The effect of homogenisation pressure on the microstructure of milk during evaporation and drying: particle-size distribution, electronic scanning microscopy, water activity and isotherm.

Homogenisation is a widely used technique in manufacturing powdered milk with a direct impact on product solubility, and the homogenisation pressure is a central attribute of this process. We aimed to understand the effect of increasing homogenisation pressures (0/0, 15/5, and 75/5 MPa, 1st/2nd stages) on particle-size distribution during homogenised whole milk powder manufacture and rehydration of the final product. The fluid milk was thermally treated, homogenised, concentrated by rotary evaporation, and then dried using a spray dryer. Particle size (Dv90) was monitored at all stages of the manufacturing process. The final product (milk powder) was analysed using particle-size distribution, electronic scanning microscopy, water activity, and isotherms. The results demonstrated that increasing the homogenisation pressure leads to milk powder with smaller particle size when rehydrated (Dv90 values: 6.08, 1.48 and 0.64 µm for 0, 20 and 80 MPa, respectively). Furthermore, the volume (%) of the particles in the 'sub-micro' region (smaller than 1.0 µm) presented an inversely proportional profile to the homogenisation pressure (homogenised fluid milk: 86.1, 29.3 and 2.4%; concentrated milk: 86.1, 26.5 and 5.7%, and reconstituted milk powder: 84.2, 31.8 and 10.9%). Surprisingly, this pattern was not observed in the SPAN value (which corresponds to the width or range of the size distribution based on the volume). Additionally, the increase in the homogenisation pressure did not affect the sorption isotherm pattern. These results demonstrate that increasing the homogenisation pressure decreases the particle size of the reconstituted powdered milk, indicating the potential for future studies on how this phenomenon affects its physicochemical and final product properties.

Decreased corneal subbasal nerve fiber length and density in diabetic dogs with cataracts using in vivo confocal microscopy.

OBJECTIVE: To determine whether there is a difference in corneal sensitivity and corneal subbasal nerve plexus (CSNP) morphology in cataractous dogs with diabetes mellitus (DM) versus without DM. ANIMALS STUDIED: Twenty six domestic dogs with cataracts of various breeds presented for phacoemulsification, 13 with DM and 13 without DM. PROCEDURE: The inclusion criteria for the study were dogs with bilateral cataracts and no clinical evidence of corneal disease. The diabetic group had documented hyperglycemia and was currently treated with insulin. The non-diabetic group had no evidence of DM on examination and bloodwork. Complete ophthalmic examination, corneal esthesiometry, and in vivo confocal microscopy of the CSNP was performed for both eyes of each dog. The CSNP was evaluated using a semi-automated program and statistically analyzed. RESULTS: The mean (±SD) CSNP fiber length was significantly decreased in diabetic (3.8 ± 3.0 mm/mm2 ) versus non-diabetic (6.7 ± 1.9 mm/mm2 ) dogs. Likewise, the mean (±SD) fiber density was significantly decreased in diabetic (8.3 ± 3.1 fibers/mm2 ) versus non-diabetic (15.5 ± 4.9 fibers/mm2 ) dogs. The corneal touch threshold was significantly reduced in diabetic (2.1 ± 0.8 cm) versus non-diabetic (2.8 ± 0.4 cm) dogs. There was a non-significant trend towards subclinical keratitis in diabetic (9/13) versus non-diabetic (4/13) dogs. CONCLUSIONS: Morphological and functional abnormalities of the CSNP were present in dogs with DM, including decreased fiber length, fiber density, and corneal sensitivity. These findings are consistent with diabetic neuropathy and could contribute to clinically significant corneal complications after cataract surgery.

Nocardia and Streptomyces keratitis in dogs: In vivo detection of filamentous bacteria by confocal microscopy.

OBJECTIVE: To describe the clinical features of dogs with Nocardia and Streptomyces keratitis, including the results of in vivo confocal microscopy examinations. ANIMAL STUDIED: A 15-year-old, male-castrated, miniature Schnauzer was presented with a multilobulated, cystic, pink, ulcerated corneal mass with surrounding dense leukocyte infiltrates. Cytologic evaluation of a corneal scraping identified pyogranulomatous inflammation and filamentous bacteria. Nocardia nova was cultured from corneal samples. Anterior lamellar keratectomy was performed to excise the affected corneal region and histopathologic evaluation confirmed the diagnosis of pyogranulomatous keratitis. A 10-year-old, male-castrated, Yorkshire terrier was presented for evaluation of a chronic anterior stromal corneal ulcer associated with a brown corneal plaque. Cytologic evaluation of a corneal scraping identified suppurative inflammation and filamentous bacteria. A Streptomyces sp. was cultured from corneal samples. The keratitis in both dogs resolved with therapy. PROCEDURES: In vivo confocal microscopy examination of the corneal lesions in both dogs revealed dense accumulations of leukocytes and clusters of hyperreflective, slender, branching bacterial structures that were approximately 1.5-2.0 µm in diameter and 25-50 µm in length. Confocal microscopy imaging of the Nocardia isolate in vitro, and ex vivo canine corneas experimentally infected with the bacteria, was performed to corroborate the in vivo findings. The morphology of the filamentous bacteria was similar between the in vivo, in vitro, and ex vivo confocal microscopy examinations. CONCLUSIONS AND CLINICAL RELEVANCE: Nocardia and Streptomyces spp. can be associated with infectious keratitis in dogs. In vivo detection of filamentous bacteria in the cornea can be accomplished by confocal microscopy.

IN VIVO CONFOCAL MICROSCOPY FOR CHARACTERIZATION OF MYCOTIC KERATITIS IN OWLS (BUBO SCANDIACUS, STRIX VARIA) AND A WOODCOCK (SCOLOPAX MINOR): THREE CASES.

This case series describes the use of in vivo confocal microscopy in the diagnosis and treatment of mycotic keratitis in two owls (one Bubo scandiacus, one Strix varia) and one woodcock (Scolopax minor). Each bird was at increased risk of fungal infection due to recent injury or stress. Ophthalmic findings in all birds included blepharospasm, ocular discharge, ulcerative keratitis, white or yellow corneal plaques, and anterior uveitis. Fungal hyphae were identified in corneal samples from all three eyes examined cytologically and in all three eyes by using in vivo confocal microscopy. Aspergillus fumigatus was isolated from a corneal culture in one bird. Despite medical treatment, progressive ocular disease prompted enucleation in two birds. Fungal hyphae were detected by histopathology in one of the two enucleated eyes. In vivo confocal microscopy aided the diagnosis of fungal keratitis in all birds and was the only diagnostic method that allowed immediate, real-time quantification of the extent (area and depth) and severity of mycotic keratitis.

Multimodal ocular imaging of known and novel corneal stromal disorders in dogs.

BACKGROUND: Imaging features obtained with Fourier-domain optical coherence tomography (FD-OCT) and in vivo confocal microscopy (IVCM) for corneal stromal disorders have been sparsely reported in dogs. This case report is a compilation of imaging features for three cases of different stromal disorders of the canine cornea which have not yet been reported elsewhere. CASE PRESENTATION: Lipid deposition in case 1 appeared as needle-shaped hyperreflective lines along the collagen lamellae, which correlated histologically with lipid clefts. In case 2, glycosaminoglycan accumulation by mucopolysaccharidosis type 1 caused diffuse stromal hyperreflectivity and depletion of keratocytes on IVCM and was associated with secondary corneal degeneration presumed to be calcium deposition. In case 3, posterior corneal stromal opacities in the absence of ocular inflammation were identified. Hyperreflective particles were scattered in the middle and posterior corneal stroma on FD-OCT. With IVCM, hyperreflective deposits were identified within keratocytes and the number of enlarged keratocytes containing hyperreflective deposits increased towards the posterior stroma. The bilateral, non-inflammatory nature and unique appearance with IVCM is most consistent with a posterior stromal dystrophy reminiscent of pre-Descemet corneal dystrophy described in humans. CONCLUSIONS: In vivo multimodal corneal imaging facilitated instantaneous microstructural analysis and may be valuable in the differential diagnosis of corneal stromal disorders in veterinary clinical practice. The non-specific nature of imaging findings occurs in some conditions such as mucopolysaccharidosis, thus in vivo corneal imaging should be complemented with other gold standard methods of definitive diagnosis.

Leishmania (L.) infantum BH401 strain induces classic renal lesions in dogs: Histological and confocal microscopy study.

Extracellular matrix (ECM) alterations in visceral leishmaniasis are related mainly to collagen deposition (fibropoiesis). In canine visceral leishmaniasis (CVL), an intense fibrosis associated to chronic inflammation in organs such as kidneys is described. However, renal fibropoiesis has not been described in natural or experimental infections with L. (L.) infantum. We aimed to characterize renal nephropathies by histology and confocal microscopy comparing renal lesions in dogs naturally and experimentally infected with L. (L.) infantum. Sixty-two mixed-breed symptomatic dogs naturally infected with L. (L.) infantum, sixteen beagles experimentally infected with two strains of L. infantum (eleven dogs with the BH400 strain and five dogs with the BH401 strain), and five uninfected beagles (controls) were used. Samples were stained with hematoxylin & eosin for routine histology. Congo red was used to visualize amyloid protein deposits, periodic acid-Schiff to identify glomerular basal membrane anomalies, Masson's trichrome for collagen deposits, and Jones' methenamine silver to reveal membranous glomerulonephropathy. Immunohistochemistry was used to identify Leishmania amastigotes, and confocal microscopy was used for macrophage characterization (L1/calprotectin and CD163 antigen receptors). The most common lesions were chronic glomerular and interstitial nephritis, which was found in all naturally infected dogs and dogs experimentally infected with L. infantum strain BH401 but not with the BH400 strain. Glomeruloesclerosis was the main lesion presented in all BH401 group. Morphometric analysis revealed positive correlation of renal glomeruli tufts with cellular expression of L1/calprotectin and CD163 antigens. Leishmania infantum strain BH401 shows pathogenicity that may be sufficient to induce classic chronic visceral renal leishmaniasis.

X-ray computerized microtomography and confocal Raman microscopy as complementary techniques to conventional imaging tools for the microstructural characterization of Cheddar cheese.

This study explored the use of X-ray computerized microtomography (micro-CT) and confocal Raman microscopy to provide complementary information to well-established techniques, such as confocal laser scanning microscopy (CLSM), for the microstructural characterization of cheese. To evaluate the potential of these techniques, 5 commercial Cheddar cheese samples, 3 with different ripening times and 2 with different fat contents, were analyzed. Confocal laser scanning microscopy was particularly useful to describe differences in fat and protein distribution, especially between the 2 samples with different fat contents. The quantitative data obtained through image analysis correlated well with the nutritional information provided in the product labels. Conversely, micro-CT was more advantageous for studying the size and spatial distribution of microcrystals present within the cheese matrix. Two types of microcrystals were identified that differed in size, shape, and X-ray attenuation. The smallest, with a diameter of approximately 10 to 20 µm, were more abundant in the samples and presented a more uniform roundish shape and higher X-ray attenuation. Larger and more heterogeneous crystals with diameters reaching 50 µm were also observed in scarcer numbers and showed lower X-ray attenuation. Confocal Raman microscopy was useful not only for identifying the distribution of all these components but also allowed comparing the presence of micronutrients such as carotenoids in the cheeses and provided compositional information on the crystals detected. Small and large crystals were identified as calcium phosphate and calcium lactate, respectively. Overall, using micro-CT, confocal Raman microscopy, and CLSM in combination generated novel and complementary information for the microstructural and nutritional characterization of Cheddar cheese. These techniques can be used to provide valuable knowledge when studying the effect of milk composition, processing, and maturation on the cheese quality attributes.

Applications of in vivo confocal microscopy in the management of infectious keratitis in veterinary ophthalmology.

In vivo confocal microscopy (IVCM) is a relatively new ocular imaging technique that permits morphological and quantitative assessment of the living cornea on the cellular level. The applications for IVCM in clinical ophthalmology are numerous and diverse. There are several advantages inherent to IVCM over standard diagnostic techniques currently used to confirm a diagnosis of infectious keratitis in veterinary ophthalmology. With IVCM, images can be viewed in real-time providing immediate diagnostic information. Traumatic corneal sampling techniques are avoided, and the procedure can be repeated as frequently as is clinically indicated without risk of corneal tissue damage. Both superficial and deep corneal lesions can be evaluated by IVCM in an atraumatic fashion. Microorganism viability is not required for their detection and specialized diagnostic laboratory assay procedures are not necessary. Many larger infectious agents can be directly identified within corneal lesions by IVCM, including fungi and parasites such as Acanthamoeba spp. In other situations, such as bacterial infectious crystalline keratopathy, the biological systems associated with the microorganism can be detected within the cornea. The current resolution of IVCM is inadequate to directly visualize some corneal infectious agents, such as herpesviruses, but host responses and virus-infected epithelial cells can be identified. This review summarizes the current knowledge and applications of IVCM in the management of infectious keratitis in veterinary ophthalmology, including its use in animals with bacterial, fungal, parasitic, and viral keratitis.

Canine endotheliitis: Clinical characteristics, advanced imaging features, and treatment.

OBJECTIVE: To describe the clinical findings, multimodal corneal imaging features and treatment in canine patients diagnosed with endotheliitis. ANIMALS STUDIED: Four canine patients met inclusion criteria for bilateral corneal disease with endothelial inflammation and secondary corneal edema that responded to topical anti-inflammatory treatment. METHODS: The patients selected underwent a complete ophthalmic examination with emphasis on the cornea including ultrasound pachymetry (USP), Fourier-domain optical coherence tomography (FD-OCT), in vivo confocal microscopy (IVCM), and digital slit lamp photography. RESULTS: All patients in this study demonstrated thickened corneas due to edema with USP and FD-OCT. With IVCM, mild to severe polymegathism and pleomorphism of corneal endothelial cells, reduced endothelial cell density, hyperreflective keratic precipitates (KPs), and extracellular debris as well as hyporeflective pseudoguttata were observed. With FD-OCT, hyperreflective KPs were commonly observed on the inferior cornea. Clinical examination and advanced imaging results were consistent with a diagnosis of endotheliitis. All patients initially responded to topical anti-inflammatory treatment and required continued therapy; two patients also received topical netarsudil, a rho-associated coiled-coil kinase inhibitor. CONCLUSION: Endotheliitis should be considered for canine patients with bilateral edema that is most severe in the inferior cornea. Careful inspection of Descemet's membrane-endothelial complex should be performed for KPs or inflammatory debris. Chronic administration of topical anti-inflammatories may be necessary to prevent flare-ups of endotheliitis.

Retinal structural and microvascular abnormalities in retinal dysplasia imaged by OCT and OCT angiography.

OBJECTIVE: To describe the in vivo structural characteristics of multifocal and geographic retinal dysplasia visualized with advanced retinal imaging including confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomography (OCT), en face OCT, and the novel vascular imaging technique OCT angiography (OCTA). DOGS STUDIED AND PROCEDURES: Two dogs were diagnosed with unilateral multifocal or geographic retinal dysplasia and underwent advanced retinal imaging under general anesthesia at the Retinal Disease Studies Facility of the University of Pennsylvania. RESULTS: In both cases, the morphological pattern of the lesions was similar including outer retinal folds that invaginated and formed tubular retinal rosettes, surrounding a central inner retinal thickening (multifocal) or plaque (geographic). The two dogs had multiple vascular anomalies in the lesions such as increased tortuosity, abnormal change of vessel diameter including aneurysms and capillary network disruption. We also identified increased autofluorescence by AF cSLO with short wavelength light source (488 nm and barrier filter at 500 nm), and several areas of photoreceptor loss associated with the lesions. CONCLUSION: The use of OCTA allowed the identification of microvascular abnormalities associated with multifocal and geographic retinal dysplasia in two dogs. To our knowledge, this is the first report where the dye-free OCTA technique is used to study vascular lesions in canine retinas.

Assessment of colorectal anastomosis perfusion with confocal laser endomicroscopy − an experimental study

Introduction: Early diagnosis of complicated healing of colorectal anastomosis can increase the chance for salvage surgery and thus reduce overall morbidity. Confocal laser endomicroscopy (CLE) enables in vivo assessment of tissue perfusion without disturbing its integrity. This experimental study evaluates the potential of CLE for postoperative monitoring of colorectal anastomosis. Methods: A hand-sewn colorectal anastomosis was performed in 9 pigs. The animals were subsequently divided into groups with normal (N=3) and ischemic anastomosis (N=6). Microscopic signs of hypoperfusion were evaluated postoperatively at regular intervals using CLE. Results: Uneven saturation of the images was evident in the group with ischemic anastomosis. The epithelium had inhomogeneous edges and more numerous crypt branching was visible. Tissue oedema quantified as the number of crypts per visual field was already more extensive at the first measurement after induction of ischemia. There was also a significant difference between the values measured before and 10 minutes after ischemia ­ 8.7±1.9 vs. 6.0±1.1 (p=0.013). Conclusion: Postoperative monitoring of the colorectal anastomosis using CLE enables prompt detection of perfusion disorders.

Differential expression and localization of TRPV channels in the mature sperm of Anas platyrhynchos.

Sperm cells perform precise chemotactic and thermotactic movement which is crucial for fertilization. However, the key molecules involved in detection of different chemical and physical stimuli which guide the sperm during navigation are not well understood. Ca2+ -signalling mediated by ion channels seem to play important role in motility and other fertility parameters. In this work, we explored the endogenous localization pattern of TRPV channels in the mature spermatozoa of avian species. Using sperm from white pekin duck (Anas platyrhynchos) as the representative avian model, we demonstrate that duck sperm endogenously express the thermosensitive channels TRPV1, TRPV2, TRPV3, TRPV4, and highly Ca2+ -selective channels TRPV5 and TRPV6 in specific yet differential locations. All of these TRPV channels are enriched in the sperm tail, indicating their relevance in sperm motility. Interestingly, the TRPV3 and TRPV4 channels are present in the mitochondrial region. Calcium selective TRPV5 channel is exclusively present in sperm tail and is most abundant among the TRPV channels. This is the first report describing the endogenous presence of TRPV2 and TRPV3 channels in the sperm of any species. Using confocal imaging and super-resolution imaging, we demonstrate that though the TRPV channels are evolutionarily closely related, they have distinct localization pattern in the duck sperm, which could impact their role in fertilization.

Intestinal immunity of dogfish Scyliorhinus canicula spiral valve: A histochemical, immunohistochemical and confocal study.

The present study describes histochemical and immunohistochemical characteristics of the spiral valve and its associated lymphoid tissue (GALT) in the dogfish Scyliorhinus canicula. The mucosal surface of the spiral valve represents the first line of defense against pathogens coming from the external environment through food. Epithelial, mucus and immune cells play a key role in controlling the inflammatory response. Valve intestine of S. canicula had many folds lined by simple columnar cells and goblet cells, which later reacted positive to PAS, AB and AB-PAS, histochemical stains differentiated the different types of mucins; lectin histochemistry (PNA and WGA), detected neutral and acid mucins secreted that plays an important role in protection against invading pathogens. Integrin α5ß1 was expressed in enterocytes that line the valve's folds with greater marking in the apical part of the cells. Laminin was found on the apical side of the epithelium, in fibrillar and cellular elements of the lamina propria and in the muscularis mucosa. In the spiral valve gut-associated lymphoid tissue (GALT) has been studied. For the first time, massive leucocytes aggregates were identified by confocal immunofluorescence techniques, using the following antibodies: TLR2, S100, Langerin/CD207. Our results expand knowledge about Dogfish valve intestine giving important news in understanding comparative immunology.

Evidence for the facultative intracellular behaviour of the fish pathogen Vibrio ordalii.

Vibrio ordalii is an extracellular, Gram-negative bacterium that produces vibriosis in salmonids. While pathogenesis is not fully understood, this bacterium has numerous likely genes for adhesion, colonization, invasion factors and, as recently suggested, intracellular behaviour. Therefore, this study aimed to clarify possible intracellular behaviour for V. ordalii Vo-LM-18 and ATCC 33509T in the fish-cell lines SHK-1 and CHSE-214. Confocal microscopy revealed Vo-LM-18 and ATCC 33509T inside cytoplasm in both fish-cell lines at 4 hr post-inoculation (hpi). At 8 and 16 hpi, the proportion of fish cells invaded by both strains increased. Moreover, intracellular V. ordalii were observed after 8 hpi inside mouse embryonic fibroblasts (MEF), demonstrating that entry was not due to a cellular phagocytosis process. Flow cytometry confirmed immunocytochemistry results, with both V. ordalii evidencing statistically significant differences in the number of infected cells between 8 and 16 hpi. Interestingly, V. ordalii infection did not significantly damage fish cells, as determined by LDH liberation. Viable counts at 8 hpi detected, on average for both lines, 176 ± 47 CFU/ml of culturable intracellular Vo-LM-18 and ATCC 33509T cells. These in vitro findings support the facultative intracellular behaviour of V. ordalii and may be of importance for understanding pathogenicity and survival in aquatic environments.

In vivo confocal microscopy characteristics of equine epithelial and subepithelial nonulcerative keratomycosis.

OBJECTIVE: To describe the in vivo confocal microscopy features of horses with epithelial and subepithelial nonulcerative keratomycosis. ANIMALS STUDIED: Four horses with a clinical diagnosis of epithelial or subepithelial keratomycosis. PROCEDURES: Horses were examined on one or more occasions by in vivo laser scanning confocal microscopy of the cornea. Confocal microscopic examination characteristics were correlated with clinical, cytological, and histopathological findings for the horses. RESULTS: All horses had an irregular corneal epithelial surface during slit-lamp biomicroscopy examination. Epithelial or subepithelial corneal opacities were present in multifocal or diffuse patterns. Positive rose bengal corneal staining was present focally or diffusely in all cases. Fungal hyphae were detected in cytological or histopathological corneal samples from all horses. Aspergillus, Fusarium, and Penicillium spp. were cultured from corneal samples. Confocal microscopy detected hyphae diffusely distributed over the axial cornea in horses with epithelial clinical disease. Fungal hyphae were present in all layers of the corneal epithelium and associated with disorganized and sloughing epithelial cells with minimal leukocytes. Subepithelial keratomycosis was correlated with focal, dense accumulations of hyphae in the immediate subepithelial anterior stroma that were surrounded by moderate numbers of leukocytes. Two horses were examined by confocal microscopy on multiple occasions during the course of medical therapy, and fungal hyphae were observed to migrate from the epithelium into the subepithelial stroma as the clinical corneal disease progressed. CONCLUSIONS: With in vivo confocal microscopy, both epithelial and subepithelial keratomycosis appear as unique clinical entities. Equine epithelial keratomycosis is a potential precursor to subepithelial keratomycosis.

Respiratory explants as a model to investigate early events of contagious bovine pleuropneumonia infection.

Contagious bovine pleuropneumonia (CBPP) is a severe disease caused by Mycoplasma mycoides subsp. mycoides (Mmm). Knowledge on CBPP pathogenesis is fragmented and hampered by the limited availability of laboratory animal and in vitro models of investigation. The purpose of the present study is to assess respiratory explants as useful tools to study the early stages of CBPP. Explants were obtained from trachea, bronchi and lungs of slaughtered cattle, tested negative for Mycoplasma spp. and for the major bacterial and viral respiratory pathogens. The interaction of Mmm with explant cells was studied by immunohistochemistry (IHC), double-labelling indirect immunofluorescence (DLIIF) and laser scanning confocal microscopy (LSCM). Mmm capability to survive and proliferate within the explants was evaluated by standard microbiological procedures. Finally, the putative cellular internalization of Mmm was further investigated by the gentamicin invasion assay. IHC and DLIIF indicated that Mmm can colonize explants, showing a marked tropism for lower airways. Specifically, Mmm was detected on/inside the bronchiolar and alveolar epithelial cells, the alveolar macrophages and the endothelial cells. The interaction between Mmm and explant cells was abolished by the pre-incubation of the pathogen with bovine anti-Mmm immune sera. Mmm was able to survive and proliferate in all tracheal, bronchial and lung explants, during the entire time course of the experiments. LSCM and gentamicin invasion assay both confirmed that Mmm can enter non-phagocytic host cells. Taken together, our data supports bovine respiratory explants as a promising tool to investigate CBPP, alternative to cattle experimental infection.

In vivo confocal microscopy for detection of subconjunctival Onchocerca lupi infection in a dog.

A seven-year-old male castrated mixed-breed dog was diagnosed with bilateral subconjunctival masses. In vivo confocal microscopy facilitated visualization of Onchocerca lupi adult nematodes and their characteristic cuticular morphology. Long, thin, white nematodes were extracted during excisional biopsy. Histopathologic and parasitologic evaluation confirmed the diagnosis of O. lupi. In addition to surgical debulking of the parasitic granulomas, the dog received systemic doxycycline, prednisone, and ivermectin therapy. In vivo confocal microscopy was repeated one year after initial diagnosis, and no remaining nematodes were visible. To the authors' knowledge, this is the first report of use of in vivo confocal microscopy as a noninvasive diagnostic and monitoring tool for canine onchocerciasis.

Characterization of a specific monoclonal antibody against immunoglobulin light kappa/L1 chain in olive flounder (Paralichthys olivaceus).

Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11]. Three newly identified sequences of the Ig light chain from olive flounder were classified according to their isotypes. Subsequent analyses revealed that M7C3-4 was able to specifically detect lymphocytes expressing one of the κ chains (Igκ-a) in olive flounder. Interestingly, Igκ-a+ B cells were more abundant in spleen and trunk-kidney than in peripheral blood, indicating a distribution different from that of IgM+ B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder.

Three-dimensional visualization of green fluorescence protein-labelled Edwardsiella tarda in whole Medaka larvae.

The invasive fish pathogen Edwardsiella tarda is common in aquatic environments and causes the environmentally and economically destructive emphysematous putrefactive disease called edwardsiellosis. In order to understand the organism's infection pathway, medaka larvae (Oryzias latipes) were immersion-infected with E. tarda labelled with green fluorescence protein (GFP) and then visualized in three dimensions under confocal laser microscopy and light-sheet fluorescence microscopy. Confocal microscopy revealed GFP-labelled E. tarda in the mouth, head, gill bridges, gill cover, skin, membrane fin, gastrointestinal tract and air bladder, and in the caudal vein, somite veins, caudal artery and caudal capillaries. Light-sheet microscopy additionally showed GFP-labelled E. tarda in the pharyngeal cavity, muscle of the pectoral fin and cardiac atrium and ventricle. These findings suggest that during its infection of fish, E. tarda initially adheres to, and invades, the epithelial cells of the skin, gills and gastrointestinal tract (through the pharyngeal cavity); E. tarda then enters the blood vessels to access organs, including the air bladder and heart.

Molecular characterization and confocal laser scanning microscopic study of Pygidiopsis macrostomum (Trematoda: Heterophyidae) parasites of guppies Poecilia vivipara.

Pygidiopsis macrostomum and Ascocotyle (Phagicola) pindoramensis (Digenea: Heterophyidae) parasitize guppies as intermediate hosts and, respectively, fish-eating mammals or birds as definitive hosts. Heterophyids have zoonotic potential, and molecular studies associated with morphological and ecological aspects have helped to clarify their taxonomy and phylogeny. Poecilia vivipara naturally parasitized by metacercariae of both species (100% prevalence) exhibit no external signs of parasitism. In this work, four new sequences of P. macrostomum (18S rDNA, 28S rDNA and ITS2 rDNA) and one new sequence of A. (P.) pindoramensis (mtDNA cox-1) are presented. Phylogeny reconstructions linked P. macrostomum to other heterophyids, but the separation of the Heterophyidae and Opisthorchiidae remains unclear. Additionally, we used indirect immunocytochemistry and the phalloidin-fluorescence techniques allied with confocal laser scanning microscopy to describe muscular and neuronal structures of P. macrostomum. A complex arrangement of muscular fibres is associated with the tegument, suckers, gut and reproductive system. Radial fibres around the ventral sucker are thick, branched and extend to the body wall. High-resolution confocal imaging revealed a typical digenean muscular arrangement and important heterophyid morphological traits. These data will support future control measures to reduce the parasitism in guppies reared in fish farming systems, especially for aquarium and experimental purposes.