David N. Phalen

Encephalitozoon hellem in Two Eclectus Parrots (Eclectus roratus): Identification from Archival Tissues

ABSTRACT Members of the phylum Microspora are obligate, intracellular, single‐celled parasites identified in a wide range of vertebrate and invertebrate hosts. Only a few cases of microsporidial infections have been documented in psittacine birds including peach‐faced, masked, and Fischers lovebirds (Agapornis roseicollis, A. personata, and A. fischeri. respectively), budgerigars (Melopsittacus undulatus), and a double yellow‐headed Amazon parrot (Amazona ochrocephala). Parasite identification has typically been limited to phylum or genus, and no avian species of microsporidia has clearly been described. In this report, microsporidia were identified in the kidney and intestine of a new host, the eclectus parrot (Eclectus roratus). Parasites were identified as Encephalitozoon hellem using morphologic, ultrastructural, and small subunit ribosomal RNA gene sequence data obtained from archived tissues. This parasite species was first identified in immunocompromised humans and may be a potential zoonotic pathogen. The epidemiology and prevalence of this parasite in humans and birds should be further explored.

A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife.

Highlights • The importance of Neospora caninum-associated disease in wildlife is reviewed.• There are only 12 reports of clinical neosporosis in wildlife species to date.• The best practice guidelines to follow for reporting wildlife cases of neosporosis are presented.

A Suspected Parasite Spill-Back of Two Novel Myxidium spp. (Myxosporea) Causing Disease in Australian Endemic Frogs Found in the Invasive Cane Toad

Infectious diseases are contributing to the decline of endangered amphibians. We identified myxosporean parasites, Myxidium spp. (Myxosporea: Myxozoa), in the brain and liver of declining native frogs, the Green and Golden Bell frog (Litoria aurea) and the Southern Bell frog (Litoria raniformis). We unequivocally identified two Myxidium spp. (both generalist) affecting Australian native frogs and the invasive Cane toad (Bufo marinus, syn. Rhinella marina) and demonstrated their association with disease. Our study tested the identity of Myxidium spp. within native frogs and the invasive Cane toad (brought to Australia in 1935, via Hawaii) to resolve the question whether the Cane toad introduced them to Australia. We showed that the Australian brain and liver Myxidium spp. differed 9%, 7%, 34% and 37% at the small subunit rDNA, large subunit rDNA, internal transcribed spacers 1 and 2, but were distinct from Myxidium cf. immersum from Cane toads in Brazil. Plotting minimum within-group distance against maximum intra-group distance confirmed their independent evolutionary trajectory. Transmission electron microscopy revealed that the brain stages localize inside axons. Myxospores were morphologically indistinguishable, therefore genetic characterisation was necessary to recognise these cryptic species. It is unlikely that the Cane toad brought the myxosporean parasites to Australia, because the parasites were not found in 261 Hawaiian Cane toads. Instead, these data support the enemy-release hypothesis predicting that not all parasites are translocated with their hosts and suggest that the Cane toad may have played an important spill-back role in their emergence and facilitated their dissemination. This work emphasizes the importance of accurate species identification of pathogens relevant to wildlife management and disease control. In our case it is paving the road for the spill-back role of the Cane toad and the parasite emergence.

Isolation and characterization of an avian isolate of Encephalitozoon hellem

Members of the phylum Microspora are a group of unusual, obligate intracellular eukaryotic parasites that infect a wide range of hosts. However, there are a limited number of microsporidial infections reported in avian hosts, and no parasite species has been defined as an avian pathogen. A microsporidian organism was recovered from the droppings of a clinically normal peach-faced lovebird (Agapornis roseicollis) and established in in vitro culture. Intermittent parasite spore shedding was documented over a 2-month period using calcofluor M2R staining of cloacal swabs. The organism was identified as Encephalitozoon hellem based on protein and antigenic profiles and molecular sequencing of the small subunit and internal transcribed spacer regions of the ribosomal RNA gene.

Museum material reveals a frog parasite emergence after the invasion of the cane toad in Australia

BackgroundA parasite morphologically indistinguishable from Myxidium immersum (Myxozoa: Myxosporea) found in gallbladders of the invasive cane toad (Bufo marinus) was identified in Australian frogs. Because no written record exists for such a parasite in Australian endemic frogs in 19th and early 20th century, it was assumed that the cane toad introduced this parasite. While we cannot go back in time ourselves, we investigated whether material at the museum of natural history could be used to retrieve parasites, and whether they were infected at the time of their collection (specifically prior to and after the cane toad translocation to Australia in 1935).ResultsUsing the herpetological collection at the Australian Museum we showed that no myxospores were found in any animals (n = 115) prior to the cane toad invasion (1879-1935). The green and golden bell frog (Litoria aurea), the Perons tree frog (Litoria peronii), the green tree frog (Litoria caerulea) and the striped marsh frog (Limnodynastes peronii) were all negative for the presence of the parasite using microscopy of the gallbladder content and its histology. These results were sufficient to conclude that the population was free from this disease (at the expected minimum prevalence of 5%) at 99.7% confidence level using the 115 voucher specimens in the Australian Museum. Similarly, museum specimens (n = 29) of the green and golden bell frog from New Caledonia, where it was introduced in 19th century, did not show the presence of myxospores. The earliest specimen positive for myxospores in a gallbladder was a green tree frog from 1966. Myxospores were found in eight (7.1%, n = 112) frogs in the post cane toad introduction period.ConclusionAustralian wildlife is increasingly under threat, and amphibian decline is one of the most dramatic examples. The museum material proved essential to directly support the evidence of parasite emergence in Australian native frogs. This parasite can be considered one of the luckiest parasites, because it has found an empty niche in Australia. It now flourishes in > 20 endemic and exotic frog species, but its consequences are yet to be fully understood.

Detection and Heterogeneity of Herpesviruses Causing Pacheco's Disease in Parrots

ABSTRACT Pachecos disease (PD) is a common, often fatal, disease of parrots. We cloned a virus isolate from a parrot that had characteristic lesions of PD. Three viral clones were partially sequenced, demonstrating that this virus was an alphaherpesvirus most closely related to the gallid herpesvirus 1. Five primer sets were developed from these sequences. The primer sets were used with PCR to screen tissues or tissue culture media suspected to contain viruses from 54 outbreaks of PD. The primer sets amplified DNA from all but one sample. Ten amplification patterns were detected, indicating that PD is caused by a genetically heterogeneous population of viruses. A single genetic variant (psittacid herpesvirus variant 1) amplified with all primer sets and was the most common virus variant (62.7%). A single primer set (23F) amplified DNA from all of the positive samples, suggesting that PCR could be used as a rapid postmortem assay for these viruses. PCR was found to be significantly more sensitive than tissue culture for the detection of psittacid herpesviruses.

Prevalence of Microsporidian Spores Shed by Asymptomatic Lovebirds: Evidence for a Potential Emerging Zoonosis

Abstract Several reports implicate Encephalitozoon hellem, a microsporidian parasite first described in humans and later in birds, as the causative agent of severe disease in immunocompromised patients. This study was conducted to assess the prevalence of microsporidian spores shed in the droppings of lovebirds. During a 7-month period, a total of 198 apparently healthy lovebirds from 8 flocks in Texas were sampled, including 113 peach-faced lovebirds (Agapornis roseicollis), 32 masked lovebirds (Agapornis personata), and 53 Fischers lovebirds (Agapornis fischeri). Smears made from cloacal swab samples of individual birds were stained with calcofluor white M2R stain and examined for the presence of spores. Microsporidian spores were identified in the droppings of 25% of the lovebirds sampled. Peach-faced and masked lovebirds were more likely to shed spores than were Fischers lovebirds (χ2 = 15.905, P < .001). No difference in spore shedding was found between juvenile and adult birds (χ2 = 0.157, P = .692; odds ratio = 1.659; 95% CI, 0.736–3.736). Lovebirds (n = 111) were also assayed for psittacine beak and feather disease virus by polymerase chain reaction (PCR) testing of individual blood samples. Lovebirds that were PCR positive for psittacine beak and feather disease virus (n = 23) were approximately 3 times more likely to shed microsporidian spores than were lovebirds that were PCR negative (χ2 = 3.981, P = .046; odds ratio = 2.6; 95% CI, 1.013–6.754). These results suggest that companion birds without obvious clinical illness commonly shed microsporidian spores. These birds may be a source of spore contamination for opportunistic infections in humans.

Obstructive Respiratory Disease in Prairie Dogs with Odontomas

The clinical manifestations of odontomas in prairie dogs are described. Familiarity with this disease is important because it is common, and the signs of this disease mimic other respiratory disorders.

Growth and Metabolic Characterization of Macrorhabdus Ornithogaster

Macrorhabdus ornithogaster (M. ornithogaster) is an anamorphic ascomycetous yeast found only in the stomach of birds. Infection is often benign but has also been associated with disease in some species of birds under some circumstances. In vitro efforts to grow M. ornithogaster have been largely unsuccessful. In this report, multiple liquid and solid media of varying pH, sugar concentration, and fetal bovine serum (FBS) concentrations, incubated at various temperatures in room air or microaerophilic conditions, were examined for their ability to support the growth of M. ornithogaster, obtained from a budgerigar (Melopsittacus undulatus). Optimum growth conditions were found to be Basal Medium Eagles, pH 3 to 4, containing 20% FBS, and 5% glucose or sucrose under microaerophilic conditions at 42°C. Using these conditions, M. ornithogaster was repeatedly passaged without loss of viability. Polyclonal isolates of M. ornithogaster consistently assimilated glucose, sucrose, and trehalose. M. ornithogaster did not grow with prolonged exposure to atmospheric oxygen, but growth in microaerophilic conditions was moderately enhanced by preincubation with atmospheric oxygen for 24 hours. An isolate of M. ornithogaster was found to be infective to day-old chickens, reduce their rate of weight gain, and induce a mild to moderate heterophilic inflammation of the isthmus. M. ornithogaster was reisolated from the chicks 7 days after infection, fulfilling Kochs postulates. A 761-bp sequence of 18S rDNA from this isolate was compared to the originally reported M. ornithogaster sequence and was found to be 97% identical.

Genetic Diversity in Twenty Variants of the Avian Polyomavirus

To determine if different pathotypes of the avian polyomavirus (APV) exist and to compare the genomes of APVs originating from different geographic areas, dates, and species of birds, the partial sequences of 18 APVs were determined. New viral sequences were compared with three published APV sequences. Two of the new viruses had identical sequences. Forty point mutations were found at 31 loci. A 27-bp deletion was found in the VP2 and VP3 open reading frames of one virus. A duplication of the putative origin of replication and adjacent enhancer region was previously reported in one APV. Smaller duplications involving the origin in one APV and a second enhancer region in another were discovered. All duplications were in tissue culture-adapted viruses, suggesting they occurred during the isolation process. Excluding duplications and the deletion, maximum variation between viruses was small (11 bp). A maximum parsimony tree was constructed that contained three major branches. The three earliest isolates were on separate branches. The European viruses were confined to branch I, but APVs from the United States were on all three branches. Lovebird, budgerigar, and macaw APVs were also on each of the three branches, suggesting that species-specific pathotypes have not developed. Most nonsynonymous mutations occurred in a small portion of the VP2 and VP3 open reading frames, demonstrating a selection for these mutations. That a glycine at VP2 221 will inhibit virus replication in chicken embryo fibroblasts (CEFs) has been previously reported. In contrast, six of seven of the new APVs isolated in CEFs had a glycine at VP2 221.