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NOTICE
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This report is free to be used by anyone, but, please, give credit to the people who did the original research. There is a literature cited section (with links, where applicable) at the end for that purpose.
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Establishing an Effective Testing Method for the
Detection of C. psittaci in Avian Subjects
by Terry Simmers Jr.
Advanced Placement Biology
Waynesboro Area Senior High School
Mr. Dennis Bechard
Abstract
Current tests for the presence and classification of Chlamydia strains are unreliable and often difficult to carry out effectively (Dalhausen and Radabaugh, undated). Absence of simple, reliable tests for this infection, specifically C. psittaci, has led to a race in the development of one. Some of the methods currently under investigation for their accuracy and ease of use include biological, morphological, and DNA hybridization. The investigation of these methods has proved little more than futile, causing some scientists to reevaluate previous methods and try to find applicable solutions to their downfalls. The work of Bendheim, Naveh, and Karen (undated) shows the reliability and versatility of the ELISA IC test, which is currently the most widely used.
Chlamydia is classified as a zoonotic infection (affects and is able to be spread by humans and other primates as well as at least one other order of the animal kingdom) (United States Department of Health and Human Services, 1997). This extreme versatility of a potentially dangerous infection is feared and generally questioned by members of the scientific profession. Some of the methods for detection currently under investigation, such as genetic analysis, could prove useful for one species, but inadequate for the next (Everett and Andersen, 1997). Because of this, a method for verification is needed which relies on chemical or physical rather than genetic presence.
Originally thought to be different than those affecting humans, the C. psittaci strands of this bacteria have recently been reclassified as members of the species C. pneumoniae (Everett and Andersen, 1997). Organisms closely related to Chlamydia, such as Simkania, have been discovered and morphologically classified into the same genus as Chlamydia. This reclassification (along with their ability to affect humans) has put them into the same genus as the strains of Chlamydia long known for causing symptoms similar to that of pneumonia. With this reclassification and new knowledge comes an inherent fear of the disease and an associated rush to find an effective method of detection.
Chlamydiosis, also referred to as Psittacosis, reproduces by infecting a host cell (Dahlhausen and Radabaugh, date unknown). Generally, the first area involved in this infection is the lining of the digestive or respiratory tracts. During this localized phase, the chlamydial organism enters the host cell, undergoes a translation, and produces between 100-500 chlamydial bodies per cell. Approximately 48 hours after infection the cell may lyse, releasing the chlamydial bodies into the area around the cell where they each find new cells and begin the process over again. There are occasions when lysing does not occur, leaving a cell which becomes chronically infected for long periods of time. The time between infection and clinical disease can vary from as little as 42 days to as much as several years. Diagnosis of incubation periods is difficult due to the persistent, asymptomatic infections and the dormant nature of this bacteria.
Another major problem in the diagnosis of Chlamydiosis is the large number of symptoms which it can be directly responsible for (The Tufts Veterinary Diagnostic Lab Newsletter, 1996). These symptoms include: 1) a chronic infection with respiratory or intestinal signs, 2) an acute infection often causing death, 3) a cyclic infection with recurring illness and remission, and 4) a carrier state where no disease is apparent, but the bird may still be shedding the organism. In humans (where the disease caused by C. psittaci is referred to as psittacosis, parrot fever or parrot disease due to the natural ability of all of the strains of chlamydia to infect and affect humans), it causes symptoms similar to those of a fever and abnormal respiratory difficulties resulting from an infection within the lungs (See Figure 1, note the blurring caused by the density of the infection) which, if gone untreated, may eventually become deadly. Due to this zoonotic potential, it is important to attain early diagnosis.
In a 1994 study of known carriers of the bacteria conducted by Andersen, pharyngeal swabs proved to be the most effective method for the isolation of C. psittaci in avian subjects. This method produced an 80.4% detection rate in cockatiels and a 93.1% detection rate in turkeys. The less effective method studied, cloacal swabs, only produced a 33.3% detection rate in cockatiels and a 77.7% detection rate in turkeys. Because none of these methods yielded a 100% detection rate, Andersen suggests using multiple forms of detection for more reliable results.
In a study conducted by Dahlhausen and Radabaugh (1999), a commercially available test offered by Research Associates Laboratory (RAL) was evaluated to try to determine the infection time in a typical psittacine, in this case the cockatiel. The RAL test was developed to quantify the amplification product derived from a conserved major outer membrane protein (MOMP) gene segment in the avian strands of C. psittaci. The study was conducted on six cockatiels selected based on their normal findings in examination, choanal and cloacal cultures, complete blood counts, and direct and flotation fecal examination. The birds were placed in a controlled infectivity environment where they were all infected in the same manner at the same time. Tests using the RAL molecular-based assay test were conducted on days 5, 10, and 15 after inoculation. On day 5, all the birds showed positive tests on the choanal swabs and negative on the cloacal and blood tests. On day 10, all birds showed positive on the choanal swabs, two birds showed positive tests on the cloacal swabs, and all still showed negative on the blood tests. On day 15, all birds showed positive on the choanal, cloacal, and blood test results. The fact that all the birds retained a negative blood test result on days 5 and 10 probably means that the infection was still in its localized phase and had not yet become systematic. With all the samples showing positive for all birds on day 15, it can be assumed that the infection had become systematic and viable. This study relied heavily on the effectivenessof the tests.
In a 1993 study by Bendheim, Keren, and Naveh, cloacal swabs and blood samples were obtained from eighty-two psittacine birds, forty-eight of which were suspected to be infected with C. psittaci based on clinical symptoms including oculo or nasal discharge, sinusitis, and/or air sacculitis. These samples were tested using an Immunocomb (IC) enzyme linked immunosorbent assay methods (ELISA) test and immunoflourescence (IF) microscopy. ELISA tests rely on the binding of a monoclonal antibody to a specific antigen of the chlamydial organism. Immunoflourescence tests rely on the combination of chlamydial bodies with flourescein-staining which will identify elementary bodies in a test sample. The samples collected for use with the IC ELISA test were sent to Biogal Galed Labs for IC testing to be completed in the Chlamydia Research & Development Laboratory. development of the tests and the reading of the results were done according to the enclosed instruction manual. Tests which yielded a color reaction exceeding that of the negative control (see Table 4)were considered positive (+). A result that was comparative to the negative control was considered negative (-). Results from the tests of the forty-eight suspected carriers are shown in Table 1. The sensitivity of this test was 94.6% ( 35/37 x 100% ) and the specificity was 81.1% ( 9/11 x 100% ).
The remaining thirty-four samples were collected from birds having no outward appearance suggesting infection with C. psittaci (Bendheim, Naveh, and Karen, 1993). The results from these tests can be viewed in Table 2. The sensitivity of this test was 100% ( 3/3 x 100%) and the specificity was 83.9% ( 26/31 x 100%).
A summation of tables one and two is available in table three (Bendheim, Naveh, and Karen, 1993), including results from all eighty-two birds. The results of this study were compiled after the results for each test were reported independently (this was done to ensure an accurate and varied evaluation environment). The overall sensitivity of the test was 95.0% ( 38/40 x 100% ), leaving a 5.0% “false” negative result. The overall specificity of the test was 83.3% ( 35/42 x 100% ), leaving a 16.7% “false” positive result.
In comparing these figures to those of other tests, it is important to remember that the ELISA IC and IF tests are testing two different pathophysiologic parameters of infection which are not always accompanied by one another (Bendheim, Naveh, and Karen, 1993). In the case of the IF test, the only manner in which the disease is correctly identified is if the animal is currently shedding the organism (United States Department of Health and Human Services, 1997). This is usually the result of extreme temperatures, overcrowding, transport, under-abundance of food or other maladaptive or otherwise stressful environmental conditions. In the case of the ELISA IC test, a considerable number of the results will falsely read negative due to a cross-reaction of the testing agents with other bacteria in the environment.
Since it’s introduction into Israel and Europe in 1995, the ELISA IC test kit has become more widely used by veterinarians for diagnosing the Chlamydia bacteria (Bendheim, Naveh , and Karen, 1993). This kit has been found to be a reliable test for many psittacine and non-psittacine species as seen in Table 4. It seems interesting to note that the psittacines with greater intensities are all of African and South American origin, while those with lesser intensities are all of Australian and Southern Pacific origin (personal observance, 1999).
The ELISA test is the easiest to use (in comparison with its accuracy) of all the tests which have currently been developed or are in development that do not rely on the DNA of the specimen in question to diagnose the presence of C.psittaci (United States Department of Health and Human Services, 1997). For these reasons, it has become the more widely used test in avian veterinary applications. The effective detection of this infection could be an important link to the prevention of it in humans and subsequently be an important step to its elimination in urban areas where it is a problem due to the prevalence of pigeon feces in areas which are frequently occupied by humans.
Figure 1. X-ray of 35-year-old female infected with Chlamydiosis (Maddison, 1998).
Table 1. Results from the tests of suspected carriers of C. psittaci (after Bendheim, Naveh, and Karen, 1993)
| Immunocomb |
Immunoflourescence (+) |
Immunoflourescence (-) |
Totals |
| + |
35 |
2 |
37 |
| - |
2 |
9 |
11 |
| Totals |
37 |
11 |
48 |
Table 2. Results from the tests of unsuspected carriers of C. psittaci (after Bendheim, Naveh, and Karen, 1993).
| Immunocomb |
Immunoflourescence (+) |
Immunoflourescence (-) |
Totals |
| + |
3 |
5 |
8 |
| - |
0 |
26 |
26 |
| Totals |
3 |
31 |
34 |
Table 3. Cumulative results from the testing for C. psittaci (after Bendheim, Naveh, and Karen, 1993).
| Immunocomb |
Immunoflourescence (+) |
Immunoflourescence (-) |
Totals |
| + |
38 |
7 |
45 |
| - |
2 |
35 |
37 |
| Totals |
40 |
42 |
82 |
Table 4. Intensity of testing on psittacine and non-psittacine birds (after Bendheim, Naveh, and Karen, 1993)
| Psittacine Birds |
Color Intensity (*) |
Non-Psittacine Birds |
Color Intensity (*) |
| African Grey Parrot |
+++ |
Turkey |
+++ |
| Macaw |
+++ |
Peacock |
+++ |
| Timneh Grey Parrot |
+++ |
Pheasant |
+++ |
| Conure |
+++ |
Guinea Fowl |
+++ |
| Amazon Parrot |
+++ |
Ostrich |
++ |
| Cockatoo |
+++ |
Quail |
++ |
| Rosella |
+++ |
Mynah Bird |
++ |
| Lovebird |
++ |
Owl |
++ |
| Parakeet (Budgerigar) |
++ |
Black Kite |
++ |
| Princess Parakeet |
++ |
Vulture |
++ |
| Lorikeet |
++ |
Toucan |
++ |
| Cockatiel |
++ |
Pigeon |
+ |
|
|
Pelican |
+ |
|
Swan |
+ |
|
Eagle |
+ |
|
Starling |
+ |
(*) = Note: The color intensity is stated in comparison with the African Grey Parrot.
Literature Cited
Andersen, Arthur A. (1994). Comparison of Pharyngeal, Fecal, and Cloacal Samples for the Isolation of Chlamydia psittaci from Experimentally Infected Cockatiels and Turkeys [Online]. [Abstract]. Available: here [1999, January 5].
Bendheim, U., A. Naveh, and E. Karen. (no date). Antibody testing for Chlamydia psittaci using a rapid ELISA-KIT. In University of Georgia College of Veterinary Medicine Website [Online]. Available: here [1999, January 7].
Dahlhausen, B. and Radabaugh, C. S. (no date). Detection of Chlamydia psittaci Infection in Pet Birds Using a Molecular-Based Diagnostic Assay [Online]. Available: here [1999, January 9].
Everett, K. D. E. and A. A. Andersen. 1997. The Ribosomal Intergenetic Spacer and Domain I of the 23S rRNA Gene Are Phylogenic Markers for Chlamydia spp. International Journal of Systematic Bacteriology 47(2):461-473.
Maddison, Ian. (1998). In Skiagram - the plain film site [Online]. Available: here [1999, January 12].
Tufts Veterinary Diagnostic Lab Newsletter, The. (1996). Avian Psittacosis. (Winter 1996), [Online]. Available: here [1999, January 13].
United States Department of Health and Human Services. (1997). Compendium of psittacosis control, 1997. Morbidity and Mortality Weekly Report. [Online]. 46(28):1-13. Available: here [1999, February 6].
© 1999, Terry L. Simmers Jr.
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