Quantitation of immune response gene expression and cellular localisation of interleukin-1beta mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD)

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, beta-actin. Interleukin-1beta (IL-1beta) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1beta mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1beta mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1beta-specific biotinylated cRNA probe. Expression of IL-1beta mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1beta at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1beta is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1beta mRNA expression during infection.     

Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: Characterization of lung pathology,persistence of variable surface protein antigens and local immune response

Background

Mycoplasma bovis is associated with pneumonia in calves characterized by the development of chronic caseonecrotic lesions with the agent persisting within the lesion. The purposes of this study were to characterize the morphology of lung lesions, examine the presence of M. bovis variable surface protein (Vsp) antigens and study the local immune responses in calves after infection with M. bovis strain 1067.

Methods

Lung tissue samples from eight calves euthanased three weeks after experimental infection with M. bovis were examined by bacteriology and pathology. Lung lesions were evaluated by immunohistochemical (IHC) staining for wide spectrum cytokeratin and for M. bovis Vsp antigens and pMB67 antigen. IHC identification and quantitative evaluation of CD4⁺ and CD8⁺ T lymphocytes and immunoglobulin (IgG1, IgG2, IgM, IgA)-containing plasma cells was performed. Additionally, expression of major histocompatibility complex class II (MHC class II) was studied by IHC.

Results

Suppurative pneumonic lesions were found in all calves. In two calves with caseonecrotic pneumonia, necrotic foci were surrounded by epithelial cells resembling bronchial or bronchiolar epithelium. In all calves, M. bovis Vsp antigens were constantly present in the cytoplasm of macrophages and were also present extracellularly at the periphery of necrotic foci. There was a considerable increase in numbers of IgG1- and IgG2-positive plasma cells among which IgG1-containing plasma cells clearly predominated. Statistical evaluation of the numbers of CD4⁺ and CD8⁺ T cells, however, did not reveal statistically significant differences between inoculated and control calves. In M. bovis infected calves, hyperplasia of bronchus-associated lymphoid tissue (BALT) was characterized by strong MHC class II expression of lymphoid cells, but only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II.

Conclusions

The results from this study show that infection of calves with M. bovis results in various lung lesions including caseonecrotic pneumonia originating from bronchioli and bronchi. There is long-term persistence of M. bovis as demonstrated by bacteriology and immunohistochemistry for M. bovis antigens, i.e. Vsp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from local downregulation of antigen presenting mechanisms and an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins.     

Properties of macrophages and lymphocytes appearing in rat renal fibrosis followed by repeated injection of cisplatin

Properties of macrophages and lymphocytes appearing in renal fibrosis remains to be investigated. F344 rats were injected once a week with cisplatin (2 mg/kg body weight) for 8 weeks and examined at post-final injection weeks 1, 3, 6, 9, and 12. Rats developed progressive renal fibrosis at weeks 1 to 6 as fibrosis-progress phase, and subsequent amelioration at weeks 9 and 12. CD68⁺ M1-macrophages and major histocompatibility complex (MHC) class II⁺ macrophages remarkably increased persistently, whereas CD163⁺ M2-macrophages slightly increased. MHC class II⁺/CD68⁺ and MHC class II⁺/CD163⁺ macrophages were present, indicating that MHC class II⁺ macrophages might have both functions of M1- and M2-macrophages. In the fibrosis-progress phase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ for M1-factors, and transforming growth factor (TGF)-β1 and IL-10 for M2-factors tended to increase; tissue injury by M1 and fibrosis by M2 might have occurred simultaneously. Lots of CD4⁺ and CD8⁺ T cells appeared in close relation with MHC class II⁺ macrophages, and mainly CD4⁺ T cells formed aggregations. In the lymphocyte aggregates collected by laser microdissection, expression of IL-17A (for Th17 cells) and forkhead box P3 (FoxP3) (for Treg) significantly increased at weeks 1 and 6, respectively; presumably, Th17 cells might be involved in tissue injury, whereas Treg might be related to fibrosis amelioration. These results suggested that macrophages and T cells may contribute interrelatedly to renal fibrosis.     

Factors affecting disease manifestation of toxocarosis in humans: Genetics and environment

Toxocara canis is regarded as the main cause of human toxocarosis but the relative contribution of T. cati is probably underestimated; serological and other diagnostic methods used in most studies of this zoonotic disease do not distinguish between the two parasites. The definitive hosts for T. canis are caniidae. Pups generally have higher infection rates than adult animals and are a major source of eggs in the environment. Humans usually acquire T. canis infection by accidental ingestion of embryonated eggs or encapsulated larvae from the environment or contaminated food, such infections may lead to visceral larva migrans (VLM), ocular larva migrans (OLM) or covert toxocarosis (CT). Although a mixed Th1- and Th2-mediated immunological response, particularly with high levels of IgE and eosinophilia is observed, the underlying mechanisms of molecular and immunopathogenesis for the development of the symptomatic syndromes of VLM, OLM, or of asymptomatic CT are largely unclear. Studies have indicated that immunological defences against various infectious diseases may be highly influenced by complex interactions of environmental and host genetic factors e.g. MHC class I and II, also known as human leucocyte antigen (HLA). Toxocara spp. infections are associated with a polarized CD4⁺ Th2 response with high IgE levels and eosinophilia, mediated mainly by HLA class II molecules. Associations have been made between HLA class II and pathological severity and host genetic effects on exposure to infection. Recent research suggests Foxp3⁺ CD4⁺CD25⁺-expressing T regulatory (Treg) cells play a role in regulation of the immunopathology of granulomas in experimental toxocaral granulomatous hepatitis and in enhanced expression of TGF-β1, which is an important factor for the local survival and function of Treg observed during T. canis invasion in the mouse small intestine, liver, muscle, and brain. Since the potential susceptibility loci HLA class II molecules, are considered involved in the regulation of a Th2-dominant immunity which is highly controlled by Foxp3⁺ CD4⁺CD25⁺ Treg cells by stimulation through TGF-β1, which thus provides a beneficial environment to T. canis larvae but severe injuries to local organs. However, TGF-β1 variant Leu10Pro known to be involved in disease severity warrants further elucidation as this too may have a role in the severity of human toxocarosis. Exploration of TGF-β1 polymorphism, Foxp3⁺ CD4⁺CD25⁺ Treg cells, and MHC polymorphisms may allow insight into the contribution made by environmental and genetic factors in influencing disease syndrome type and severity in humans with toxocarosis.     

Major histocompatibility class II expression in the normal canine cornea and in canine chronic superficial keratitis

OBJECTIVE: To compare the expression of major histocompatibility complex (MHC) class II antigen in the corneas of normal dogs and dogs affected with chronic superficial keratitis (CSK). METHODS: MHC class II expression was determined in frozen sections of normal canine cornea and cornea from lesions of CSK by immunohistochemistry using a monoclonal antibody directed against the canine MHC class II molecule. Langerhans cell phenotype was determined morphologically and by histochemical determination of ATPase activity. To determine the influence of gamma interferon on expression of MHC class II molecules by corneal cells, corneal explants were cultured with the cytokine and MHC class II expression determined as above. RESULTS: Numerous MHC class II-expressing cells were demonstrated within the stroma and epithelium of the normal corneal limbus and conjunctival epithelium while very little MHC class II expression was detected in the central region of normal canine cornea. In limbal and conjunctival epithelium, cells expressing MHC class II antigen showed ATPase activity, suggesting that they were Langerhans cells. Corneas from dogs with CSK showed MHC class II expression associated with stromal cells, some of which exhibited a dendritic morphology while most were lymphocytic. Corneal epithelial cells within the lesion also aberrantly expressed MHC class II. Corneal explants expressed MHC class II to varying degrees after differing periods of incubation with the cytokine gamma interferon. CONCLUSIONS: While the normal central cornea has little MHC class II expression, aberrant expression occurs in CSK, associated with secretion of gamma interferon by infiltrating CD4-expressing lymphocytes. Although this change is likely to be a secondary feature of the CSK lesion, increased MHC class II expression may play a part in perpetuating the corneal inflammation seen in the disease.     

A study of dendritic cell and MHC class II expression in dogs with immunomodulatory-responsive lymphocytic-plasmacytic pododermatitis

The term immunomodulatory-responsive lymphocytic-plasmacytic pododermatitis (ImR-LPP) has previously been proposed to denote a sub-population of dogs with idiopathic pododermatitis. The objective of this study was to investigate dendritic cell (DC) and MHC class II antigen expression in lesional skin of dogs with ImR-LPP (n = 47). Median epidermal CD1c⁺ cell counts were 37.8 and 12.5 mm⁻¹ in ImR-LPP dogs and healthy controls (n = 27), respectively (P < 0.01), while the corresponding dermal cell counts were 180.9 and 45.0 mm⁻², respectively (P < 0.01).Intra-epidermal clusters of DCs were observed in 18/47 dogs with ImR-LPP. Median epidermal MHC class II⁺ cell counts were 32.5 and 10.5 mm⁻¹ in ImR-LPP dogs and healthy controls, respectively (P < 0.01), while the corresponding dermal cell counts were 216.9 and 46.9 mm⁻², respectively (P < 0.01). Dermal MHC class II⁺ staining was primarily associated with DCs (47/47 dogs), mononuclear inflammatory cells (45/47), fibroblast-like cells (19/47) and vascular endothelium (14/47). The DC hyperplasia and increased MHC class II expression in lesional ImR-LPP skin are consistent with enhanced antigen presentation, and suggest that both parameters may contribute to the pathogenesis of ImR-LPP through the priming and activation of CD4⁺ T cells. Equally, it is possible that the enhanced DC numbers observed in this study may contribute to the immunoregulation of steady-state pathology in lesional ImR-LPP skin through additional expanded, although as yet unresolved, mechanisms.     

Phenotypic and functional analysis of bovine peripheral blood dendritic cells before parturition by a novel purification method

Dendritic cells (DCs) are specialized antigen presenting cells specializing in antigen uptake and processing, and play an important role in the innate and adaptive immune response. A subset of bovine peripheral blood DCs was identified as CD172a⁺/CD11c⁺/MHC (major histocompatibility complex) class II⁺ cells. Although DCs are identified at 0.1%–0.7% of peripheral blood mononuclear cells (PBMC), the phenotype and function of DCs remain poorly understood with regard to maintaining tolerance during the pregnancy. All cattle used in this study were 1 month before parturition. We have established a novel method for the purification of DCs from PBMC using magnetic‐activated cell sorting, and purified the CD172a⁺/CD11c⁺ DCs, with high expression of MHC class II and CD40, at 84.8% purity. There were individual differences in the expressions of CD205 and co‐stimulatory molecules CD80 and CD86 on DCs. There were positive correlations between expression of cytokine and co‐stimulatory molecules in DCs, and the DCs maintained their immune tolerance, evidenced by their low expressions of the co‐stimulatory molecules and cytokine production. These results suggest that before parturition a half of DCs may be immature and tend to maintain tolerance based on the low cytokine production, and the other DCs with high co‐stimulatory molecules may already have the ability of modulating the T‐cell linage.     

Clinical and Pathological Effects of the Polyopisthocotylean Monogenean,Gamacallum macroura in White Bass

Abstract

An aquaculture research facility experienced high mortality rates in white bass Morone chrysops associated with a monogenean infestation of the gills, but not in striped bass Morone saxatilis in the same facility. All mortalities had pale gills. Monogeneans, identified as Gamacallum macroura (MacCallum and MacCallum 1913) Unnithan 1971, were found on the gills. Pale-gilled and healthy white bass were selected with no particular attention to condition for venipuncture and euthanasia for postmortem examination, including parasite counts from gills. The median packed cell volume (PCV) of fish with gill pallor was 12.5% (range 9–37%) while PVC of fish with more normal color was 30% (27–33%). Association between the PCV and gill pallor score was statistically significant, as was the association between PCV and the number of monogeneans found on the gills of each fish. Median estimated white blood cell count of fish with gill pallor, at 12.05 × 10^3/μL (range 3.8–24.7), was significantly lower than of apparently healthy fish: 24.7 × 10³/μL (17.3–31.5). Histopathology of the gill arches of pale-gilled fish revealed multifocal moderate to severe branchitis, focal areas of dilated hyperplastic lamellae occluded by fibrin, and monogeneans attached to the lamellae. Fish that were apparently healthy had grossly similar histologic lesions, but at lower frequency and severity.

Received May 27, 2011; accepted July 12, 2012     

Induction and Evaluation of Proliferative Gill Disease in Channel Catfish Fingerlings

Abstract

Proliferative gill disease (PGD) was first reported in channel catfish Ictalurus punctatus at commercial farms in 1981 and is caused by the myxozoan parasite Henneguya ictaluri. The disease affects the gills and is characterized by severe branchial inflammation, epithelial hyperplasia, lamellar fusion, and lysis of chondrocytes. Presumptive diagnosis is based on the presence of lytic areas in the cartilage of the primary lamellae on microscopic examination and is confirmed histologically by the presence of the organism. In these trials, PGD was induced by exposing channel catfish fingerlings to fresh or aged infectious water collected from a pond containing fish diagnosed with severe PGD. The severity of disease was graded by histological scoring and microscopic examination of wet mounts to determine the percentage of gill filaments containing chondrolytic lesions. Exposure of fish to infectious pond water was shown to produce pathological lesions consistent with PGD, and the percentage of gill filaments containing chondrolytic lesions was positively correlated with histological scoring of gill pathology. The number of trophozoite stages in the gills was shown to increase with the severity of the disease. In most cases, however, parasitic cells were not observed in tissue samples with chondrolytic lesions during the early stages of infection. These observations indicate that pathology and lysis of chondrocytes can occur prior to detection of the organism by histopathology. Exposing fish to infectious pond water that was aged for 1 d produced negligible gill pathology and implies that the infectivity of the H. ictaluri actinospore stage is short lived. Removing fish from the source of infection promoted repair of damaged gill tissue; within 14 d of fish transfer to clean water, gill pathology associated with the acute infection was negligible.     

Necrotic processes in the gill tissue of carp caused by changes in the aquatic environment

Histological findings show that various pathogenic processes are involved in the cases of clinically detected branchionecrosis. The changes in gills occurring at toxic branchionecrosis are a necrobiotic process in their nature; in gill lamellae this process manifests itself as a swelling of the respiratory epithelium and as dilation of lamellar sinuses, and in stratified epithelium of gill arch and filaments it takes on the form of the hypertrophy and hyperplasia of epithelial cells. In severe cases the swollen epithelium forms deposits filling up also the interlamellar space. Toxic branchionecrosis is a term referring to a disease when the pathological processes are caused by ammonia intoxication and autointoxication, the main role being played by pH value. At experimental ammonia intoxication the changes in gills are about the same as in toxic branchionecrosis but the symptoms are not so pronounced. Changes characteristic of spherosporosis involve severe necrotic processes in the respiratory epithelium as well as in the stratified epithelium of gills, and the epithelium harbours different developmental stages of spherospores. The spherospores are easily detected when stained with alcian blue at pH 2.5. The changes in the gills of carp naturally invaded by spherospores with additional ammonia intoxication correspond to findings characteristic of toxic branchionecrosis: degeneration of stratified and respiratory epithelium of gills combined with necrotic changes caused by the developmental stages of spherospores. When water is highly oxygenated with chlorinated lime, large cells with eosinophilic granules severely multiply mainly in the stratified epithelium of gills; this process is considered as an inflammatory process. Parasitic invasions by Trichodina and Chilodonella induce local inflammatory reactions with the swelling of the respiratory epithelium.     

Gill epithelium of an angler catfish,Chaca chaca (Siluriformes,Chacidae): Enzyme and glycoprotein histochemistry

A series of histochemical techniques have been employed to localize alkaline phosphatase, acid phosphatase, non-specific esterase, catalase and peroxidase; and to visualize and characterize glycoprotein (GPs) moieties in the epithelium of gill arch, gill filaments and secondary lamellae of an angler catfish Chaca chaca. The epithelium of gill arch and gill filament shows strong alkaline phosphatase activity in the deeper layer epithelial cells; strong non-specific esterase activities in the outer layer epithelial cells; and weak acid phosphatase activity throughout the epithelium. The activity of these enzymes in the secondary lamellae is weak. The catalase and peroxidase show strong activities in the blood cells of the secondary lamellae. Various classes of GPs have been identified and characterized in the mucous secretions of the gill epithelium of C. chaca. These include—GPs with oxidizable vicinal diols, GPs with sialic acid residues without O-acyl substitution and GPs with O-sulphate esters. The functional significance of different enzymes in gill epithelium and the GPs in the mucus secreted on the surface has been discussed with the physiology of the gills in relation to the characteristic habit and habitat of the fish.     

Effect of Indomethacin on the Development of Proliferative Gill Disease

Abstract

After parenteral treatment with the cyclooxygenase inhibitor indomethacin, channel catfish Ictalurus punctatus were exposed to mature spores of an Aurantiactinomyxon sp. demonstrated to be the etiological agent of proliferative gill disease (PGD). Fish that received indomethacin at a dose of 2.0 or 5.0 mg/kg body weight within 0.5 h before exposure to the myxozoan and again at 24 h postexposure had significantly (P < 0.05) less severe gill lesions 7 d after exposure than fish that received the drug vehicle alone. Fish that received 0.5 mg indomethacin/kg had moderately severe lesions. All fish were confirmed to be infected with the organism associated with PGD by microscopic examination of gills 4 or 7 d postexposure. These results suggest that products of the cyclooxygenase pathway (e.g., prostaglandins) participate in the pathophysiologic host response to PGD.     

Isolation and characterization of equine nasal mucosal CD172a+ cells

The nasal mucosa surface is continuously confronted with a broad variety of environmental antigens, ranging from harmless agents to potentially harmful pathogens. This area is under rigorous control of professional antigen presenting cells (APCs), such as dendritic cells (DCs) and macrophages. Mucosal APCs play a crucial role in inducing primary immune responses and the establishment of an immunological memory. In the present study, a detailed characterization of CD172a⁺ cells, containing the APCs residing in the equine nasal mucosa was performed for the first time. CD172a⁺ cells were isolated from collagenase-treated equine nasal mucosa fragments by MACS. Expression of surface markers was determined by flow cytometry and functional analysis was done by measuring the uptake of FITC conjugated ovalbumin (FITC-OVA). Cell surface phenotype of the isolated cells was as follows: 90% CD172a⁺, 30% CD1c⁺, 46% CD83⁺, 42% CD206⁺ and 28% MHC II⁺. This clearly differs from the phenotype of blood-derived monocytes: 96% CD172a⁺, 4% CD1c⁺, 11% CD83⁺, 9% CD206⁺, 72% MHC II⁺ and blood monocyte derived DCs: 99% CD172a⁺, 13% CD1c⁺, 30% CD83⁺, 51% CD206⁺ and 93% MHC II⁺. The CD172a⁺ nasal mucosal cells were functionally able to endocytose FITC-OVA but to a lesser degree than monocyte-derived DCs. Together, these results demonstrate that the isolated CD172a⁺ nasal mucosal cells resemble immature DCs in the nasal area.     

Structural and Ultrastructural Differentiation of Cell Types in the Gills of the Tench (Tinca tinca, L.)

This study used 22 tench of 25-30 cm in length and weighing roughly 200 g. After sacrifice, the gill arch was extracted and fixed in 5% glutaraldehyde for structural, ultrastructural and morphometrical study. Tench gills consist of primary lamellae which are perpendicular to numerous secondary lamellae in which the respiratory barrier is located. A study was made of the structures which make up that barrier and of the multi-layered epithelium situated in the interlamellar space, which is closely linked to maintaining of internal balance in the fish.     

Gill Infection Model for Columnaris Disease in Common Carp and Rainbow Trout

Challenge models generating gill lesions typical for columnaris disease were developed for the fry of both Common Carp Cyprinus carpio and Rainbow Trout Oncorhynchus mykiss by means of an immersion challenge and Flavobacterium columnare field isolates were characterized regarding virulence. Carp inoculated with highly virulent isolates revealed diffuse, whitish discoloration of the gills affecting all arches, while in trout mostly unilateral focal lesions, which were restricted to the first two gill arches, occurred. Light microscopic examination of the gills of carp exposed to highly virulent isolates revealed a diffuse loss of branchial structures and desquamation and necrosis of gill epithelium with fusion of filaments and lamellae. In severe cases, large parts of the filaments were replaced with necrotic debris entangled with massive clusters of F. columnare bacterial cells enwrapped in an eosinophilic matrix. In trout, histopathologic lesions were similar but less extensive and much more focal, and well delineated from apparently healthy tissue. Scanning and transmission electron microscopic observations of the affected gills showed long, slender bacterial cells contained in an extracellular matrix and in close contact with the destructed gill tissue.

This is the first study to reveal gill lesions typical for columnaris disease at macroscopic, light microscopic, and ultrastructural levels in both Common Carp and Rainbow Trout following a challenge with F. columnare. The results provide a basis for research opportunities to examine pathogen–gill interactions.

Received January 10, 2014; accepted July 27, 2014     

Histopathological findings,phenotyping of inflammatory cells,and expression of markers of nitritative injury in joint tissue samples from calves after vaccination and intraarticular challenge with Mycoplasma bovis strain 1067

Background

The pathogenesis of caseonecrotic lesions developing in lungs and joints of calves infected with Mycoplasma bovis is not clear and attempts to prevent M. bovis-induced disease by vaccines have been largely unsuccessful. In this investigation, joint samples from 4 calves, i.e. 2 vaccinated and 2 non-vaccinated, of a vaccination experiment with intraarticular challenge were examined. The aim was to characterize the histopathological findings, the phenotypes of inflammatory cells, the expression of class II major histocompatibility complex (MHC class II) molecules, and the expression of markers for nitritative stress, i.e. inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT), in synovial membrane samples from these calves. Furthermore, the samples were examined for M. bovis antigens including variable surface protein (Vsp) antigens and M. bovis organisms by cultivation techniques.

Results

The inoculated joints of all 4 calves had caseonecrotic and inflammatory lesions. Necrotic foci were demarcated by phagocytic cells, i.e. macrophages and neutrophilic granulocytes, and by T and B lymphocytes. The presence of M. bovis antigens in necrotic tissue lesions was associated with expression of iNOS and NT by macrophages. Only single macrophages demarcating the necrotic foci were positive for MHC class II. Microbiological results revealed that M. bovis had spread to approximately 27% of the non-inoculated joints. Differences in extent or severity between the lesions in samples from vaccinated and non-vaccinated animals were not seen.

Conclusions

The results suggest that nitritative injury, as in pneumonic lung tissue of M. bovis-infected calves, is involved in the development of caseonecrotic joint lesions. Only single macrophages were positive for MHC class II indicating down-regulation of antigen-presenting mechanisms possibly caused by local production of iNOS and NO by infiltrating macrophages.     

Cellular composition of granulomatous lesions in gut-associated lymphoid tissues of goats during the first year after experimental infection with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) causes lesions in naturally and experimentally infected ruminants which greatly differ in severity, cellular composition and number of mycobacteria. Morphologically distinct lesions are already found during the clinically inapparent phase of infection. The complex local host response and number of MAP were characterized at the initial sites of lesions, organized gut-associated lymphoid tissue, in experimentally infected goats. Tissues were collected at 3, 6, 9 and 12 month post-inoculation (mpi) from goat kids that had orally received 10 times 10 mg of bacterial wet mass of MAP (JII-1961). The cellular composition of lesions in Peyer's patches in the jejunum and next to the ileocecal valve was evaluated in 21 MAP-inoculated goats, where lesions were compared with unaltered tissue of six control goats. CD68⁺, CD4⁺, CD8⁺, γδ T lymphocytes, B lymphocytes and plasma cells, MHC class II⁺ and CD25⁺ cells were demonstrated by immunohistochemistry in serial cryostat sections.At 3 mpi, extensive granulomatous infiltrates predominated, consisting of numerous epitheloid cells admixed with many CD4 and γδ T lymphocytes. Only single MAP were detected. This indicates a strong cellular immune reaction able to control MAP infection. γδ T lymphocytes were markedly increased in this type of lesion which may reflect their important role early in the pathogenesis of paratuberculosis. At 9 and 12 mpi, divergent lesions were observed which may reflect different outcomes of host–pathogen interactions. In five goats, minimal granulomatous lesions were surrounded by extensive lymphoplasmacytic infiltrates and no MAP were detected by immunohistochemistry. This was interpreted as effective host response that was able to eliminate MAP locally. In three goats, decreased numbers of lymphocytes, but extensive granulomatous infiltrates with numerous epitheloid cells containing increased numbers of mycobacteria were seen. This shift of the immune response resulted in uncontrolled mycobacterial multiplication. Focal and multifocal circumscribed granulomatous infiltrates of mainly epitheloid cells may represent sites of new infection, since they were observed in goats at all times after inoculation. Their presence in goats with minimal granulomatous lesions surrounded by extensive lymphoplasmacytic infiltrates may indicate that despite the local clearance, the infection may be perpetuated.The complex cellular immune reactions postulated for the pathogenesis of paratuberculosis were demonstrated at the local sites of infection. These early host–pathogen interactions are most likely essential for the eventual outcome of the MAP infection.     

First record of Chilodonella hexasticha (Ciliophora: Chilodonellidae) in Brazilian cultured fish: A morphological and pathological assessment

Chilodonelids are small ciliated protozoans found worldwide and can be dangerous in culture conditions. This study presents morphometric data on the ciliate Chilodonella that is found in cultured Nile tilapia (Oreochromis niloticus), native bait fish tuvira (Gymnotus aff. inaequilabiatus) and native pacu (Piaractus mesopotamicus) and includes a histopathological assessment of the changes that occur in the pacu. For parasitic diagnosis, skin and gill samples were scraped onto slides, dried at room temperature, stained with Giemsa or impregnated with silver nitrate, and the measurements were obtained from photomicrographs. In the diseased pacu, the first gill arch was collected and fixed in a 10% buffered formalin solution for histopathological analysis. Parasite specimens from the different collection sites were identified morphologically as C. hexasticha Kiernik (1909). Diseased fish exhibited depigmentation, skin ulceration, scale loss, excessive mucus production and gill lesions. Histopathological analysis of pacu gills displayed epithelial proliferation with mononuclear inflammatory infiltrate, hemorrhages, and scattering necrosis. In Brazilian-farmed fish this is the first record of C. hexasticha, which has great pathogenic potential in cultured freshwater species. In addition, two new hosts are presented.     

Reference ranges of hematology and lymphocyte subsets in healthy Korean native cattle (Hanwoo) and Holstein dairy cattle

There are no accurate reference ranges for hematology parameters and lymphocyte subsets in Korean native beef cattle (Hanwoo). This study was performed to establish reliable reference ranges of hematology and lymphocyte subsets using a large number of Hanwoo cattle (n = 350) and to compare differences between Hanwoo and Holstein dairy cattle (n = 334). Additionally, age‐related changes in lymphocyte subsets were studied. Bovine leukocyte subpopulation analysis was performed using mono or dual color flow cytometry. The leukocyte subpopulations investigated in healthy cattle included: CD2⁺ cells, sIgM⁺ cells, MHC class II⁺ cells, CD3⁺CD4⁺ cells, CD3⁺CD8⁺ cells, and WC1⁺ cells. Although Hanwoo and Holstein cattle are the same species, results showed several differences in hematology and lymphocyte subsets between Hanwoo and Holstein cattle. This study is the first report to establish reference ranges of hematology and lymphocyte subsets in adult Hanwoo cattle.     

MHC class II expression by follicular keratinocytes in canine demodicosis--an immunohistochemical study

MHC class II proteins present fragments of extra cellular antigen to stimulate CD4(+) T lymphocytes. Aim of this study was the detection of MHC class II antigens on different cutaneous cells in canine demodicosis. Histopathological and immunohistochemical examination of skin biopsies from 44 dogs with demodicosis is reported. The control group consisted of skin biopsies taken from 10 necropsied dogs without obvious skin lesions. The immunohistological assessment of the MHC class II expression revealed MHC class II proteins on different cell types of infiltrating inflammatory cells, i.e. APCs (antigen-presenting cells), macrophages, T lymphocytes and B lymphocytes. The plasma cells, however, only showed expression in 32 (73%) of 44 cases. Generally it was noticeable that most plasma cells but never all of them expressed MHC class II. Neutrophils, mast cells and eosinophils were MHC class II negative. Furthermore, in 39 biopsies (89%) from dogs with demodicosis MHC class II positive follicular keratinocytes were found. The control group did not show MHC class II expression on epithelial cells. Concerning the endothelial cells, a total of 25 biopsies (57%) showed MHC class II expression in which different vascular plexuses were affected by staining. This examination shows that MHC class II expression in the skin of dogs suffering form demodicosis is elevated. Especially the MHC class II expression by follicular keratinocytes seems to be conspicuous. We hypothesize that this is in association with the development and the maintenance of follicular inflammation.