom, Chlamydiae phylum, Chlamydiae class, Chlamy‐ diales order, Chlamydiaceae family) is part of the genus *Chlamydophila*, where *Cp. abortus*, *Cp.*

Avian chlamydiosis induced by this Gram-negative obligate intracellular bacteria [2] is tradi‐ tionally known as ornithosis or psittacosis [3-5]. It is an infectious disease capable of infecting domestic and wild birds [6,7], being the Psittacidae family where most occurrences are report‐

*Chlamydophila psittaci* infects primarily birds [9], but mammals, including humans, are also sus‐ ceptible of infection [10-11]. In fact, zoonotic status of this organism is largely described and

*Chlamydophila psittaci* can be found in bird feathers, excrements and blood, whether or not these

Psittacosis can be transmitted by vertical and horizontal via [2]. The agent is excreted on faeces and ingested from the food or inhaled via aerosols [13]. At the lungs of newly infected animals, the organism gets an infecting status becoming capable to replicate and causing clinical signs of

**•** The elementary body, which is small, spherical, of about 0.2-0.3 mm in diameter;

**•** The reticular body, wider, of about 0.5-2.0 mm, which is able to replicate by binary division;

**•** And the intermediate body, with 0.3-2.0 mm in diameter, seen in infected host cells [18,20].

© 2013 Morais et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Morais et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

*caviae*, *Cp. felis*, *Cp. pecorum* and *Cp. pneumoniae* are the other five species of bacteria [1].

**Chapter 5**

## **Psittacosis**

and Type-1 Diabetes Mellitus. PLoS ONE. 2009; 4(9):e7109. http://dx.doi.org/

LA. Specific Immunoassays Confirm Association of Mycobacterium avium Subsp. paratuberculosis with Type-1 but Not Type-2 Diabetes Mellitus. PLoS ONE. 2009;

are specific tags of Mycobacterium avium subsp. paratuberculosis infection in type I

[194] Rosu V, Ahmed N, Paccagnini D, Gerlach G, Fadda G, Hasnain SE, Zanetti S, Sechi

[195] Cossu A, Rosu V, Paccagnini D, Cossu D, Pacifico A, Sechi LA. MAP3738c and MptD

4(2):e4386. http://dx.doi.org/10.1371%2Fjournal.pone.0004386.

diabetes mellitus. Clinical Immunology. 2011;141(1) 49-57.

10.1371%2Fjournal.pone.0007109.

128 Insights from Veterinary Medicine

João Morais, Ana Cláudia Coelho and Maria dos Anjos Pires

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53720

## **1. Introduction**

*Chlamydophila psittaci* (Bacteria kingdom, Chlamydiae phylum, Chlamydiae class, Chlamy‐ diales order, Chlamydiaceae family) is part of the genus *Chlamydophila*, where *Cp. abortus*, *Cp. caviae*, *Cp. felis*, *Cp. pecorum* and *Cp. pneumoniae* are the other five species of bacteria [1].

Avian chlamydiosis induced by this Gram-negative obligate intracellular bacteria [2] is tradi‐ tionally known as ornithosis or psittacosis [3-5]. It is an infectious disease capable of infecting domestic and wild birds [6,7], being the Psittacidae family where most occurrences are report‐ ed [8].

*Chlamydophila psittaci* infects primarily birds [9], but mammals, including humans, are also sus‐ ceptible of infection [10-11]. In fact, zoonotic status of this organism is largely described and emphasizes the scientific reports worldwide [12-15].

*Chlamydophila psittaci* can be found in bird feathers, excrements and blood, whether or not these animals are showing clinical signs of disease [16,17].

Psittacosis can be transmitted by vertical and horizontal via [2]. The agent is excreted on faeces and ingested from the food or inhaled via aerosols [13]. At the lungs of newly infected animals, the organism gets an infecting status becoming capable to replicate and causing clinical signs of disease [18,19].

There are three morphologically distinct forms of *Chlamydophila*:

© 2013 Morais et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Morais et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Parrots are often infected animals but they show no signs of disease unless they are stressed out [21], and thus can be a source of contamination to other birds and mam‐ mals, so as the man [3,4,22]. Infections in turkeys have been described long ago, with a mortality range of 5-40% without any treatment [23], and more recently in ducks, with a mortality of 30%, which was highlighted as an economic problem and health impairment [24]. The mortality rate in humans is low if treatment is appropriate. Therefore, setting up a quick prognosis is essential [20,23].

Later, Everett and collaborators (1999), proposed a new classification, where the *Chlamy‐ dophila* genus would contain six species: *Chlamydophila pneumoniae, Chlamydophila peco‐ rum, Chlamydophila abortus, Chlamydophila felis, Chlamydophila caviae* and *Chlamydophila psittaci* [1]. This taxonomic classification was accepted and is still used worldwide [17].

Psittacosis

131

http://dx.doi.org/10.5772/53720

The aim of this study was to discuss phylogeny, epidemiology, clinical signs, pathology, di‐ agnostic techniques, treatment, prevention and public health concerns in psittacosis with

Bacteria kingdom consists of different phyla, including the Chlamydiae, holding the class with the same name. The order Chlamydiales belongs to the class Chlamydiae and the fami‐ ly Chlamydiaceae to the order described. The genus *Chlamydophila* belongs to the family Chlamydiaceae and accordingly with the National Center For Biotechnology Information Taxonomy Browser (www.ncbi.nlm.nih.gov), it has six species of bacteria [1]. The Chlamy‐

In the genus *Chlamydophila, Chlamydophila abortus* is a bacterium whose disease is se‐ vere, especially in small ruminants [37], but it is also described as an important zoonot‐

*Chlamydophila pneumoniae* has been primarily described as an infection in humans but later has been reported in other animals as well, such as mammals, marsupials, reptiles and am‐ phibians [41-44]. The transmission of *Cp. pneumoniae* among animals and humans was not yet described; however, Myers and collaborators proved that human could be infected from

*Chlamydophila caviae* was found in animals such as rabbits, guinea pigs, horses, cats and dogs [45-48]. In 2006, it was reported the identification of this bacteria in humans, raising the pos‐

*Chlamydophila felis* affects cats mainly under one year old and is very often associated with conjunctivitis [49-52]. Although there is evidence that this bacterium can cause keratocon‐ junctivitis in humans, there is still little evidence that can create systemic disease or severe

*Chlamydophila pecorum* affects several small and large mammals such as ruminants, swine and koalas [54-59], but little or no studies have been published to date that can prove human

*Chlamydophila psittaci* is the causative agent of Psittacosis and is capable of infecting domes‐ tic and wild birds [6, 60], but also reptiles [61] and mammals such as man [10,11]. This bacte‐ rium is definitely an important zoonosis and such potential has been widely reported across

diae genus contains three species: *C. trachomatis, C. suis* and *C. muridarum* [1].

animal isolated bacteria following adaptation to the new host [44].

sibility that it has a zoonotic potential, which has not been yet clarified [45].

special attention due to *Chlamydophila psittaci* infection.

ic disease [38-40].

pneumonia in the man [53].

infection from this agent.

the years [12-15].

**2. Phylogeny and biology of Chlamydiacea family**

Clinical symptoms are very extensive in number and vary depending on the serotype of the bacteria and the animal affected. General infections may cause fever, anorexia, lethargy, diar‐ rhoea and sometimes shock or even death. Chlamydiosis in psittacines is mostly chronic and cause conjunctivitis, enteritis, sacculitis, pneumonitis, hepatomegaly and droppings can range from green to yellow-green in colour [20]. Clinical signs in humans range from unapparent to severe systemic disease with interstitial pneumonia and encephalitis [12,20]. Signs of disease are headache, chills, malaise and myalgia with possible respiratory involvement. Infection of pigeons by *Chlamydophila* is vast and well reported [15]. These animals are a major spreader of the disease, and present clinical signs such as conjunctivitis, blepharitis and rhinitis [20]. *Chla‐ mydophila psittaci* antibodies can be detected by several laboratory methodologies. The comple‐ ment fixation test (CFT) is the most frequently used test [25,26]. Other serological tests such as PCR (polymerase chain reaction) and ELISA are used as well for its diagnosis [8,22,27].

Psittacosis or "parrot fever" was documented for first time in 1879, when Jakob Ritter descri‐ bed an epidemic and unusual pneumonia, associated with exposure to tropical birds in sev‐ en Switch individuals causing flu-like symptoms and pneumonia [28]. The term "psittacosis" (from the Latin word for parrot - *psittacus*) was firstly used by Morange, in 1895, when trans‐ mission of an infectious agent to humans from parrots was notorious [29-30].

Investigations of chlamydiosis began in 1907 when microorganisms within intracytoplasmic vacuoles in conjunctival scraping cells from humans with trachoma were found. Trachoma is a chronic infectious disease of the conjunctiva and cornea [18] a disease well known since ancient civilizations [31]. These organisms were named *Chlamydozoa*, from the characteristic shape of the mantle or "*chlamys*" in Greek [32].

A psittacosis outbreak occurred during the winters of 1929 and 1930 in Europe and in the United States. The causative agent of psittacosis was isolated from birds and infected hu‐ mans and the source of contamination traced to parrots of the genus *Amazona*, originated in South America [33]. At the same time, a microorganism causing lymphogranuloma venere‐ um (LGV) was isolated in humans [34]. Until then, it was believed avian psittacosis was re‐ stricted to *Psittaciformes* birds [18]. However, a number of studies demonstrated a much higher number of species concerned or potentially infected by this microorganism. In fact, Meyer and Eddie, in 1932, described a case of transmission of human psittacosis from do‐ mestic fowl [35] and Haagen and Mauer, six years later, reported an infection in fulmar (*Fulmarus glacialis)*. Pinkerton and Swank, in 1940, proved the existence of this agent in do‐ mestic pigeons, as Wolins, in 1948, described the same disease in ducks [33]. Later studies of this matter enhanced the idea that psittacosis was not restricted to parrots, reporting infec‐ tions in humans by contact with other affected birds [18,36].

Later, Everett and collaborators (1999), proposed a new classification, where the *Chlamy‐ dophila* genus would contain six species: *Chlamydophila pneumoniae, Chlamydophila peco‐ rum, Chlamydophila abortus, Chlamydophila felis, Chlamydophila caviae* and *Chlamydophila psittaci* [1]. This taxonomic classification was accepted and is still used worldwide [17].

The aim of this study was to discuss phylogeny, epidemiology, clinical signs, pathology, di‐ agnostic techniques, treatment, prevention and public health concerns in psittacosis with special attention due to *Chlamydophila psittaci* infection.

## **2. Phylogeny and biology of Chlamydiacea family**

Parrots are often infected animals but they show no signs of disease unless they are stressed out [21], and thus can be a source of contamination to other birds and mam‐ mals, so as the man [3,4,22]. Infections in turkeys have been described long ago, with a mortality range of 5-40% without any treatment [23], and more recently in ducks, with a mortality of 30%, which was highlighted as an economic problem and health impairment [24]. The mortality rate in humans is low if treatment is appropriate. Therefore, setting

Clinical symptoms are very extensive in number and vary depending on the serotype of the bacteria and the animal affected. General infections may cause fever, anorexia, lethargy, diar‐ rhoea and sometimes shock or even death. Chlamydiosis in psittacines is mostly chronic and cause conjunctivitis, enteritis, sacculitis, pneumonitis, hepatomegaly and droppings can range from green to yellow-green in colour [20]. Clinical signs in humans range from unapparent to severe systemic disease with interstitial pneumonia and encephalitis [12,20]. Signs of disease are headache, chills, malaise and myalgia with possible respiratory involvement. Infection of pigeons by *Chlamydophila* is vast and well reported [15]. These animals are a major spreader of the disease, and present clinical signs such as conjunctivitis, blepharitis and rhinitis [20]. *Chla‐ mydophila psittaci* antibodies can be detected by several laboratory methodologies. The comple‐ ment fixation test (CFT) is the most frequently used test [25,26]. Other serological tests such as

PCR (polymerase chain reaction) and ELISA are used as well for its diagnosis [8,22,27].

mission of an infectious agent to humans from parrots was notorious [29-30].

Psittacosis or "parrot fever" was documented for first time in 1879, when Jakob Ritter descri‐ bed an epidemic and unusual pneumonia, associated with exposure to tropical birds in sev‐ en Switch individuals causing flu-like symptoms and pneumonia [28]. The term "psittacosis" (from the Latin word for parrot - *psittacus*) was firstly used by Morange, in 1895, when trans‐

Investigations of chlamydiosis began in 1907 when microorganisms within intracytoplasmic vacuoles in conjunctival scraping cells from humans with trachoma were found. Trachoma is a chronic infectious disease of the conjunctiva and cornea [18] a disease well known since ancient civilizations [31]. These organisms were named *Chlamydozoa*, from the characteristic

A psittacosis outbreak occurred during the winters of 1929 and 1930 in Europe and in the United States. The causative agent of psittacosis was isolated from birds and infected hu‐ mans and the source of contamination traced to parrots of the genus *Amazona*, originated in South America [33]. At the same time, a microorganism causing lymphogranuloma venere‐ um (LGV) was isolated in humans [34]. Until then, it was believed avian psittacosis was re‐ stricted to *Psittaciformes* birds [18]. However, a number of studies demonstrated a much higher number of species concerned or potentially infected by this microorganism. In fact, Meyer and Eddie, in 1932, described a case of transmission of human psittacosis from do‐ mestic fowl [35] and Haagen and Mauer, six years later, reported an infection in fulmar (*Fulmarus glacialis)*. Pinkerton and Swank, in 1940, proved the existence of this agent in do‐ mestic pigeons, as Wolins, in 1948, described the same disease in ducks [33]. Later studies of this matter enhanced the idea that psittacosis was not restricted to parrots, reporting infec‐

up a quick prognosis is essential [20,23].

130 Insights from Veterinary Medicine

shape of the mantle or "*chlamys*" in Greek [32].

tions in humans by contact with other affected birds [18,36].

Bacteria kingdom consists of different phyla, including the Chlamydiae, holding the class with the same name. The order Chlamydiales belongs to the class Chlamydiae and the fami‐ ly Chlamydiaceae to the order described. The genus *Chlamydophila* belongs to the family Chlamydiaceae and accordingly with the National Center For Biotechnology Information Taxonomy Browser (www.ncbi.nlm.nih.gov), it has six species of bacteria [1]. The Chlamy‐ diae genus contains three species: *C. trachomatis, C. suis* and *C. muridarum* [1].

In the genus *Chlamydophila, Chlamydophila abortus* is a bacterium whose disease is se‐ vere, especially in small ruminants [37], but it is also described as an important zoonot‐ ic disease [38-40].

*Chlamydophila pneumoniae* has been primarily described as an infection in humans but later has been reported in other animals as well, such as mammals, marsupials, reptiles and am‐ phibians [41-44]. The transmission of *Cp. pneumoniae* among animals and humans was not yet described; however, Myers and collaborators proved that human could be infected from animal isolated bacteria following adaptation to the new host [44].

*Chlamydophila caviae* was found in animals such as rabbits, guinea pigs, horses, cats and dogs [45-48]. In 2006, it was reported the identification of this bacteria in humans, raising the pos‐ sibility that it has a zoonotic potential, which has not been yet clarified [45].

*Chlamydophila felis* affects cats mainly under one year old and is very often associated with conjunctivitis [49-52]. Although there is evidence that this bacterium can cause keratocon‐ junctivitis in humans, there is still little evidence that can create systemic disease or severe pneumonia in the man [53].

*Chlamydophila pecorum* affects several small and large mammals such as ruminants, swine and koalas [54-59], but little or no studies have been published to date that can prove human infection from this agent.

*Chlamydophila psittaci* is the causative agent of Psittacosis and is capable of infecting domes‐ tic and wild birds [6, 60], but also reptiles [61] and mammals such as man [10,11]. This bacte‐ rium is definitely an important zoonosis and such potential has been widely reported across the years [12-15].

All species of the genera *Chlamydophila* and Chlamydiae have one or more serotypes, whose sequence are known [62-63] or are still under intense study [64].

Some studies have shown the existence of hemispherical-shaped projections of the cyto‐ plasm on the surface of the elementary bodies and ridge-shaped projections of the reticular forms for this bacterium [76-78]. These projections range from the bacteria to the surface of the membrane inclusion and it is speculated that can possess pores of type III secretion [32].

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Infection of *Chlamydophila psittaci* begins with the attachment of the elementary body to the host cell and subsequent parasite-mediated endocytosis [79]. The bacterium binds to the cell membrane receptor of the host cell, often associated with a cytoplasm protein known as cla‐ thrin [20]. This connection stimulates the cell to emit pseudopods; these increase in size until all bacteria have been surrounded, forming a vacuole delimited by clathrin, referred to as inclusion [18, 80]. In the vacuole, the bacterium has a biphasic cycle (Figure 1), alternating

Once inside the vacuole, the EB are redistributed from the periphery of the cell and join in the region of the Golgi apparatus, which corresponds to the MTOC (Microtubule Organiz‐ ing Centre) [19, 80]. The non-acid inclusions are firstly too small, allowing to avoid its fusion with lysosomes, and are formed by endocytic constituents of the plasma membrane of the host cell such as proteins and lipids [19, 80]. All of these conditions lead to a more efficient

On the first hours, the elementary bodies initiate to differentiate into reticular bodies. These forms of *Chlamydophila* are not infectious, have a non-condensed nucleoid and a greater size than EB [19, 65]. From eight to ten hours of cycle, the reticular bodies start to replicate by binary fission, remaining in contact with the inclusion membrane [20, 65]. In this moment, it's possible to see numerous mitochondria surrounding the inclusion [18, 20], possibly due to protein motors function as kinesins [81]. *Cp. psittaci* may not be able to generate as much energy in the form of ATP as other species of the genus, so a deeper association with the host cell mitochondria is necessary for the bacteria to recruit the necessary ATP by an alter‐ native mechanism [20]. The migration of nutrients from the host cell into the inclusion is es‐ sential. Amino acids and nucleotides cross the inclusion membrane [20, 82]. Since the vacuole is not in the vicinity of lysosomes leads to the assumption that the releasing of nu‐ trients by fusion with endocytic vesicles is unlikely [20]. It is known that exocytic vesicles containing sphingomyelin fuse with the membrane of the inclusion, delivering nutrients and lipids to the bacteria [80]. It was demonstrated that the inclusion membrane is permeable to small molecules of a molecular weight ranging from 100-520 Da [83,84]. After releasing the nutrients into the lumen of the bacterium by passive diffusion, membrane-specific carriers/ transporters located in the membrane would facilitate the entry of new nutrients and would be indispensable for the entry of molecules larger than 520 Da, but such carriers have not

Throughout the cycle, the size of the vesicles increases, as they accumulate growing num‐ bers of bacteria inside [20, 84]. The surface of the inclusion membrane also increases, while it intercepts biosynthetic transport pathways of the host cell membrane and acquires the abili‐ ty to fuse with a subset of Golgi derived vesicles [20, 84]. Within 20 hours of cycle, reticular

between states of elementary body and reticular body [19].

survival and infection by the bacterium [20].

been yet described [20].

**c.** Pathogenesis

#### **a.** *Chlamydophila psittaci*

*Chlamydophila psittaci* is negative to Gram test bacterium [2, 49, 57], having a cytoplasmic membrane and an outer three-layer membrane [65-66], with a significant cell wall fraction insoluble to ionic detergents [67]. In bacteria of the genus *Chlamydophila*, this portion is refer‐ red to as COMC (*Chlamydia* outer membrane complex), which is composed of MOMP (major outer membrane protein) and other small proteins [68]. MOMP is composed of cysteine-rich proteins and takes approximately 60% of the total weight of the outer membrane [69]. This constituent is important to maintain the integrity and rigidity of the bacteria, and the main antigen to host immune system [70].

#### **b.** *Chlamydophila psittaci* biology

*Chlamydophila bacteria* have three distinct forms during its life cycle: elementary body (EB), reticular body (RB) and intermediate body (IB) [19,20, 71].

The EB is small, electro-dense, spherical and about 0.2 to 0.3 mm in diameter [18,20]. The outer membrane is composed of proteins, lipids, lipopolysaccharides and proteins. However, unlike the Gram-negative bacteria, this species is devoid of muramic acid [72]. This is an active form of the bacterium capable of binding with the host cell allowing to reach its inside [19]. This first form is characterized by a highly electro-dense nucleoid [71, 73], located in the periphery and clearly separated from the cytoplasm [19, 71]. The Hc1 histone maintains the chromatin highly condensed [74]. Within the cell, the elemen‐ tary body increases in size to form the reticular body, which is the metabolic active in‐ tracellular form of the organism [19, 20]. At this stage, the chromatin is dispersed, since the bacteria begin a process of transcription [20, 75].

The RB is of about 0.5 to 2.0 mm in diameter and its inner and outer membranes are relative‐ ly nearby, thereby reducing the virtual space between them [18, 20]. This form of the bacteri‐ um has the ability to split by binary fission, resulting in new RB [19, 65]. During this process, intermediate bodies can be observed within the host cell, measuring between 0.3 and 1.0 mm in diameter [18, 20].

This third form of the bacterium (IB) has a very specific presentation with an electro-dense core nucleoid surrounded by fibres dispersed radially [65, 73]. In the periphery, there exists an agglomerate of cytoplasmic granules, separated from the core by a translucent area [71]. When the replication process is over, several elementary forms are observed, which can be somewhat condensed, depending on their conformations [20]. The less condensed are more immature form, have further fibrous elements in the granular cytoplasm, bearing for the electro-dense nucleoid, which will become progressively highly condensed [71]. The nucle‐ oid is close to the inner membrane [20].

The mature elementary bodies are condensed, having a homogeneous oval shaped nucleoid, irregular or an elongated, separated from the cytoplasmic organisms by a well visible elec‐ tro-transparent area [20, 75].

Some studies have shown the existence of hemispherical-shaped projections of the cyto‐ plasm on the surface of the elementary bodies and ridge-shaped projections of the reticular forms for this bacterium [76-78]. These projections range from the bacteria to the surface of the membrane inclusion and it is speculated that can possess pores of type III secretion [32].

#### **c.** Pathogenesis

All species of the genera *Chlamydophila* and Chlamydiae have one or more serotypes, whose

*Chlamydophila psittaci* is negative to Gram test bacterium [2, 49, 57], having a cytoplasmic membrane and an outer three-layer membrane [65-66], with a significant cell wall fraction insoluble to ionic detergents [67]. In bacteria of the genus *Chlamydophila*, this portion is refer‐ red to as COMC (*Chlamydia* outer membrane complex), which is composed of MOMP (major outer membrane protein) and other small proteins [68]. MOMP is composed of cysteine-rich proteins and takes approximately 60% of the total weight of the outer membrane [69]. This constituent is important to maintain the integrity and rigidity of the bacteria, and the main

*Chlamydophila bacteria* have three distinct forms during its life cycle: elementary body (EB),

The EB is small, electro-dense, spherical and about 0.2 to 0.3 mm in diameter [18,20]. The outer membrane is composed of proteins, lipids, lipopolysaccharides and proteins. However, unlike the Gram-negative bacteria, this species is devoid of muramic acid [72]. This is an active form of the bacterium capable of binding with the host cell allowing to reach its inside [19]. This first form is characterized by a highly electro-dense nucleoid [71, 73], located in the periphery and clearly separated from the cytoplasm [19, 71]. The Hc1 histone maintains the chromatin highly condensed [74]. Within the cell, the elemen‐ tary body increases in size to form the reticular body, which is the metabolic active in‐ tracellular form of the organism [19, 20]. At this stage, the chromatin is dispersed, since

The RB is of about 0.5 to 2.0 mm in diameter and its inner and outer membranes are relative‐ ly nearby, thereby reducing the virtual space between them [18, 20]. This form of the bacteri‐ um has the ability to split by binary fission, resulting in new RB [19, 65]. During this process, intermediate bodies can be observed within the host cell, measuring between 0.3 and 1.0

This third form of the bacterium (IB) has a very specific presentation with an electro-dense core nucleoid surrounded by fibres dispersed radially [65, 73]. In the periphery, there exists an agglomerate of cytoplasmic granules, separated from the core by a translucent area [71]. When the replication process is over, several elementary forms are observed, which can be somewhat condensed, depending on their conformations [20]. The less condensed are more immature form, have further fibrous elements in the granular cytoplasm, bearing for the electro-dense nucleoid, which will become progressively highly condensed [71]. The nucle‐

The mature elementary bodies are condensed, having a homogeneous oval shaped nucleoid, irregular or an elongated, separated from the cytoplasmic organisms by a well visible elec‐

sequence are known [62-63] or are still under intense study [64].

reticular body (RB) and intermediate body (IB) [19,20, 71].

the bacteria begin a process of transcription [20, 75].

**a.** *Chlamydophila psittaci*

132 Insights from Veterinary Medicine

antigen to host immune system [70].

**b.** *Chlamydophila psittaci* biology

mm in diameter [18, 20].

oid is close to the inner membrane [20].

tro-transparent area [20, 75].

Infection of *Chlamydophila psittaci* begins with the attachment of the elementary body to the host cell and subsequent parasite-mediated endocytosis [79]. The bacterium binds to the cell membrane receptor of the host cell, often associated with a cytoplasm protein known as cla‐ thrin [20]. This connection stimulates the cell to emit pseudopods; these increase in size until all bacteria have been surrounded, forming a vacuole delimited by clathrin, referred to as inclusion [18, 80]. In the vacuole, the bacterium has a biphasic cycle (Figure 1), alternating between states of elementary body and reticular body [19].

Once inside the vacuole, the EB are redistributed from the periphery of the cell and join in the region of the Golgi apparatus, which corresponds to the MTOC (Microtubule Organiz‐ ing Centre) [19, 80]. The non-acid inclusions are firstly too small, allowing to avoid its fusion with lysosomes, and are formed by endocytic constituents of the plasma membrane of the host cell such as proteins and lipids [19, 80]. All of these conditions lead to a more efficient survival and infection by the bacterium [20].

On the first hours, the elementary bodies initiate to differentiate into reticular bodies. These forms of *Chlamydophila* are not infectious, have a non-condensed nucleoid and a greater size than EB [19, 65]. From eight to ten hours of cycle, the reticular bodies start to replicate by binary fission, remaining in contact with the inclusion membrane [20, 65]. In this moment, it's possible to see numerous mitochondria surrounding the inclusion [18, 20], possibly due to protein motors function as kinesins [81]. *Cp. psittaci* may not be able to generate as much energy in the form of ATP as other species of the genus, so a deeper association with the host cell mitochondria is necessary for the bacteria to recruit the necessary ATP by an alter‐ native mechanism [20]. The migration of nutrients from the host cell into the inclusion is es‐ sential. Amino acids and nucleotides cross the inclusion membrane [20, 82]. Since the vacuole is not in the vicinity of lysosomes leads to the assumption that the releasing of nu‐ trients by fusion with endocytic vesicles is unlikely [20]. It is known that exocytic vesicles containing sphingomyelin fuse with the membrane of the inclusion, delivering nutrients and lipids to the bacteria [80]. It was demonstrated that the inclusion membrane is permeable to small molecules of a molecular weight ranging from 100-520 Da [83,84]. After releasing the nutrients into the lumen of the bacterium by passive diffusion, membrane-specific carriers/ transporters located in the membrane would facilitate the entry of new nutrients and would be indispensable for the entry of molecules larger than 520 Da, but such carriers have not been yet described [20].

Throughout the cycle, the size of the vesicles increases, as they accumulate growing num‐ bers of bacteria inside [20, 84]. The surface of the inclusion membrane also increases, while it intercepts biosynthetic transport pathways of the host cell membrane and acquires the abili‐ ty to fuse with a subset of Golgi derived vesicles [20, 84]. Within 20 hours of cycle, reticular bodies continue their replication and each body can give rise to a thousand of new bacteria [20]. As this process occurs, the inclusion becomes increasingly overloaded with bacteria and as such they are obligated to leave the inclusion [19, 20]. This may be the reason for the reticular bodies to turn into intermediate bodies and new infectious elementary bodies, which occurs about 36 hours after infection [19]. During the final stage, at 50 hours of cycle, the host cell and the inclusion undergo a process of lysis or, very often, the elementary bod‐ ies are released by reverse endocytosis [20], which leaves the host cell intact, allowing per‐ sistence of a chronic and silent infection [19].

After treatment with cytokines, antibiotics or restriction to particular nutrients [19, 85], this cycle may undergo a modification with the emergence of "persistent" bacteria [85-86]. This kind of bacterium does not complete its continuing transformation from reticular bodies to infectious elementary form. Instead, it remain with low metabolic activity [19]. These RB are morphologically aberrant, appearing with a dilated oval shape within inclusions of small size [20, 85-86]. Excessive accumulation of chromosomes is due to the continuous DNA rep‐ lication of the bacteria that lack the ability to divide [20]. These persistent forms of *Cp. psitta‐ ci* are associated with chronic infections, and it has been shown that aberrant reticular bodies can quickly develop into normal forms and subsequently into infective elementary bodies [85-86]. The way this bacteria model passes through such transformations between aberrant forms and normal forms of reticular bodies is not yet completely understood [75, 87-88].

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135

http://dx.doi.org/10.5772/53720

Parrots were the first animals linked to *Cp. psittaci* in 1929-1930 and 1930-1938 when they were confirmed to be the source of outbreaks of psittacosis [33]. However, thereafter it became clear that this disease was not confined to these birds. In 1939, the agent was isolated from two pigeons in South Africa and later two new cases of psittacosis arose in citizens who contacted with pigeons in the U.S. [89]. Ducks and turkeys are given as possible sources of infection since the early fifties, although in the seventies there was a large decline in the prevalence of bacteria, but without ever ceasing [20]. Confirmation of new U.S. cases of chlamydiosis in turkeys in the following decade [20] and in Europe, al‐ ready in the nineties [90,91], proved the continuing problematic status of this disease. In the last thirty years it was reported several cases in humans who have contracted the disease through direct contact with ducks [92-95] and more recently the number of these

Studies on *Chlamydophila'*s prevalence increased and psittacosis was classified as an en‐ demic disease in Belgium [97-98] and other European countries, such as France and

Psittacines and pigeons are the most disturbing cases, being the range of prevalence within the first, 16% to 81% and mortality frequently above 50% [99-101]. Other studies have shown that parrots are the largest sources of *Cp. psittaci*, especially when they are

The wild pigeons have a broad seropositivity that ranges from 12.5% up to 95.6%. These da‐ ta were obtained from studies conducted from 1966 to 2005 [20,104-105]. The seropositivity in this species is alarming, once they live in urban and rural areas throughout the world, in close contact with human [60]. Carrier pigeons are reported as having a lower seropositivity

Kaleta and Taday (2003) reported that seabirds are more often infected with *Cp. psittaci* than other birds like chickens, quails and pheasants, but virtually all species of birds can

**3. Epidemiology**

cases increased dramatically [96].

than wild pigeons, between 35.9 and 60% [20].

Germany [97].

in captivity [102-103].

**Figure 1.** Chlamydia life cycle. Infection begins with the attachment of the elementary bodies (EB) to the surface of target epithelial cells. These cells promote a pseudopod formation to engulf the EB. Inside the cytoplasm this bacterium inhibits the fusion of the vesicle with the cell lysosomes. The nascent inclusion is accompanied by the transition from EBs to reticu‐ late bodies (RB). Late in the cycle, RBs replicate by binary fission to generate both RBs and intermediate bodies (IB). At this stage, antigenic proteins are exposed into the cell surface. An elongated, aberrant RB could be formed at this time with an arrest on chlamydia cycle originating a persistent infection, or continuing the cycle. The various intracytoplasmic inclusions with bacterium inside, can also be fused in this phase, and the agent develop into intermediate bodies (IB), before DNA condensation and RB transformation into a newly EB. The mature inclusion increases in size with EB formation, until be‐ coming infectious and released into the extracellular space to continue a new intracellular cycle. N – nucleus; G – Golgi ap‐ paratus: EB – elementary bodies; RB – reticulate bodies; IB – intermediate bodies.

After treatment with cytokines, antibiotics or restriction to particular nutrients [19, 85], this cycle may undergo a modification with the emergence of "persistent" bacteria [85-86]. This kind of bacterium does not complete its continuing transformation from reticular bodies to infectious elementary form. Instead, it remain with low metabolic activity [19]. These RB are morphologically aberrant, appearing with a dilated oval shape within inclusions of small size [20, 85-86]. Excessive accumulation of chromosomes is due to the continuous DNA rep‐ lication of the bacteria that lack the ability to divide [20]. These persistent forms of *Cp. psitta‐ ci* are associated with chronic infections, and it has been shown that aberrant reticular bodies can quickly develop into normal forms and subsequently into infective elementary bodies [85-86]. The way this bacteria model passes through such transformations between aberrant forms and normal forms of reticular bodies is not yet completely understood [75, 87-88].

## **3. Epidemiology**

bodies continue their replication and each body can give rise to a thousand of new bacteria [20]. As this process occurs, the inclusion becomes increasingly overloaded with bacteria and as such they are obligated to leave the inclusion [19, 20]. This may be the reason for the reticular bodies to turn into intermediate bodies and new infectious elementary bodies, which occurs about 36 hours after infection [19]. During the final stage, at 50 hours of cycle, the host cell and the inclusion undergo a process of lysis or, very often, the elementary bod‐ ies are released by reverse endocytosis [20], which leaves the host cell intact, allowing per‐

**Figure 1.** Chlamydia life cycle. Infection begins with the attachment of the elementary bodies (EB) to the surface of target epithelial cells. These cells promote a pseudopod formation to engulf the EB. Inside the cytoplasm this bacterium inhibits the fusion of the vesicle with the cell lysosomes. The nascent inclusion is accompanied by the transition from EBs to reticu‐ late bodies (RB). Late in the cycle, RBs replicate by binary fission to generate both RBs and intermediate bodies (IB). At this stage, antigenic proteins are exposed into the cell surface. An elongated, aberrant RB could be formed at this time with an arrest on chlamydia cycle originating a persistent infection, or continuing the cycle. The various intracytoplasmic inclusions with bacterium inside, can also be fused in this phase, and the agent develop into intermediate bodies (IB), before DNA condensation and RB transformation into a newly EB. The mature inclusion increases in size with EB formation, until be‐ coming infectious and released into the extracellular space to continue a new intracellular cycle. N – nucleus; G – Golgi ap‐

paratus: EB – elementary bodies; RB – reticulate bodies; IB – intermediate bodies.

sistence of a chronic and silent infection [19].

134 Insights from Veterinary Medicine

Parrots were the first animals linked to *Cp. psittaci* in 1929-1930 and 1930-1938 when they were confirmed to be the source of outbreaks of psittacosis [33]. However, thereafter it became clear that this disease was not confined to these birds. In 1939, the agent was isolated from two pigeons in South Africa and later two new cases of psittacosis arose in citizens who contacted with pigeons in the U.S. [89]. Ducks and turkeys are given as possible sources of infection since the early fifties, although in the seventies there was a large decline in the prevalence of bacteria, but without ever ceasing [20]. Confirmation of new U.S. cases of chlamydiosis in turkeys in the following decade [20] and in Europe, al‐ ready in the nineties [90,91], proved the continuing problematic status of this disease. In the last thirty years it was reported several cases in humans who have contracted the disease through direct contact with ducks [92-95] and more recently the number of these cases increased dramatically [96].

Studies on *Chlamydophila'*s prevalence increased and psittacosis was classified as an en‐ demic disease in Belgium [97-98] and other European countries, such as France and Germany [97].

Psittacines and pigeons are the most disturbing cases, being the range of prevalence within the first, 16% to 81% and mortality frequently above 50% [99-101]. Other studies have shown that parrots are the largest sources of *Cp. psittaci*, especially when they are in captivity [102-103].

The wild pigeons have a broad seropositivity that ranges from 12.5% up to 95.6%. These da‐ ta were obtained from studies conducted from 1966 to 2005 [20,104-105]. The seropositivity in this species is alarming, once they live in urban and rural areas throughout the world, in close contact with human [60]. Carrier pigeons are reported as having a lower seropositivity than wild pigeons, between 35.9 and 60% [20].

Kaleta and Taday (2003) reported that seabirds are more often infected with *Cp. psittaci* than other birds like chickens, quails and pheasants, but virtually all species of birds can contract the disease, even without apparent symptoms, such as for instance, the cranes and seagull. Psittacosis in arthropods can be detected, however without initiation of in‐ fection in these animals [106].

General clinical signs of disease and highlights of the most common within the species are listed on table 1, being generally flu-like symptoms, CNS disorders, pericarditis, sacculitis

**Psittacines** Anorexia, diarrhoea, difficulty breathing, sinusitis, conjunctivitis, yellowish droppings and, perhaps,

Chronic Infection - lameness, stiff neck, opisthotonos, tremor and convulsions.

production, conjunctivitis, sinusitis, sneezing and mortality between 10 and 30%.

*Chlamydophila psittaci* post-mortem lesions are not specific enough to be able to differentiate this disease from other systemic diseases [113]. The severity of injury depends on several factors including the virulence of serotype, the susceptibility and age of the host, the form

In most cases of psittacosis, lesions are limited to three structures: spleen, liver and air sacs [113]. Table 2 summarizes the post-mortem lesions in these specific organs and points addi‐

**Clinical signs**

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Difficulty breathing, fever, lethargy, anorexia, ruffled feathers, diarrhoea, oral and nasal discharges, decreased egg laying, polyuria, pericarditis, sacculitis, pneumonia, lateral nasal adenitis, peritonitis,

Acute Infection - anorexia, diarrhoea, conjunctivitis, rhinitis, swollen eyelids and a decrease in flight

D serotype of the bacteria - Anorexia, cachexia, diarrhoea gelatinous yellow-green, low egg

Agitation, unsteady gait, conjunctivitis, serous to purulent nasal discharge and depression.

and occasionally shock and death, signs of infection [18, 20,113].

hepatitis and splenitis, occasionally shock and death.

Signs appear only when there is another competitor disease.

B serotype of the bacteria - Anorexia and green manure

CNS disorders.

performance.

**Ducks** It affects mostly the young ones.

**Chickens** Blindness, anorexia and occasionally death.

**Table 1.** General clinical signs of disease (adapted from [18,20,113].

and time of exposure and the presence of concurrent diseases [18].

tional possible lesions of *Cp. psittaci* found in animals [18, 108,113].

**Overall**

**Species**

**Pigeons**

**Turkeys**

Although the horizontal transmission is the most common way of infection [24], the vertical transmission was also described [18].

*Chlamydophila psittaci* are most excreted in the faeces [21, 107], nasal [12] and oral dis‐ charges of infected animals [108]. Sareyyupoglu and collaborators (2007) indicates that excretion of the bacteria may occur intermittently in sub-clinical infections for a long time, being activated in situations of stress such as transportation and handling [21] or nutritional restrictions and egg laying [108]. Bacteria are found in the powder of feathers [106], in excrements, secretions and respiratory exudates from infected animals, and when dry, become spread in the air [20-21] being the aerosol transmission extremely fea‐ sible [109]. The contact of non infected animals with bacteria or its proximity to animals with *Cp. psittaci* gain greater importance, being this an essential criteria for the develop‐ ment of new infections [22].

Animals become infected by ingestion [110] or inhalation of the bacteria [13, 60], and the iso‐ lation of this agent is much more substantial from choanal and throat swabs collection rath‐ er than from faeces collection [111], especially in the early stages of the disease [20]. Thus, contamination by aerosol exudates must be considered the primary form of infection [89].

Birds who share contaminated water are also susceptible of infection [18], as well as predators eating carcasses contaminated with *Cp. psittaci* [108]. The nest environment is also very susceptible to disease transmission, since this is a place where are deposited loads debris that may contain multiple bacteria [20]. Granivorous animals, such as pi‐ geons or pheasants, that are often found in corrals or stables contaminated with faeces, and also grain storage areas can become infected by inhalation of dust from grain or from aerosols of faeces [18, 20].

Ectoparasites such as fleas, mites and lice may also serve as vectors for the transmission of disease from animal to animal [112].

## **4. Clinical signs and pathology**

After 4 hours of infection via aerosol, bacteria can be found in the respiratory system of the animal [113].

This disease may be acute, sub-acute, chronic or subclinical [113], being the last one found when the animal is showing no signs of disease, and elimination of the bacteria can occur intermittently due to stress [21], inadequate nutrition or other diseases [108]. These animals are persistently infected and named as "source of infection" to other animals [20]. The acute form, in turn, is a generalized form, affecting all the organs of the animal [113].

General clinical signs of disease and highlights of the most common within the species are listed on table 1, being generally flu-like symptoms, CNS disorders, pericarditis, sacculitis and occasionally shock and death, signs of infection [18, 20,113].

contract the disease, even without apparent symptoms, such as for instance, the cranes and seagull. Psittacosis in arthropods can be detected, however without initiation of in‐

Although the horizontal transmission is the most common way of infection [24], the vertical

*Chlamydophila psittaci* are most excreted in the faeces [21, 107], nasal [12] and oral dis‐ charges of infected animals [108]. Sareyyupoglu and collaborators (2007) indicates that excretion of the bacteria may occur intermittently in sub-clinical infections for a long time, being activated in situations of stress such as transportation and handling [21] or nutritional restrictions and egg laying [108]. Bacteria are found in the powder of feathers [106], in excrements, secretions and respiratory exudates from infected animals, and when dry, become spread in the air [20-21] being the aerosol transmission extremely fea‐ sible [109]. The contact of non infected animals with bacteria or its proximity to animals with *Cp. psittaci* gain greater importance, being this an essential criteria for the develop‐

Animals become infected by ingestion [110] or inhalation of the bacteria [13, 60], and the iso‐ lation of this agent is much more substantial from choanal and throat swabs collection rath‐ er than from faeces collection [111], especially in the early stages of the disease [20]. Thus, contamination by aerosol exudates must be considered the primary form of infection [89].

Birds who share contaminated water are also susceptible of infection [18], as well as predators eating carcasses contaminated with *Cp. psittaci* [108]. The nest environment is also very susceptible to disease transmission, since this is a place where are deposited loads debris that may contain multiple bacteria [20]. Granivorous animals, such as pi‐ geons or pheasants, that are often found in corrals or stables contaminated with faeces, and also grain storage areas can become infected by inhalation of dust from grain or

Ectoparasites such as fleas, mites and lice may also serve as vectors for the transmission of

After 4 hours of infection via aerosol, bacteria can be found in the respiratory system of the

This disease may be acute, sub-acute, chronic or subclinical [113], being the last one found when the animal is showing no signs of disease, and elimination of the bacteria can occur intermittently due to stress [21], inadequate nutrition or other diseases [108]. These animals are persistently infected and named as "source of infection" to other animals [20]. The acute

form, in turn, is a generalized form, affecting all the organs of the animal [113].

fection in these animals [106].

136 Insights from Veterinary Medicine

ment of new infections [22].

from aerosols of faeces [18, 20].

disease from animal to animal [112].

**4. Clinical signs and pathology**

animal [113].

transmission was also described [18].

**Table 1.** General clinical signs of disease (adapted from [18,20,113].

*Chlamydophila psittaci* post-mortem lesions are not specific enough to be able to differentiate this disease from other systemic diseases [113]. The severity of injury depends on several factors including the virulence of serotype, the susceptibility and age of the host, the form and time of exposure and the presence of concurrent diseases [18].

In most cases of psittacosis, lesions are limited to three structures: spleen, liver and air sacs [113]. Table 2 summarizes the post-mortem lesions in these specific organs and points addi‐ tional possible lesions of *Cp. psittaci* found in animals [18, 108,113].

The samples for subsequent isolation of *Cp. psittaci* must be handled carefully to prevent the loss of infectivity of bacteria during transport and handling, but also due to its high zoonotic potential [89]. Thus, the safety of the operator must be respected (use of gloves, gown and mask) and the samples and swabs are placed in SPG (sucrose phosphate glutamate) trans‐ port medium, which is also suitable for bacteria of the Rickettsia genus [89, 114]. SPG is

Psittacosis

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http://dx.doi.org/10.5772/53720

SPG is also used for samples dilution and freezing, if they will be processed with adelay of four or more days after harvest; otherwise samples should not be frozen [89, 114]. Freezing of material must be performed within the first 24 hours, in a stabilized 7.2 pH phosphatebuffered saline medium and kept at a minimum temperature of -20 ° C [89, 114]. Spencer and Johnson (1983) report that *Cp. psittaci* can survive in SPG to just over 30 days at 4 ° C, but their infectivity decreased by 1.55% [115]. Detection of *Cp. psittaci* in the blood, liver, spleen and kidney is only possible two whole days after, whereas in faeces is only possible

*Chlamydophila psittaci* can be identified by direct observation of the organism using different staining techniques such as the Giemsa, Stamp and Gimenez stainings [116]. Giemsa stain‐ ing could be performed from faeces, exudates, or liver and spleen cytologies [20]. Although cytological staining can be useful and quick, it is less sensitive and less specific than immu‐ nochemical staining or molecular detection methods [18, 20]. With Giemsa staining the methanol fixed material can be observed under the microscope. Inside the cells a basophilic inclusion composed by EB and IB located near the nucleus is found in infected cells [117]. Hayashi visualized bacteria using this technique, once isolated from organs such as liver, spleen, kidney, heart and intestine [118]. This technique is a powerful tool in the *Chlamydia*

This staining is often used, although it is not specific to this bacterium and has reduced sen‐ sitivity. Other agents beyond *Chlamydophila* (for instance, *Coxiella burnetii, Helicobacter pylori*)

are stained red with Gimenez staining in contrast to background in light green [119].

Sucrose 74.6 g / L K2HPO4 1.237 g / L Glutamic Acid 0.721 g / L Fetal calf serum 10% Streptomycin 100 µg / mL Vancomycin 100 µg / mL Nnystatin 50 µg / mL Gentamicin 50 µg / mL

formed according to Table 3 [115].

**Table 3.** Composition of SPG [115].

beyond the 72nd hour [113].

Giemsa cytological staining

Gimenez staining

**b.** *Chlamydophila psittaci* identification

diagnosis, but it requires a vast experience from the observer.

**Table 2.** Post-mortem lesions in specific organs (Adapted from [18,108,113]).

## **5. Diagnostic techniques**

There are several available methods for the diagnosis of *Chlamydophila psittaci*, including the agent direct visualization with a specific staining technique, isolation of the agent followed by agent identification, detection of bacteria specific antigen or genes in samples and, final‐ ly, serology tests that identify antibodies against this organism [20,89].

**a.** Harvesting and storage of samples

Sampling should be carried out aseptically to avoid contaminating bacteria [89]. At nec‐ ropsy, the main structures to isolate this bacterium are the air sacs, spleen, pericardium, heart, areas of hyperemia of the intestine, liver and kidney. On live animals the choanal, oropharyngeal and cloacal swabs are the principal material for *Chlamydophila psittaci* search [52,89,111]. Other samples can be taken from blood, conjunctiva and peritoneal exudates [20].

The samples for subsequent isolation of *Cp. psittaci* must be handled carefully to prevent the loss of infectivity of bacteria during transport and handling, but also due to its high zoonotic potential [89]. Thus, the safety of the operator must be respected (use of gloves, gown and mask) and the samples and swabs are placed in SPG (sucrose phosphate glutamate) trans‐ port medium, which is also suitable for bacteria of the Rickettsia genus [89, 114]. SPG is formed according to Table 3 [115].

#### **Table 3.** Composition of SPG [115].

**Spleen**

138 Insights from Veterinary Medicine

**Liver**

**Air Sacs**

**Injuries and/or changes in other**

exudates [20].

**organs**

**Size**: Increased (splenomegaly).

**Size**: Increased (hepatomegaly).

other organs show no alterations

**Colour**: Colour ranging from yellow to green.

mononuclear cell and heterophils infiltrate.

with fibrinous or fibrinopurulent exudate.

**Kidney** Acute necrosis and nephritis with inflammatory infiltrate of mixed type.

Pneumonia although not frequent is possible.

ly, serology tests that identify antibodies against this organism [20,89].

Infectious pericarditis, myocarditis, adenitis and peritonitis.

Inflammation of adrenals and gonads sporadically found.

Medium increase in granulocytic cells series in spinal cord.

There are several available methods for the diagnosis of *Chlamydophila psittaci*, including the agent direct visualization with a specific staining technique, isolation of the agent followed by agent identification, detection of bacteria specific antigen or genes in samples and, final‐

Sampling should be carried out aseptically to avoid contaminating bacteria [89]. At nec‐ ropsy, the main structures to isolate this bacterium are the air sacs, spleen, pericardium, heart, areas of hyperemia of the intestine, liver and kidney. On live animals the choanal, oropharyngeal and cloacal swabs are the principal material for *Chlamydophila psittaci* search [52,89,111]. Other samples can be taken from blood, conjunctiva and peritoneal

**Consistency**: soft.

**Consistency**: friable.

surfaces.

Brain injuries are rare.

**Table 2.** Post-mortem lesions in specific organs (Adapted from [18,108,113]).

**Intestine** Lymphocytic enteritis.

**5. Diagnostic techniques**

**a.** Harvesting and storage of samples

**Colour:** blackish, sometimes with greyish white necrotic foci and petechial haemorrhages.

Increased macrophages numbers and decreased number of lymphocytes.

Increased activity of Kupffer cells, stimulated by hemosiderin accumulation.

Perivascular sheaths of macrophages in the arterioles transforming the architecture of the organ.

**Acute infection** – often the presence of a multifocal necrosis is the only sign of disease, and the

**Subacute or chronic infections** - hyperplasia of the biliary ducts and sinus histiocytosis with

**Acute and subacute infections** – changes in thickness of the membranes that were covered

Chronic infections - presence of pyogranulomatous infections or diffuse granulomas on serous

SPG is also used for samples dilution and freezing, if they will be processed with adelay of four or more days after harvest; otherwise samples should not be frozen [89, 114]. Freezing of material must be performed within the first 24 hours, in a stabilized 7.2 pH phosphatebuffered saline medium and kept at a minimum temperature of -20 ° C [89, 114]. Spencer and Johnson (1983) report that *Cp. psittaci* can survive in SPG to just over 30 days at 4 ° C, but their infectivity decreased by 1.55% [115]. Detection of *Cp. psittaci* in the blood, liver, spleen and kidney is only possible two whole days after, whereas in faeces is only possible beyond the 72nd hour [113].

#### **b.** *Chlamydophila psittaci* identification

#### Giemsa cytological staining