ATOXOPLASMA MEDICAL PROTOCOLS RECOMMENDED BY THE PASSERINE ATOXOPLASMA WORKING GROUP
Terry M. Norton, DVM, Dipl. ACZM1, Donald L. Neiffer, VMD2, Bob Seibels9, Keith Benson, DVM9, Denise McAloose, VMD, Dipl. ACVP6, Dominic Travis, DVM, MS10, Ellis Greiner, PhD3, Kenneth Latimer, DVM, PhD4, Dipl. ACVP, Susan E. Little, DVM, PhD5, Jacqueline M. Zdziarski, DVM7, Mark Schrenzel, DVM, PhD, Dipl. ACVP8, Bruce Rideout, DVM, PhD, Dipl. ACVP8, Martin Vince9, Ned Gentz, DVM, Dipl. ACZM11
1Wildlife Conservation Society, St. Catherine Island Wildlife Survival Center,
182 Camelia Road, Midway, GA 31320, Ph: 912-884-5005, Fax: 912-884-5007,
10,000, Lake Buena Vista, Florida 32830, Ph: 407-938-2719, Fax: 407-939-6391,
3University of Florida, Department of Pathobiology, College of Veterinary Medicine, 2015 SW 16th Ave, Building 1017, Rm V3-240, Gainesville, Florida 32610, Ph: 352-392-4700 ext. 5861, Fax: 352-392-8351, E-mail: email@example.com
4University of Georgia, College of Veterinary Medicine, Department of Pathology, Athens, GA 30602, Ph: 706-542-5844, Fax: 706-542-5828, Email: firstname.lastname@example.org;
5Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602, Ph: 706-542-8447,
706-542-0059, Email: email@example.com
6Wildlife Conservation Society/Bronx Zoo, Department of Pathology, 2300 Southern Boulevard, Bronx, NY 10460-1090, Email: firstname.lastname@example.org
7Brookfield Zoo/Chicago Zoological Society, Department of Animal Health, 3300 Golf Road, Brookfield, IL 60513 1095, Email: email@example.com, Ph: 708-485-0262 ext. 504
8San Diego Zoo, Department of Pathology, 2920 Zoo Drive, San Diego, CA 92101, Email: firstname.lastname@example.org
9Riverbanks Zoo and Botanical Garden, 500 Wildlife Parkway, Columbia, SC 29210, email@example.com, firstname.lastname@example.org, email@example.com
10Davee Center for Veterinary Epidemiology, Lincoln Park Zoo, 2001 N. Clark St., Chicago, IL 60614, 312-742-7225 firstname.lastname@example.org
11Albuquerque Biological Park, 903 10th Street SW, Albuquerque, NM 87102-4029, Email: email@example.com, Ph: 505-764-6261.
The following document is the edited output from a workshop on Atoxoplasma held at the Eastern Regional AZA conference in Columbia, SC on March 27, 2003. Participants included a panel of veterinarians, researchers, and avian curators, each of whom gave a presentation focusing on a particular aspect of the organism: etiology; diagnosis; treatment; and implications for captive avian management programs. The stated goal of the workshop was to produce a “white paper” summarizing the most current information available on Atoxoplasma. It is the hope of the authors that this document will encourage both veterinarians and curators in North America to establish protocols with some measure of consistency to deal with what is now generally recognized as a ubiquitous disease.
Atoxoplasma constitutes an extremely difficult and controversial parasitic pathogen to manage in captive passerine birds. Currently, zoological institutions manage this parasite in a variety of ways, making it difficult to conduct long term reproduction and conservation programs in passerine bird species. The following recommendations and information represent an effort to bring some consistency to the management of Atoxoplasma in passerine bird species. Each institution should have a management team in place to evaluate the risks and benefits of bringing Atoxoplasma-positive birds into the collection. Treatment protocols discussed have been successful in managing Atoxoplasma in a variety of passerine species. Please refer to http://www.riverbanks.org/subsite/aig/baliopen.htm for more detailed information on atoxoplasmosis.
RECOMMENDATIONS FOR ATOXOPLASMA MANAGEMENT IN PASSERINE BIRDS
I. General comments
1. Any zoological institution housing passerines in the typical manner (natural substrates, planted enclosures, mixed species) probably should be considered to have Atoxoplasma in their collection. Although facilities may claim to be “Atoxoplasma free”, the more likely explanation is that effective screening for the organism has not been performed.
2. Atoxoplasma is present in all outside aviaries.
3. All parent-reared passerines should be considered to have been exposed to an Atoxoplasma contaminated environment whether captive hatched or wild-caught.
4. Indoor enclosures that have housed passerines (other than hand-reared passerines as described below) and have not been disinfected, including removal of substrate, are considered to be Atoxoplasma contaminated.
1. While the organism may be species specific, interspecies transmission certainly occurs.
2. There is concern that free-ranging North American passerine birds may be infecting some captive specimens housed outdoors.
3. Pilot Study: Comparative measurements of the oocysts of superb starling (Spreo superbus) and Bali mynah Atoxoplasma revealed that the superb starling oocysts were slightly smaller. A pilot study (Norton and Greiner, unpublished data) was conducted with 1 superb starling as a control bird and 1 superb starling as an experimental bird at North Carolina Zoological Park. The experimental bird was given the Bali mynah Atoxoplasma oocysts by gavage. The oocysts essentially passed through the gastrointestinal tract but did not disseminate to other parts of the body. Dexamethasone was given to the bird to cause immunosuppression and it was rechallenged, but the organism still just passed through the gastrointestinal tract and was not detected on serial buffy coat smears.
4. Further interspecies transmission studies are needed, with larger numbers of birds and more species, in order to gain more insight into the species specificity of this organism.
5. Species documented with Atoxoplasma (See Appendices and references for further information):
a. UGA Study: Amakihi, Apapane, Bali mynah, black-headed weaver, blue dacnis, blue-faced honeyeater, canary, fairy bluebird, golden crested mynah, honeycreepers (purple, red-legged), liwi, house sparrow, Montezuma oropendula, starling (emerald, golden breasted, superb), tanagers (blue gray, green and gold, paradise, rufous-crowned, scarlet rumped, silver beaked, turquoise), violaceous euphonia.
b. Brookfield Zoo Study: 18 tanager species (9 Genera) were included in this study
c. Disney Study: golden-breasted starling and golden crested mynah
d. Greiner’s Study: Sturnidae: Bali mynah, superb starling, gold breasted starlings, gold crested mynah, Indian hill mynah; Timaliidae: black-throated laughing thrush, yellow-throated laughing thrush; Ploceidae: black-headed weaver; Troglodytidae: cactus wren; Thraupidae: silver-beaked tanager; Pittidae: hooded pitta
6. Species-specific susceptibility to the organism
i. There are probably numerous other examples of morbidity and mortality variability in closely related passerine species.
ii. One example of species variation in susceptibility has been noted by the staff at Disney’s Animal Kingdom. Golden-breasted starlings have experienced mortality from the organism, whereas the golden crested mynah have not.
1. The effect of atoxoplasmosis on a given collection depends on the species housed; reproductive efforts/programs for the particular species; facility design and maintenance; and enclosure location (indoor or outdoor). Consequently, no two institutions should be expected to have identical plans or protocols for managing Atoxoplasma.
2. Discussions should occur between husbandry and veterinary departments regarding risks and benefits of reproductive programs and species exhibition versus potential Atoxoplasma infection.
3. Responsibility for the management of Atoxoplasma should be shared by both the husbandry and veterinary teams.
4. All protocols and management plans for Atoxoplasma should be re-evaluated regularly.
5. Increased Atoxoplasma-associated mortality or morbidity in a newly-acquired or reproducing species should prompt an immediate re-evaluation of protocols to address the situation.
Questions to be considered when bringing a passerine species or individual bird into a collection for breeding
1. What is the species’ susceptibility to clinical atoxoplasmosis?
2. Are the birds considered exposed to Atoxoplasma?
3. Has Atoxoplasma-associated mortality been documented at the sending institution? In the species of concern?
4. If the answer is no, what type of screening for the disease occurs at the sending institution?
5. Has this species been maintained previously at the receiving institution? Has there been Atoxoplasma-associated disease in adults and/or chicks/fledglings at this institution?
6. Receiving institution: for species deemed highly susceptible to clinical atoxoplasmosis, can modifications be made to the current aviary management system that would reduce risk to acceptable levels? Note: in situations where infection causes 100% mortality at a particular institution, acquisition may not be acceptable under any situation. Certain species may be deemed highly susceptible under the current aviary management system at the receiving institution. These species may not be accepted for that reason.
· One tool available for assessing disease processes on captive populations is the electronic records system, MedARKS. However, in order for the information to be easily searchable, it would be advantageous to identify MedARKS records with studbook or ARKS identifiers. Without this information in the medical record, it is difficult to formulate an animal’s life history across institutions.
Example of application: Currently, a review of the historic medical records of over 500 Bali mynahs is under way. Publications are being planned to both 1) summarize the clinical signs and health parameters associated and not associated with atoxoplasmosis, and 2) relate the medical information to that found in the studbook in order to determine the effects of this parasite on the entire population, including chick and adult mortality and survival rates. This project should be finished at the end of 2004. This study has been greatly delayed by the effort required to match both paper and electronic records from birds across both time and multiple institutions.
1. Oocysts are not always present in feces of clinically affected birds.
2. If Isospora-like coccidia are found in the feces of a Bali mynah, it is most likely Atoxoplasma. Hundreds of fecal samples have been performed on Bali mynahs and on only 2 occasions has another species of coccidia been observed.
3. The oocysts of Isospora and Atoxoplasma cannot be distinguished from each other via light microscopy. Both have 2 sporocysts each with 4 sporozoites. Systemic stages must be identified to confirm that the passerine bird is infected with Atoxoplasma.
4. To definitively rule out fecal shedding, feces should be collected over several days and placed in 3-5% potassium dichromate (PDC). Periodic aeration of the PDC and feces allows the organism to sporulate in approximately 96 hours thus enhancing identification. Pooled fecal samples over 24 hrs or multiple fecal exams are alternatives methods to rule out fecal shedding.
5. A definitive diagnosis of atoxoplasmosis cannot be made only by performing a fecal examination and finding Isospora sp. This is an important part of the diagnostic workup, but needs to be combined with other diagnostic tests listed below for a definitive diagnosis.
1. Atoxoplasma can be found on blood smears in mononuclear cells.
2. Buffy coat smears concentrate the white blood cells, thus increasing the likelihood of finding the organism when compared to routine blood smears. Unfortunately, making buffy coat smears is an art, thus always submit a normal blood smear with the buffy coat smears for insurance.
3. Only a very small percentage of adult Bali mynahs shedding oocysts in the feces have been positive on buffy coat smear evaluations.
4. Positive buffy coat smears are much more common in juvenile birds with clinical signs of atoxoplasmosis (Partington et al., 1989).
5. Multiple buffy coat smears are recommended to rule out atoxoplasmosis in symptomatic birds.
6. See Appendix 1 for Greiner unpublished information on correlations between buffy coat smears and fecal samples for positive diagnosis of oocysts and systemic stages of Atoxoplasma.
1) Advantages of assay
· Can be applied to multiple sample types including whole blood, feces, and tissue although usefulness depends on disease status, species affected, and sample type being tested.
· Aids in diagnosis of cases where clinical signs or microscopic lesions are suggestive of Atoxoplasma but organisms are not observed.
· Potential application in evaluating efficacy of chemotherapeutic protocols and husbandry practices designed to reduce incidence of infection.
· Provides data to further evaluate species-specificity and diversity of Atoxoplasma spp.
· Useful as a screening test for determining incidence/prevalence of atoxoplasmosis in a particular species or collection.
Useful in evaluating breeding individuals for the
purpose of assessing need for prophylactic
treatment during brooding or for consideration of pulling eggs for hand-rearing
2) Limitations of assay
· Only confirms presence of organism - cannot be used to identify Atoxoplasma-free birds.
· The commercially available UGA PCR has the potential to detect other Isospora spp. The San Diego Zoo PCR test will not confuse Atoxoplasma with Isospora. They are genetically distinct.
· Especially problematic in fecal samples based on experience by the UGA research group.
q Sensitivity in fecal sample is poor.
q Oocysts must be present to successfully amplify.
failure of amplification in the presence of oocysts on fecal flotation
including clinical cases with disseminated atoxoplasmosis.
3) See Appendix 2 for summaries of recent or ongoing Atoxoplasma PCR studies and UGA Atoxoplasma PCR sample submission protocol.
1) Cytology may be more reliable than histopathology for detecting the circulating stages. Histopathology is very good at picking up the intestinal stages.
2) Liver and spleen are preferred samples for detecting circulating stages of the organism.
1. Atoxoplasmosis can be diagnosed by histopathology.
2. Given the typical small size of biopsies taken in avian species along with the multifocal distribution of hepatic lesions associated with Atoxoplasma infection, histopathology may only reveal normal hepatic tissue, resulting in a false negative diagnosis.
3. Hepatic pathology associated with Atoxoplasma infection often includes lymphohistiocytic and heterophilic hepatitis. However, this type of lesion can be seen with other etiologic agents. Consequently, the presence of this type of lesion without observable Atoxoplasma organisms does not represent a positive diagnosis of atoxoplasmosis.
1. Most common gross pathological findings are an enlarged liver and spleen with pinpoint foci of necrosis, an edematous pancreas, and a fluid filled intestinal tract.
2. In the majority of birds, histopathology reveals inflammatory foci (most commonly in the liver, spleen, and lung) with intracytoplasmic zoites. In addition, atypical lymphoid or other proliferative lesions (possible neoplastic processes) are seen in association with the organism in a few species.
3. Many other organs may be involved.
4. A thorough necropsy should be performed on passerine birds that die.
5. Recommendations for necropsy: collect a small section of all major tissues (heart, lung, airsac (on a piece of paper towel), thymus, bursa, crop, proventriculus, multiple sections of intestine, pancreas, kidney, adrenal gland, gonad, oviduct, muscle, bone marrow, and brain) in 10% buffered formalin. Impression smears of liver and spleen should be made after gently blotting cut surface on a paper towel and fixed.liver and spleen.
A definitive diagnosis of atoxoplasmosis can only be made by directly visualizing the organism on buffy coat smears, cytological, or histopathological specimens. However, the San Diego Zoo pathologists have seen unusual morphologic manifestations of atoxoplasmosis that could only be definitively diagnosed by PCR.
A. Sufachlorpyrazine (ESB3)
§ Has been successful in reducing mortality from Atoxoplasma in canaries and Bali mynahs.
§ Sulfachlorpyrazine in the water has significantly reduced or totally cleared fecal oocyst shedding for an extended period of time in Bali mynahs. If fecal shedding is inhibited, chick mortality will be decreased or eliminated.
§ It is unlikely that drug can completely clear a bird of Atoxoplasma.
§ Sulfachlorpyrazine only affects the intestinal stages of the parasite.
§ The treatment regimen using sulfachlorpyrazine in the Bali mynah and several other passerine bird species shedding the organism and long term prevention is as follows: mix 1 gram of the 30% powder in 1 liter of water and use this as the only source of drinking water for 5 days; no drug for 3 days; then repeat the 5 day treatment; this cycle is repeated 4 times, constituting one treatment. The birds should be treated three times per year if possible. Important times to schedule preventive treatments are during the breeding season and when parents are feeding chicks through fledging. A vitamin supplement containing vitamin B6 should be utilized during the treatment period. If the regular diet already contains adequate levels of vitamin B6 then this may not be necessary.
B. Vetisulid (sulfachlorpyridazine) can be used as a replacement for sulfachlorpyrazine (ESB3), which is not available in the US. The dose used is 300 mg/liter of water. Follow the same general protocol and frequency as ESB3.
§ Recent study using Vetisulid treatment in one sturnid species Golden-breasted starling (Cosmopsarus regius)) at Disney (D. Neiffer)
1. Treatment parameters
a. Dosed at 300 mg/L water
b. Treated water was sole source on treatment days.
c. Treatment period lasted 32 days.
d. Treatment period consisted of four cycles.
e. Each cycle consisted of 5 days of treatment followed by 3 days without.
f. Birds pre-tested 1-5 days before treatment and re-tested 5-7 days after last treatment day.
a. 2/5 birds positive by buffy coat pre-treatment, 0/5 positive by buffy coat post-treatment.
b. 4/5 birds had Isospora oocysts both pre- and post-treatment. However, quantitative estimates changed from moderate-few oocysts pre-treatment to few-rare oocysts post-treatment
c. 2/2 birds were liver PCR positive both pre- and post-treatment.
d. 5/5 birds negative by PCR on whole blood (EDTA) pre- and post-treatment.
a. PCR testing has limited, but potential use in evaluating success of treatment.
b. Birds that are liver PCR positive are not expected to convert to negative following treatment.
c. Even if treatment kills organisms, expect that DNA will persist long term or forever.
d. This has ramifications for claims on treatment efficacy and Atoxoplasma status.
e. For birds that are blood PCR positive, conversion to negative blood PCR test following treatment may indicate clearance of circulating organisms (more diagnostic testing needs to be conducted on blood PCR positive individuals).
C. Toltrazaril (Baycox)
§ Baycox has been successful in reducing mortality from Atoxoplasma in canaries and several other passerine birds.
§ No published documentation that the birds were cleared of the organism and it is unlikely that drug can completely clear a bird of Atoxoplasma.
§ Toltrazuril appears to have some affect on systemic stages of the disease (Dorrenstein, pers comm.). It is currently approved in the US for use on Equine protozoal myelitis.
§ Toltrazuril has not been used extensively in the Bali mynah, but has been used in several other passerine bird species. Further studies are needed using toltrazuril in a more controlled manner to demonstrate its efficacy against systemic atoxoplasmosis.
D. Diclazuril has been effective in treating systemic avian toxoplasmosis and sarcocystis, and thus may have an effect on systemic atoxoplasmosis.
***Treatment minimizes shedding but is not a cure. It may actually be of benefit to have a mild infestation in order to stimulate the immune system. Birds that have not been exposed to the organism previously may be more susceptible to developing systemic disease. More clinical and controlled treatment trials are needed utilizing various available drugs to manage atoxoplasmosis.***
A. Husbandry practices are an extremely important part of the management of this parasite.
B. Handrearing chicks to prevent exposure to Atoxoplasma
1. Hand-rearing may be a viable method of prevention under certain circumstances.
2. For the purposes of this discussion, “hand-reared” refers to chicks hatched from eggs that were pulled and cleaned for incubation prior to piping.
3. A negative liver PCR test supports the Atoxoplasma-free status in hand-reared chicks as long as they have been kept out of a contaminated environment.
4. A negative liver PCR test in a parent-reared chick does not rule-out exposure as it is unknown how long after infection with Atoxoplasma that a conversion to positive liver PCR occurs.
5. Concerns regarding hand-rearing and Atoxoplasma
c. More controlled studies are needed to evaluate this issue.
C. Diagnostic recommendations for preshipment and quarantine:
· CBC and chemistry panel with emphasis on liver values
· whole body x-ray
· fecal bacterial culture
· fecal exam for parasites
· Atoxoplasma diagnostics described above; treat if indicated or discuss situation with receiving institution so that treatment can be performed while bird is in quarantine
· Unless liver biopsy with PCR is performed with negative results and the bird is housed in sterile conditions following biopsy, any claim of negative atoxoplasma status is highly questionable
· A neonatal examination is recommended shortly after fledging occurs, as the bird is being transferred to a new enclosure.
· The examination should include a body weight, a thorough physical exam, complete blood count, buffy coat smear for Atoxoplasma assessment, serum biochemistry panel (if possible), and fecal examination.
· If the fledgling bird appears unthrifty, the Atoxoplasma diagnostic protocol should be followed.
· Regarding screening for Atoxoplasma infection in healthy fledglings, if buffy coat exam is negative, then it is recommended to use liver PCR testing (rather than fecal exam and blood (EDTA) PCR to confirm negative status. If the buffy coat exam is positive then this confirms the positive Atoxoplasma status and in most birds is less invasive than liver biopsy.
D. Birds being used for display vs. a reproductive program:
1. Birds in large multi-species aviaries are generally difficult to treat on a regular basis in a stress free manner and it is usually best to use this situation for displaying birds rather than for intensive reproduction programs.
2. Passerine birds that are positive for Atoxoplasma and are in a breeding program will be easier to manage in smaller enclosures that can be periodically broken down and sanitized. Furthermore, it is much easier to treat these birds routinely in the drinking water with the various therapeutics that are currently available.
3. The impact of subclinical infections are unknown, but the following are some possible scenarios:
§ Decline in condition and increased disease susceptibility
§ Negative impact on breeding success
§ Clinical disease in birds that are stressed, old, or that become ill from other causes.
§ Transmission to juveniles
§ Mild infections may stimulate the immune system and prevent systemic atoxoplasmosis.
Appendix 1—(Greiner); Unpublished information on correlations between buffy coat smears and fecal samples for positive diagnosis of oocysts and systemic stages of Atoxoplasma:
· Sturnidae: Bali mynah 397 positive in feces, 170 negative, 27 positive on buffy coat, 268 negative; superb starling: 47 positive on fecal, 13 negative, 5 positive on buffy coat and 20 negative; gold breasted starlings: 28 positive and 8 negative on feces, 5 positive on buffy coat and 20 negative; gold crested mynah: 1 positive on feces and 11 negative, 33 positive on buffy coat and 31 negative: Indian hill mynah: 0 positive on feces and 1 negative, buffy coat 1 positive and 0 negative.
· Timaliidae: Black-throated laughing thrush: 0 positive and 2 negative on feces, 4 positive and 3 negative on buffy coat; yellow-throated laughing thrush: 4 positive and 0 negative on feces, 4 positive and 9 negative on buffy coat.
· Ploceidae: Black-headed weaver: 1 positive on impression smear
· Troglodytidae: cactus wren: 4 positive and 0 negative on feces, 1 positive and 2 negative on buffy coat.
· Thraupidae: silver-beaked tanager: 1 positive and 3 on feces, 3 positive and 3 negative on buffy coat.
· Pittidae: hooded pitta: no feces, 1 positive on buffy coat.
Appendix 2-- Summaries of recent or ongoing Atoxoplasma PCR studies and UGA Atoxoplasma PCR sample submission protocol.
Introduction: The current knowledge of polymerase chain reaction (PCR) diagnostics as it pertains to Atoxoplasmosa in passerine birds comes from efforts by researchers at the San Diego Zoo and University of Georgia CVM.
Study 1: Information currently available from the San Diego Zoo Study on necropsy specimens
Host species Range
· Fifty birds of 23 species were PCR positive.
· 47/50 had a postmortem diagnosis of atoxoplamosis.
· 27/50 had sexual stages in small intestine.
· 19/33 had positive postmortem lung/spleen smears.
· 6 birds of 4 species had isosporoid oocysts in the feces (less fecal samples were evaluated).
· In most instances, each avian species had a unique genotype of the parasite. However, house sparrows were found to be infected only with the Bali mynah parasite, so they did not have their own unique genotype.
· Some species, and individuals within a species, had multiple genotypes (as determined from 16s and 23s rRNA gene sequences).
· 28 unique parasite genotypes were detected in the 23 avian species
· The average genetic distance between Atoxoplasma organisms is 2.19% (with a std dev 0.937, max 4.946, min 0.414).
· The closest genetic relative to Atoxoplasma in GenBank is Eimeria tenella.
· The average genetic distance (% difference in nucleotides) between Atoxoplasma organisms in this study and E. tenella is 9.127%.
· The parasites are therefore monophyletic (i.e. are more closely related to each other than to any other parasite).
· While each species of bird had a unique Atoxoplasma genotype, different avian species sometimes had parasites that were very closely related (99.6%).
· These closely related genotypes could be substrains of the same Atoxoplasma species.
· The other genotypes that are 2-4.9% different, are probably truly different species of Atoxoplasma.
· Atoxoplasma organisms in general appear to be host-specific. However, horizontal transmission between avian species has almost certainly occurred.
· It is possible that wild Northern house sparrows are transmitting Atoxoplasma to collection birds, however, transmission studies would be required to prove this.
· The blood forms (monocyte merozoites) of Atoxoplasma are genetically identical to the isosporoid oocysts seen in feces.
· No evidence for mite transmission has been found by PCR.
· Some individual birds have parasite genotypes that differ substantially enough to represent different species of Atoxoplasma.
· Horizontal transmission between different passerine species probably occurs.
· The risk of horizontal transmission between divergent taxonomic groups of birds may occur based on the data from this study, but appears to be less frequent and more unpredictable. Transmission studies are still needed to confirm and investigate this issue further.
· Wild sparrows may pose a risk to captive birds such as Bali mynahs, but this risk is unpredictable and must be balanced against costs and other risks. Further investigation is needed to confirm this.
Study 2: Information currently available from the University of Georgia research group using PCR to identify Atoxoplasma spp.
PCR assay design
· 18s rDNA-----550 bp fragment-------424 bp fragment
· Designed to amplify 18S rDNA fragment of Isospora spp.
· Used on systemic stages to amplify Atoxoplasma fragment.
PCR Assays on Clinical Samples
· 122 samples out of 303 were positive (40.3%)
o 192 blood/plasma samples with 61 positive (31.8%)
o 65 liver samples with 40 positive (61.5%)
o 46 other tissues with 21 positive (45.7%)
Species represented (refer to section 1. B. 5. for details)
Advantages of assay
· Aids diagnosis in cases where clinical signs or microscopic lesions are suggestive, but organisms not seen.
· Potential application in evaluating efficacy of chemotherapeutic protocols and husbandry practices designed to reduce incidence of infection.
· Provides data to further evaluate species- specificity and diversity of Atoxoplasma spp.
· PCR assay currently available on a fee-for-service basis through diagnostic parasitology laboratory at UGA firstname.lastname@example.org.
Limitations of assay
· Potential to detect other Isospora spp is especially problematic in fecal samples.
· Sensitivity in fecal samples poor
o Oocysts must be present to successfully amplify.
o Occasional failure of amplification even when oocysts are present on fecal flotation.
· Only confirms presence of organism, cannot be used to identify Atoxoplasma-free birds.
· PCR assay best suited to confirm suspected cases on histopathology when organisms cannot be directly identified by microscopic examination
Study 3: Atoxoplasmosis in Tanagers: Zdziarski, Adkesson, Little
o Survey conducted: 22 institutions responded, 4 of these reported Atoxoplasma infections in tanagers
q 18 tanager species (9 Genera) were included in this study
q Also identified atoxoplasma in 6 non-tanager spp
q 89 individual birds
o Whole blood was taken and evaluated from 60 birds: 21 of 60 (35%) were positive for Atoxoplasma.
o Fecal samples were obtained from 8 birds: 1 pooled fecal sample from 2 birds was positive for Atoxoplama and 3 birds had negative fecal samples but were positive for Atoxoplasma on blood samples.
o Tissue samples from 32 birds: 20 of 32 (62.5% ) birds were positive on various tissue samples.
q Samples were submitted to Dr. Susan Little at UGA, where a nested PCR assay to amplify a fragment of 18S rRNA gene of Isospora spp was performed.
q Recommended PCR testing for tanagers based on this study include:
o Test any bird showing non-specific signs of illness
o Screen whole blood specimens from live birds and tissue from post-mortem samples to determine prevalence
o Identify status of breeding birds
o Many apparently healthy adults have subclinical infections
o PCR of whole blood or liver tissue samples are the method of choice
Study 4: Application of “Atoxoplasma” Diagnostic Tests at Disney’s Animal Kingdom, Preliminary Findings, Donald L. Neiffer, VMD
· Species involved: golden-breasted starling (GBS) (Cosmopsarus regius) and golden crested mynah (GCM) (Ampeliceps coronatus)
· Mortality due to atoxoplasmosis in GBS population, but not in GCM population--demonstrates differences in avian species susceptibility to the organism.
· All testing was antemortem and all individuals were clinically normal.
· PCR testing was performed at UGA CVM.
· Whole blood (EDTA) PCR: negative in most cases even when Atoxoplasma zoites detected in buffy coat smears. May have value in identifying at risk birds, as a prognostic indicator, and as a means of evaluating response to therapy (i.e. positive test converts to negative during or following treatment).
· Fecal PCR:
o Limited value for screening or prognosis.
o Negative in all cases including:
§ Cases where Isospora oocysts were detected in feces. Feces were collected over 72 hrs and placed in potassium dichromate and then aerated to allow sporulation. Sheather’s sugar solution was used to concentrate the oocysts.
§ Clinical cases with disseminated atoxoplasmosis including invasion of the gastrointestinal tract.
· Liver PCR
§ Represented the most sensitive test in this study
· Liver PCR was positive in all cases where positive results were found using other diagnostic tests.
· A positive result should be considered to at least represent previous infection and likely represents sequestered organisms/latent infection.
§ Other possible uses:
· Confirming Atoxoplasma-free status in hand-reared chicks.
· Evaluating treatment efficacy.
· Liver PCR vs Liver biopsy findings
o Use of liver biopsies to detect Atoxoplasma infection in the absence of liver PCR testing will result in a high number of false negatives.
§ Histopathology in cases of Atoxoplasma infection often reveals lymphohistiocytic and heterophilic hepatitis without visualization of the organism. A positive liver PCR test in conjunction with typical histological changes confirms a diagnosis of Atoxoplasma.
§ Given the typical small size of biopsies taken in avian species along with the multifocal distribution of hepatic lesions associated with Atoxoplasma infection, histopathology may only reveal normal hepatic tissue.
ATOXOPLASMA PCR SAMPLE COLLECTION PROTOCOL
PCR testing for Atoxoplasma is now available on a fee-for-service basis from the diagnostic parasitology laboratory at the University of Georgia. The cost to the submitting institution is $25 for the first sample + $10 for each additional sample submitted at the same time. Charges will be billed to the submitter through the diagnostic parasitology laboratory. A submission form is available for you to print and use at http://www.riverbanks.org/subsite/aig/bmsubfrm.htm, or you may obtain a hard copy form by FAXing a request to Dr. Susan Little at (706) 542-0059.
Preferred samples for testing include whole blood and fresh or frozen liver, spleen, or small intestine. However, other tissues may also be used for Atoxoplasma PCR testing. Submit 0.3-0.5 ml of whole blood in EDTA (purple top) tubes. Place blood tubes in whirl-pak or small ziplock bags prior to final packaging for shipment. Submit tissues without fixative or preservative in individual 1.5 ml plastic microcentrifuge tubes or small whirl pack bags (one tissue/tube or bag). Ship all specimens overnight with plenty of ice or ice packs. Alternatively, blood and tissue can be frozen overnight at –20°C to –70°C and shipped frozen for overnight delivery at a later date.
Fecal samples have been less rewarding for Atoxoplasma PCR assay. However, fecal samples can be examined by simple flotation and PCR if desired. Collect as much feces as possible (24-hour collection) and place in 1.5 ml microcentrifuge tubes, small serum tubes (red top), or similar container. Place the primary collection tubes in whirl-pak or small ziplock bags prior to final packaging for shipment. Ship all specimens overnight with plenty of ice or ice packs. Alternatively, feces can be frozen overnight at –20°C or –70°C and shipped frozen for overnight delivery at a later date.
Blood, tissue, and feces may all be frozen for several weeks and the samples submitted in batches to facilitate ease of submission and minimize shipping costs.
The PCR assay can also be useful in identifying Atoxoplasma in paraffin-embedded tissues with lesions. If desired, a cover letter requesting the return of the blocks once sections have been cut for PCR may be included.
To avoid thawing during shipment and subsequent leaking of packaging, please do not ship fresh or frozen material after 12 noon on Thursday or 36 hours before any standard holidays when UGA may be closed. If shipping fresh feces or fresh blood, please hold refrigerated until next possible shipment date (usually Monday). Fresh tissues can be frozen for shipment the following week. Samples should be sent, together with a thorough description of the case (MedARKS pathology and clinical notes will be fine). Make sure that all samples are labeled with the studbook number, accession number, date and species. Please call, email, or FAX prior to sending a package.
If you have any questions about submission of samples, please call Dr. Little at
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