AMYLOIDOSIS IN CAPTIVE EUROPEAN EASTERN BONGO (TRAGELAPHUS EURYCERUS ISAACI): PREVALENCE, PREDICTIVE FACTORS, ORGAN PREDILECTION, AND SERUM AMYLOID A CONCENTRATIONS.

Amyloidosis is frequently identified during postmortem examination of captive eastern bongo (Tragelaphus eurycerus isaaci) in the European Endangered Species Programme (EEP). However, its significance and etiopathogenesis are poorly understood. The objective of this study was to investigate the prevalence of amyloidosis within this population and identify potential predictive factors for the presence of disease. Postmortem reports obtained from 24 EEP institutions were analyzed and assessed for evidence of amyloidosis. Seventy-two individuals had histopathological assessment performed after gross postmortem examination and were included in the study. Further histopathological analysis was performed on Congo red-stained slides from 26 individuals, and organ predilection sites were identified. Immunohistochemical analysis was performed in six individuals to identify the type of amyloid present. Serum amyloid A (SAA) analysis was performed on blood samples from 34 individuals, and concentrations in affected and unaffected individuals were compared. Amyloidosis was reported in 26 animals (36%). The association between the presence of amyloidosis and sex, age, or body condition was not statistically significant. However, amyloidosis was not identified in any individuals under the age of 6 yr. The presence of chronic inflammatory conditions was the only statistically significant predictive factor for the presence of amyloidosis (P = 0.03). Chronic inflammatory conditions present included nephritis, enteritis, and pneumonia. The majority of affected animals presented with amyloid deposition in multiple organs, with the liver and kidneys being most commonly affected. Immunohistochemistry confirmed the presence of AA amyloid. The association between the presence of amyloidosis and SAA values measured on a single occasion was not statistically significant. This study identified a high prevalence of amyloidosis within the captive European eastern bongo population associated with chronic inflammatory conditions. Antemortem diagnosis of amyloidosis remains challenging, and this study indicates that SAA protein concentrations are not a reliable indicator for the presence of amyloidosis.

Fungal microbiomes are determined by host phylogeny and exhibit widespread associations with the bacterial microbiome.

Interactions between hosts and their resident microbial communities are a fundamental component of fitness for both agents. Though recent research has highlighted the importance of interactions between animals and their bacterial communities, comparative evidence for fungi is lacking, especially in natural populations. Using data from 49 species, we present novel evidence of strong covariation between fungal and bacterial communities across the host phylogeny, indicative of recruitment by hosts for specific suites of microbes. Using co-occurrence networks, we demonstrate marked variation across host taxonomy in patterns of covariation between bacterial and fungal abundances. Host phylogeny drives differences in the overall richness of bacterial and fungal communities, but the effect of diet on richness was only evident in the mammalian gut microbiome. Sample type, tissue storage and DNA extraction method also affected bacterial and fungal community composition, and future studies would benefit from standardized approaches to sample processing. Collectively these data indicate fungal microbiomes may play a key role in host fitness and suggest an urgent need to study multiple agents of the animal microbiome to accurately determine the strength and ecological significance of host-microbe interactions.

COELOMIC STEATITIS IN TENTACLED SNAKES (ERPETON TENTACULATUM).

A retrospective study revealed seven cases of coelomic steatitis in adult tentacled snakes (Erpeton tentaculatum), including two males and five females, between May 2014 and August 2020. Common clinical signs included death after unusual floating, generalized weakness, inappetence, reduced body condition, coelomic distension, and reproductive pathology in females. Hematology of one specimen revealed marked monocytosis and lymphocytosis with mild heterophilia (chronic and active inflammation). Gross examination identified variable degrees of intracoelomic fat necrosis in all snakes. Consistent histopathologic features included necrotic adipocytes, lipid saponification, lipofuscin/ceroid deposition, granulomatous inflammation, and multinucleated giant cells (Langhans type). Three females exhibited intralesional yolk fluid associated with periovarian steatitis. Hepatic lipidosis was the second most frequent pathologic finding. Thawed frozen lesser sand eels (Ammodytes tobianus) were fed during this period, stored in vacuum-sealed or opened packets at -18°C (frozen). After the death of the last specimen, vitamin E concentrations and peroxide values of the diet were analyzed. For the sealed and opened frozen batches, respectively, vitamin E concentrations were 0.71 and 0.49 mg/100 g (compared with 4 to 8 mg/100 g in average, fresh, raw mixed eel species samples) and peroxide values were 62.5 and 48.6 meq/kg (exceeding the acceptable peroxide values of 8 meq/kg for fish oils). This case study represents the first report of coelomic steatitis in tentacled snakes of unconfirmed etiology but with a putative association with feeding a long-term frozen-stored sand eel diet containing low vitamin E concentrations and fish oils with high peroxide values at time of analysis.

SUCCESSFUL TREATMENT OF CLINICAL ORTHOPOXVIRUS INFECTION IN A GIANT ANTEATER (MYRMECOPHAGA TRIDACTYLA).

An anorexic 5-yr-old female giant anteater (Myrmecophaga tridactyla) developed multifocal ulcerative and vesicular lesions affecting the rostrum, oral cavity, and tongue. Disseminated skin lesions were also found on the body, affecting the feet, flanks, and genital area. Polymerase chain reaction confirmed a systemic viremic orthopoxvirus infection. Cowpox virus was considered to be the only likely etiological agent. Intensive supportive treatment, including daily fluid therapy, force-feeding, and anti-inflammatory administration achieved a successful outcome after 3 wk. To the authors' knowledge, this is the first time a giant anteater with severe orthopoxvirus lesions has survived the disease. This unique case discusses current and possible future therapeutic and prophylactic options for the treatment of orthopoxvirus infections in giant anteaters and other nondomestic animal species.

Evaluation of full S1 gene sequencing of classical and variant infectious bronchitis viruses extracted from allantoic fluid and FTA cards.

Sequence variability in the S1 gene determines the genotype of infectious bronchitis virus (IBV) strains. A single RT-PCR assay was developed to amplify and sequence the full S1 gene for six classical and variant IBVs (M41, D274, 793B, IS/885/00, IS/1494/06 and Q1) enriched in allantoic fluid (AF) or the same AF inoculated onto Flinders Technology Association (FTA) cards. Representative strains from each genotype were grown in specific-pathogen-free eggs and RNA was extracted from AF. Full S1 gene amplification was achieved using primer A and primer 22.51. Products were sequenced using primers A, 1050+, 1380+ and SX3+ to obtain short sequences covering the full gene. Following serial dilutions of AF, detection limits of the partial assay were higher than those of the full S1 gene. Partial S1 sequences exhibited higher-than-average nucleotide similarity percentages (79%; 352 bp) compared to full S1 sequences (77%; 1756 bp), suggesting that full S1 analysis allows greater strain differentiation. For IBV detection from AF-inoculated FTA cards, four serotypes were incubated for up to 21 days at three temperatures, 4°C, room temperature (approximately 24°C) and 40°C. RNA was extracted and tested with partial and full S1 protocols. Through partial sequencing, all IBVs were successfully detected at all sampling points and storage temperatures. In contrast, using full S1 sequencing it was not possible to amplify the gene beyond 14 days or when stored at 40°C. Data presented show that for full S1 sequencing, a substantial amount of RNA is needed. Field samples collected onto FTA cards are unlikely to yield such quantity or quality. ABBREVIATIONS: AF: allantoic fluid; CD50: ciliostatic dose 50; FTA: Flinders Technology Association; IB: infectious bronchitis; IBV: infectious bronchitis virus.

UROLITHIASIS IN A GROUP OF VISAYAN WARTY PIGS (SUS CEBIFRONS NEGRINUS).

Four cases of obstructive urolithiasis occurred in male Visayan warty pigs (Sus cebifrons negrinus) during a 12-mo period. One animal died, two were euthanized, and one was treated successfully with a tube cystotomy procedure and a subsequent urinary acidification diet. Uroliths from two cases of urethral obstruction were analyzed and confirmed as calcium carbonate. A fifth nonobstructive case was suspected in an adult female in which calcium carbonate crystalluria was diagnosed, and that case was resolved with medical management. Possible causes of these uroliths included reduced water intake, increased calcium in the diet through use of lucerne hay, and concurrent urinary tract infections. Changes to the diet and access to water were correlated with cessation of further cases, and no recurrence has been seen to date. To the authors' knowledge, this is the first report of calcium carbonate urolithiasis and the first use of a tube cystotomy in a nondomestic pig species.

PRELIMINARY CHARACTERIZATION OF DILATED CARDIOMYOPATHY IN A CAPTIVE POPULATION OF BANDED MONGOOSES (MUNGOS MUNGO).

Between 2006 and 2015, a high incidence of dilated cardiomyopathy (DCM) was diagnosed in a captive population of banded mongooses (Mungos mungo) at Chester Zoo, United Kingdom. The aim of this study was to characterize DCM in these mongooses in order to raise awareness of this condition and help inform management and clinical decisions. Prospective clinical assessments, including echocardiography, radiography, and cardiac biomarkers, were carried out in four mongooses remaining in the collection. Radiographs from 15 mature mongooses were reviewed and cardiac size and metrics assessed. Ten postmortem reports and the histologic sections from nine of these cases were reviewed for cardiac lesions. Echocardiographic findings were consistent with a diagnosis of preclinical DCM in one out of the four cases assessed, and it was considered equivocal in a second case. Taurine levels were within normal limits for domestic carnivores. Radiographs in seven mongooses showed right-sided or generalized cardiomegaly. The width of the heart in intercostal spaces and vertebral-tracheal angle on the lateral view were the most-discriminatory radiographic variables for diagnosis of cardiac disease. At necropsy, there was gross pathological evidence consistent with DCM in seven out of 10 mongooses examined. Histopathologically, mild multifocal fibrosis and rare intermyofiber edema were observed. This study provides preliminary evidence that DCM occurs in captive banded mongoose, but etiology and wider prevalence need to be determined.

Enhancement of wildlife disease surveillance using multiplex quantitative PCR: development of qPCR assays for major pathogens in UK squirrel populations.

Rapid development in polymerase chain reaction (PCR) technology has revolutionised the speed and accuracy of many diagnostic assays. However, comparatively few wildlife epidemiological studies use quantitative PCR (qPCR) for pathogen detection, even fewer employ an internal control, to ensure confidence in negative results, and PCR's ability to multiplex and therefore detect several targets in a single reaction is underutilised. Here, we describe the development of two multiplex qPCR assays for the red and grey squirrel that detect the pathogens squirrelpox virus (SQPV) and adenovirus in squirrels (SADV), both of which cause mortality in the red squirrel. Both assays use a section of the squirrel phosphoglycerate kinase gene as an endogenous internal control that identifies and compensates for both, inadequate sampling or PCR inhibition. Tests on infected squirrel tissue demonstrate that simple swab samples (particularly from distal antebrachial skin) are sufficient to detect and identify the relative quantity of SQPV DNA in both squirrel species, while rectal swabs and blood cell pellets can be used to reliably indicate SADV infection. These assays are sensitive and specific with an endogenous internal control providing confidence in negative results and allowing comparison across laboratories. Using such assays should prove advantageous in wildlife studies with limited resources while allowing the maximum data yield.

Histopathological characterization and grading of chronic enterocolitis in Sulawesi crested macaques (Macaca nigra).

Sulawesi crested macaques (Macaca nigra) (SCMs) are critically endangered and frequently suffer from chronic intestinal disease in captivity. Often, despite routine diagnostic investigations and confirmation of intestinal inflammation, an aetiology cannot be identified, leading to a non-specific categorization as chronic enterocolitis rather than an aetiological diagnosis. This study evaluates the histological features of gastrointestinal tissues from 23 SCMs, comparing animals with a clinical history suggestive of chronic enterocolitis (n = 14) with those without gastrointestinal clinical signs (n = 9). Tissues were graded according to the Nancy index (NI), a scoring system used in human medicine to evaluate disease activity in ulcerative colitis, a common form of human inflammatory bowel disease (IBD). Additionally, inflammatory cells in the colonic lamina propria were visually identified by type, counted and subsequently compared between diseased and control animals. Moderate to severe lymphoplasmacytic inflammation and structural changes were most common in the colons of affected SCMs, whereas histopathological changes were absent or mild in all examined small intestine (n = 17) and stomach (n = 11) tissues. The colonic NI had a significant positive correlation with clinical disease severity and 57% (n = 8) of animals with clinical signs had a NI grade of ≥2, consistent with moderate to severe, active IBD. Half of SCMs with recurrent rectal prolapse (n = 6) had a NI grade of 0, suggesting that intestinal inflammation is not always part of this condition's pathogenesis. The numbers of colonic lymphocytes, plasma cells, neutrophils, macrophages and total leucocytes were significantly higher in diseased animals. This study validated the use of the NI in SCMs, enabling a more standardized histopathological evaluation of the colon in this species.

Management of severe respiratory tract disease caused by human respiratory syncytial virus and Streptococcus pneumoniae in captive chimpanzees (Pan troglodytes).

Chimpanzees (Pan troglodytes) are susceptible to many viral and bacterial pathogens of human origin. This case series reports an acute outbreak of respiratory disease due to human respiratory syncytial virus and Streptococcus pneumoniae in a single group of 30 captive chimpanzees. Both pathogens are potentially zoonotic. The diagnosis was made antemortem and enabled a targeted response to the outbreak; but it more importantly, prompted improvements to the disease surveillance, biosecurity for risk mitigation and risk communication protocols within the zoo. A defined zoonotic disease risk communication pathway provides a model for management and compliance requirements for other collections.

A non-invasive feather-based methodology for the detection of blood parasites (Haemosporida).

Blood parasite (haemosporidian) infections are conventionally detected using blood samples; this implies capturing and handling birds to obtain them, which induces stress and causes pain. Feathers have blood vessels, and some blood could be preserved in the feather's shaft after moulting. We used feather DNA for detecting haemosporidians by PCR testing in diverse scenarios. First, haemosporidian DNA was detected in feathers from carcasses of infected birds, proving the feasibility of the approach. Storage temperature affected DNA recovery, with maximum retrieval and haemosporidian detection at the lowest temperature (- 20 °C). All feather types from infected birds kept at optimal conditions yielded haemosporidian DNA. Parasite detection by PCR was correlated with DNA yield, which was significantly higher in heavier birds, flight feathers, and more feathers per pool. Lastly, haemosporidians were detected employing feathers moulted from wild and captive birds to estimate infection prevalence. We show for the first time that using blood from feather shafts for haemosporidian detection can be an advantageous and less invasive alternative to blood sampling if feathers are optimally preserved. This method could contribute to uncovering haemosporidian infections in endangered and elusive birds, and it might facilitate routine screening in captive birds, thereby improving infection detection, prevention, and control.

Cowpox in zoo and wild animals in the United Kingdom.

Cowpox virus is considered to be a re-emerging zoonotic pathogen and a public health threat due to increasing numbers of cases in humans and animals in Europe over the past decade, including within the United Kingdom (UK). We present epidemiological data and diagnostic features of 27 recent, naturally occurring cowpox cases in zoo and wild animals across the UK, including the first reports of cowpox in two snow leopards (Panthera uncia), a Bengal tiger (Panthera tigris tigris), three Chilean pudus (Pudu puda), a Malayan tapir (Tapirus indicus) and a Eurasian otter (Lutra lutra), and the first reports of Orthopoxvirus infection in a lar gibbon (Hylobates lar), a Southern tamandua (Tamandua tetradactyla) and an aardvark (Orycteropus afer). This study provides a detailed overview of cowpox infections in a wide range of non-domestic animal species, presents a range of methods for diagnosis and demonstrates the value of retrospective analysis of pathology surveillance in revealing epidemiological links.

Application of next-generation sequencing technologies in virology.

The progress of science is punctuated by the advent of revolutionary technologies that provide new ways and scales to formulate scientific questions and advance knowledge. Following on from electron microscopy, cell culture and PCR, next-generation sequencing is one of these methodologies that is now changing the way that we understand viruses, particularly in the areas of genome sequencing, evolution, ecology, discovery and transcriptomics. Possibilities for these methodologies are only limited by our scientific imagination and, to some extent, by their cost, which has restricted their use to relatively small numbers of samples. Challenges remain, including the storage and analysis of the large amounts of data generated. As the chemistries employed mature, costs will decrease. In addition, improved methods for analysis will become available, opening yet further applications in virology including routine diagnostic work on individuals, and new understanding of the interaction between viral and host transcriptomes. An exciting era of viral exploration has begun, and will set us new challenges to understand the role of newly discovered viral diversity in both disease and health.

Elephant Endotheliotropic Herpesvirus 4 and Clostridium perfringens Type C Fatal Co-Infection in an Adult Asian Elephant (Elephas maximus).

Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) is an acute, often fatal, multisystemic hemorrhagic disease and one of the most significant causes of mortality of Asian elephants in captivity. Most fatal cases of EEHV-HD are associated with EEHV1A and EEHV1B in juveniles. This case report describes the clinical and pathological features of a fatal co-infection of Clostridium perfringens type C and EEHV-HD, caused by EEHV4, in an adult female Asian elephant. Although fatal clostridial enterotoxemia has been occasionally reported in elephants, this report highlights the importance of having both EEHV-HD and clostridial enterotoxemia as potential differential diagnoses in cases of widespread tissue necrosis and internal hemorrhage in elephants, regardless of the animal age group, due to their macroscopic similarities, frequent co-occurrence and cumulative morbid potential.

Interspecies comparative morphological evaluation of the corneal epithelial stem cell niche: a pilot observational study.

BACKGROUND: The corneal and limbal morphology relevant to corneal epithelial maintenance in ten different species was examined using histological methods. OBJECTIVES: The presence of a Bowman's layer, limbal epithelial cell, and superficial stromal morphology was examined in the following species to evaluate the differences in corneal thickness and epithelium: Java sparrows, frogs, macaws, spoonbills, red pandas, penguins, horses, Dobermans, orangutans, and humans. METHODS: Corneal sections (4 µm) were obtained from ten ocular globes from three different animal classes: Aves, Amphibia, and Mammalia. All sections were stained with hematoxylin and eosin and periodic acid-Schiff reaction. After microscopy, all stained slides were photographed and analyzed. RESULTS: Significant morphological differences in the corneal and limbal epithelia and their underlying stroma between species were observed. The number of corneal epithelial cell layers and the overall corneal epithelial thickness varied significantly among the species. The presence of a Bowman's layer was only observed in primates (orangutans and humans). Presumed supranuclear melanin caps were noted in four species (orangutans, macaws, red pandas, and horses) in the limbal basal epithelial layer (putative site of corneal epithelial stem cells). The melanin granules covered the apex of the cell nucleus. CONCLUSIONS: Supranuclear melanin capping has been described as a process within the epidermis to reduce the concentration of ultraviolet-induced DNA photoproducts. Similarly, there may be a relationship between limbal stem cell melanin capping as a protective mechanism against ultra-violet radiation.

Novel adenovirus associated with necrotizing bronchiolitis in a captive reindeer (Rangifer tarandus).

Adenoviruses cause a range of major diseases across many diverse animal species including ruminants. They are classified into six genera in the family Adenoviridae. In deer species, two adenoviruses are currently recognized: deer adenovirus 1 in the Atadenovirus genus, and deer adenovirus 2 in the Mastadenovirus genus. Deer adenovirus 1 causes adenovirus haemorrhagic disease with high fatality in black-tailed and mule deer in North America. Conversely, deer adenovirus 2 was incidentally detected from a healthy white-tailed deer fawn, but experimentally it has been shown to cause pyrexia, cough and moderate to severe haemorrhage. Here, we detected a novel adenovirus, reindeer adenovirus 1, from lung lesions of a 5-year-old male reindeer (Rangifer tarandus). This animal presented with aspiration pneumonia and necrotizing bronchiolitis following a period of clinical weakness, nasal discharge and wasting. Histopathological examination of the lung revealed large intranuclear basophilic inclusions associated with the areas of necrotizing bronchiolitis. Next generation sequencing of the lung tissue identified a novel mastadenovirus with close similarity to deer adenovirus 2 and bovine adenovirus 3. To our knowledge, this is the first report of a deer mastadenovirus associated with necrotizing bronchiolitis in captive reindeer.

Molecular and epidemiological surveillance of Plasmodium spp. during a mortality event affecting Humboldt penguins (Spheniscus humboldti) at a zoo in the UK.

In 2017, a mortality event affected Humboldt penguins at Chester Zoo (UK), which coincided with the diagnosis of avian malaria (AM) in some birds. AM is found worldwide wherever a competent mosquito vector is present, but the disease is particularly severe in penguins and other species that originate from non-endemic regions. To better understand the role of AM and manage its threat to penguin collections, Plasmodium was surveyed through PCR at Chester Zoo in mosquitoes, penguins, and dead free-living wild birds during and around the mortality event. Additional sequences were obtained from penguin fatalities from four other UK zoological collections. All sequences were integrated into phylogenetic analyses to determine parasite species and lineages. In total, 753/6459 positive mosquitoes were recorded (11.7% prevalence), reaching a weekly peak of 30% prevalence in mid-summer. Among penguin fatalities at Chester Zoo, several penguins presented signs and lesions compatible with AM; nevertheless, exoerythrocytic meronts were identified in only one case and Plasmodium spp. was identified in 5/22 birds. Phylogenetic analysis revealed at least five parasite cytb lineages of three Plasmodium species (P. matutinum, P. relictum and P. vaughani) circulating in mosquitoes at Chester Zoo; however, infections in free-living wild birds and penguins were only from P. matutinum. Plasmodium matutinum was confirmed as the cause of death of one penguin and was highly suspected to be the cause of death of another three. The lineage LINN1 was associated with 4/5 penguin infections. AM had a key role in the penguin multicausal mortality event. Understanding the risk of AM to penguin collections at Chester Zoo and elsewhere requires long-term surveillance to examine the association between Plasmodium infection and penguin mortality and the variability in parasite virulence. Surveillance of Plasmodium spp. in mosquitoes and local birds provides information about the parasite's transmission cycle locally, and could warn about infection risks to species of interest, which is essential for efficient disease control and prevention.

Host immune response to infectious bronchitis virus Q1 in two commercial broiler chicken lines.

This study investigated the pathogenesis of infectious bronchitis virus (Gammacoronavirus) strain Q1 in two commercial broiler chicken lines, and the host immune response to infection. Chicks from each line were grouped into either infected or control. Following Q1 infection at day-old, fast (Line-A) and slow (Line-B) growing chicks were monitored for clinical signs and body weights. At 3, 7, 9, 14, 21 and 28 days post infection (dpi), five birds were humanely euthanised, and trachea, kidney and proventriculus tissues were collected for quantitative RT-PCR and histopathology. Blood was collected weekly to determine IBV-specific ELISA antibody titres. Q1 infection significantly reduced the body weights of Line-A chicks at 14 and 21 dpi, but there were no significant differences in Line-B. Through qRT-PCR, significantly higher viral loads were found in the trachea, proventriculus and kidney tissues of Line-A chicks at 7-9 dpi. At day-old and at 28 dpi, the mean antibody titre in Line-B was notably higher than Line-A. Significant IFN-α mRNA expression was noted in the trachea and kidneys of Line-A, whereas no change occurred in Line-B. Chicks in Line-B, compared to those in Line-A, demonstrated a tissue-dependent increase of IFN-ß, TLR3, IL-1ß and IL-6 and LITAF gene transcription responses to IBV Q1. It appears that the level of maternal antibodies, growth rates, and other inherent host genetic factors could have influenced the differences in viral loads and immune responses.

Detection of squirrel poxvirus by nested and real-time PCR from red (Sciurus vulgaris) and grey (Sciurus carolinensis) squirrels.

BACKGROUND: Squirrel poxvirus (SQPV) is highly pathogenic to red squirrels (Sciurus vulgaris), and is a significant contributing factor to the local extinction of the species in most parts of England and Wales, where infection is endemic in Eastern grey squirrel (Sciurus carolinensis) populations. Although a nested PCR assay has been used successfully to study the epidemiology of SQPV, samples have a long processing time and the assay is not quantifiable. RESULTS: This project describes the design and optimization of a real-time PCR for SQPV. Comparison with the nested PCR showed the real-time assay to be more sensitive by one log and able to detect approximately 144 genome copies per mg of tissue. CONCLUSIONS: The real-time PCR has been used to quantify viral genome load in tissues from diseased and apparently healthy red and grey squirrels, and suggests that the titre of virus in tissues from diseased red squirrels is considerably higher than that found even in a grey squirrel with cutaneous lesions.

Nectarivorous Bird Emphysematous Ingluvitis (NBEI): A Novel Disease in Loriinae Birds Associated With Clostridium perfringens Infection.

A retrospective study revealed ten cases of emphysematous ingluvitis in Loriinae birds from two zoological collections between 2009 and 2020. Common clinical features were sudden death with gas distention of the crop, subcutaneous cervical emphysema and poor body condition, but also included collapse, hypothermia and abandonment. Macroscopic examination revealed moderate crop enlargement, distention and thickening with minimal intraluminal content, and moderate to severe submucosal to transmural gas-filled cysts (emphysema). Histopathology identified widespread transmural multifocal to coalescing empty pseudo-cystic cavities with lytic necrosis, pyo-/granulomatous inflammatory infiltrates, epithelial ulceration, parakeratotic hyperkeratosis, epithelial ballooning degeneration, and occasional intralesional rod-shaped bacteria. The lesion may have impaired the birds' ability to ingest food, resulting in suboptimal body condition. Necrotizing to granulomatous aspiration pneumonia was also a feature in some cases. Anaerobic bacterial culture of four crops identified Clostridium perfringens with associated toxin genes for alpha and occasionally beta2 toxin (cpa and cpb2 genes respectively), by PCR analysis of bacterial isolates cultured from fresh or frozen tissue. C. perfringens was identified as the common etiological agent of emphysematous ingluvitis in crop and/or liver (six out of ten birds), and type A was confirmed in five birds. C. perfringens was not detected in the crop nor liver of two unaffected Loriinae birds. This is the first publication that characterizes nectarivorous bird emphysematous ingluvitis (NBEI), attributes C. perfringens as an etiological agent, and highlights this novel disease as an important cause of death in Loriinae birds, particularly in nestling and fledgling stage of development, but also in older lorikeets and lories.