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Retrospective Evaluation of Clinical Signs and Gross Pathologic Findings in Birds Infected With Mycobacterium genavense.

Abstract: Mycobacterium genavense is regarded as the primary cause of mycobacteriosis in passerine and psittacine birds kept in captivity. Mycobacterium genavense is a potential zoonotic pathogen; therefore, early antemortem detection in birds is needed. In humans, infections with M genavense are found predominantly in immunocompromised people. To investigate clinical signs and pathologic lesions and to determine the prevalence of coinfections in birds infected with M genavense, we reviewed records of 83 birds in which DNA from M genavense had been detected via real-time polymerase chain reaction. To evaluate clinical signs in birds presented as patients, results of standardized examinations of 60 birds and radiographic results from 37 birds were investigated. Necropsy results of 82 of the 83 birds were evaluated, including results of additional parasitologic, bacteriologic, and virologie examinations. Birds included in the study comprised 15 species in the orders Passeriformes, Psittaciformes, Coliiformes, Columbiformes, Coraciiformes, and Ciconiiformes. A wide range of clinical manifestations were documented, including neurologic disorders, ocular manifestations, and gastrointestinal signs. Of the 60 birds examined clinically, 15% showed no clinical signs. Coinfections with a wide range of pathogens were detected in 52% (43 of 83) of the tested birds. Coinfections included Macrorhabdus ornithogaster, circovirus, Polyomavirus, avian bornavirus, adenovirus, Mycobacterium avium ssp. avium/silvaticum. Mycoplasma species, Salmonella species, Escherichia coli, Aspergillus species, and various parasites. The high number of coinfections may reflect an impaired immune status in the birds examined. These results also suggest a broad host range for M genavense, and the existence of various clinical signs that may be strongly associated with coinfections with other pathogens.

Key words: Mycobacterium genavense, clinical signs, coinfections, real-time PCR, avian

Introduction

Mycobacterium genavense is a slow-growing, fastidious mycobacterium, which is very difficult to grow. (1,2) At the beginning of the 1990s, it was identified for the first time in pet birds as an agent of mycobacteriosis, (3) a common disease, (4) whereas, for many years, Mycobacterium avium subspecies avium had been regarded as the main and prevalent causal pathogen. Meanwhile, M genavense has been described as the primary cause of mycobacteriosis in, at minimum, psittacine and passerine birds, (5-10) Infections with this agent have also been described in a spheniscine bird. (11) Gallinaceous, piscine, and coracine birds have been reported as being infected with noncultivable or difficult-togrow mycobacteria, which may have been M genavense. (12) Infections in humans, (2) and occasionally in mammals (ferrets [Mustela putorius], (13) chinchillas [Chinchilla lanigera], (14) a horse,15 a cat, (16) a grizzled giant squirrel [Ratufa macroura], (17) a monkey [Cercopithecus diana], (18) a lemur [Lemur macaco mayottensis], (19) a dwarf rabbit [Oryctolagus cuniculus], (20) and dogs (21)), have been reported for this bacterial species as well.

Descriptions of clinical signs associated with M genavense include sudden death with no previous signs of disease, (3,10,12) nonspecific clinical signs that include weight loss and diarrhea, (9,10,22,23) neurologic signs, (3,9,10,19,24,25) alterations of the eyelids, (9,10) and respiratory disorders. (3,10) It has, however, also been detected in the feces of clinically healthy parrots. (26)

The nonspecific postmortem picture of an enlarged liver with a lack of macroscopically visible foci seems to be characteristic of M genavense infections in pet birds, but granulomatous tubercles have also been found in infected birds. (3,4,6,9)

Although a few descriptions of infections in immunocompetent people exist, (27) M genavense was found to be especially pathogenic for people with immunosuppression, for example, with an infection and HIV. (2,28) Whether diseases caused by M genavense occur more frequently in birds with impaired immune status or in association with coinfections is not known. Only a few reports mention possible coinfections with M genavense in birds: an individual Eurasian goldfinch (Carduelis carduelis) was additionally infected with Polyomavirus, (25) a zebra finch (Taeniopygia guttata) was additionally infected with circovirus, (28) and a coinfection of mycobacteria (species was not determined) with Macrorhabdus ornithogaster was seen in a canary (Serinus canaria). (29)

The main goal of this explorative study was to investigate the occurrence of M genavense in various pet bird species and to raise awareness of the many different presentations of clinical signs associated with M genavense and possible coinfections in naturally infected pet birds.

Materials and Methods

All avian patients diagnosed with M genavense infections by DNA detection in a clinic in Germany between 2011 and 2016 were selected for this study. In total, that included 83 birds, belonging to 6 zoological orders and 26 species (Table 1), naturally infected with M genavense. The birds originated from 58 different sources. They were kept by owners as pets or originated from zoological gardens and were presented to this hospital as clinical patients (60 birds; 72%) or submitted for necropsy (23 birds; 28%). Retrospectively, the recorded bird species, the clinical signs, and the results of radiographs, necropsy, histologic, parasitologic, bacteriologie, mycologie, and virologie examinations were assessed. Because all conducted examinations depended on the owners' willingness for investigation, not all examinations were performed in every bird.

M genavense real-time polymerase chain reaction testing

From every bird (n = 83), samples were collected from a standardized range of organs (heart, liver, lungs, trachea, kidneys, spleen, intestine, pancreas, proventriculus, ventriculus, brain, and integument) and homogenized with a Precellys tissue homogenizer (PeqLab Biotechnologie, Erlangen, Germany). The DNA was extracted from homogenized material with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Tissue was left to lyse for a minimum of 3 hours at 55[degrees]C. Realtime polymerase chain reaction (PCR) assays were conducted with the forward primer 5'-AAA CAG CGT CAG GAA ATC-3' and the reverse primer 5'-GTG GGA CGA AGA TGT AGT-3' as well as the TaqMan probe 5'-FAM-AAC CGC TAT CTA CAT CCG CAG-TAMRA-3', targeting the hypothetical 21-kDa protein gene. (30) The PCR assays were performed with the QuantiFast Pathogen PCR +IC Kit (Qiagen), which contains an internal control assay for PCR inhibition and includes 0.4 [micro]M of each M genavense primers and 0.16 [micro]M of the M genavense probe and was performed in a Mx3000P real-time PCR device (Agilent Technologies, Santa Clara, CA, USA). Reactions were analyzed with the software MxPro (Agilent) and were evaluated as a positive result if the fluorescence curves of the M genavense assay were sigmoid, and the fluorescence values exceeded the threshold values calculated by the software for each run. (8) The sensitivity of the M genavense realtime PCR was previously determined in a blinded study as [10.sup.5] bacteria [g.sup.-1] feces by spiking the feces of pigeons (Columbia livia f. domestica) known to be free of M genavense with defined numbers of cultured bacteria. (8) Five different M genavense reference strains were correctly identified with this assay. (8) During specificity testing in that blinded evaluation study, 15 reference strains of 9 mycobacterial species and M avium subspecies different from M genavense as well as fecal samples without mycobacteria were tested, and all were correctly identified as having no positive signals. (8)

Clinical investigations

In the 60 birds presented as patients, clinical signs were recorded based on a standardized examination protocol. The case history, body condition as determined by palpation of the pectoral muscles and by the body weights, and the general condition of the birds, including behavior, feather quality, and palpation of the body (including crop and abdomen), were assessed. Radiographs (HF 400 A, Gierth, Riesa, Germany) were performed in 37 birds, and for 16 of those birds (43%), additional radiographs with barium sulfate contrast medium were performed according to standard procedures.

Necropsy examinations

The birds included in the pathologic examination of this study were either euthanatized for animal welfare reasons or died naturally. Necropsy was performed on 82 of the 83 birds (99%). Of those birds, 23 (28%) had been submitted for necropsy only; therefore, results from a standardized clinical investigation were not available from those birds. The sex of 78 birds (95%) was recorded.

Histologic examinations were conducted in 6 of the 83 birds (7%; 3 budgerigars [Melopsittacus undulatus], 1 zebra finch, 1 vitelline masked weaver [Ploceus vitellinus], and 1 gouldian finch [Erythrura gouldiae]). Organs were fixed in formalin and embedded in paraffin, followed by staining of slices with hematoxylin and eosin, periodic acid-Schiff reaction, and Ziehl-Neelsen, following standard protocols.

Other diagnostic testing

Direct cytologic examinations of smears from feces, ingesta (from the intestine), and mucus (from the proventriculus) were performed in all 83 birds to detect parasites and M ornithogaster.

Liver, heart, and lungs of 66 birds (80%) were examined for bacteria by standard culture procedures. Columbia and colistin-nalidixic acid agar plates (Oxoid, Thermo Scientific, Wesel, Germany) were used as selective agar plates for gram-positive bacteria, whereas gram-negative bacteria were selected with eosin methylene blue agar plates (Oxoid). Culture was performed at 37[degrees]C for a minimum of 24 hours. Bacteria were identified morphologically by colony color, form, and Gram's stain features, and biochemically with the help of catalase, oxidase, and Analytical Profile Index tests (bioMerieux, Marcy l'Etoile, France). The quantity of bacteria in the clinical samples was estimated with a 3-phase streaking technique, and only bacteria occurring in high quantities (cultured in all 3 streaks) were considered relevant and were included in the evaluation. Bacteria at lower intensities were not evaluated because their roles as coinfections were regarded as unclear without histologic examination. In addition, liver and intestine samples were investigated for salmonella according to DIN EN ISO standard 6579:2002/ Amd 1:2007. If an infection with fungi was suspected (n = 5; 6%), fungal culture was performed with Sabouraud agar plates (Oxoid).

The PCR assays were performed depending on the case history, organ lesions, and the owners' willingness for further investigations. Organ samples included heart, liver, lungs, kidneys, spleen, proventriculus, ventriculus, intestine, and brain. The RNA was extracted from organs taken at necropsy with the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions, and reverse transcriptase-PCR assays were conducted for avian bornavirus (n = 11; 13%). (31,32) The DNA was extracted with the DNeasy Blood and Tissue Mini Kit. Additionally, PCR assays were performed for circovirus (33) (n = 33; 40%), Polyomavirus (34) (n = 32; 39%), adenovirus35 (n = 9; 11%), and Mycoplasma species (n = 5; 6%), (36) according to previously published protocols. Fifty-eight birds (70%), mainly belonging to the orders Psittaciformes and Passeriformes, were also examined via real-time PCR for M avium subspecies avium/silvaticum: (37)

Statistical analysis

Statistical analyses were performed with BiAS for Windows (version 11, Epsilon-Verlag, Hochheim Darmstadt, Germany). The general and body condition of psittacine and passerine birds were compared by the Mann-Whitney U test for 2 X k ordered tables. (38) The clinical signs and pathologic lesions of psittacine and passerine birds were compared by determining the odds ratios. Associations between the effects of the 3 pathogens--circovirus, Polyomavirus, and M ornithogaster--on single parameters of the pathologic lesions and clinical signs were determined by Fisher exact test, and the magnitude of risks were assessed by determining the odds ratios. An odds ratio > 3 supported by a significant P value (P < .05) on the Fisher exact test was interpreted as a strong indication that the examined pathogen was associated with a higher risk of the pathologic lesion or clinical signs. If the odds ratio was > 3 and the Fisher exact test was not significant (P > 0.5), results were interpreted as indicating possible increased risk of lesions or signs, but the sample size likely was too small to show significance.

Results

Infections with M genavense were detected in 26 species of birds belonging to the orders Passeriformes, Psittaciformes, Coliiformes, Columbiformes, and Ciconiiformes, which were all included in this study (Table 1). Prevailing species were budgerigars (n = 28; 34%), zebra finches (n = 10; 12%), Eurasian goldfinches (n = 5; 6%), vitelline masked weavers (n = 5; 6%), red-fronted parakeets (?Cyanoramphus novaezelandiae', n = 5; 6%), and canaries (n = 4; 5%).

Clinical signs

Of the 60 birds examined clinically, the general condition was assessed as good in 18 birds (30%), reduced in 21 birds (35%), and severely reduced in 21 birds (35%). In the psittacine birds examined clinically (n = 38; 63%), the general condition was good in 8 birds (21%), reduced in 14 birds (37%) and severely reduced in 16 birds (42%). Passerine birds examined (n = 22; 37%) had a good general condition in 10 cases (45%), a reduced general condition in 7 cases (32%), and a severely reduced general condition in 5 cases (23%). The difference in general condition between psittacine and passerine birds was significant (P < .05; Fig 1). The body condition of the birds was rated as good in 21 of the 60 birds (35%), reduced in 25 birds (42%) and cachectic in 14 birds (23%). There was no significant difference in body condition between psittacine and passerine birds.

No clinical signs were observed in 9 of the 60 clinically examined birds (15%); however, most birds exhibited several different clinical signs. The most common clinical signs detected in the 60 birds were feather disorders (n = 18; 31%), dyspnea (n = 17; 28%), ocular manifestations (n = 12; 20%), brittle beak and/or claws (n = 12; 20%), falling over or falling from the perch (n = 12; 20%), and polyuria (n = 10; 16%). A list of all clinical signs is shown in Table 2. Feather disorders included discoloration or abnormalities in growth (such as stress lines). Ocular manifestations affected mainly the adnexal structures with periocular swellings (subconjunctival granulomas/tubercles), as shown in Figure 2. Purulent conjunctivitis, lens luxation, disseminated chorioretinitis, and areas of retinal hyperpigmentation were also found.

In 17 of the 60 birds (28%), neurologic signs, such as paralysis, incoordination, seizures, or circular movement, were detected. Digestive system disorders, such as vomiting or regurgitation, diarrhea, undigested feces, and increased food uptake, were present in 11 of the 60 birds (19%). Significant differences in the presented clinical signs between psittacine and passerine birds were not detected.

Radiographic findings

The most common findings in the 37 birds examined radiographically were loss of the hourglass waist of the heart-liver shadow (n = 28; 76%), shadowing of the lungs (n = 23; 60%), shadowing or displacement of the air sacs because of enlarged liver shadow (n = 23; 60%), displacement of the ventriculus (n = 19; 51%), prominent kidneys (n = 18; 49%), and arthrosis (n = 13; 35%). Eight birds (22%) showed signs of osteolysis.

Radiographs with contrast media revealed dilated intestines in 10 of 16 birds (62%), and in 3 of those birds, prominent intestinal villi were revealed by the sawtooth-like appearance of the intestinal wall.

Pathologic findings

In 42 of the 82 examined birds (51%), only minor or no gross pathologic lesions of the internal organs were identified. The main gross pathologic findings were (in various degrees) enlarged liver (n = 38; 46%), dilated intestines (n = 30; 37%), and reddish wet lung (n = 22; 27%). White, firm nodules were found in 21% of the examined birds (n = 17), and 4% of birds (n = 3) had only local lesions of the eyes (mostly subconjunctival granulomatosis). Five birds (6%) were diagnosed as obese on pathologic examination. The most common necropsy findings are shown in Table 3. Significant differences between psittacine and passerine birds were found concerning enlarged livers (odds ratio [OR], 5.0; 95% CI, 1.8-13.5; P < .001) and occurrence of white, firm nodules (OR, 3.8; 95% CI, 0.96-14.6; P = .03).

Of the 78 birds in which the sex was recorded, 49% (n = 38) were male and 51% (n = 40) were female, indicating an equal sex ratio for the examined birds. The sex distribution in psittacine (male 50%, female 50%) and passerine (male 47%, female 53%) birds was similar.

Findings in the 6 of the 83 birds examined histologically revealed pathologic disorders of the livers in all 6 birds (100%; 3 fatty liver degeneration [50%]; 1 necrotic hepatitis [17%]; and 2 hepatosis with foamy macrophages [33%]), renal tubulonephroses in 4 birds (67%), and pneumonia in 3 birds (50%). Three birds (50%) had enteritis, and 1 of those birds (33%) showed prominent intestinal villi. In 2 of the 6 birds (33%), a necrotic splenitis was found, and 1 bird (17%) showed signs of immunosuppression. Further pathologic disorders included encephalopathy (2 birds; 33%), ingluvitis (1 bird; 17%), severe polyserositis from bacterial septicemia (1 bird; 17%), and a severe osteodystrophia fibrosa as well as inflammation of the uropygial gland (1 bird; 17%).

Coinfections

Coinfections were detected in 43 of the 83 birds (52%). Fungal coinfections were detected in 20 of the 83 birds (47%). In 17 of the 83 investigated birds (20%), M ornithogaster was found, and in 3 birds of 5 (60%), Aspergillus species were found.

Viral coinfections were detected in 20 of the 83 birds (24%). Eleven of 33 birds (33%; 6 zebra finches [55%], 2 blue rock thrushes [Monticola solitaries', 18%], 1 rufous bush robin [Cercotrichas galactotes; 9%], 1 red-fronted parakeet (9%), and 1 Syrian serin [Serinus syriacus; 9%]) were infected with circovirus; 8 of 32 birds (25%; 4 Eurasian goldfinches [50%], 3 zebra finches [38%], and 1 Gouldian finch [12%]) had Polyomavirus; 2 of 11 birds (18%; 1 canary [50%] and 1 cockatiel [Nymphicus hollandicus [50%]) had avian bornavirus; and 2 of 9 birds (22%; 1 vitelline masked weaver [50%] and 1 zebra finch [50%]) had adenovirus. Because not all Mycobacterium-positive birds in this study were investigated for viral infections, a minimum infection rate of only 24% could be calculated for the 83 birds (n = 20).

Coinfections with parasites were detected in 11 of 83 birds (13%). Six birds were infected with coccidia (55%), 2 birds with Knemidocoptes species (18%), 2 birds with Sternostoma tracheacolum (18%), and single birds with Trichomonas species, Ascaridia species, and cestoda (9% each).

Bacterial coinfections were detected in 9 of 66 examined birds (14%; corresponding to at least 11% of the total 83 birds). This included infections with Escherichia coli, Enterococcus species, Pseudomonas aeruginosa, and Klebsiella oxytoca. Salmonella species was detected in 1 of the 66 birds (2%) and 1 in 5 birds (20%) had a coinfection with Mycoplasma species.

Coinfections with M avium subspecies avium/ silvaticum were detected in 2 fruit doves (Ptilinopus porphyreus and Ptilinopus regina), which corresponds to 3% of the 58 birds tested for this pathogen.

Results of statistical analyses revealed that birds infected with Polyomavirus were more likely to have feather disorders (OR, 10.7; 95% CI, 1.0-115; Fisher exact test, P= .047) than birds with negative results for Polyomavirus. Birds with positive results for M ornithogaster were significantly more likely than birds with negative results to present with vomiting and/or regurgitation (OR, 14; 95% CI, 1.5-140; P = .02). Birds infected with Polyomavirus tended to have a greater risk of a brittle beak and/or nails (OR, 9.5; 95% CI, 1.1-83; P = .06) than uninfected birds had, although results were not significant. The results of this explorative study indicate that coinfections with circovirus, Polyomavirus, and M ornithogaster are associated with greater risks of several pathologic lesions and clinical signs (Table 4).

Three of the 83 birds (4%; 1 Amazon parrot, 1 budgerigar, and 1 canary, 33% each) presented with diarrhea. In 2 of the 3 birds (66%), coinfections with other pathogens were detected. The budgerigar had an additional infection with M ornithogaster, and the canary had additional infections with cestodes, Sternostoma tracheacolum, and Aspergillus species.

Discussion

This study is, to our knowledge, the first report of M genavense infections in coliiform and ciconiiform birds. Infection of coraciiform birds with M genavense, which was suspected in another study, (12) was proven, widening the host range of M genavense. To date, infections have mainly been reported in passerine and psittacine birds kept in captivity, (5-10) which is consistent with the results of this study. Nevertheless, this study shows that natural infections may occur in other host species.

The comparisons between psittacine and passerine birds revealed interesting results. The general condition of birds with mycobacteriosis differed significantly between psittacine and passerine birds. Passerine birds presented with a much better general condition than did psittacine birds. In addition, significant differences in gross pathologic findings were found between psittacine and passerine birds. Enlarged livers and the occurrence of white, firm nodules (granulomas) were found more frequently in psittacine birds. These results might suggest that psittacine and passerine bird orders differ in their reaction to M genavense infections.

Concerning the clinical signs and necropsy findings, the results presented here confirm that the body condition of naturally infected birds can vary widely and that birds in good body condition or even obese birds can be infected with M genavense. Mycobacteriosis in birds, in general, caused by M avium subspecies avium, has been described as a chronic wasting disease that leads to emaciation. (39) Only a few reports describe that the body condition of companion birds with mycobacteriosis can be good.40-41 However, because the birds in this study were naturally infected, they may have been in an early stage of infection, when body condition was not yet affected.

A broad range of different clinical signs was exhibited by the 60 birds examined clinically in this study, and 15% (9) of the birds presented with no clinical signs. Diarrhea was present in only 3 birds (5%), although dilated intestines were commonly found at necropsy (37%). Of the 60 birds examined clinically in this study, 17 (28%) had neurologic disorders. Neurologic signs have been often described in birds infected with M genavense, (3-10,19,24) emphasizing that neurologic disorders are common in infected birds. In addition, 20% of the 60 birds (n = 12) had vision disorders, ocular lesions, or both; thus, there may be a link between ocular lesions and infection with M genavense. In previous studies, infection with M genavense have been associated with severely swollen eyelids. (9,10) About 1.8% of ocular lesions in birds have been described as caused by mycobacterial infections. (42) A complete ophthalmologic examination of all the eyes of birds in this study would have been interesting because a significant number of microscopic lesions may have been present.

Because of the wide variety of clinical signs and the nonspecific radiographic results (suggesting only organ enlargement) in birds, the diagnosis of M genavense infection in clinical cases is difficult. A combination of those findings with results of other diagnostic tools, such as hematologic and biochemical testing, plasma protein electrophoresis, Ziehl-Neelsen stains of biopsy, or aspirate samples taken from bone marrow, liver, or spleen or of fecal smears--as suggested for diagnosing infections with M avium subspecies avium (43)-might be essential for the diagnosis of infections with M genavense as well.

In necropsies performed in this study, nonspecific findings, such as dilated intestines and enlarged livers were commonly seen. Granulomas were found in 21% (17 of 82) of the birds naturally infected with M genavense. In 51% of the birds examined in this study (42 of 82), only mild or no gross pathologic lesions were found. This supports the lack of pathologic lesions described in other studies (6) and confirms the difficulties in recognizing infections with M genavense at necropsy. The histologic findings in this study were not further evaluated because of the small sample size.

Influences of the bird species involved on the clinical or pathologic manifestations were not detected. However, these birds were suffering from natural infections and determining which signs were due to infection with M genavense and which were due to other pathogens was not possible. The results provided in this study clearly indicate that the pathogens M ornithogaster, Polyomavirus, and circovirus are associated with greater risk of some clinical signs and pathologic lesions. Additionally, the birds were from various owners and thus kept under diverse conditions, and other noninfectious factors, such as habitat and diet, which were not considered, might also have affected the clinical picture. The variety of clinical signs presented here may thus have been associated with factors other than M genavense infection.

The frequency of coinfections detected in the birds in this study was surprising. Infections with at least 1 other pathogen were detected in 43 birds. Because the birds were only partly examined for varying coinfections, an exact infection rate cannot be calculated, but a minimum prevalence of 52% can be concluded, a rather high value that might be even higher if every bird had been examined for every pathogen.

The high percentage of coinfections found suggests that there may be an association of coinfections and infection with M genavense. For example, most viral coinfections in this study were due to circovirus (11 of 33 birds; 35%) and Polyomavirus (8 of 32 birds; 25%). Circovirus infections have been associated with immune suppression in birds (44-46) Therefore, birds infected, especially with circovirus or Polyomavirus, might have been more susceptible to infections with M genavense.

Many birds (20%; 17 of 83) infected with M genavense were additionally infected with M ornithogaster. Because fungal agents, in general, are thought to be secondary rather than primary pathogens, those infections may have occurred secondary to infection with M genavense. That is in contrast to the viral infections, which we hypothesized were the primary pathogens.

In general, coinfections with bacteria did not seem to have a high significance in this study because only 9 out of 66 birds (14%) had confirmed bacterial infections on culture. Mixed infections with M avium subspecies avium/silvaticum were detected via real-time PCR in 2 fruit doves (3% of the 58 tested birds). Such mixed infections of M avium and M genavense have been reported in a Touit species (9) and in a canary, (8) as well as in people. (47)

The relevance of coinfections in relationship to M genavense infections is unclear. Coinfections may indicate a state of immunosuppression in birds, which predisposes them to an increased susceptibility for infection with M genavense. Further studies are needed to evaluate that hypothesis.

Results of this study suggest that infections with mycobacteria in psittacine and passerine birds may be widely underdiagnosed because of nonspecific clinical signs and pathologic findings, similar to results presented in other studies, (4,7,8) and the common occurrence of coinfections. Passerine and psittacine birds infected with M genavense differed significantly in general condition as well as in the occurrence of enlarged livers and white, firm nodules (granulomas) at necropsy. Further investigations, including more individual birds of different species and additional diagnostic testing, might clarify whether host species-specific differences occur in response to infection with M genavense.

Acknowledgments: We thank Miriam Schiller, Sabrina Fellner, and Lisa Koch for excellent technical assistance.

Anna Schmitz, Dr Med Vet, Monika Rinder, PD, Dr Med Vet, Susanne Thiel, Dr Med Vet, Andrea Peschel, Dr Med Vet, Kristina Moser, Dr Med Vet, Sven Reese, PD, Dr Med Vet, and Rudiger Korbel, Prof Dr Med Vet Dipl ECZM

From the Center for Clinical Veterinary Medicine. Clinic for Birds, Small Mammals, Reptiles and Ornamental Fish. Faculty of Veterinary Medicine, Ludwig-Maximilians-Universitat Munchen, Sonnenstrasse 18, D-85764 Oberschleissheim. Germany (Schmitz, Rinder, Thiel. Peschel. Moser, Korbel); and the Department of Veterinary Sciences, Institute of Anatomy, Histology, and Embryology, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universitat Munchen, Veterinarstr 13, D80539 Munchen, Germany (Reese).

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Caption: Figure 1. The 60 birds examined for general condition, included 38 psittacine (63%) and 22 passerine (37%) birds. There was a significant, opposed difference for general conditioning in the passerine birds compared with psittacine birds (P < .05).

Caption: Figure 2. Red-fronted parakeet with Chemosis conjunctiva and severe palpebral swellings because of subconjunctival granulomatosis and consecutive swelling of the eyelids.
Table 1. Bird species and number of birds by species included
in a study of clinical signs and pathologic findings in birds
(N = 83) naturally infected with Mycobacterium genavense.

Order             Common name               Scientific name

Psittaciformes    Budgerigar                Melopsittacus undulatus
  (n = 45; 54%)   Red-fronted parakeet      Cyanoramphus novaezelandiae
                  Cockatiel                 Nymphicus hollandicus
                  Jamaican Amazon           Amazona collaria
                  Rainbow lorikeet          Triclioglossus haematodus
                  Peach capitula            Agapornis roseicollis
                  Blue-bellied parrot       Triclaria malachitacea
                  Blue-fronted Amazon       Amazona aestiva
                    parrot
                  Lovebird                  Agapornis species
                  Red-lored Amazon parrot   Amazona autumnalis
                  Swift parrot              Lathamus discolor
Passeriformes     Zebra finch               Taeniopygia guttata
  (n = 33; 40%)   Eurasian goldfinch        Carduelis carduelis
                  Vitelline masked weaver   Ploceus vitellinus
                  Canary                    Serinus canaria
                  Gouldian finch            Erythrura gouldiae
                  Blue rock thrush          Monticola solitarius
                  Black headed canary       Serinus alario
                  Red fody                  Foudia madagascariensis
                  Rufous bush robin         Cercotrichas galactotes
                  Syrian serin              Serinus syriacus
Columbiformes     Pink-headed fruit dove    PtiUnopus porphyreus
  (n = 2; 2%)     Rose-crowned fruit dove   Ptilinopus regina
Ciconiiformes     Boat-billed heron         Cochlearius cochlearius
  (n = 1; 1%)
Coliiformes       Mousebird                 Colius species
  (n = 1; 1%)
Coraciiformes     Northern carmine          Merops nubicus
  (n = 1; 1%)       bee-eater

Order             Common name               No. (%)

Psittaciformes    Budgerigar                28 (34)
  (n = 45; 54%)   Red-fronted parakeet       5 (6)
                  Cockatiel                  2 (2)
                  Jamaican Amazon            2 (2)
                  Rainbow lorikeet           2 (2)
                  Peach capitula             1 (1)
                  Blue-bellied parrot        1 (1)
                  Blue-fronted Amazon        1 (1)
                    parrot
                  Lovebird                   1 (1)
                  Red-lored Amazon parrot    1 (1)
                  Swift parrot               1 (1)
Passeriformes     Zebra finch               10 (12)
  (n = 33; 40%)   Eurasian goldfinch         5 (6)
                  Vitelline masked weaver    5 (6)
                  Canary                     4 (5)
                  Gouldian finch             3 (4)
                  Blue rock thrush           2 (2)
                  Black headed canary        1 (1)
                  Red fody                   1 (1)
                  Rufous bush robin          1 (1)
                  Syrian serin               1 (1)
Columbiformes     Pink-headed fruit dove     1 (1)
  (n = 2; 2%)     Rose-crowned fruit dove    1 (1)
Ciconiiformes     Boat-billed heron          1 (1)
  (n = 1; 1%)
Coliiformes       Mousebird                  1 (1)
  (n = 1; 1%)
Coraciiformes     Northern carmine           1 (1)
  (n = 1; 1%)       bee-eater

Table 2. Clinical signs in birds that were presented for
examination and were confirmed as naturally infected
with Mycobacterium genavense (n = 60).

Clinical signs                     Results, No. (%)

Feather disorders                      18 (30)
Dyspnea                                17 (28)
Vision disorders and eye lesions       12 (20)
Brittle beak and/or nails              12 (20)
Falling over                           12 (20)
Polyuria                               10 (17)
No clinical signs                       9 (15)
Lameness and/or paralysis               8 (13)
Incoordination                          6 (10)
Vomiting and/or regurgitation           5 (8)
Swellings or granulomas                 4 (7)
Inability to fly                        4 (7)
Egg binding                             3 (5)
Diarrhea                                3 (5)
Undigested feces                        2 (3)
Epileptic fits                          2 (3)
Discolored feces                        2 (3)
Increased food uptake                   2 (3)
Circular movement                       1 (2)

Table 3. Frequencies of the most common
(n [greater than or equal to] 4) pathologic lesions
by gross examination of all birds submitted for v
(n = 82), subdivided by order (Psittaciformes [n = 44; 54%],
Passeriformes [n = 33; 40%], and other species [n = 5; 6%]).
Percentages are by subcategory.

Pathologic lesions              Total (n = 82)    Psittaciformes
                                   No. (%)       (n = 44) No. (%)

Enlarged liver                     38 (46)           27 (61)
Dilated intestine                  30 (37)           15 (34)
Reddish wet lung                   22 (27)           10 (23)
Enlarged spleen                    19 (23)           12 (27)
White firm nodules                 17 (21)           12 (27)
  in intestine                      6 (7)             4 (9)
  in body cavity                    4 (5)             2 (5)
  in liver                          4 (5)             2 (5)
  in spleen                         4 (5)             3 (7)
  in bone                           4 (5)             2 (5)
  in air sac                        1 (1)             1 (2)
  in proventriculus                 1 (1)             1 (2)
  in nares                          1 (1)             0 (0)
  in kidney                         1 (1)             1 (2)
  in ear                            1 (1)             1 (2)
Dilated proventriculus             14 (17)           11 (25)
Liver discoloration                11 (13)            3 (7)
No lesions                         11 (13)            5 (11)
Pale/beige spleen                   8 (10)            6 (14)
Periocular swelling                 8 (10)            5 (11)
Liver white/yellow patches          7 (9)             3 (7)
Liver brittle                       7 (9)             5 (11)
Liver compact/indurated             7 (9)             7 (16)
Marked renal tissue                 6 (7)             3 (7)
Hemorrhages proventriculus          6 (7)             6 (14)
Intestine filled with gas           6 (7)             2 (5)
Kidney beige color                  6 (7)             2 (5)
Villous hypertrophy intestine       5 (6)             5 (11)

Pathologic lesions               Passeriformes     Other (n = 5)
                                (n = 33) No. (%)      No. (%)

Enlarged liver                       8 (24)           3 (60)
Dilated intestine                   12 (36)           3 (60)
Reddish wet lung                    10 (30)           1 (20)
Enlarged spleen                      5 (15)           2 (40)
White firm nodules                   3 (9)            2 (40)
  in intestine                       1 (3)            1 (20)
  in body cavity                     1 (3)            1 (20)
  in liver                           0 (0)            2 (40)
  in spleen                          0 (0)            1 (20)
  in bone                            2 (6)            0 (0)
  in air sac                         0 (0)            0 (0)
  in proventriculus                  0 (0)            0 (0)
  in nares                           1 (3)            0 (0)
  in kidney                          0 (0)            0 (0)
  in ear                             0 (0)            0 (0)
Dilated proventriculus               3 (9)            0 (0)
Liver discoloration                  4 (12)           4 (80)
No lesions                           6 (18)           0 (0)
Pale/beige spleen                    1 (3)            1 (20)
Periocular swelling                  3 (9)            0 (0)
Liver white/yellow patches           2 (6)            2 (40)
Liver brittle                        1 (3)            1 (20)
Liver compact/indurated              0 (0)            0 (0)
Marked renal tissue                  3 (9)            0 (0)
Hemorrhages proventriculus           0 (0)            0 (0)
Intestine filled with gas            4 (12)           0 (0)
Kidney beige color                   2 (6)            2 (40)
Villous hypertrophy intestine        0 (0)            0 (0)

Table 4. Risk estimations of the association, as determined
by Fisher exact test (P value) and magnitude of effect
(odds ratio [OR]) of the pathogens circovirus, Polyomavirus,
and Macrorhabdus ornithogaster with pathologic lesions and
clinical signs in birds (n = number positive/number tested)
coinfected with Mycobacterium genavense.

                                      Circovirus
                                      (n = 11/33)

  Lesions and clinical signs            OR     P value

Pathologic lesions
  Enlarged liver                       1.7       .69
  Brittle liver                       -- (a)     --
  Discoloration liver                  1.1       1.0
  Liver white/yellow patches            --       --
  Pale/beige spleen                    2.3       .54
  Enlarged spleen                      1.9       .65
  Dilated proventriculus                --       --
  Hemorrhages proventriculus            --       --
  Dilated intestine                    0.20      .22
  Intestine filled with gas             --       --
  White firm nodules                   0.38      .64
  Marked renal tissue                  1.1       1.0
  Kidney beige color                   2.3       .54
  Reddish wet lung                     2.2       .42
  Periocular swelling                  1.6       .64
  No lesions                           0.54      .69
Clinical signs
  Falling over                         2.6       .13
  Incoordination                        --       --
  Disability to fly                     --       --
  Dyspnea                              3.8     .18 (b)
  Polyuria                              --       --
  Vision disorders and eye lesions     0.31      .63
  Vomiting and/or retching              --       --
  Diarrhea                              --       --
  Egg binding                           --       --
  Feather disorders                    1.7       .66
  Brittle beak and/or nails            0.40      .63
  No clinical signs                    0.76      1.0

                                       Polyomavirus
                                        (n = 8/32)

  Lesions and clinical signs           OR    P value

Pathologic lesions
  Enlarged liver                      0.26     .39
  Brittle liver                        --       --
  Discoloration liver                 1.2      1.0
  Liver white/yellow patches           --       --
  Pale/beige spleen                   3.9    .40 (b)
  Enlarged spleen                      --       --
  Dilated proventriculus               --       --
  Hemorrhages proventriculus           --       --
  Dilated intestine                   0.83     1.0
  Intestine filled with gas           4.3    .21 (b)
  White firm nodules                  0.52     1.0
  Marked renal tissue                  --       --
  Kidney beige color                   --       --
  Reddish wet lung                    0.43     .65
  Periocular swelling                 0.86     1.0
  No lesions                          2.2      .39
Clinical signs
  Falling over                         --       --
  Incoordination                       --       --
  Disability to fly                    --       --
  Dyspnea                              --       --
  Polyuria                            4.2    .22 (b)
  Vision disorders and eye lesions    0.67     1.0
  Vomiting and/or retching             --       --
  Diarrhea                             --       --
  Egg binding                          --       --
  Feather disorders                   10.7   .047 (c)
  Brittle beak and/or nails           9.5    .06 (b)
  No clinical signs                    --       --

                                      M ornithogaster
                                        (n = 17/83)

  Lesions and clinical signs           OR     P value

Pathologic lesions
  Enlarged liver                      0.56      .42
  Brittle liver                       0.62      1.0
  Discoloration liver                  --       --
  Liver white/yellow patches          0.62      1.0
  Pale/beige spleen                    --       --
  Enlarged spleen                     0.16      .10
  Dilated proventriculus              1.7       .47
  Hemorrhages proventriculus          2.0       .60
  Dilated intestine                   1.7       .40
  Intestine filled with gas           2.0       .60
  White firm nodules                  0.19      .11
  Marked renal tissue                 4.4     ,10 (b)
  Kidney beige color                  4.2     .19 (b)
  Reddish wet lung                    1.2       .78
  Periocular swelling                 0.52      1.0
  No lesions                          1.6       .70
Clinical signs
  Falling over                        0.48      .48
  Incoordination                      0.52      1.0
  Disability to fly                   0.91      1.0
  Dyspnea                             1.2       .76
  Polyuria                            2.1       .43
  Vision disorders and eye lesions    1.5       .72
  Vomiting and/or retching             14     .02 (c)
  Diarrhea                            1.4       1.0
  Egg binding                         6.1     .17 (b)
  Feather disorders                   1.6       .53
  Brittle beak and/or nails           0.89      1.0
  No clinical signs                   0.35      0.7

(a) Not applicable.

(b) Association between the agent and pathologic lesion
or clinical sign appears to be present, but the sample
size was too small to show significance.

(c) Significant association between pathogen
and pathologic lesion or clinical sign.
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Title Annotation:Original Study
Author:Schmitz, Anna; Rinder, Monika; Thiel, Susanne; Peschel, Andrea; Moser, Kristina; Reese, Sven; Korbel
Publication:Journal of Avian Medicine and Surgery
Date:Sep 1, 2018
Words:7187
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