Feline Facial Spindle Cell Tumors in 29 Cats: Histomorphological and Immunohistochemical Characterization.

Soft tissue tumors/sarcomas (STSs) in felines, encompassing a variety of mesenchymal tumors with similar histomorphological features, present diagnostic challenges due to their diverse cellular origins and the overlap with other tumor types such as feline sarcoid. This study aimed to delineate the clinical, histomorphological, and immunohistochemical characteristics of 34 feline facial spindle cell tumors affecting 29 cats, including testing for bovine papillomavirus type 14 (BPV14), the virus causing feline sarcoids. Only five out of 12 tumors previously diagnosed as feline sarcoids based on histomorphology were confirmed by PCR for BPV14, underscoring the importance of comprehensive diagnostic approaches to accurately distinguish between STSs and feline sarcoids. This study shows that most facial spindle cell tumors were compatible with peripheral nerve sheath tumors (PNSTs) based on positive immunohistochemical staining for Sox10 and other immunohistochemical markers such as GFAP, NSE, and S100. Some of these tumors displayed as multiple independent masses on the face or as erosive and ulcerative lesions without obvious mass formation, an atypical presentation and an important highlight for general practitioners, dermatologists, and oncologists. This study also describes periadnexal whorling of neoplastic cells as a novel histomorphologic finding in feline facial PNSTs and emphasizes Sox10 as a useful complementary immunohistochemical marker for the diagnosis of facial PNST in cats, providing valuable insights for veterinary pathologists.

Unilateral segmental uterine horn hypoplasia with fallopian tube aplasia in a guinea pig (Caviaporcellus).

A 2-year-3-month-old female guinea pig (Cavia porcellus) was referred for lethargy, weight gain and severe abdominal pain. Ultrasound examination revealed thickening and enlargement of the left uterine horn and an unremarkable right uterine horn. The animal died during ovariohysterectomy due to respiratory and cardiac arrest. Necropsy revealed unilateral segmental uterine horn hypoplasia and aplasia of the fallopian tube of the corresponding side. This is the first report of a congenital uterine malformation in the guinea pig and we describe the clinical and pathological presentation of segmental uterine horn hypoplasia and fallopian tube aplasia. Congenital uterine malformations are rare in guinea pigs but should be considered as differential diagnoses for the more common acquired uteropathies.

Voluntary exercise does not always suppress lung cancer progression.

Physical exercise can lower lung cancer incidence. However, its effect on lung cancer progression is less understood. Studies on exercising mice have shown decreased ectopic lung cancer growth through the secretion of interleukin-6 from muscles and the recruitment of natural killer (NK) cells to tumors. We asked if exercise suppresses lung cancer in an orthotopic model also. Single-housed C57Bl/6 male mice in cages with running wheels were tail vein-injected with LLC1.1 lung cancer cells, and lung tumor nodules were analyzed. Exercise did not affect lung cancer. Therefore, we also tested the effect of exercise on a subcutaneous LLC1 tumor and a tail vein-injected B16F10 melanoma model. Except for one case of excessive exercise, tumor progression was not influenced. Moderately exercising mice did not increase IL-6 or recruit NK cells to the tumor. Our data suggest that the exercise dose may dictate how efficiently the immune system is stimulated and controls tumor progression.

Exploring the epidemiological role of the Eurasian lynx (Lynx lynx) in the life cycle of Toxoplasma gondii.

Toxoplasma gondii is a successful coccidian parasite able to infect all warm-blooded animals and humans, causing one of the most common zoonoses worldwide. The Eurasian lynx (Lynx lynx) is one of the feline potential hosts of T. gondii in Switzerland, but little is known about its epidemiological role as a definitive or intermediate host. Serum samples from 183 Eurasian lynx collected from 2002 to 2021 were tested for antibodies to T. gondii by ELISA, IFAT and in case of inconclusive results, immunoblot. Antibodies to T. gondii were found in 150 of 183 (82%) Eurasian lynx. Older age, good health status and a low-altitude habitat were found to be significant predictors for seropositivity. T. gondii oocysts were detected in 3 of 176 (1.7%) faecal samples, indicating the Eurasian lynx as a definitive host. In addition, T. gondii DNA was detected in skeletal muscle (7/88), heart muscle (2/26) and/or brain tissue (2/36) from 10 different lynx by real-time PCR. In one animal, a T. gondii-like tissue cyst was observed in heart muscle and confirmed as T. gondii by immunohistochemistry (1/20) and real-time PCR. With an adapted nested-PCR-multilocus-sequence typing (MLST) and in silico restriction-fragment-length-polymorphism analysis (RFLP) approach two different T. gondii genotypes were detected: a lineage II variant (ToxoDB #3) in three animals (two oocyst samples and one heart muscle sample) and a novel genotype exhibiting both type II and III alleles in a further animal (skeletal muscle). The present results indicate that T. gondii infection is widespread in the Swiss lynx population. The Eurasian lynx may contribute to environmental contamination with oocysts and is able to harbour the parasite in different tissues. Genotyping revealed the presence of both a common T. gondii lineage in Europe and a previously unknown genotype and thus shedding more light on the complex molecular epidemiology of T. gondii.

High prevalence rates of Toxoplasma gondii in cat-hunted small mammals - Evidence for parasite induced behavioural manipulation in the natural environment?

Toxoplasma gondii causes one of the most frequent parasitic infections in vertebrates on earth. The present study aimed to assess the occurrence of T. gondii infection in cat-hunted wild small mammals, and to determine the circulating T. gondii genotypes in cat prey. There is evidence suggesting that T. gondii may manipulate rodents' behaviour enhancing transmission to their definitive feline host by facilitating predation. Given that most studies focusing on rodent behavior have been performed under laboratory conditions, we tested this hypothesis in the natural environment. We analysed 157 cat-hunted wild small mammals of six different species from Switzerland. Brain and skeletal muscle samples from each animal were tested for T. gondii DNA by PCR, and positive samples were genotyped using a multilocus sequence typing approach, including 10 genetic markers. Additionally, to evaluate exposure to cat faeces, the presence of Taenia taeniaeformis metacestodes was investigated at necropsy. The prevalence of T. gondii in cat-hunted Arvicola amphibius s.l. was 11.1% (7/63), 14.6% (7/48) in Apodemus spp., 13.6% (3/22) in Myodes glareolus, 6.7% (1/15) in Crocidura russula, and 0% in Microtus arvalis (0/8) and Sorex sp. (0/1). All completely genotyped T. gondii parasites, exhibited the ToxoDB #3 genotype, a Type II variant. We additionally analysed 48 trap-captured A. amphibius s.l., which all tested negative for T. gondii infection, contrasting with the higher prevalence in cat-hunted A. amphibius s.l. (0% vs. 11.1%; p = 0.0176). Furthermore, T. taeniaeformis was detected in both groups, indicating widespread contamination with cat faeces in the sampled areas. These results provide evidence that T. gondii infected rodents are at higher risk to be predated by cats and therewith support the behaviour manipulation hypothesis.

Erythropoietin receptor regulates tumor mitochondrial biogenesis through iNOS and pAKT.

Erythropoietin receptor (EPOR) is widely expressed in healthy and malignant tissues. In certain malignancies, EPOR stimulates tumor growth. In healthy tissues, EPOR controls processes other than erythropoiesis, including mitochondrial metabolism. We hypothesized that EPOR also controls the mitochondrial metabolism in cancer cells. To test this hypothesis, we generated EPOR-knockdown cancer cells to grow tumor xenografts in mice and analyzed tumor cellular respiration via high-resolution respirometry. Furthermore, we analyzed cellular respiratory control, mitochondrial content, and regulators of mitochondrial biogenesis in vivo and in vitro in different cancer cell lines. Our results show that EPOR controls tumor growth and mitochondrial biogenesis in tumors by controlling the levels of both, pAKT and inducible NO synthase (iNOS). Furthermore, we observed that the expression of EPOR is associated with the expression of the mitochondrial marker VDAC1 in tissue arrays of lung cancer patients, suggesting that EPOR indeed helps to regulate mitochondrial biogenesis in tumors of cancer patients. Thus, our data imply that EPOR not only stimulates tumor growth but also regulates tumor metabolism and is a target for direct intervention against progression.

Iron- and erythropoietin-resistant anemia in a spontaneous breast cancer mouse model.

Anemia of cancer (AoC) with its multifactorial etiology and complex pathology is a poor prognostic indicator for cancer patients. One of the main causes of AoC is cancer-associated inflammation that activates mechanisms, commonly observed in anemia of inflammation, whereby functional iron deficiency and iron-restricted erythropoiesis are induced by increased hepcidin levels in response to raised levels of interleukin-6. So far only a few AoC mouse models have been described, and most of them did not fully recapitulate the interplay of anemia, increased hepcidin levels and functional iron deficiency in human patients. To test if the selection and the complexity of AoC mouse models dictates the pathology or if AoC in mice per se develops independently of iron deficiency, we characterized AoC in Trp53floxWapCre mice that spontaneously develop breast cancer. These mice developed AoC associated with high levels of interleukin-6 and iron deficiency. However, hepcidin levels were not increased and hypoferremia coincided with anemia rather than causing it. Instead, an early shift in the commitment of common myeloid progenitors from the erythroid to the myeloid lineage resulted in increased myelopoiesis and in the excessive production of neutrophils that accumulate in necrotic tumor regions. This process could not be prevented by either iron or erythropoietin treatment. Trp53floxWapCre mice are the first mouse model in which erythropoietin-resistant anemia is described and may serve as a disease model to test therapeutic approaches for a subpopulation of human cancer patients with normal or corrected iron levels who do not respond to erythropoietin.

Epitheliocystis in Greater Amberjack: Evidence of a Novel Causative Agent, Pathology, Immune Response and Epidemiological Findings.

Epitheliocystis is a fish gill disease caused by a broad range of intracellular bacteria infecting freshwater and marine fish worldwide. Here we report the occurrence and progression of epitheliocystis in greater amberjack reared in Crete (Greece). The disease appears to be caused mainly by a novel Betaproteobacteria belonging to the Candidatus Ichthyocystis genus with a second agent genetically similar to Ca. Parilichlamydia carangidicola coinfecting the gills in some cases. After a first detection of the disease in 2017, we investigated epitheliocystis in the following year's cohort of greater amberjack juveniles (cohort 2018) transferred from inland tanks to the same cage farm in the open sea where the first outbreak was detected. This cohort was monitored for over a year together with stocks of gilthead seabream and meagre co-farmed in the same area. Our observations showed that epitheliocystis could be detected in greater amberjack gills as early as a month following the transfer to sea cages, with ionocytes at the base of the gill lamellae being initially infected. Cyst formation appears to trigger a proliferative response, leading to the fusion of lamellae, impairment of gill functions and subsequently to mortality. Lesions are characterized by infiltration of immune cells, indicating activation of the innate immune response. At later stages of the outbreak, cysts were no longer found in ionocytes but were observed in mucocytes at the trailing edge of the filament. Whole cysts appeared finally to be expelled from infected mucocytes directly into the water, which might constitute a novel means of dispersion of the infectious agents. Molecular screening indicates that meagre is not affected by this disease and confirms the presence of previously described epitheliocystis agents, Ca. Ichthyocystis sparus, Ca. Ichthyocystis hellenicum and Ca. Similichlamydia spp., in gilthead seabream. Prevalence data show that the bacteria persist in both gilthead seabream and greater amberjack cohorts after first infection.

Iron Regulation in Elderly Asian Elephants (Elephas maximus) Chronically Infected With Mycobacterium tuberculosis.

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection.

Sarcocystis infection in red deer (Cervus elaphus) with eosinophilic myositis/fasciitis in Switzerland and involvement of red foxes (Vulpes vulpes) and hunting dogs in the transmission.

Red deer (Cervus elaphus) carcasses showing grey-greenish discolouration have been increasingly observed in the canton of Grisons, Switzerland. We investigated whether Sarcocystis infections were associated with this pathology, and whether wild and domestic canids were involved in their transmission. Meat from affected red deer (n = 26), faeces and intestines from red foxes (Vulpes vulpes) (n = 126), and faeces from hunting dogs (n = 12) from the region, were analysed. Eosinophilic myositis and/or fasciitis were diagnosed in 69% of the deer, and sarcocysts were observed in 89% of the animals. Molecular typing targeting a ~700bp variable region of the 18S rRNA gene revealed Sarcocystis hjorti in 73%, S. venatoria/S. iberica in 54%, S. linearis/S. taeniata in 12%, S. pilosa in 8% and S. ovalis in 4% of the deer samples. No inflammatory changes were observed in red deer carcasses with normal appearance (n = 8); however, sarcocysts were observed in one sample, and S. hjorti, S. venatoria/S. iberica or S. silva DNA was detected in five samples. Sarcocystis oocysts/sporocysts were observed in 11/106 faecal and 6/20 intestinal fox samples, and in 2/12 canine samples. Sarcocystis tenella (n = 8), S. hjorti (n = 2), S. gracilis (n = 2), and S. miescheriana (n = 1) were identified in foxes, and S. gracilis (n = 2), S. capreolicanis (n = 1) and S. linearis/S. taeniata (n = 1) in dogs. This study provides first molecular evidence of S. pilosa and S. silva infection in red deer and S. linearis/S. taeniata in dogs and represents the first record of S. ovalis transmitted by corvids in Central Europe. Although Sarcocystis species infecting red deer are not regarded as zoonotic, the affected carcasses can be declared as unfit for human consumption due to the extensive pathological changes.

Native parasite affecting an introduced host in aquaculture: cardiac henneguyosis in the red seabream Pagrus major Temminck & Schlegel (Perciformes: Sparidae) caused by Henneguya aegea n. sp. (Myxosporea: Myxobolidae).

BACKGROUND: Henneguya Thélohan, 1892 (Myxobolidae) is one of the most species-rich genera of myxosporean parasites infecting fish. Although common in nature, there are few reports of these parasites causing important disease in aquaculture. In this paper, we describe a new species of Henneguya infecting Pagrus major (Temminck & Schlegel), a fish host introduced to the Mediterranean Sea from Japan in the late 1980s. RESULTS: Large plasmodia of the parasite were found in the bulbus arteriosus and in the ventricle of the infected fish. Spores were found mainly in the kidney and heart and were accompanied by melanized macrophages or vascular intimal proliferation mixed with a mild non-suppurative response, respectively. Comparisons of morphometric data for spore and polar capsule length and width, suggest a unique combination of features in the newly described species. Molecular analysis, based on 18S rDNA sequence of the parasite, followed by phylogenetic analysis, indicated that the parasite described here is a novel species of Henneguya, clustered with the marine congeneric species. CONCLUSIONS: Henneguya aegea n. sp. infects in aquaculture P. major, a host introduced as eggs to the Mediterranean from Japan. Despite the high host specificity of the myxobolid parasites, H. aegea n. sp. seems to be able to use P. major as a host and propagate successfully, causing morbidity and mortality. This could result in spillback of the new species from high density cultured non-native P. major to native fish hosts.

Clinical and histologic outcome in a dog surviving massive hepatic necrosis.

This report describes the clinical and histologic recovery of a 2-year-old mixed-breed dog presented with hypovolemic shock, markedly increased serum alanine amino transferase activity, and hemoabdomen. Emergency exploratory surgery revealed a friable liver with multiple capsule hemorrhages necessitating removal of the left lateral lobe. Histologic evaluation showed acute massive hepatic necrosis with centrilobular and midzonal distribution. The dog survived, and all monitored laboratory values normalized within 7 weeks. A liver biopsy taken 8 weeks after presentation revealed normal hepatic architecture with a few, randomly distributed neutrophilic foci. Follow-up included intermittent determination of liver variables including liver function tests for a period of 7 years. The dog's health status, and all test results remained normal during this time. Complete recovery and good long-term quality of life after life-threatening acute liver failure secondary to massive hepatic necrosis is possible in dogs.

First report of nodular skin lesions caused by Mycobacterium nebraskense in a 9-year-old cat.

CASE SUMMARY: A 9-year-old cat was referred with multiple, raised, ulcerative skin nodules in the region of the neck and dorsal head. Histopathological findings of a biopsied nodule were granulomatous dermatitis and panniculitis without multinucleated giant cells or caseous necrosis. In addition, by Ziehl-Neelsen staining numerous acid-fast intracellular bacilli were observed within the lesions. Mycobacterial culture showed growth of rough scotochromogenic colonies after 3 weeks of incubation. Molecular characterisation of the isolate identified Mycobacterium nebraskense as the cause of the infection. No phenotypic resistance was detected for the antimycobacterial agents tested. The cat was successfully treated with a combination of surgical excision and a 12 week course of antimicrobial therapy, including rifampicin combined with clarithromycin. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this is the first documented case of mycobacterial granulomatous dermatitis and panniculitis due to M nebraskense infection in a cat. The successful surgical and antimycobacterial treatment regimen is described.

Tuberculosis in Swiss captive Asian elephants: microevolution of Mycobacterium tuberculosis characterized by multilocus variable-number tandem-repeat analysis and whole-genome sequencing.

Zoonotic tuberculosis is a risk for human health, especially when animals are in close contact with humans. Mycobacterium tuberculosis was cultured from several organs, including lung tissue and gastric mucosa, of three captive elephants euthanized in a Swiss zoo. The elephants presented weight loss, weakness and exercise intolerance. Molecular characterization of the M. tuberculosis isolates by spoligotyping revealed an identical profile, suggesting a single source of infection. Multilocus variable-number of tandem-repeat analysis (MLVA) elucidated two divergent populations of bacteria and mixed infection in one elephant, suggesting either different transmission chains or prolonged infection over time. A total of eight M. tuberculosis isolates were subjected to whole-genome sequence (WGS) analysis, confirming a single source of infection and indicating the route of transmission between the three animals. Our findings also show that the methods currently used for epidemiological investigations of M. tuberculosis infections should be carefully applied on isolates from elephants. Moreover the importance of multiple sampling and analysis of within-host mycobacterial clonal populations for investigations of transmission is demonstrated.

Ca. Similichlamydia in Epitheliocystis Co-infection of Gilthead Seabream Gills: Unique Morphological Features of a Deep Branching Chlamydial Family.

The Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum constitutes a broad range of organisms with an intriguing array of ultrastructural morphologies, including intracellular membranes and compartments and their corresponding complex genomes encoding these forms. The phylum Chlamydiae are all obligate intracellular bacteria and, although much is already known of their genomes from various families and how these regulate the various morphological forms, we know remarkably little about what is likely the deepest rooting clade of this phylum, which has only been found to contain pathogens of marine and fresh water vertebrates. The disease they are associated with is called epitheliocystis; however, analyses of the causative agents is hindered by an inability to cultivate them for refined in vitro experimentation. For this reason, we have developed tools to analyse both the genomes and the ultrastructures of bacteria causing this disease, directly from infected tissues. Here we present structural data for a member of the family Ca. Similichlamydiaceae from this deep-rooted clade, which we have identified using molecular tools, in epitheliocystis lesions of gilthead seabream (Sparus aurata) in Greece. We present evidence that the chlamydial inclusions appear to develop in a perinuclear location, similar to other members of the phylum and that a chlamydial developmental cycle is present, with chlamydial forms similar to reticular bodies (RBs) and elementary bodies (EBs) detected. Division of the RBs appeared to follow a budding process, and larger RBs with multiple condensed nucleoids were detected using both transmission electron microscopy (TEM) and by focused-ion beam, scanning electron microscopy (FIB-SEM). As model hosts, fish offer many advantages for investigation, and we hope by these efforts to encourage others to explore the biology of fish pathogens from the PVC.

Experimental Toxoplasma gondii infections in pigs: Humoral immune response, estimation of specific IgG avidity and the challenges of reproducing vertical transmission in sows.

Ten pregnant sows were experimentally inoculated per os with T. gondii in order to investigate vertical and galactogenic transmission of the parasite and the evolution and maturation of the specific IgG humoral response in the sows and piglets. Five seronegative sows received 104T. gondii (CZ isolate clone H3) sporulated oocysts during late-pregnancy (Exp. 1), three sows received 104 oocysts during mid-pregnancy (Exp. 2) and three sows from Exp. 1 (and two seronegative sows) were re-inoculated with 105 oocysts during a further pregnancy (late-pregnancy) (Exp. 3). Besides, six 4.5 week-old piglets inoculated per os with 5×103 oocysts were also included in the serological investigations. All animals seroconverted (PrioCHECK Toxoplasma Ab porcine ELISA, Prionics, Switzerland) by 2-3 weeks post inoculation (wpi) and remained seropositive for at least 38 weeks or until euthanasia. Four chronically infected sows from Exp. 1 and 2 were serologically monitored during a further pregnancy and no reactivation, but a decrease of the antibody levels was observed at farrowing (Exp. 4). In all experiments, the specific IgG-avidity was initially low, increased during the course of infection and after re-inoculations. An avidity index (AI) ≥40% could be used to rule out recent infections (<8 weeks) in most (15 of 16) animals. In some piglets (18.6% of 70) delivered by inoculated sows (Exp. 1 and 2), maternal antibodies were still detectable at 2 months (but not by 3 months) of age, with constant high avidity values, comparable to those of the dams at farrowing. In all experiments, the sows remained asymptomatic and delivered non-infected offspring at term. A total of 208 normal and 5 stillborn piglets delivered by the inoculated sows (Exp. 1-4) tested serologically negative before colostrum uptake. Placentas (n=88) from all sows and tissues (brain, liver, lung, heart, and masseter muscle) from 56 delivered piglets were analysed histopathologically and by real-time PCR for T. gondii with negative results. Colostrum and milk samples from all sows were negative by real-time PCR for T. gondii DNA. In addition, no seroconversion was observed in 16 piglets from seronegative dams that were transferred to infected dams one day after birth to detect a possible infection through colostrum or milk during the suckling period. Although vertical transmission of T. gondii was demonstrated in naturally infected pigs, many factors involved in the outcome of vertical transmission and congenital toxoplasmosis in pigs are still unknown.

A Zebrafish Model for Chlamydia Infection with the Obligate Intracellular Pathogen Waddlia chondrophila.

Obligate intracellular chlamydial bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum are important pathogens of terrestrial and marine vertebrates, yet many features of their pathogenesis and host specificity are still unknown. This is particularly true for families such as the Waddliacea which, in addition to epithelia, cellular targets for nearly all Chlamydia, can infect and replicate in macrophages, an important arm of the innate immune system or in their free-living amoebal counterparts. An ideal pathogen model system should include both host and pathogen, which led us to develop the first larval zebrafish model for chlamydial infections with Waddlia chondrophila. By varying the means and sites of application, epithelial cells of the swim bladder, endothelial cells of the vasculature and phagocytosing cells of the innate immune system became preferred targets for infection in zebrafish larvae. Through the use of transgenic zebrafish, we could observe recruitment of neutrophils to the infection site and demonstrate for the first time that W. chondrophila is taken up and replicates in these phagocytic cells and not only in macrophages. Furthermore, we present evidence that myeloid differentiation factor 88 (MyD88) mediated signaling plays a role in the innate immune reaction to W. chondrophila, eventually by Toll-like receptor (TLRs) recognition. Infected larvae with depleted levels of MyD88 showed a higher infection load and a lower survival rate compared to control fish. This work presents a new and potentially powerful non-mammalian experimental model to study the pathology of chlamydial virulence in vivo and opens up new possibilities for investigation of other members of the PVC superphylum.

Emerging pathogens of gilthead seabream: characterisation and genomic analysis of novel intracellular ß-proteobacteria.

New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel ß-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

Molecular characterization and virus neutralization patterns of severe, non-epizootic forms of feline calicivirus infections resembling virulent systemic disease in cats in Switzerland and in Liechtenstein.

Feline calicivirus (FCV) infections are associated with oral ulceration, chronic stomatitis and a limping syndrome. Epizootic outbreaks of virulent systemic disease (VSD) have been reported in the USA and Europe. Here, the molecular characterization and neutralization patterns of FCV isolates from cases of severe, non-epizootic infection associated with skin ulceration and edema are presented. Samples from eleven symptomatic cats, four in-contact cats and 27 cats with no contact with symptomatic cats were collected and tested for FCV, feline herpesvirus-1 (FHV-1), feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). Phylogenetic analyses based on the capsid (VP1) gene of FCV and virus neutralization with antisera raised against four FCV vaccine strains were performed. Nine kittens and two adult cats in two shelters and two veterinary clinics in four geographically distinct locations in Switzerland and Liechtenstein were affected. The cats showed fever, tongue and skin ulceration, head and paw edema, and occasionally jaundice, generalized edema and dyspnea. All symptomatic cats tested FCV-positive but were negative for FHV-1, FeLV and FIV, with the exception of one FIV-positive kitten. All kittens of one litter and both adult cats died. The disease did not spread to cats in the environment. Cats in the environment displayed phylogenetically distinct, but related, FCV strains. Virus neutralization patterns suggested that some cases might have been potentially prevented by vaccination with the optimal vaccine strain. In conclusion, clinicians should be aware of severe, non-epizootic forms of FCV infections with initial clinical presentations similar to VSD.

Environmental marine pathogen isolation using mesocosm culture of sharpsnout seabream: striking genomic and morphological features of novel Endozoicomonas sp.

Aquaculture is a burgeoning industry, requiring diversification into new farmed species, which are often at risk from infectious disease. We used a mesocosm technique to investigate the susceptibility of sharpsnout seabream (Diplodus puntazzo) larvae to potential environmental pathogens in seawater compared to control borehole water. Fish exposed to seawater succumbed to epitheliocystis from 21 days post hatching, causing mortality in a quarter of the hosts. The pathogen responsible was not chlamydial, as is often found in epitheliocystis, but a novel species of the γ-proteobacterial genus Endozoicomonas. Detailed characterisation of this pathogen within the infectious lesions using high resolution fluorescent and electron microscopy showed densely packed rod shaped bacteria. A draft genome sequence of this uncultured bacterium was obtained from preserved material. Comparison with the genome of the Endozoicomonas elysicola type strain shows that the genome of Ca. Endozoicomonas cretensis is undergoing decay through loss of functional genes and insertion sequence expansion, often indicative of adaptation to a new niche or restriction to an alternative lifestyle. These results demonstrate the advantage of mesocosm studies for investigating the effect of environmental bacteria on susceptible hosts and provide an important insight into the genome dynamics of a novel fish pathogen.