Journal of Avian Medicine and Surgery 28(4):297–303, 2014 Ó 2014 by the Association of Avian Veterinarians

Retrospective Studies

Gastrointestinal Cryptosporidiosis in Captive Psittacine Birds in the United States: A Case Review Michelle L. Ravich, DVM, Dipl ABVP, Drury R. Reavill, DVM, Dipl ABVP, Dipl ACVP, Laurie Hess, DVM, Dipl ABVP, April L. Childress, and James F. X. Wellehan Jr, DVM, MS, PhD, Dipl ACZM, Dipl ACVM Abstract: Histopathologic analysis performed on tissue samples from 34 captive psittacine birds over a 14-year period demonstrated gastrointestinal cryptosporidiosis. Although 8 birds had died unexpectedly, at least 10 showed gastrointestinal signs before death. Most birds had other significant pathologic lesions, unrelated to cryptosporidiosis, in which case Cryptosporidium was likely a secondary pathogen. However, in 11 birds the only significant histopathologic finding was gastrointestinal cryptosporidiosis. In 2 lovebird (Agapornis species) cases, direct sequencing of proventricular samples confirmed the presence of Cryptosporidium avian genotype III. A description and analysis of the pathologic findings in these 34 birds, as well as a review of avian gastrointestinal cryptosporidiosis, is provided. Key words: Cryptosporidium, proventriculus, pathology, parasitology, psittacine birds

causes of cryptosporidiosis in people, along with Cryptosporidium hominis.8 Cryptosporidium serpentis is well known in snakes, in which it causes hypertrophic gastritis, often characterized by weight loss, regurgitation, and inappetance,9 whereas Cryptosporidium muris predominantly affects rodents but does not cause disease in snakes.10 Both C serpentis and C muris are commonly found in snake feces, emphasizing the clinical importance of species identification. However multiple species have been shown to be capable of jumping across vertebrate host classes: C serpentis has been found in cattle11; Cryptosporidium meleagridis, first described in turkeys, is a human pathogen12; C parvum has been identified in ray-finned fish13,14; and an unnamed Cryptosporidium genotype first identified in cockatiels (Nymphicus hollandicus) has been reported in association with cloacitis and cystitis in a green iguana (Iguana iguana).15 The presence of Cryptosporidium in birds was first described by Tyzzer in 1929,3 although a specific species was not named. In 1955, a similar organism was isolated in turkeys and was named C meleagridis.2,3 Since then, infections caused by Cryptosporidium have been observed in more than 30 avian species.16 There are 3 primary avian

Introduction The genus Cryptosporidium consists of coccidia in the suborder Eimeriorina, family Cryptosporidiidae, and can be pathogenic in numerous birds, reptiles, and mammals.1–3 There are many species of Cryptosporidium with significantly different clinical implications. Morphologic species identification is not reliable, and sequence-based techniques are needed to differentiate species.4 The known species of Cryptosporidium form 2 clades, one with gastric tropism and one with primarily intestinal tropism, indicating that site of infection has had greater long-term fidelity than host species.5 However, most Cryptosporidium species studied often prefer specific clades of hosts. For example, Cryptosporidium parvum is a frequent cause of diarrhea in calves,6 and it has also been associated with self-limiting diarrhea in cats.7 Cryptosporidium parvum is also one of the main From the Veterinary Center for Birds & Exotics, 709 Bedford Rd, Bedford Hills, NY 10507, USA (Ravich, Hess); Zoo and Exotic Pathology Service, 2825 KOVR Dr, West Sacramento, CA 95605, USA (Reavill); and the University of Florida College of Veterinary Medicine, PO Box 100126, 2015 SW 16th Ave, Gainesville, FL 32608, USA (Childress, Wellehan).

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species of Cryptosporidium (C meleagridis, C baileyi, and C galli),17 as well as at least 12 published but unnamed avian genotypes such as genotypes I–IV (identified in numerous avian species), the Eurasian woodcock (Scolopax rusticola) genotype, and the black duck (Anas rubripes) genotype.2,18 Although C meleagridis was initially discovered in turkeys, it affects a wide range of avian species, including parrots, chickens, partridges, and certain columbiform birds.2,17 It has also been reported in dogs, cattle, pigs, rabbits, and rodents, and it is zoonotic.2,12,19 Cryptosporidium meleagridis primarily infects the small intestine, large intestine, and bursa, leading to enteritis and death.2 Cryptosporidium baileyi was initially isolated from broiler chickens; however, it has also been found in a wide range of avian hosts, including parrots, turkeys, geese, ducks, falcons, gulls, cranes, and cormorants.2 Infections from C baileyi have been found in diverse anatomic locations in avian species, including the lower gastrointestinal tract (small and large intestines, ceca, cloaca), respiratory tract (trachea, air sacs), urinary system, bursa, and conjunctiva. Respiratory infections with this organism can cause high mortality, especially in broiler chickens. Cryptosporidium baileyi is closely related to avian genotypes I, II, and V. Cryptosporidium galli was isolated from chickens and primarily affects the proventriculus, and infections have also been reported in turquoise parrots (Neophema pulchella), flamingos (Phoenicopterus ruber), hornbills (Buceros rhinoceros), and certain passerine birds. 2 Cryptosporidium galli is closely related to other gastric species, including C muris and C serpentis, as well as avian genotype IV. Avian genotype III has been reported in Australia and Japan, and avian genotype V has also been reported in Japan.16,20 Pathologic associations have been documented with identified Cryptosporidium species in psittacine birds. Cryptosporidium meleagridis was described in a nestling Indian ring-necked parrot (Psittacula krameri) in Australia.16 The parrot presented for lethargy and slow crop emptying and was ultimately euthanatized. Histopathologic analysis confirmed oocysts in the small intestine and showed intestinal pathologic lesions of hyperplasia and necrosis. Cryptosporidium galli was reported in Brazil in a cockatiel that demonstrated lethargy for approximately 1 year before death.21 A proventricular smear demonstrated Cryptosporidium oocysts with Kinyoun acid-fast staining, and Cryptosporidium oocysts were present in fecal samples as well. Lastly, Cryptosporidium avian

genotype III was isolated in numerous peach-faced lovebirds (Agapornis roseicollis) in Japan.20 All birds demonstrated gastrointestinal signs of weight loss and chronic vomiting. Histopathologic lesions were found within the proventriculus and isthmus. In the United States, although cryptosporidiosis has been described in parrots, to our knowledge, Cryptosporidium species identification has not been reported in psittacine birds.22,23 Several studies have also documented the presence and prevalence of cryptosporidia in fecal samples from psittacine birds. A study performed on pet shop birds in China found Cryptosporidium avian genotype III in 2 cockatiels and Cryptosporidium avian genotype V in 6 cockatiels.17 Cryptosporidium avian genotype II was identified in a wild white-eyed parakeet (Aratinga leucophthalma) in a similar study performed in Brazil.24 Ng et al25 demonstrated the presence of Cryptosporidium species in numerous psittacine birds in Australia. Avian genotype II was found in Major Mitchell’s cockatoos (Cacatua leadbeateri), cockatiels, eclectus parrots (Eclectus roratus), a galah (Eolophus roseicapilla), and a sun conure (Aratinga solstitialis), whereas avian genotype III was found in cockatiels, princess parrots (Polytelis alexandrae), a galah, and a sun conure. Lastly, C galli was found in turquoise parrots.25 Cryptosporidium species have direct life cycles, with oocysts shed in the feces or expelled from the respiratory tract by infected hosts. The oocysts are infective once shed and are transmitted by either ingestion or inhalation. Once inside the new host, sporozoites are released and penetrate epithelial cells, primarily within the gastrointestinal and respiratory tracts, but the urinary system, bursa, and conjunctiva are also possible targets.1–3 The purpose of this retrospective study is to describe Cryptosporidium infection in 34 captive psittacine birds in the United States, including clinical signs as well as location and severity of organ systems affected. Materials and Methods Over a period of 14 years, histopathologic analysis performed on tissue samples collected from 34 psittacine birds demonstrated gastrointestinal Cryptosporidium infection or contained lesions consistent with gastrointestinal cryptosporidiosis. The birds included 12 cockatiels, 18 lovebirds (2 A roseicollis, 16 species unknown), and 4 parrotlets (Forpus genus, species unknown), all from different regions throughout the United States. The birds ranged in age from 1 month to

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16 years old. All birds were either kept as pets or were being raised by breeders or pet stores to be sold as pets. The diets and housing conditions of the birds were unknown. Aside from 1 lovebird in which a surgical biopsy was taken from the proventriculus, the birds were either found dead or were euthanatized, and either the entire body or collected tissue samples were placed in formalin for analysis. In 1 lovebird, only the proventriculus and ventriculus were submitted, and in 2 lovebirds, only the proventriculus, ventriculus, and liver were submitted. All tissue samples obtained at postmortem examination or at surgery were preserved in 10% neutral buffered formalin solution for routine processing. Paraffinembedded tissues were sectioned at approximately 5 lm, mounted on glass slides, and stained with hematoxylin and eosin. All histopathologic samples were analyzed by the Zoo and Exotic Pathology Service (West Sacramento, CA). In 2 lovebird cases, tissues were submitted for polymerase chain reaction (PCR) analysis for Cryptosporidium. One case consisted of recut, unstained sections of the proventriculus from the formalin-fixed paraffin-embedded tissues, and the second was a frozen section of proventriculus. DNA was extracted (DNeasy Blood and Tissue Kit, Qiagen, Valencia, CA, USA), and amplification with consensus primers for the Cryptosporidium 18S rRNA gene was performed according to previously described methods.5 Direct sequencing was performed, with all products sequenced in both directions. Predicted homologous nucleotide sequences of the 18S rRNA gene were aligned with the MAFFT (multiple alignment using fast Fourier transform) program.26 Theileria ovis (GenBank accession JQ737135) was designated as the outgroup. Bayesian analysis of the nucleotide alignment was performed with MrBayes 3.1.227 on the CIPRES server28 with gamma distributed rate variation and a proportion of invariant sites and a general timereversible model. The first 25% of 2 000 000 iterations were discarded as a burn-in. Maximum likelihood analyses of each alignment were performed with RAxML on the CIPRES server,29 with gamma-distributed rate variation and a proportion of invariant sites. Bootstrap analysis was used to test the strength of the tree topology.30 Results Cryptosporidium, or pathologic lesions consistent with cryptosporidiosis, were documented in 34 psittacine birds. The birds represented both Old

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World (12 cockatiels and 18 lovebirds) and New World (4 parrotlets) species. Eight birds died acutely, with no antemortem clinical signs. At least 10 birds exhibited gastrointestinal signs of lime-green stool, chronic or intermittent regurgitation, weight loss, or fungal or bacterial crop infections. Of these 10 birds, 8 (1 cockatiel and 7 lovebirds) had chronic gastrointestinal signs. A few birds had only nonspecific signs before death, including weakness, fluffed appearance, decreased appetite, or lethargy. Other birds had presented with specific problems, including lockjaw, aspiration pneumonia, seizures, and egg binding. In 3 cases the proventriculus appeared abnormal on radiographs; in 1 cockatiel, survey radiographs demonstrated a suspect dilated proventriculus, and in 2 lovebirds, a barium study demonstrated a thickened proventricular wall. At postmortem examination, an abnormal proventriculus (either dilated, thickened, or both) was observed in 9 birds (2 cockatiels and 7 lovebirds), and dilated intestines were observed in 2 birds (1 cockatiel and 1 lovebird). Histopathologic lesions associated with Cryptosporidium were proventricular glandular hyperplasia with intraluminal protozoa, proventriculitis, lymphocytic plasmacytic ventriculitis, and nonsuppurative enteritis with mucosal protozoa. Proventricular cryptosporidiosis with or without ventricular cryptosporidiosis was identified in 31 birds, whereas only 3 cockatiels had intestinal cryptosporidiosis without observation of gastric Cryptosporidium organisms. However, 1 of these 3 cockatiels had gastric megabacteriosis (Macrorhabdus ornithogaster) and intraluminal yeast, and another had severe diffuse gastric megabacteriosis. In 11 birds (3 cockatiels, 7 lovebirds, and 1 parrotlet), cryptosporidial lesions were the most significant pathologic findings, and either no other pathologic lesions were observed or the other changes were considered insignificant. However, not all tissues were submitted in all cases. In at least 17 birds, Cryptosporidium was considered likely to be a secondary pathogen because a probable primary cause of death was identified by histopathologic analysis. Two birds (one 2 months and one 4 months old) had intraluminal foreign material in the lung, interpreted as aspiration. Three birds had glomerular intranuclear inclusions, most consistent with adenovirus. One lovebird with a history of feather loss was positive for psittacine beak and feather disease (circovirus) by DNA in situ hybridization of feathered skin sections. Other postmortem diagnoses were cholangiohepatitis, atherosclerosis, tubu-

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Figure 1. Hyperplasia of the ductal epithelium of a proventricular gland from a lovebird with cryptosporidiosis. * Lumen; bar ¼ 100 lm (hematoxylin and eosin; 310).

Figure 2. Protozoa identified as cryptosporidia present on the ductal epithelium of proventricular glands (arrow) from a lovebird. Bar ¼ 50 lm (hematoxylin and eosin; 340).

lointerstitial nephritis, membranoproliferative glomerulopathy, pneumonia, encephalitis, and enteritis with intraluminal ascarids. Many of the birds had other gastrointestinal infections as well. Seven birds (6 cockatiels and 1 lovebird) had proventricular megabacteriosis, and 1 of those cockatiels also had intraluminal gastric yeast and ingluvial yeast with pseudohyphae. Another cockatiel had ingluvial yeast with pseudohyphal proliferation as well. Both of these cockatiels with ingluvial pseudohyphae were less than 1 month old, and it is known that at least one of them was being tube fed. One cockatiel had enteritis with flagellated intraluminal protozoa, most consistent with either Trichomonas or Hexamita species on the basis of histologic evaluation with hematoxylin and eosin staining. Histopathologically, the typical lesion of cryptosporidiosis was extensive hyperplasia of the ductal epithelium of the proventricular glands, resulting in some distortion of the glandular architecture caused by hyperplastic changes (Fig 1). Variable numbers of oval to round amphophilic protozoa-like organisms were closely adhering to the lining of ductal and surface epithelial cells (Fig 2). In the 2 lovebird cases where identification of Cryptosporidium species by PCR and sequencing was performed, both samples resulted in products that were 765 base pairs after primers were edited out and were 100% identical to an unnamed Cryptosporidium species from a cockatiel described as avian genotype III (GenBank accession HM116385). A representative sequence was submitted to GenBank (accession KJ661334). Phylo-

genetic analysis found that this isolate clustered with other gastric-tropic Cryptosporidium species (Fig 3). Discussion Significant diversity exists in Cryptosporidium species found in birds. Different species appear to have significant differences in site tropism and host fidelity (including zoonotic potential), and it can reasonably be extrapolated that significant differences in pathogenicity are likely. Treatment of diverse cryptosporidial species of birds as a single entity is likely to result in poor clinical responses. Knowledge of site of infection of a given Cryptosporidium species is also important for diagnostic sampling. Gastric lavage was found to be a better sample for detection of C serpentis (a gastric species) than cloacal swabs31; this would not be expected for a species with an affinity for the intestine. Our phylogenetic analysis is in agreement with most other published studies. It can be seen that the Cryptosporidium species that infect intestines form a clade (Fig 3). Gastric tropism appears to be a basal state. It can also be seen that there is no deep-level segregation of Cryptosporidium species between host classes, implying that host jumping might not be a rare event. Host fidelity appears to differ significantly, with several species capable of infecting more than one host class. Among the species using avian hosts, C meleagridis presents the known zoonotic risk; knowledge of host species is needed to assess risk.

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Figure 3. Bayesian phylogenetic tree of MAFFT alignment of homologous 18S rRNA nucleotide sequences. Theileria ovis (GenBank accession JQ737135), a non-Cryptosporidium apicomplexan, was used as the outgroup. Confidence of the tree topology obtained is shown by Bayesian posterior probabilities to the left of the slash or above in bold and maximum likelihood bootstrap values to the right or below. The sample from this study is marked with an arrow. Site of infection, when known, and host clade are shown in columns on the right. GenBank accession numbers follow sequence names.

Avian Cryptosporidium is typically considered to be a secondary, opportunistic pathogen. In healthy animals, infection can be subclinical, changing to clinical disease when animals become immunosuppressed.1,12 This is potentially the case in many of the birds in our study, as approximately 50% had obvious primary causes of death. Additionally, in this study, most cryptosporidial lesions were confined to the proventriculus and ventriculus. Similar clinical signs and pathologic findings have been demonstrated previously in psittacine birds. A study in Japan involving peach-faced lovebirds found that out of 37 lovebirds positive for Cryptosporidium oocysts in fecal samples, 54% displayed gastrointestinal signs, including de-

creased appetite, weight loss, melena, and chronic vomiting.20 In most symptomatic birds in that study, contrast radiographs revealed thickened proventricular walls, and proventricular mucosal hypertrophy was noted in 3 birds at postmortem examination. Cryptosporidium avian genotype III was isolated from 13 of those birds. These findings are very similar to the findings observed in our study. Most birds in our study were lovebirds, at least 7 of which demonstrated chronic gastrointestinal signs. Additionally, in 2 lovebirds, contrast radiographs demonstrated thickened proventricular walls, and 78% (7/9) of birds with an abnormal proventriculus observed at postmortem examination were lovebirds as well.

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Finally, 2 lovebirds were confirmed to have cryptosporidia by PCR analysis, and direct sequencing confirmed Cryptosporidium avian genotype III. Given these similarities, it seems plausible that lovebirds may be prone to proventricular cryptosporidiosis, with Cryptosporidium avian genotype III as a primary cause of death. Because this was a retrospective study primarily involving postmortem examination and histopathologic results, further investigation into the initial clinical signs, diagnostic results, and potential treatment of these lovebirds was not possible. Also, in 4 lovebirds, limited tissues were submitted (only proventriculus, with or without ventriculus, and liver), potentially resulting in a sampling bias. Moreover, although morphologically all cases had proventricular Cryptosporidium, species identification was only performed in 2 cases. Our study supports other findings that some Cryptosporidium species may cause gastrointestinal disease in psittacine birds. Only 3 birds in our study demonstrated intestinal cryptosporidiosis without gastric involvement; however, 2 of those birds had severe gastric lesions from other infectious agents (M ornithogaster and yeast), possibly obscuring identification of initial or concurrent Cryptosporidium infection. Based on the cases analyzed in this study, it would be prudent to evaluate for cryptosporidiosis in parrots exhibiting gastrointestinal signs, especially in chronic cases. Fecal analysis by oocyst identification after acidfast staining or concentration by sugar flotation constitutes a logical first step toward antemortem diagnosis.3,32 Biopsy and histologic examination will demonstrate pathogenicity and site tropism.3 Positive acid-fast staining without histologic analysis should be followed up with sequence-based identification to determine site tropism, host range, and overall risk. The primary limitation of this study was that, given its retrospective nature, a thorough analysis of each specimen was not possible. Often, all tissues for a given bird were not submitted, preventing complete analysis for other disease processes. Additionally, most cases did not have specific species or genotype identification by sequencing. This study illustrates numerous cases of gastrointestinal cryptosporidiosis in psittacine birds. Results suggest that Cryptosporidium was a contributor to or the potential sole cause of death in these birds. Moreover, Cryptosporidium avian genotype III was sequenced for the first time in the United States, from samples from 2 lovebird specimens. The specimens in this study emphasize

the importance of evaluating for Cryptosporidium in the antemortem diagnostic approach to psittacine birds with gastrointestinal signs, especially lovebirds and cockatiels. Further studies are needed to determine both the prevalence of specific Cryptosporidium species and genotypes in parrots and the potential for Cryptosporidium to act as a primary pathogen in psittacine birds. References 1. Doneley RJT. Bacterial and parasitic diseases of parrots. Vet Clin North Am Exot Anim Pract. 2009;12(3):417–432. 2. Ryan U. Cryptosporidium in birds, fish and amphibians. Exp Parasitol. 2010;124(1):113–120. 3. Sr´eter T, Varga I. Cryptosporidiosis in birds—a review. Vet Parasitol. 2000;87(4):261–279. 4. Fall A, Thompson RCA, Hobbs RP, Morgan-Ryan U. Morphology is not a reliable tool for delineating species within Cryptosporidium. J Parasitol. 2003;89(2):399–402. 5. Griffin C, Reavill DR, Stacy BA, et al. Cryptosporidiosis caused by two distinct species in Russian tortoises and a pancake tortoise. Vet Parasitol. 2010;170(1–2):14–19. 6. O’Handley RM, Cockwill C, McAllister TA, et al. Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea. J Am Vet Med Assoc. 1999;214(3):391–396. 7. Bush JM, Speer B, Opitz N. Disease transmission from companion parrots to dogs and cats: what is the real risk? Vet Clin North Am Small Anim Pract. 2011;41(6):1261–1272. 8. Palmer CJ, Xiao L, Terashima A, et al. Cryptosporidium muris, a rodent pathogen, recovered from a human in Per´u. Emerg Infect Dis. 2003;9(9):1174– 1176. 9. Paiva PR, Grego KF, Lima VMF, et al. Clinical, serological, and parasitological analysis of snakes naturally infected with Cryptosporidium serpentis. Vet Parasitol. 2013;198(1–2):54–61. 10. Fayer R, Graczyk TK, Cranfield MR. Multiple heterogenous isolates of Cryptosporidium serpentis from captive snakes are not transmissible to neonatal BALB/c mice (Mus musculus). J Parasitol. 1995;81(3):482–484. 11. Chen F, Qiu H. Identification and characterization of a Chinese isolate of Cryptosporidium serpentis from dairy cattle. Parasitol Res. 2012;111(4):1785– 1791. 12. Pedraza-Diaz S, Amar CF, McLauchlin J, et al. Cryptosporidium meleagridis from humans: molecular analysis and description of affected patients. J Infect. 2001;42(4):243–250. 13. Reid A, Lymbery A, Ng J, et al. Identification of novel and zoonotic Cryptosporidium species in marine fish. Vet Parasitol. 2010;168(3):190–195.

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Gastrointestinal cryptosporidiosis in captive psittacine birds in the United States: a case review.

Histopathologic analysis performed on tissue samples from 34 captive psittacine birds over a 14-year period demonstrated gastrointestinal cryptosporid...
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