Diagnosis of chlamydial infection in pet birds: comparison of cloacal-swab culture and peroxidase-antiperoxidase methods

Cloacal swabs were examined using a peroxidase-antiperoxidase (PAP) method that employs a monoclonal antibody to chlamydiae. The specificity and sensitivity of cloacal-swab PAP examination and the prevalence of chlamydiosis were calculated using both culture and tissue PAP evaluation as standards. The prevalence of chlamydial infections was 9.3% as determined by culture and 46.3% as judged by tissue PAP evaluation. Cloacal-swab PAP examination yielded a sensitivity of 71.4% and specificity of 85.5% when compared with culture results and a sensitivity of 81% and specificity of 85.7% compared with tissue PAP results. Culture had a sensitivity of 47.4% and specificity of 100% compared with tissue PAP results. Similarly, the efficiency of cloacal-swab PAP test was about 84% compared with culture results and tissue PAP. The efficiency of culture compared with tissue PAP was 73.1%.     

Diagnosis of avian chlamydiosis: specificity of the modified Giménez staining on smears and comparison of the sensitivity of isolation in eggs and three different cell cultures.

For the diagnosis of chlamydiosis in dead and live birds different methods were compared for their sensitivity and specificity. The specificity of the modified Giménez staining and the direct immunofluorescence (DIF) test for direct demonstration of Chlamydia psittaci in organ, cloacal and/or conjunctival smears was examined. The sensitivity of the isolation of Chlamydia psittaci in 6 days embryonated specific pathogen free (SPF) chicken eggs, Buffalo Green Monkey (BGM) cell line, McCoy cell line and Vero cell line was compared. On smears, the direct immunofluorescence test was more specific than the modified Giménez staining. The concordance between the results of both detection methods was 80%. The BGM cell culture was the most sensitive artificial host for isolation of Chlamydia psittaci, followed by the embryonated eggs, the Vero cell line and the McCoy cell line. The concordance between the results of isolation in BGM cell culture and eggs was 96.5%, while it was 86% between the results of isolation in BGM cell culture and Vero cell culture and only 65.5% between the results of isolation in BGM cell culture and McCoy cell culture. For dead bird species, chlamydiosis could be diagnosed more often using DIF on smears than with isolation. The concordance between the results of the DIF on smears and isolation followed by DIF was 91%.     

Development and field validation of a Mycoplasma iowae real-time polymerase chain reaction assay

A Mycoplasma iowae real-time polymerase chain reaction (PCR) assay using primers and probes targeting the 16S rRNA gene was developed and field-validated in this study. The assay specifically identified M. iowae with a detection limit of 80 colony-forming units (cfu) per turkey cloacal swab sample (3.2 cfu per PCR reaction). It was validated by testing 154 field turkey cloacal swab samples in parallel with culture isolation. The diagnostic sensitivity of the PCR was 97.6%, and the specificity was 95.5%. The real-time PCR developed in this study is a rapid, sensitive, and cost-effective alternative to culture isolation for detecting M. iowae from cloacal swab samples.     

Enigmatic psittacine chlamydiosis: results of serotesting and isolation attempts, 1978 through 1983, and considerations for the future

From 1978 through 1983, chlamydiosis was diagnosed by isolation of Chlamydia psittaci from various types of psittacine birds. The organism was isolated from 126 (30.4%) of 414 tissue specimens, with the percentages ranging from 12.5% (budgerigars) to 42.8% (cockatiels), excluding 2 parakeets with 1 isolation (50%). From 1,035 cloacal swab/feces specimens, 51 (4.9%) isolations were made, ranging from 1.4% from African grays (1 of 70) to 27.8% from lovebirds (5 of 18). Positive direct microscopic examination of stained (Gimenez method) tissue impressions correlated with positive isolation at a rate of 79.2% and those found negative by direct examination had a correlation of 87.5%. Direct complement-fixation testing was done on 3,485 sera. Forty-six were unsatisfactory for testing due to their being anticomplementary or reacting with control antigen. The distribution of titers ranged from 2,008 (57.6%) at 8 to 76 (2.2%) at greater than or equal to 256. In serotests and isolation attempts from the same bird, there was 42.8% agreement between titers greater than or equal to 32 and positive isolation. One cockatiel with a complement-fixation titer of 16 yielded a positive isolation, whereas other types of birds with a less than or equal to 16 titer were negative.     

Viremia, virus shedding, and antibody response during natural avian polyomavirus infection in parrots

OBJECTIVE: To determine rapidity of spread and onset and duration of viremia, virus shedding, and antibody production in parrots naturally infected with avian polyomavirus (APV). DESIGN: Case series. ANIMALS: 92 parrots in 2 aviaries. PROCEDURE: Blood samples were obtained from parrots naturally exposed to APV during a 3- to 4-month period for determination of serum virus neutralizing antibody and detection of viral DNA. Nestlings from the next year's hatch were monitored for APV infection. RESULTS: The first indication of inapparent infection was viremia, which developed simultaneously with or was followed within 1 week by cloacal virus shedding and antibody production. Cloacal virus shedding continued after viremia ceased. During viremia, viral DNA was detected continuously in blood samples. Viral DNA was detected in serial cloacal swab specimens in most birds, but it was detected inconsistently in 6 birds and not detected in 3 birds, even though these birds were viremic. Duration of cloacal virus shedding was < or = 4.5 months. In 1 aviary, prevalence of infection was 88% and dissemination of virus through the 3-room building required 4.5 months. In the second aviary, a single-room nursery, prevalence of infection was > or = 90%. For all affected birds, infection could be detected 18 days after the first death. CONCLUSIONS AND CLINICAL RELEVANCE: If a single sampling is used for polymerase chain reaction detection of viral DNA, blood and cloacal swab specimens are required. In nestling nonbudgerigar parrots, cloacal virus shedding may persist for 4.5 months. Management protocols alone are sufficient to prevent introduction of APV into a nursery.     

Chlamydophila psittaci in free-living Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil

Chlamydophila psittaci (C. psittaci) infection was evaluated in 77 free-living nestlings of Blue-fronted Amazon parrots (Amazona aestiva) and Hyacinth macaws (Anodorhynchus hyacinthinus) in the Pantanal of Mato Grosso do Sul, Brazil. Tracheal and cloacal swab samples from 32 wild parrot and 45 macaw nestlings were submitted to semi-nested PCR, while serum samples were submitted to complement fixation test (CFT). Although all 32 Amazon parrot serum samples were negative by CFT, cloacal swabs from two birds were positive for Chlamydophila DNA by semi-nested PCR (6.3%); these positive birds were 32 and 45 days old. In macaws, tracheal and cloacal swabs were positive in 8.9% and 26.7% of the samples, respectively. Complement-fixing antibodies were detected in 4.8% of the macaw nestlings; macaw nestlings with positive findings were between 33 and 88 days old. These results indicate widespread dissemination of this pathogen in the two evaluated psittacine populations. No birds had clinical signs suggestive of chlamydiosis. To the best of our knowledge, this is the first report on C. psittaci in free-living Blue-fronted Amazon parrots and Hyacinth macaws in Brazil.     

Infection with Mycobacterium simiae complex in four captive Micronesian kingfishers

CASE DESCRIPTION: 4 captive adult Micronesian kingfishers (Halcyon cinnamomina cinnamomina) at 3 zoologic institutions were examined routinely or because of dyspnea or lethargy. CLINICAL FINDINGS: All birds had marked hepatomegaly. Two birds had dyspnea caused by compression of air sacs by the enlarged liver, and 1 bird had generalized weakness and lethargy. Three birds had distended coelomic cavities, and 3 birds were thin or had lost weight. There were no consistent abnormalities in blood analytes. Results of most ancillary diagnostic tests such as acid-fast staining of cloacal or fecal swab specimens and culture of feces for acid-fast bacteria were negative. Results of examination of hepatic biopsy specimens in 2 of 4 birds were suggestive of mycobacteriosis. TREATMENT AND OUTCOME: 3 birds died or were euthanized soon after diagnosis. One kingfisher was isolated and monitored for 4 months without treatment and died during anesthesia for disease monitoring. Postmortem histologic examination revealed histiocytic hepatitis and acid-fast bacteria in all 4 birds. Bacteriologic culture of liver specimens yielded Mycobacterium simiae complex in all 4 birds. CLINICAL RELEVANCE: Infection with M simiae complex should be considered in ill Micronesian kingfishers, and further monitoring is warranted to determine whether this is an emerging pathogen in this species.     

Pneumococcal antigen detection in cerebrospinal fluid: a comparative study on counter immunoelectrophoresis,latex agglutination and coagglutination

The sensitivity, specificity, accuracy and predictive values of counter immunoelectrophoresis (CIE), latex agglutination (LA) and coagglutination (CoAg) tests were compared for detection of pneumococcal antigen in cerebrospinal fluid (CSF) of patients suspected of meningitis. A total of 95 CSF samples comprising 15 culture proven, 47 clinically suspected but culture negative cases of meningitis and 33 controls were screened by above tests. Among three tests, LA was found to have high sensitivity and moderately high negative predictive value than CIE and CoAg tests. However, CIE had slightly better specificity than LA and CoAg tests. Accuracywise CIE and LA tests were comparable than CoAg test. CIE and LA tests had high positive predictive value than CoAg test.     

Experimental infection of rosellas (Platycercus eximius) with velogenic viscerotropic Newcastle disease virus (VVNDV)

Plaque-purified and non-plaque-purified velogenic viscerotropic Newcastle disease viruses (VVNDVs) were inoculated into golden-mantled rosellas (Platycercus eximius). VVNDV produced acute clinical disease in this species: all birds died within 6 days postexposure. There was no difference between the two inoculation groups in clinical signs. Seven tissues and five tissue swabs were collected from each of 15 birds. The VVNDV concentration in each specimen was titrated, and the concentrations were compared. The lung and trachea had the highest concentrations of virus in both the tissue suspensions and the swab suspensions. The average virus concentrations of the lung were 10(5.9) 50% embryo lethal doses (ELD50) per 0.1 ml for the tissue suspension and 10(4.9) for the swab. The average virus concentrations of the trachea were 10(5.6) ELD50 per 0.1 ml of tissue suspension and 10(4.6) for the swab.     

Evaluation of an antimicrobial resistance monitoring program for Campylobacter in poultry by simulation

An ideal national resistance monitoring program should deliver a precise estimate of the resistance situation for a given combination of bacteria and antimicrobial at a low cost. To achieve this, decisions need to be made on the number of samples to be collected at each of different possible sampling points. Existing methods of sample size calculation can not be used to solve this problem, because sampling decisions do not only depend on the prevalence of resistance and sensitivity and specificity of resistance testing, but also on the prevalence of the bacteria, and test characteristics of isolation of these bacteria. Our aim was to develop a stochastic simulation model that optimized a national resistance monitoring program, taking multi-stage sampling, imperfect sensitivity and specificity of diagnostic tests, and cost-effectiveness considerations into account. The process of resistance testing of Campylobacter spp. isolated from cloacal swab samples from poultry was modeled using a Markov Chain Monte Carlo model. Different sampling scenarios on the number of flocks to be tested, the number of birds from each flock, and the number of campylobacter colonies submitted to susceptibility testing were evaluated regarding the precision of the resulting prevalence estimate. Precision of the prevalence estimate was defined as the absolute difference between apparent and true prevalence of resistance. A partial budget approach was utilized to find the most cost-effective combination of samples to obtain a defined precision of the prevalence estimate. For a sampling scenario testing 100 flocks, five birds per flock, and one campylobacter colony per sample, the median error of the prevalence estimate was 2.5%, and 95% of the simulations resulted in an error of 7% or less. When the total number of samples was kept constant, maximizing the number of flocks tested, and only testing one bird per flock resulted in the most precise prevalence estimate. Submitting more than one campylobacter colony to resistance testing did not improve the prevalence estimate. Partial budget analysis indicated that the most cost-effective strategy was testing of two birds per flock, and submitting one colony per sample to resistance testing.     

Processing postmortem specimens with C18-carboxypropylbetaine and analysis by PCR to develop an antemortem test for Mycobacterium avium infections in ducks.

Mycobacterium avium is the causative agent of the avian mycobacteriosis commonly known as avian tuberculosis (ATB). This infection causes disseminated disease, is difficult to diagnose, and is of serious concern because it causes significant mortality in birds. A new method was developed for processing specimens for an antemortem screening test for ATB. This novel method uses the zwitterionic detergent C18-carboxypropylbetaine (CB-18). Blood, bone marrow, bursa, and fecal specimens from 28 ducks and swabs of 20 lesions were processed with CB-18 for analysis by smear, culture, and polymerase chain reaction (PCR). Postmortem examination confirmed nine of these birds as either positive or highly suspect for disseminated disease. The sensitivities of smear, culture, and PCR, relative to postmortem analysis and independent of specimen type, were 44.4%, 88.9%, and 100%, respectively, and the specificities were 84.2%, 57.9%, and 15.8%, respectively. Reductions in specificity were due primarily to results among fecal specimens. However, these results were clustered among a subset of birds, suggesting that these tests actually identified birds in early stages of the disease. Restriction fragment length polymorphism mapping identified one strain of M. avium (serotype 1) that was isolated from lesions, bursa, bone marrow, blood, and feces of all but three of the culture-positive birds. In birds with confirmed disease, blood had the lowest sensitivity and the highest specificity by all diagnostic methods. Swabs of lesions provided the highest sensitivity by smear and culture (33.3% and 77.8%, respectively), whereas fecal specimens had the highest sensitivity by PCR (77.8%). The results of this study indicate that processing fecal specimens with CB-18, followed by PCR analysis, may provide a valuable first step for monitoring the presence of ATB in birds.     

Detection of pigeon circovirus in cloacal swabs: implications for diagnosis, epidemiology and control

Pigeon circovirus (picv) was detected in cloacal swab samples by means of a newly-developed, sensitive pcr. An initial investigation of 17 Belgian racing pigeons aged up to eight months showed that rates of detection of 88 per cent and above were achieved using samples of cloacal swab, blood and bursa of Fabricius. The sampling of 15 caged pigeons six times when they were from three to 31 weeks of age indicated that picv infections were more readily detected in cloacal swabs than in blood, and that the virus could be detected in cloacal swabs for longer periods after infection than in blood. picv infections were detected in cloacal swabs from 38 of 47 young pigeons aged from two to 31 weeks, from 12 racing lofts, which had clinical signs including diarrhoea and weight loss, regurgitation and respiratory signs. Samples from birds from two infected lofts indicated that picv could be detected in some birds for at least 27 weeks. Although nine of 14 pigeons aged from 32 to 45 weeks were virus-positive, picv was detected in only one of 18 adult pigeons that originated from four infected lofts.     

A survey for paramyxoviruses in caged birds, wild birds, and poultry in New Zealand

AIMS: To determine the presence of avian paramyxovirus (APMV) types 1, 2, and 3 in caged and wild birds, and APMV-2 and -3 in poultry in New Zealand. METHODS: Blood samples collected from caged (231) and wild birds (522) from various regions of New Zealand in 1997-99 were tested by haemagglutination inhibition (HI) test for antibodies to APMV types 1, 2, and 3. Blood samples collected from 1778 commercial poultry in 1996-99 were tested for APMV-2 and APMV-3 antibodies and the samples that reacted with APMV-3 antigen were tested for antibodies to APMV-1. Isolation of APMV was attempted from cloacal swabs collected from 116 of the caged birds and 175 of the wild birds sampled. RESULTS: Antibodies to APMV types 1, 2, and 3 were detected in 4.8, 1.7, and 2.6%, respectively, of caged bird samples. The majority of these caged birds were 'exotic' or 'fancy' poultry breeds. Amongst wild birds, 4.2% had titres to APMV-2 and over half of these were passerine birds; 1.7% of the samples had titres to APMV-1 and 0.8% to APMV-3 antigen. No virus was isolated from any of the cloacal swabs tested. Of the 1778 poultry serum samples tested, only 5 reacted with APMV-3 antigen and these were later found to be cross-reactions to APMV-1. No reactions were detected with APMV-2 antigen. CONCLUSIONS: APMV-1 is present in caged birds, wild birds, and poultry of New Zealand. There is no conclusive evidence of the presence of APMV-2 and APMV-3 in poultry or APMV-3 in wild birds. The results do not provide conclusive evidence for the presence of APMV-2 in wild birds in New Zealand.     

A virulent mono-eukaryotic culture of Histomonas meleagridis is capable of inducing fatal histomonosis in different aged turkeys of both sexes, regardless of the infective dose

Histomonosis (syn. histomoniasis) is a parasitic disease which affects predominately turkeys but also other avian species. Concurrent with the ban of therapeutic and prophylactic substances, the disease, caused by the flagellated protozoon Histomonas meleagridis, is more frequently reported. Due to somewhat diverse results reported in the past, a well-characterized culture was used in the present study to investigate the possible influence of certain parameters on the outcome of the disease. For this study, turkeys were infected with different doses of the mono-eukaryotic culture Histomonas meleagridis/Turkey/Austria/2922-C6/04 using birds of both sexes at various ages. All study groups consisted of 14 birds, of which 10 birds were directly infected via the cloacal route and four birds were kept as in-contact birds. This scheme was used to investigate the pathogenicity of the cloned isolate in 1-day-old and 14-day-old turkeys. In 8-week-old turkeys, only eight birds out of 12 were infected. When 1-day-old and 8-week-old turkeys were infected with 10(4) histomonads per bird, all turkeys died between 11 and 21 days postinfection or had to be euthanatized due to their poor condition. In a group of 14 poults, infective doses of either 10 histomonads (100 histomonads among 10 birds) or 10(3) histomonads per bird had hardly any influence on the first notification of clinical signs. However, even though the onset of clinical signs and mortality was delayed with the lower dose, none of the birds survived the infection. As a consequence, no differences were noticed between male and female turkeys using the mono-eukaryotic culture of Histomonas meleagrigis/Turkey/Austria/2922-C6/04 in the current experimental setting.     

A rapid monoclonal immunofluorescence assay for Chlamydia psittaci in fecal smears from psittacine birds

One hundred two fecal specimens from psittacine birds submitted to Veterinary Laboratory Services of the California Department of Food and Agriculture at Petaluma were screened for Chlamydia psittaci by a direct immunofluorescence assay using a fluorescein-labeled monoclonal antibody conjugate specific for Chlamydia sp. Results were compared with those obtained by isolation of chlamydia in cultures of McCoy mouse cells. The relative specificity of the direct fluorescent antibody test on fecal smears was 98.9% and the relative sensitivity was 62.5%. The results of this study suggested that the direct fluorescent antibody test was highly specific, and it proved to be a useful same-day antemortem diagnostic test for birds with symptomatic chlamydial infection. The use of centrifugation in the cell culture assay was found to significantly enhance the level of chlamydial infection in cell culture.     

Diagnosis of avian mycobacteriosis: comparison of culture,acid-fast stains,and polymerase chain reaction for the identification of Mycobacterium avium in experimentally inoculated Japanese quail (Coturnix coturnix japonica)

In this study we compared culture, acid-fast stains, and polymerase chain reaction (PCR) for the detection of acid-fast organisms in fecal and tissue samples from Japanese quail (Coturnix coturnix japonica) that were experimentally inoculated intravenously with Mycobacterium avium. For culture, three different culture media (modified Herrold egg yolk with mycobactin; Lowenstein-Jensen [L-J]; and L-J with cyclohexamide, naladixic acid, and lincomycin) were tested to determine which medium had the greatest success in isolating mycobacteria. Acid-fast staining methods included Zichl-Neelsen (Z-N) and Truant. The PCR assay detected mycobacterial DNA with primers specific for the 65-kD heat shock protein gene. Culture was considered the "gold standard." Compared with other culture media, L-J yielded more positive cultures and greater numbers of colonies on positive tubes, and incubation times were shorter. Mycobacterium avium was isolated from all of the harvested tissue samples (liver, spleen, and intestine) of inoculated birds. Mycobacteria were isolated from 53% (69/130) of fecal samples from inoculated birds. As the disease advanced, fecal culture was positive on more culture days, indicating that the culture-positive rate was higher later in the course of the disease. Compared with culture, all of the laboratory methods had 100% specificity for the tissue samples. Sensitivities for the tissue samples were 82.6% (Z-N), 95.7% (Truant), and 100% (PCR). For the fecal samples, the specificity was >95% for all methods. Sensitivities compared with fecal culture were 7.2% (Z-N), 30.4% (Truant), and 20.3% (PCR). Tissue and fecal samples from the two control birds were negative for acid-fast organisms by any method. These results were comparable with clinical cases of avian mycobacteriosis where culture and PCR of tissue samples seem to be the most sensitive and specific laboratory tests and evaluation of fecal samples still remains challenging. On the basis of the results of this study, identification of mycobacteria in fecal samples from Japanese quail can be optimized by repeated cultures and Truant acid-fast staining of fecal smears.