Viral shedding and environmental dispersion of two clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in experimentally infected mule ducks: implications for environmental sampling.

High pathogenicity avian influenza viruses (HPAIVs) have caused major epizootics in recent years, with devastating consequences for poultry and wildlife worldwide. Domestic and wild ducks can be highly susceptible to HPAIVs, and infection leads to efficient viral replication and massive shedding (i.e., high titres for an extended time), contributing to widespread viral dissemination. Importantly, ducks are known to shed high amounts of virus in the earliest phase of infection, but the dynamics and impact of environmental contamination on the epidemiology of HPAIV outbreaks are poorly understood. In this study, we monitored mule ducks experimentally infected with two H5N8 clade 2.3.4.4b goose/Guangdong HPAIVs sampled in France in 2016-2017 and 2020-2021 epizootics. We investigated viral shedding dynamics in the oropharynx, cloaca, conjunctiva, and feathers; bird-to-bird viral transmission; and the role of the environment in viral spread and as a source of samples for early detection and surveillance. Our findings showed that viral shedding started before the onset of clinical signs, i.e., as early as 1 day post-inoculation (dpi) or post-contact exposure, peaked at 4 dpi, and lasted for up to 14 dpi. The detection of viral RNA in aerosols, dust, and water samples mirrored viral shedding dynamics, and viral isolation from these environmental samples was successful throughout the experiment. Our results confirm that mule ducks can shed high HPAIV titres through the four excretion routes tested (oropharyngeal, cloacal, conjunctival, and feather) while being asymptomatic and that environmental sampling could be a non-invasive tool for early viral RNA detection in HPAIV-infected farms.

Membrane expression of the estrogen receptor ERα is required for intercellular communications in the mammary epithelium.

17ß-Estradiol induces the postnatal development of mammary gland and influences breast carcinogenesis by binding to the estrogen receptor ERα. ERα acts as a transcription factor but also elicits rapid signaling through a fraction of ERα expressed at the membrane. Here, we have used the C451A-ERα mouse model mutated for the palmitoylation site to understand how ERα membrane signaling affects mammary gland development. Although the overall structure of physiological mammary gland development is slightly affected, both epithelial fragments and basal cells isolated from C451A-ERα mammary glands failed to grow when engrafted into cleared wild-type fat pads, even in pregnant hosts. Similarly, basal cells purified from hormone-stimulated ovariectomized C451A-ERα mice did not produce normal outgrowths. Ex vivo, C451A-ERα basal cells displayed reduced matrix degradation capacities, suggesting altered migration properties. More importantly, C451A-ERα basal cells recovered in vivo repopulating ability when co-transplanted with wild-type luminal cells and specifically with ERα-positive luminal cells. Transcriptional profiling identified crucial paracrine luminal-to-basal signals. Altogether, our findings uncover an important role for membrane ERα expression in promoting intercellular communications that are essential for mammary gland development.

Pathobiology of highly pathogenic H5 avian influenza viruses in naturally infected Galliformes and Anseriformes in France during 2015-2016.

In late 2015, an epizootic of Highly Pathogenic Avian Influenza (H5Nx) was registered in Southwestern France, including more than 70 outbreaks in commercial poultry flocks. Phylogenetic analyses suggested local emergence of H5 viruses which differed from A/goose/Guangdong/1/1996 clade 2.3.4.4b lineage and shared a unique polybasic cleavage site in their hemagglutinin protein. The present work provides an overview of the pathobiological picture associated with this epizootic in naturally infected chickens, guinea fowls and ducks. Upon necropsy examination, selected tissues were sampled for histopathology, immunohistochemistry and quantitative Real Time Polymerase Chain Reaction. In Galliformes, HPAIVs infection manifested as severe acute systemic vasculitis and parenchymal necrosis and was associated with endothelial expression of viral antigen. In ducks, lesions were mild and infrequent, with sparse antigenic detection in respiratory and digestive mucosae and leukocytes. Tissue quantifications of viral antigen and RNA were higher in chickens and guinea fowls compared to duck. Subsequently, recombinant HA (rHA) was generated from a H5 HPAIV isolated from an infected duck to investigate its glycan-binding affinity for avian mucosae. Glycan-binding analysis revealed strong affinity of rHA for 3'Sialyl-LacNAc and low affinity for Sialyl-LewisX, consistent with a duck-adapted virus similar to A/Duck/Mongolia/54/2001 (H5N2). K222R and S227R mutations on rHA sequence shifted affinity towards Sialyl-LewisX and led to an increased affinity for chicken mucosa, confirming the involvement of these two mutations in the glycan-binding specificity of the HA. Interestingly, the rHA glycan binding pattern of guinea fowl appeared intermediate between duck and chicken. The present study presents a unique pathobiological description of the H5 HPAIVs outbreaks that occurred in 2015-2016 in Southwestern France.

Multiple follicular abnormalities in a 1-year old cat consistent with basaloid follicular hamartomas.

BACKGROUND: In humans, basaloid follicular hamartomas are benign follicular tumours, that can be solitary or multiple, in which case they show autosomal dominant inheritance. HYPOTHESIS/OBJECTIVES: This study describes clinical and histopathological findings observed in a young cat, which could be consistent with basaloid follicular hamartomas. CASE DESCRIPTION: Multiple follicular abnormalities, consistent with cutaneous diffuse basaloid follicular hamartomas, were observed in skin samples from a one-year old neutered domestic short hair cat. Clinical signs were diffuse symmetrical alopecia with exaggerated skin markings (ventral abdomen, thorax and medial aspects of the limbs) and intense follicular-centred thickening (face and feet). Microscopic lesions were characterised by multiple proliferative follicular abnormalities in all samples. The epidermis showed a very irregular surface with the follicles filled with variably pigmented keratin. The epithelial walls of the follicles had multiple small hyperplastic basaloid cells foci. In the superficial dermis under the epidermis and around the follicles, fibroblastic spindle-shaped mesenchymal cells with a homogeneous moderate density were present in the collagenous connective tissue. The interfollicular epidermis was also abnormal with multiple small proliferating trichoblastic foci originating from the basal layer. RNAscope testing for feline papillomavirus was negative. CONCLUSIONS AND CLINICAL RELEVANCE: This case report provides the first evidence of clinical and histopathological findings of multiple follicular abnormalities, consistent with cutaneous diffuse basaloid follicular hamartomas in a cat.

De multiples anomalies folliculaires, compatibles avec des hamartomes folliculaires basaloïdes diffus cutanés, ont été observées dans des échantillons de peau d'un chat domestique à poils courts castré âgé d'un an. Les signes cliniques étaient une alopécie diffuse symétrique avec des marques cutanées exagérées (abdomen ventral, thorax et face médiale des membres) et un épaississement folliculaire intense (face et pieds).

Múltiplas anormalidades foliculares, consistentes com hamartomas cutâneos foliculares basaloides difusos, foram observadas em amostras de pele de um gato doméstico de pelo curto castrado de um ano de idade. Os sinais clínicos foram alopecia simétrica difusa com marcações cutâneas exuberantes (abdômen, tórax e aspecto medial dos membros) e espessamento folicular central intenso (face e patas).

Se observaron múltiples anomalías foliculares, consistentes con hamartomas foliculares basaloides difusos cutáneos, en muestras de piel de un gato doméstico de pelo corto castrado de 1 año. Los signos clínicos fueron alopecia simétrica difusa con marcas cutáneas exageradas (abdomen ventral, tórax y cara medial de las extremidades) e intenso engrosamiento de la piel centrado en los folículos (cara y pies).

Pathological investigation of high pathogenicity avian influenza H5N8 in captive houbara bustards (Chlamydotis undulata), the United Arab Emirates 2020.

At the end of 2020, an outbreak of HPAI H5N8 was registered in captive African houbara bustards (Chlamydotis undulata) in the United Arab Emirates. In order to better understand the pathobiology of this viral infection in bustards, a comprehensive pathological characterization was performed. A total of six birds were selected for necropsy, histopathology, immunohistochemistry, RNAscope in situ hybridization and RT-qPCR and nanopore sequencing on formalin-fixed and paraffin-embedded (FFPE) tissue blocks. Gross lesions included mottled and/or hemorrhagic pancreas, spleen and liver and fibrinous deposits on air sacs and intestine. Necrotizing pancreatitis, splenitis and concurrent vasculitis, hepatitis and fibrino-heterophilic peritonitis were identified, microscopically. Viral antigens (nucleoprotein) and RNAs (matrix gene) were both detected within necro-inflammatory foci, parenchymal cells, stromal cells and endothelial cells of affected organs, including the myenteric plexus. Molecular analysis of FFPE blocks successfully detected HPAI H5N8, further confirming its involvement in the lesions observed. In conclusion, HPAI H5N8 in African houbara bustards results in hyperacute/acute forms exhibiting marked pantropism, endotheliotropism and neurotropism. In addition, our findings support the use of FFPE tissues for molecular studies of poorly characterized pathogens in exotic and endangered species, when availability of samples is limited.

High pathogenicity avian influenza A (H5N1) clade 2.3.4.4b virus infection in a captive Tibetan black bear (Ursus thibetanus): investigations based on paraffin-embedded tissues, France, 2022.

High pathogenicity avian influenza viruses (HPAIVs) H5Nx of clade 2.3.4.4b have been circulating increasingly in both wild and domestic birds in recent years. In turn, this has led to an increase in the number of spillover events affecting mammals. In November 2022, an HPAIV H5N1 caused an outbreak in a zoological park in the south of France, resulting in the death of a Tibetan black bear (Ursus thibetanus) and several captive and wild bird species. We detected the virus in various tissues of the bear and a wild black-headed gull (Chroicocephalus ridibundus) found dead in its enclosure using histopathology, two different in situ detection techniques, and next-generation sequencing, all performed on formalin-fixed paraffin-embedded tissues. Phylogenetic analysis performed on the hemagglutinin gene segment showed that bear and gull strains shared 99.998% genetic identity, making the bird strain the closest related strain. We detected the PB2 E627K mutation in minute quantities in the gull, whereas it predominated in the bear, which suggests that this mammalian adaptation marker was selected during the bear infection. Our results provide the first molecular and histopathological characterization of an H5N1 virus infection in this bear species. IMPORTANCE: Avian influenza viruses are able to cross the species barrier between birds and mammals because of their high genetic diversity and mutation rate. Using formalin-fixed paraffin-embedded tissues, we were able to investigate a Tibetan black bear's infection by a high pathogenicity H5N1 avian influenza virus at the molecular, phylogenetic, and histological levels. Our results highlight the importance of virological surveillance programs in mammals and the importance of raising awareness among veterinarians and zookeepers of the clinical presentations associated with H5Nx virus infection in mammals.

Tomodensitometric and histological age-related changes in the normal feline middle and inner ear.

Deafness in cats may be due to acquired causes such as aging. Similar age-related morphological changes in the cochlea have been noted in several animal species. However, little is known about the effects of age on the morphology of the middle and inner ear in cats. The aim of the present study was to compare these structures in middle-aged and geriatric cats using computed tomography and histological morphometric analysis. Data were obtained from 28 cats, aged 3-18 years, with no hearing or neurological disorders. Computed tomography showed an increase in tympanic bulla (middle ear) volume with aging. Histological morphometric analysis revealed thickening of the basilar membrane and atrophy of the stria vascularis (inner ear) in older cats, similar to what has been observed in older humans and dogs. Nevertheless, histological procedures could be improved to provide more data for comparison with different forms of presbycusis in humans.

Thoracic Tumor Associated with a Unilateral Empyema in a Beef Cow: A Case Report.

Tumors in cows are not frequently reported in the literature. They often represent unusual findings in live animals and are incidental at slaughter with rare positive therapeutic outcomes for farmers. A 9-year-old beef cow was referred to the hospital of ruminants of the National Veterinary School of Toulouse, France. The cow started to become sick 10 days prior, and major symptoms were anorexia, arched back, tachycardia, and tachypnea associated with significantly attenuated cardiac and pulmonary sounds upon right-sided auscultation. After specific investigations, a thoracic sarcoma associated with unilateral empyema was diagnosed. The empyema was treated, and supportive treatment was only performed for the tumor. Although the sarcoma remained, clinical improvement was significant, and the cow went back to her farm of origin. After the end of the withdrawal period, the cow recovered clinically but was culled by the owners for economic reasons. The present case report offers a continuum from the initial clinical signs motivating specific investigations to interesting laboratory findings, which were confirmed post-mortem.

Validation of an RNAscope assay for the detection of avian influenza A virus.

Highly pathogenic avian influenza (HPAI) is an acute viral disease associated with high mortality and great economic losses. Immunohistochemistry (IHC) is a common diagnostic and research tool for the demonstration of avian influenza A virus (AIAV) antigens within affected tissues, supporting etiologic diagnosis and assessing viral distribution in both naturally and experimentally infected birds. RNAscope in situ hybridization (ISH) has been used successfully for the identification of a variety of viral nucleic acids within histologic samples. We validated RNAscope ISH for the detection of AIAV in formalin-fixed, paraffin-embedded (FFPE) tissues. RNAscope ISH targeting the AIAV matrix gene and anti-IAV nucleoprotein IHC were performed on 61 FFPE tissue sections obtained from 3 AIAV-negative, 16 H5 HPAIAV, and 1 low pathogenicity AIAV naturally infected birds, including 7 species sampled between 2009 and 2022. All AIAV-negative birds were confirmed negative by both techniques. All AIAVs were detected successfully by both techniques in all selected tissues and species. Subsequently, H-score comparison was assessed through computer-assisted quantitative analysis on a tissue microarray comprised of 132 tissue cores from 9 HPAIAV-infected domestic ducks. Pearson correlation of r = 0.95 (0.94-0.97), Lin concordance coefficient of ρc = 0.91 (0.88-0.93), and Bland-Altman analysis indicated high correlation and moderate concordance between the 2 techniques. H-score values were significantly higher with RNAscope ISH compared to IHC for brain, lung, and pancreatic tissues (p ≤ 0.05). Overall, our results indicate that RNAscope ISH is a suitable and sensitive tool for in situ detection of AIAV in FFPE tissues.

Concurrent Mycobacterium genavense infection and intestinal B-cell lymphoma in a pet rabbit (Oryctolaguscuniculus).

A 6-year-old male intact pet rabbit was evaluated for chronic weight loss. A large mass was detected by palpation in the mid-abdomen and ultrasound examination suggested a jejunal location. Explorative laparotomy revealed a nodular mass within the jejunal wall. Histological examination of a biopsy revealed mycobacterial granulomatous enteritis with an atypical lymphoblastic proliferation suggestive of lymphoma. Neoplastic lymphocytes were immunopositive for Pax-5 but negative for CD3, which is diagnostic of a B-cell neoplasm. Numerous acid-fast bacteria were seen within histiocytes and identified by polymerase chain reaction as Mycobacterium genavense, which is a non-tuberculous and opportunistic mycobacterium with zoonotic potential. To the best of our knowledge, this is the first documented case of a concurrent B-cell lymphoma and M. genavense infection in a rabbit. Concomitant mycobacteriosis and lymphoma have been rarely described in animals and the coexistence of neoplasia and mycobacterial infection within the jejunum suggests a potential pathogenetic association. Interestingly, the rabbit owner worked in an anti-tuberculosis clinic, and an anthropic origin of the mycobacterial infection could not be excluded.

The feather epithelium contributes to the dissemination and ecology of clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in ducks.

Immature feathers are known replication sites for high pathogenicity avian influenza viruses (HPAIVs) in poultry. However, it is unclear whether feathers play an active role in viral transmission. This study aims to investigate the contribution of the feather epithelium to the dissemination of clade 2.3.4.4b goose/Guangdong/1996 lineage H5 HPAIVs in the environment, based on natural and experimental infections of domestic mule and Muscovy ducks. During the 2016-2022 outbreaks, H5 HPAIVs exhibited persistent and marked feather epitheliotropism in naturally infected commercial ducks. Infection of the feather epithelium resulted in epithelial necrosis and disruption, as well as the production and environmental shedding of infectious virions. Viral and feather antigens colocalized in dust samples obtained from poultry barns housing naturally infected birds. In summary, the feather epithelium contributes to viral replication, and it is a likely source of environmental infectious material. This underestimated excretion route could greatly impact the ecology of HPAIVs, facilitating airborne and preening-related infections within a flock, and promoting prolonged viral infectivity and long-distance viral transmission between poultry farms.

Causes of Mortality and Pathological Findings in European Hedgehogs (Erinaceus europaeus) Admitted to a Wildlife Care Centre in Southwestern France from 2019 to 2020.

The European hedgehog (Erinaceus europaeus) is a nocturnal, solitary and non-territorial mammal endemic across Europe and central Asia. Due to population decline in recent decades in Europe, this species was declared as protected and registered as 'Least Concern' on the International Union for Conservation of Nature Red List of Threatened Species. Previous studies pointed to the possible contribution of human activities to hedgehog mortality but few reports provide a complete overview of the pathological processes associated with mortality of the European hedgehog. The aim of this study was to identify the causes of mortality and pathological findings in 35 wild adult European hedgehogs that died spontaneously or were euthanized at the Wildlife Care Centre of the Ecole Nationale Vétérinaire de Toulouse between 2019 and 2020. The main causes of mortality were trauma (41%) including collision (9%), predation (9%), trapping (6%) and trauma of unknown origin (17%). Infectious diseases associated with systemic and local bacterial infections accounted for 34% of the cases and included cutaneous wounds (23%), mandibular abscesses and botryomycosis (23%), exudative bronchopneumonia (9%), suppurative rhinitis (7%) and otitis (3%). One hedgehog (3%) had a large subcutaneous tumour consistent with a malignant peripheral nerve sheath tumour. Parasitism was associated with mortality in 11% of cases with severe debilitation, massive infestation and active larval migration. Incidental findings included splenic and hepatic extramedullary haematopoiesis (100% and 69%, respectively), pulmonary crenosomiasis (91%), gastrointestinal capillariasis (61%), renal lipofuscinosis (59%), chronic renal infarction and interstitial nephritis (50%). These data emphasize the need for further study of hedgehog mortality and of the genetic, behavioural and environmental factors that may contribute to population decline.

A case of eagle fern (Pteridium aquilinum) poisoning on a pig farm.

BACKGROUND: Free-range pig farming represents a minor proportion of pig production in France but is attracting an increasing number of farmers because of societal expectations and the opportunity to use pasture-grazed forage. However, this type of farming faces several challenges, including biosecurity, parasitic management, and contact with wild fauna and pathogenic flora. CASE PRESENTATION: Two Gascon pigs raised on an outdoor fattening farm in the Hautes-Pyrenees department of France were submitted after sudden death for necropsy at the National Veterinary School of Toulouse. The pigs were of two different breeds but from the same group of 85 animals that had grazed on a 4-ha plot of land being used for grazing for the first time. Based on an in-depth interview with the farmer, the epidemiological information available, and the necropsy and histology examinations, a hypothesis of great eagle fern intoxication was proposed. Although the sample of animals available for diagnosis was small, the success of the administered therapy confirmed our diagnosis. It was recommended that in the short term, the animals be prevented access to the eagle fern by changing their pasture or removing the plants. Vitamin B1 and vitamin B6 were administered via feed as Ultra B® at 1 mL per 10 kg body weight per day for 2 days (providing 9 mg thiamine (vitamin B1) and 0.66 mg pyridoxine (vitamin B6) per kg body weight per day). Marked remission was observed, with 6 of 10 intoxicated animals with symptoms surviving (yielding a therapeutic success rate over 50%), but the therapy did not compensate for the loss of initial body condition. In total, of the 85 animals in the group after intoxication, 6 died, and 6 recovered. CONCLUSIONS: The significance of this report lies in the scarcity of eagle fern intoxication cases reported in the literature, though such intoxication may become a significant problem as the development of outdoor rearing continues. Thus, eagle fern intoxication should be included in the differential diagnosis of nervous system symptoms in swine. The case also emphasizes the importance of anamnesis and discussion with the farmer as an essential step to guide diagnosis.

Additional Value of Second Harmonic Generation Microscopy in the Diagnosis of Feline Collagen Type III Glomerulopathy.

A 1.5-year-old neutered female Domestic Shorthair cat was euthanized after the diagnosis of end-stage protein-losing nephropathy associated with the onset of nephrotic syndrome. At necropsy, both kidneys were diffusely pale and swollen with a granular cortex. Histologically, glomeruli had diffuse global mesangial and capillary wall expansion by homogeneous pale eosinophilic material. This material was Congo red negative, blue with Masson's trichrome stain, weakly positive with periodic acid-Schiff stain, bright red with Picrosirius red and birefringent under polarized light. Transmission electron microscopy and second harmonic generation (SHG) microscopy revealed mesangial and subendothelial collagen fibril deposition. Type III collagen deposition was confirmed by immunohistochemistry. This study provides an original and complete description of feline collagen type III glomerulopathy and emphasizes the possibility of directly diagnosing glomerular collagen deposition on unstained slides through SHG microscopy.

Viral tropism and detection of clade 2.3.4.4b H5N8 highly pathogenic avian influenza viruses in feathers of ducks and geese.

Highly Pathogenic Avian Influenza viruses (HPAIVs) display a tissue pantropism, which implies a possible spread in feathers. HPAIV detection from feathers had been evaluated for H5N1 or H7N1 HPAIVs. It was suggested that viral RNA loads could be equivalent or higher in samples of immature feather compared to tracheal (TS) or cloacal swabs (CS). We investigated the suitability of feathers for the detection of clade 2.3.4.4b H5N8 HPAIV in ducks and geese field samples. In the six H5N8 positive flocks that were included in this study, TS, CS and immature wing feathers were taken from at least 10 birds. Molecular loads were then estimated using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) targetting H5 and M genes. In all flocks, viral loads were at least equivalent between feather and swab samples and in most cases up to 103 higher in feathers. Bayesian modelling confirmed that, in infected poultry, RT-qPCR was much more likely to be positive when applied on a feather sample only (estimated sensitivity between 0.89 and 0.96 depending on the positivity threshold) than on a combination of a tracheal and a cloacal swab (estimated sensitivity between 0.45 and 0.68 depending on the positivity threshold). Viral tropism and lesions in feathers were evaluated by histopathology and immunohistochemistry. Epithelial necrosis of immature feathers and follicles was observed concurrently with positive viral antigen detection and leukocytic infiltration of pulp. Accurate detection of clade 2.3.4.4b HPAIVs in feather samples were finally confirmed with experimental H5N8 infection on 10-week-old mule ducks, as viral loads at 3, 5 and 7 days post-infection were higher in feathers than in tracheal or cloacal swabs. However, feather samples were associated with lower viral loads than tracheal swabs at day 1, suggesting better detectability of the virus in feathers in the later course of infection. These results, based on both field cases and experimental infections, suggest that feather samples should be included in the toolbox of samples for detection of clade 2.3.4.4b HPAI viruses, at least in ducks and geese.

Neu5Gc and α1-3 GAL Xenoantigen Knockout Does Not Affect Glycemia Homeostasis and Insulin Secretion in Pigs.

Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited antibodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and N-glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc-/- mice exhibit glycemic dysregulations and pancreatic ß-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities, and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies.

Disseminated mycobacteriosis manifesting as paraplegia in two Parma wallabies (Macropus parma) naturally exposed to Mycobacterium avium.

Two captive female Parma wallabies (Macropus parma) died after a history of flaccid paraplegia. On postmortem examination, granulomatous and suppurative osteomyelitis involving the left ischium and the lumbosacral region, with meningeal extension at the cauda equina, and caseonecrotic mastitis were the most significant changes. Multiple small nodules in the liver and spleen, and an enlargement of some lymph nodes with central caseous necrosis were also observed. Microscopically, a disseminated granulomatous inflammation with numerous multinucleate giant cells was seen. Numerous acid-fast bacilli were detected in macrophages, in multinucleated giant cells, and free in the central necrosis and suppurative exudate. After culture, polymerase chain reaction assays were carried out to detect the 65-kDa heat shock protein (Hsp65) and insertion sequences (IS)1245 and IS900. The causative agent was identified as Mycobacterium avium subsp. avium.

Within-day and between-day variability of transthoracic anatomic M-mode echocardiography in the awake bottlenose dolphin (Tursiops truncatus).

The use of transthoracic echocardiography in dolphins has been limited so far owing to technical and anatomical specificities. Anatomic M-mode (AMM) is a postprocessing echocardiographic technique generating M-mode studies from two-dimensional (2D) cineloops independently of the ultrasound beam orientation. The aim of the present study was to determine the within-day (repeatability) and between-day (reproducibility) variability of AMM echocardiography in awake healthy bottlenose dolphins (BN, Tursiops truncatus). Four adult BN trained to lie in left recumbency at the water surface were involved in the protocol. A total of 96 echocardiographic examinations were performed on 4 different days by a trained observer examining each BN 6 times per day. Video clips of 2D left parasternal long-axis views showing the left ventricle (LV) ventrally and the aortic root dorsally were recorded at each examination and analyzed for AMM measurements in a random order. A general linear model was used to determine the within-day and between-day coefficients of variation (CV). All examinations were interpretable allowing calculation of 10 AMM variables (i.e., end-diastolic and end-systolic ventral and dorsal LV myocardial wall thicknesses as well as LV and aortic diameters, mean aortic diameter, and LV shortening fraction). Most within- and between-day CV values (18/20) were <15%, the lowest being observed for the end-diastolic LV diameter (1.6%). In conclusion, AMM provides a simple non-invasive evaluation of heart morphology and function in the awake BN with good repeatability and reproducibility of the measurements. Further studies are required to determine the corresponding reference intervals.

Variations of N-acetylation level of peptidoglycan do not influence persistence of Lactococcus lactis in the gastrointestinal tract.

The food-grade Gram-positive bacterium, Lactococcus lactis, is recognized as a potential candidate to deliver proteins of medical interest by mucosal routes. The ability of carrier bacteria to persist and/or to lyse in the gastrointestinal tract needs to be considered to design optimal carrier strains to deliver proteins of interest at the mucosal level. Meyrand et al. (2007) have previously characterized in L. lactis, a peptidoglycan (PG) N-acetylglucosamine deacetylase (PgdA), which activity on PG influences bacterial sensitivity to lysozyme. Inactivation of pgdA gene in this bacterium, led to fully acetylated PG, resulting in a lysozyme-sensitive phenotype, whereas pgdA overexpression led to an increased degree of PG deacetylation, resulting in a lysozyme-resistant phenotype (Meyrand et al., 2007). In order to determine whether variations in L. lactis resistance to host lysozyme may influence its persistence in the GIT and its ability to deliver heterologous proteins in situ, we constructed L. lactis strains with different de-N-acetylation levels and producing a model antigen (the human papillomavirus type-16 E7 protein) and we compared the pharmacokinetics properties of these recombinant strains with that of a wild-type strain producing the same antigen in the GIT of mice. Our results show that there was no correlation between survival, at the ileum level, of bacteria intragastrically administered in mice and bacteria sensitivity or resistance to lysozyme. In addition, analysis of the E7-specific immune response evoked by the three strains after mucosal administration in mice suggest that neither lysozyme-sensitive nor lysozyme-resistant phenotype in L. lactis enhances significantly the potential of this bacterium as mucosal delivery live vector. In conclusion, our results suggest that either pgdA inactivation or pgdA overexpression in L. lactis leading to different levels of PG deacetylation does not confer any advantage in the persistence of this bacterium in the GIT and its ability to enhance host immune responses induced by delivered antigen in situ.