Lipid storage disease in 4 sibling superb birds-of-paradise (Lophorina superba).

Pedigree analysis, clinical, gross, microscopic, ultrastructural, and lipidomic findings in 4 female superb bird-of-paradise (SBOP, Lophorina superba) siblings led to the diagnosis of a primary inherited glycerolipid storage disease. These birds were the offspring of a related breeding pair (inbreeding coefficient = 0.1797) and are the only known SBOPs to display this constellation of lesions. The birds ranged from 0.75 to 4.3 years of age at the time of death. Two birds were euthanized and 1 died naturally due to the disease, and 1 died of head trauma with no prior clinical signs. Macroscopic findings included hepatomegaly and pallor (4/4), cardiac and renal pallor (2/4), and coelomic effusion (1/4). Microscopic examination found marked tissue distortion due to cytoplasmic lipid vacuoles in hepatocytes (4/4), cardiomyocytes (4/4), renal tubular epithelial cells (4/4), parathyroid gland principal cells (2/2), exocrine pancreatic cells (3/3), and the glandular cells of the ventriculus and proventriculus (3/3). Ultrastructurally, the lipids were deposited in single to coalescing or fused droplets lined by an inconspicuous or discontinuous monolayer membrane. Lipidomic profiling found that the cytoplasmic lipid deposits were primarily composed of triacylglycerols. Future work, including sequencing of the SBOP genome and genotyping, will be required to definitively determine the underlying genetic mechanism of this disease.

A Widefield Light Microscopy-Based Approach Provides Further Insights into the Colonization of the Flea Proventriculus by Yersinia pestis.

Yersinia pestis (the agent of flea-borne plague) must obstruct the flea's proventriculus to maintain transmission to a mammalian host. To this end, Y. pestis must consolidate a mass that entrapped Y. pestis within the proventriculus very early after its ingestion. We developed a semiautomated fluorescent image analysis method and used it to monitor and compare colonization of the flea proventriculus by a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Our data suggested that flea blockage results primarily from the replication of Y. pestis trapped in the anterior half of the proventriculus. However, consolidation of the bacteria-entrapping mass and colonization of the entire proventricular lumen increased the likelihood of flea blockage. The data also showed that consolidation of the bacterial mass is not a prerequisite for colonization of the proventriculus but allowed Y. pestis to maintain itself in a large flea population for an extended period of time. Taken as the whole, the data suggest that a strategy targeting bacterial mass consolidation could significantly reduce the likelihood of Y. pestis being transmitted by fleas (due to gut blockage), but also the possibility of using fleas as a long-term reservoir. IMPORTANCE Yersinia pestis (the causative agent of plague) is one of the deadliest bacterial pathogens. It circulates primarily among rodent populations and their fleas. Better knowledge of the mechanisms leading to the flea-borne transmission of Y. pestis is likely to generate strategies for controlling or even eradicating this bacillus. It is known that Y. pestis obstructs the flea's foregut so that the insect starves, frantically bites its mammalian host, and regurgitates Y. pestis at the bite site. Here, we developed a semiautomated fluorescent image analysis method and used it to document and compare foregut colonization and disease progression in fleas infected with a fully competent flea-blocking Y. pestis strain, a partially competent strain, and a noncompetent strain. Overall, our data provided new insights into Y. pestis' obstruction of the proventriculus for transmission but also the ecology of plague.

Long read sequencing revealed proventricular virome of broiler chicken with transmission viral proventriculitis.

BACKGROUND: Transmissible viral proventriculitis (TVP) causes significant economic loss to the poultry industry. However, the exact causative agents are obscure. Here we examine the virome of proventriculus from specified pathogen free (SPF) chickens that reproduced by infection of proventricular homogenate from broiler chicken with TVP using long read sequencing of the Pacific Biosciences RSII platform. The normal SPF chickens were used as control. RESULTS: Our investigation reveals a virome of proventriculitis, including three Gyrovirus genera of the Aneloviridae: Gyrovirus homsa1 (GyH1) (also known as Gyrovirus 3, GyV3) (n = 2662), chicken anemia virus (CAV) (n = 482) and Gyrovirus galga1 (GyG1) (also known as avian Gyrovirus 2, AGV2) (n = 11); a plethora of novel CRESS viral genomes (n = 26) and a novel genomovirus. The 27 novel viruses were divided into three clusters. Phylogenetic analysis showed that the GyH1 strain was more closely related to the strains from chicken (MG366592) than mammalian (human and cat), the GyG1 strain was closely related to the strains from cat in China (MK089245) and from chicken in Brazil (HM590588), and the CAV strain was more closely related to the strains from Germany (AJ297684) and United Kingdom (U66304) than that previously found in China. CONCLUSION: In this study, we revealed that Gyrovirus virome showed high abundance in chickens with TVP, suggesting their potential role in TVP, especially GyH1. This study is expected to contribute to the knowledge of the etiology of TVP.

EVALUATION OF CLINICAL DIAGNOSTICS FOR PROVENTRICULAR NEMATODIASIS DUE TO SYNHIMANTUS NASUTA IN LORIKEETS (TRICHOGLOSSUS SPP.).

In this case series, clinical investigations were pursued during a Synhimantus nasuta infection in a lorikeet (Trichoglossus spp.) flock outbreak situation to better describe and document clinical presentations. In 11 lorikeets suspected to be infected with Synhimantus based on at least one abnormal finding on their physical examination (lethargy, feather-damaging behavior on the ventrum, weight loss, pale iris), the presence of five additional parameters was documented: anemia, relative eosinophilia, increased proventricular diameter-to-keel height ratio (PKR), proventricular barium filling defect, and positive fecal occult blood detection test. A total score (X of 9) was calculated by combining all these findings. Synhimantus nasuta infection was confirmed in four of these individuals by modified Wisconsin fecal examination. Suspected cases (n = 7 of 11) presented only with low scores (1-3 of 9), whereas birds with confirmed infections (n = 4 of 11) presented with both low (1-3 of 9, n = 2 of 4) and high (6-7 of 9, n = 2 of 4) total scores. High scores were associated with clinical anemia. Fecal occult blood was present in all confirmed cases and 4 of 7 suspected cases. An enlarged proventriculus was only observed in birds with active shedding (n = 3 of 4). Follow-up evaluations after 6 mon of treatment with ivermectin and selamectin suggested complete recovery with lowered or normalized total scores. In conclusion, during an S. nasuta outbreak, a rapid physical examination helps to identify suspect cases, including individuals requiring immediate medical attention. In the absence of ova shedding, infection cannot be excluded on the basis of scarce clinical findings, but the detection of occult fecal blood and an increased PKR should raise the index of suspicion.

Medical and Surgical Management of Phaeohyphomycosis in a Kea (Nestor notabilis).

A 2.5-year-old female kea (Nestor notabilis) weighing 711 g was presented for acute lethargy, pelvic limb paraparesis, and decreased appetite. Results from a complete blood count revealed a leukocytosis (67540 cells/µL [reference interval 4200 - 37880 cells/µL]). Radiographic images revealed a mass effect within the mid coelom. The patient was provided supportive care that included antifungal medication (voriconazole 15 mg/kg PO q12h x 6 months and 10 days) and antibiotic therapy (enrofloxacin 20 mg/kg PO q12h x 27 days). A discrete 2.3 × 2.7 × 2.6 cm soft tissue mass adjacent to multiple organs was identified on contrast computed tomographic images (IsoVue 370 at 4 mL/kg IV over 2 minutes). The mass was medial and dorsal to the proventriculus, cranial to the ventriculus, caudal to the liver, and ventral to the cranial renal divisions. The mass had an irregular vascularized wall with a poorly vascularized center. Ten days after initial presentation, exploratory coeliotomy and mass removal via left lateral coeliotomy were performed. Bacterial (aerobic and anaerobic) and fungal cultures were negative. Fourteen days postsurgery, the leukocytosis was resolved. Microscopic review of the submitted tissue mass found multinucleated giant cells, macrophages, and brown fungal hyphae with irregular internal septations and some branching, leading to a diagnosis of phaeohyphomycosis. Panfungal polymerase chain reaction testing and sequencing were unsuccessful at speciation. Treatment with voriconazole was continued until behavioral, hematologic, and computed tomographic assessments indicated resolution of the problem 6 months postsurgery. No recurrence of disease has been reported 20 months following mass removal.

Comparative Features of the Upper Alimentary Tract in the Domestic Fowl (Gallus gallus domesticus) and Kestrel (Falco tinnunculus): A Morphological, Histochemical, and Scanning Electron Microscopic Study.

The avian alimentary tract has evolved into different histologic structures to accommodate the physical and chemical features of several food types and flight requirements. We compared the esophagus, proventriculus, and gizzard of the domestic fowl, Gallus gallus domesticus (GGD) and kestrels, Falco tinnunculus (FT) using immunohistochemistry and scanning electron microscopy with various stains and lectins [Dolichos biflorus agglutinin (DBA) and Ricinus communis agglutinin I (RCA120)], and α-smooth muscle actin (α-SMA). The esophagus of GGD demonstrated thickened epithelium, muscularis mucosae, and inner circular longitudinal tunica muscularis layers; moderate outer longitudinal tunica muscularis layers; and a true crop. In contrast, the esophagus of FT showed a thin epithelium, no muscularis mucosae, moderate inner longitudinal and thick outer circular tunica muscularis layers, and no true crop. In the proventriculus, the nature of the secretion in GGD was neutral, but that of FT was acidic and neutral. In the gizzard, the muscle coat of GGD by α-SMA had no muscularis mucosae, unlike FT, which had muscularis mucosae. In summary, there are many histologic differences between GGD and FT to meet their different physiologic needs, such as feeding.

Removal of foreign bodies from the proventriculus in a young golden eagle (Aquila chrysaetos).

This paper presents a clinical case report of a golden eagle (Aquila chrysaetos) with foreign bodies (stones) in its proventriculus. The case deals with the identification, management and removal of foreign objects identified in the gastrointestinal tract. A surgical removal by proventriculotomy under general anaesthesia was attempted. The surgery and the recovery were uneventful, and the follow-up after six months revealed no complications. To the best of our knowledge, there are no other reports of successful foreign body removal by proventriculotomy in the golden eagle.

Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies.

How differential levels of gene expression are controlled in post-mitotic neurons is poorly understood. In the Drosophila retina, expression of the transcription factor Defective Proventriculus (Dve) at distinct cell type-specific levels is required for terminal differentiation of color- and motion-detecting photoreceptors. Here, we find that the activities of two cis-regulatory enhancers are coordinated to drive dve expression in the fly eye. Three transcription factors act on these enhancers to determine cell-type specificity. Negative autoregulation by Dve maintains expression from each enhancer at distinct homeostatic levels. One enhancer acts as an inducible backup ('dark' shadow enhancer) that is normally repressed but becomes active in the absence of the other enhancer. Thus, two enhancers integrate combinatorial transcription factor input, feedback and redundancy to generate cell type-specific levels of dve expression and stable photoreceptor fate. This regulatory logic may represent a general paradigm for how precise levels of gene expression are established and maintained in post-mitotic neurons.

Retrospective study on transmissible viral proventriculitis and chicken proventricular necrosis virus (CPNV) in the UK.

Chicken proventricular necrosis virus (CPNV) is a recently described birnavirus, which has been proposed to be the cause of transmissible viral proventriculitis (TVP). The understanding of the epidemiology of both the virus and the disease is very limited. A retrospective investigation on TVP and CPNV in broiler chicken submissions from the UK from between 1994 and 2015 was performed with the aims of assessing the longitudinal temporal evolution of TVP and CPNV, and to review the histological proventricular lesions in the studied chickens. Ninety-nine of the 135 included submissions (73.3%) fulfilled the TVP-diagnostic criteria, while the remaining 36 submissions (26.7%) displayed only lymphocytic proventriculitis (LP). The first detection of CPNV by PCR dated from 2009. Results showed a rise in the number of both TVP and positive CPNV RT-PCR submissions from 2009 with a peak in 2013, suggesting that they may be an emerging or re-emerging disease and pathogen, respectively. Twenty-two out of the 99 submissions displaying TVP lesions (22%) and four out of the 36 (11%) submissions with LP gave positive CPNV RT-PCR results, further supporting the association between CPNV and TVP and confirming that CPNV is present in a low proportion of proventriculi that do not fulfil the TVP-diagnostic criteria. In addition, intranuclear inclusion bodies were observed in 22 of the submissions with TVP. The vast majority of these cases (21 of 22, 96%) gave negative CPNV RT-PCR results, raising the question of whether a virus other than CPNV is responsible for some of these TVP-affected cases.RESEARCH HIGHLIGHTSTVP and CPNV have been present in British broilers since at least 1994 and 2009, respectively.TVP and CPNV seem to be an emerging and re-emerging disease and pathogen, respectively.CPNV was detected in proventriculi with both TVP and LP-lesions.Viruses other than CPNV may be responsible for some TVP-affected cases.

Identification of pheasant ghrelin and motilin and their actions on contractility of the isolated gastrointestinal tract.

Motilin and ghrelin were identified in the pheasant by molecular cloning, and the actions of both peptides on the contractility of gastrointestinal (GI) strips were examined in vitro. Molecular cloning indicated that the deduced amino acid sequences of the pheasant motilin and ghrelin were a 22-amino acid peptide, FVPFFTQSDIQKMQEKERIKGQ, and a 26-amino acid peptide, GSSFLSPAYKNIQQQKDTRKPTGRLH, respectively. In in vitro studies using pheasant GI strips, chicken motilin caused contraction of the proventriculus and small intestine, whereas the crop and colon were insensitive. Human motilin, but not erythromycin, caused contraction of small intestine. Chicken motilin-induced contractions in the proventriculus and ileum were not inhibited by a mammalian motilin receptor antagonist, GM109. Neither atropine (a cholinergic receptor antagonist) nor tetrodotoxin (a neuron blocker) inhibited the responses of chicken motilin in the ileum but both drugs decreased the responses to motilin in the proventriculus, suggesting that the contractile mechanisms of motilin in the proventriculus was neurogenic, different from that of the small intestine (myogenic). On the other hand, chicken and quail ghrelin did not cause contraction in any regions of pheasant GI tract. Since interaction of ghrelin and motilin has been reported in the house musk shrew, interaction of two peptides was examined. The chicken motilin-induced contractions were not modified by ghrelin, and ghrelin also did not cause any contraction under the presence of motilin, suggesting the absence of interaction in both peptides. In conclusion, both the motilin system and ghrelin system are present in the pheasant. Regulation of GI motility by motilin might be common in avian species. However, absence of ghrelin actions in any GI regions suggests the avian species-related difference in regulation of GI contractility by ghrelin.

The morphological development of the proventriculus of Dandarawi chick: Light and electron microscopical studies.

This study was carried out on 40 chick embryos collected from incubated eggs of Dandarawi chicken (Gallus gallus domesticus) on the 5th to 19th incubation day (27 to 45 Hamburger and Hamilton, H&H stages). In addition, 15 chicks were collected on the day of hatching (stage 46 H&H), one week and two weeks post-hatching to demonstrate the histological, histochemical, and electron microscopic developmental changes of the proventriculus (of the digestive tract). Histologically, the proventriculus was observed as a narrow tube at 27 H&H stage. It was lined by pseudostratified columnar epithelium through 27-39 H&H stages and from the stage 43 till post-hatching, it was lined by simple columnar epithelium. The Lamina muscularis mucosa could be identified at stage 43. The proventricular glands were detected firstly at stage 31 and branching at stage 35. Histochemically, the surface epithelium and proventricular glands reacted positively to PAS, alcian blue and bromophenol blue from stage 31 till maturity. The glands displayed an apocrine mode of secretion at stage 39 and their cytoplasm contained abundant mitochondria, RER, secretory granules, and lipid droplets. Enteroendocrine cells could be observed among the glandular and surface epithelium at stage 45 H&H. The interstitial tissue contained fibroblasts and telocytes. The telocytes were firstly detected at stage 35 H&H and composed of a cell body and two long cell processes called telopodes. The tunica muscularis differentiated into three layers of smooth muscle fibers at stage 37 H&H. The cellular and stromal organizations of the proventriculus and their relations to the development and function were discussed.

Anatomopathological changes, quantification and distribution of Libyostrongylus spp. in regions of the proventriculus and ventriculus of naturally- and experimentally-infected ostriches.

Nematodes of the genus Libyostrongylus parasitize ostriches, causing high mortality rates. These nematodes are found in the proventriculus and ventriculus of ostriches, but little is known about their distribution and the possible anatomopathological changes they cause in the various regions of these organs. This paper describes the distribution and quantification of Libyostrongylus and pathological changes found in regions of the proventriculus and ventriculus of ostriches with high and low levels of both natural and experimental infection. Ostriches were necropsied and tissue samples from the distinct regions of both organs were analysed based on nematode counts and histopathology after staining with haematoxylin and eosin, Masson's trichrome or Alcian blue/PAS. The cranial and glandular regions of the proventriculus were the most parasitized. The ventriculus contained more nematodes in the caudal region. No macro- or microscopic pathological changes were observed in either of these organs of experimentally-infected birds. However, naturally-infected birds with high levels of infection presented proventriculus with macroscopic lesions and heterophilic infiltrates surrounding nematodes. In the glandular region of this organ, nematodes were located in the adenomeres of the secretory ducts, causing altered architecture and erosions and ulcerative lesions with damaged epithelium. Nematode eggs were found in the koilin layer of the middle and caudal regions of the ventriculus only of these birds. The pH of the regions assessed by Alcian blue/PAS staining changed from acidic in the proventriculus to more alkaline in the caudal region of the ventriculus. These data add knowledge to the biology of Libyostrongylus. RESEARCH HIGHLIGHTS The most parasitized areas were the cranial and glandular regions of the proventriculus. Naturally-infected birds with high levels of infection presented macro lesions in the proventriculus and damaged epithelium. Nematode eggs were found in the ventriculus. The proventriculus had an acidic pH, which turned alkaline towards the ventriculus.

Macrorhabdus ornithogaster Infection and Spontaneous Proventricular Adenocarcinoma in Budgerigars ( Melopsittacus undulatus).

Proventricular infection with the anamorphic, ascomycetous yeast Macrorhabdus ornithogaster and disease associated with infection (macrorhabdiosis) have been widely reported in budgerigars ( Melopsittacus undulatus). Proventricular adenocarcinoma has also been previously reported in this species. In a retrospective archival search of clinical cases submitted to a zoo animal and exotic pet pathology service between 1998 and 2013, a total of 28 128 avian submissions were identified, which included 1006 budgerigars kept in zoos or aviaries or as pets. Of these budgerigars, 177 were identified histologically as infected with M. orthithogaster at the time of necropsy. Histologic examination of tissues from budgerigars infected with M. orthithogaster identified an apparent continuum in the development of proventricular isthmus lesions associated with M. ornithogaster that included inflammation, mucosal hyperplasia, glandular dysplasia, and adenocarcinoma. Proventricular adenocarcinoma was identified histologically in 21 budgerigars. Budgerigars with proventricular adenocarcinoma were significantly more likely to have macrorhabdiosis than budgerigars without proventricular adenocarcinoma. Based on odds ratios for archival data, budgerigars were 41 times (95% confidence interval [CI], 33-52) more likely to have macrorhabdiosis than other avian species and 19 times (95% CI, 11-33) more likely to have proventricular adenocarcinoma than other birds. Budgerigars were 323 times (95% CI, 42-2490) more likely to be affected by both diseases simultaneously compared with other avian species. These findings suggest that macrorhabdiosis may be an associated factor for proventricular adenocarcinoma, although the explanation for this statistically significant correlation remains unknown and further investigation is warranted.

Influence of inclusion level of barley in wheat-based diets and supplementation of carbohydrase on growth performance, nutrient utilisation and gut morphometry in broiler starters.

1. The influence of barley inclusion level and supplementation of a multi-component non-starch polysaccharide degrading enzyme on performance and nutrient utilisation in broilers was investigated. Normal-starch hulled barley was evaluated with five levels of inclusion (0, 141, 283, 424 and 565 g/kg) in a wheat-based diet and two levels of enzyme supplementation (0 and 150 g/tonne of feed; a 5 × 2 factorial arrangement of 10 dietary treatments). All diets were equivalent in metabolisable energy and digestible amino acid contents. A total of 400, one-d old male broilers (five cages/treatment; eight birds/cage) were used in the experiment.2. Regardless of enzyme supplementation, weight gain (WG) increased up to 283 g/kg of barley and was reduced afterwards (P < 0.01). Increasing levels of barley resulted in greater (P < 0.001) gain per feed (G/F). Enzyme addition increased WG (P < 0.05) and G/F (P < 0.001) at each barley inclusion level.3. Birds fed diets with 0 and 565 g/kg barley showed the lowest and highest (P < 0.001to 0.05) digestibility for all nutrients measured, respectively. Digestibility of all nutrients was improved by enzyme supplementation at each barley inclusion level (P < 0.05). The nitrogen-corrected apparent metabolisable energy improved with increasing inclusion of barley (P < 0.001) and supplemental enzyme (P < 0.01). Increasing inclusion of barley increased the relative weight of gizzard (P < 0.001) and reduced jejunal digesta viscosity (P < 0.001). Supplemental enzyme (P < 0.001) reduced digesta viscosity.4. The optimum inclusion level of barley, with respect to growth performance, was 283 g/kg of diet. Increasing barley inclusion improved nutrient and energy utilisation, possibly through lowered digesta viscosity and better function of the gizzard. Feed efficiency and nutrient and energy utilisation can benefit from carbohydrase supplementation in barley-based diets, regardless of barley inclusion level.

Characterization of an Unusual Mesenchymal Tumor in the Proventriculus of a Free-ranging Red-tailed Hawk (Buteo jamaicensis).

An adult, female red-tailed hawk (Buteo jamaicensis) was presented for examination after being found unable to fly on a private citizen's property. Further diagnostic testing revealed a vascular, soft tissue mass arising from the serosal surface of the proximal proventriculus. Postmortem histological and immunohistochemical examination of the mass revealed an unusual mesenchymal tumor with features consistent with a leiomyoma or low-grade gastrointestinal stromal tumor.

T-cell Thymic Lymphoma With Proventricular Metastasis in a Florida Scrub Jay ( Aphelocoma coerulescens).

An adult, wild-caught, female Florida scrub jay ( Aphelocoma coerulescens) was evaluated because of an observable mass on the ventral neck. Initial physical examination and diagnostic tests were performed, which revealed a subcutaneous mass. Surgical removal of the mass was attempted, but the bird died during surgery. Results of necropsy and histopathologic evaluation identified the mass as thymic lymphoma with proventricular metastasis. Immunohistochemical staining revealed strong cytoplasmic immunoreactivity for CD3 in the thymic mass and within the predominant lymphoid population in the serosal proventricular masses, which confirmed metastasis of T-cell lymphoma. To our knowledge, this is the first report of T-cell thymic lymphoma in a wild Florida scrub jay.

Dynamic distribution and tissue tropism of avian encephalomyelitis virus isolate XY/Q-1410 in experimentally infected Korean quail.

Avian encephalomyelitis (AE) is an important infectious poultry disease worldwide that is caused by avian encephalomyelitis virus (AEV). However, to date, the dynamic distribution of AEV in quails has not been well described. Quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry (IHC) assays were used to investigate the dynamic distribution and tissue tropism of AEV in experimentally infected Korean quail. AEV was detected in the cerebrum, cerebellum, proventriculus, intestine, liver, pancreas, spleen, bursa, lung and kidney as early as 3 days post-infection (dpi). The viral loads in the proventriculus, intestine, spleen and bursa were relatively higher than in other tissues. According to the qPCR results, AEV XY/Q-1410 infection lasted for at least 60 days in infected Korean quail. Immunohistochemistry-positive staining signals of AEV antigen were analysed by Image-Pro Plus software. A positive correlation between qPCR and IHC results was identified in most tissues. Our results provide an insight into the dynamic distribution of AEV in various tissues after infection. The distinct dynamic distribution of the viral genome in Korean quail in the early and late stages of infection suggests that AEV replication is affected by antibody levels and the maturity of the immune system of the host.

Detection of transmissible viral proventriculitis and chicken proventricular necrosis virus in the UK.

Increasing evidence suggests that a new birnavirus, named chicken proventricular necrosis virus (CPNV), is the aetiological agent of transmissible viral proventriculitis (TVP). The present work aimed to explore the possible presence of both TVP and CPNV in the UK. Forty-four chickens showing TVP-compatible gross lesions were classified into three groups based on the histological lesions: (i) TVP-affected chickens: lymphocytic infiltration and glandular necrosis (n = 15); (ii) lymphocytic proventriculitis (LP)-affected chickens: lymphocytic infiltration without necrosis (n = 18); and (iii) without proventriculitis (WP): no lymphocytic infiltration or necrosis (n = 11). Nine proventriculi (seven out of 15 corresponding to TVP, and two out of 11 corresponding to LP) were positive for CPNV by reverse transcriptase polymerase chain reaction (RT-PCR). These results support the previously suggested idea of CPNV as causative agent of TVP. Moreover, these data show that CPNV can also be detected in a number of cases with LP, which do not fulfil the histological TVP criteria. Phylogenetic analysis of partial sequences of gene VP1 showed that British CPNV sequences were closer to other European CPNV sequences and might constitute a different lineage from the American CPNV. TVP cases with negative CPNV PCR results may be due to chronic stages of the disease or to the reduced PCR sensitivity on formalin-fixed paraffin-embedded tissues. However, involvement of other agents in some of the cases cannot totally be ruled out. As far as the authors are aware, this is the first peer-reviewed report of TVP as well as of CPNV in the UK, and the first exploratory CPNV phylogenetic study.

Investigation of a mortality cluster in wild adult yellow-eyed penguins (Megadyptes antipodes) at Otago Peninsula, New Zealand.

We investigated an epidemic mortality cluster of yellow-eyed penguins (Megadyptes antipodes) that involved 67 moribund or dead birds found on various beaches of the Otago Peninsula, New Zealand, between 21 January and 20 March 2013. Twenty-four carcases were examined post-mortem. Histological lesions of pulmonary, hepatic and splenic erythrophagocytosis and haemosiderosis were found in 23 of 24 birds. Fifteen birds also had haemoglobin-like protein droplets within renal tubular epithelial cells. Despite consistent histological lesions, a cause of death could not be established. Virology, bacteriology and molecular tests for avian influenza, avian paramyxovirus-1, avipoxvirus, Chlamydia psittaci, Plasmodium spp., Babesia spp., Leucocytozoon spp. and Toxoplasma gondii were negative. Tissue concentrations of a range of heavy metals (n = 4 birds) were consistent with low level exposure, while examination of proventricular contents and mucus failed to detect any marine biotoxins or Clostridium botulinum toxin. Hepatic concentrations of total polycyclic aromatic hydrocarbons (PAHs) (n = 5 birds) were similar to background concentrations of polycyclic aromatic hydrocarbons previously found in yellow-eyed penguins from the South Island of New Zealand, but there were significantly higher concentrations of 1-methylnapthelene and 2-methylnapthelene in the birds found dead in this mortality cluster. The biological significance of this finding is unclear. A temporal investigation of the epidemic did not indicate either a common source or propagative epidemic pattern. Although our investigation did not definitively implicate a toxic or infectious agent, we could not rule out causes such as toxic marine organisms or mycoplasmosis. Further investigations should therefore by carried out in the event of future mortality clusters.

Changes in expression of neuropeptides and their receptors in the hypothalamus and gastrointestinal tract of calorie restricted hens.

The list of orexigenic and anorexigenic peptides, those are known to alter feed intake, is continuously growing. However, most of them are studied in mammalian species. We aimed to investigate plasma level and mRNA expression of the pituitary adenylate cyclase-activating polypeptide (PACAP), gene expression of its receptor (PAC1), furthermore the gene expression of galanin (GAL), neuromedin U (NMU), and its two receptors (NMUR1 and NMUR2) in the hypothalamus, proventriculus, and jejunum of hens exposed to 40% calorie restriction. Feed restriction resulted in a 88% increase in mRNA and a 27% increase in peptide level of PACAP in proventriculus measured with qPCR and RIA, respectively. Increases were found in the gene expression of PAC1 (49%) and NMUR1 (63%) in the hypothalamus. Higher expressions of peptide encoding genes (76% for PACAP, 41% for NMU, 301% for NMUR1 and 308% for GAL, P < 0.05) were recorded in the jejunum of hens exposed to restricted nutrition. The results indicate that PACAP level responds to calorie restriction in the proventriculus and jejunum, but not in the hypothalamus and plasma.