Comparative histopathology of livers from Baltic cod infected with the parasitic nematode Contracaecum osculatum.

Infection levels with the parasitic nematode Contracaecum osculatum in Eastern Baltic cod have increased in the last decades. Eastern Baltic cod is transport host for this parasite that has a high affinity for the liver of the fish. The liver is a highly vital organ and damage to the liver tissue can result in reduced functionality of the organ. Previous studies have revealed that cod with high infections loads reveal impaired physiological performance, reduced nutritional condition and show signs of having a liver disease. Yet, little is known about the pathological changes and inflammatory reactions of the cod liver related to the infections. In this study, we performed histological examinations on 30 Baltic cod livers caught in the eastern part of the Baltic Sea (length; 38 ± 0.9 cm, weight; 454 ± 34.8 gram) and three Sound cod livers (length; 63 ± 2.9 cm, weight; 3396 ± 300.2 gram) to categorize the degree of inflammation and its relation to pathological changes in infected cod livers. We further investigated how C. osculatum infection levels varied with intensity of inflammation and co-infections. We found that high infection loads with C. osculatum caused severe inflammation in the liver tissue of cod and reduced fat content of the hepatocytes. Conspicuous amounts of glycogen were found in the muscle and intestinal epithelial cells of the nematodes and parasitic co-infections occurred more frequently in the most heavily infected livers.

Experimental infection trials with European North Atlantic ranavirus (Iridoviridae) isolated from lumpfish (Cyclopterus lumpus, L.).

European North Atlantic ranavirus (ENARV, Iridoviridae), is a ranavirus species recently isolated from lumpfish (Cyclopterus lumpus, L.), which are used as cleaner fish in Atlantic salmon (Salmo salar) farming in Northern Europe. This study aimed to investigate (1) the virulence of ENARV isolates from Ireland, Iceland and the Faroe Islands to lumpfish; (2) horizontal transmission between lumpfish; and (3) virulence to Atlantic salmon parr. Lumpfish were challenged in a cohabitation model using intraperitoneally (IP) injected shedders, and naïve cohabitants. IP challenge with isolates from Iceland (1.9 × 107 TCID50  ml-1 ) and the Faroe Islands (5.9 × 107 TCID50  ml-1 ) reduced survival in lumpfish, associated with consistent pathological changes. IP challenge with the Irish strain (8.6 × 105 TCID50  ml-1 ) did not significantly reduce survival in lumpfish, but the lower challenge titre complicated interpretation. Horizontal transmission occurred in all strains tested, but no clinical impact was demonstrated in cohabitants. Salmon parr were challenged by IP injection with the Irish isolate, no virulence or virus replication were demonstrated. A ranavirus qPCR assay, previously validated for fish ranaviruses, was first used to detect ENARV in tissues of both in lumpfish and Atlantic salmon. This study provides the first data on the assessment of virulence of ENARV isolates to lumpfish and salmon, guidelines for the diagnosis of ENARV infection, and poses a basis for further investigations into virulence markers.

Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss).

Piscine orthoreovirus (PRV) mediated diseases have emerged throughout salmonid aquaculture. Three PRV subtypes are currently reported as causative agents of or in association with diseases in different salmonid species. PRV-1 causes heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) and is associated with jaundice syndrome in farmed chinook salmon (Oncorhynchus tshawytscha). PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon in Japan. PRV-3 has recently been associated with a disease in rainbow trout (Oncorhynchus mykiss) characterized by anaemia, heart and red muscle pathology; to jaundice syndrome in coho salmon (Oncorhynchus kisutch). In this study, we conducted a 10-week long experimental infection trial in rainbow trout with purified PRV-3 particles to assess the causal relationship between the virus and development of heart inflammation. The monitoring the PRV-3 load in heart and spleen by RT-qPCR shows a progressive increase of viral RNA to a peak, followed by clearance without a measurable change in haematocrit. The development of characteristic cardiac histopathological findings occurred in the late phase of the trial and was associated with increased expression of CD8+, indicating cytotoxic T cell proliferation. The findings indicate that, under these experimental conditions, PRV-3 infection in rainbow trout act similarly to PRV-1 infection in Atlantic salmon with regards to immunological responses and development of heart pathology, but not in the ability to establish a persistent infection.

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV.

A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land-based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real-time RT-PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re-isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%-50% of the cohabiting lumpfish survived after 4 weeks. 80%-92% of the Atlantic salmon survived, but no viral RNA was detected by real-time RT-PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters.

Presence and genetic variability of Piscine orthoreovirus genotype 1 (PRV-1) in wild salmonids in Northern Europe and North Atlantic Ocean.

Piscine orthoreovirus genotype 1 (PRV-1) is widespread in farmed Atlantic salmon (Salmo salar L.) populations in northern Europe, Canada and Chile. PRV-1 occurs in wild fish in Norway and Canada; however, little information of its geographical distribution in wild populations is currently available, and the effect of PRV-1 infection in wild populations is currently unknown. In this study, we present the findings of a survey conducted on 1,130 wild salmonids sampled in Denmark, Sweden, Ireland, Faroe Islands, France, Belgium and Greenland between 2008 and 2017. PRV-1 is reported for the first time in wild salmonids in Denmark, Sweden, Faroe Island and Ireland. The annual PRV-1 prevalence ranged from 0% in France, Belgium and Greenland to 43% in Faroe Islands. In total, 66 samples tested positive for PRV-1, including Atlantic salmon broodfish returning to spawn and Atlantic salmon collected at the feeding ground north of Faroe Islands. The phylogenetic analysis of S1 sequences of the PRV-1 isolates obtained in this survey did not show systematic geographical distribution. This study sheds light on the spread and genetic diversity of the virus identified in populations of free-living fish and provides rationale for screening wild broodfish used in restocking programmes.

Piscine orthoreovirus infection in Atlantic salmon (Salmo salar) protects against subsequent challenge with infectious hematopoietic necrosis virus (IHNV).

Infectious hematopoietic necrosis virus (IHNV) is endemic in farmed rainbow trout in continental Europe and in various salmonid fish species at the Pacific coast of North America. IHN has never occurred in European Atlantic salmon (Salmo salar) farms, but is considered as a major threat for the European salmon industry. Another virus, Piscine orthoreovirus (PRV), is widespread in the sea phase of Atlantic salmon, and is identified as the causative agent of heart and skeletal muscle inflammation. The aim of this study was to investigate the interactions between a primary PRV infection and a secondary IHNV infection under experimental conditions. A PRV cohabitation challenge was performed with Atlantic salmon. At peak of PRV viremia the fish were challenged by immersion with an IHNV genogroup E isolate. Clinical signs and morbidity were monitored. Target organs were sampled at selected time points to assess viral loads of both pathogens. Antiviral immune response and presence of histopathological findings were also investigated. Whereas the PRV-negative/IHNV positive group suffered significant decrease in survival caused by IHNV, the PRV infected groups did not suffer any morbidity and showed negligible levels of IHNV infection. Antiviral response genes were induced, as measured in spleen samples, from PRV infected fish prior to IHNV challenge. In conclusion, PRV-infection protects Atlantic salmon against IHNV infection and morbidity, most likely by inducing a protective innate antiviral response.

The jejunal cellular responses in chickens infected with a single dose of Ascaridia galli eggs.

This histopathological study was carried out in order to investigate the cellular response in the jejunum to Ascaridia galli during the first 7 weeks of infection. Fourty-two ISA Brown chickens (7 weeks old) were infected orally with 500 embryonated A. galli eggs each while 28 chickens were left as uninfected controls. Six infected and four control chickens were necropsied at each time point 3, 7, 10, 14, 21, 28 and 42 days post-infection (dpi). Samples for histopathology were taken from three sites of the jejunoileum. Significantly higher eosinophil counts were seen in infected chickens compared to uninfected at 3, 7, 10, 14 and 28 dpi (P < 0.01). In both groups, the initial number of mast cells was high, but this high level of mast cells remained for a longer period in the infected group compared to the control group. Significantly higher counts were thus found in the infected group at 21 (P < 0.001), 28 (P < 0.01) and 42 dpi (P < 0.05). A. galli infection induced changes in the mucosal thickness as reduced villi length at 7, 10, 14, 21 and 28 dpi and in the degree of general cellular infiltration in the lamina propria of the mucosal layer. No adult worms were seen during the experiment; therefore, A. galli larvae have elicited a moderate cellular response in the lamina propria, mainly consisting of eosinophils in the early phase and later of mast cells.

Decreased water temperature enhance Piscine orthoreovirus genotype 3 replication and severe heart pathology in experimentally infected rainbow trout.

Piscine orthoreovirus genotype 3 (PRV-3) was first discovered in Denmark in 2017 in relation to disease outbreaks in rainbow trout (Oncorhynchus mykiss). While the virus appears to be widespread in farmed rainbow trout, disease outbreaks associated with detection of PRV-3 have only occurred in recirculating aquaculture systems, and has predominantly been observed during the winter months. To explore the possible effects of water temperature on PRV-3 infection in rainbow trout, an in vivo cohabitation trial was conducted at 5, 12, and 18°C. For each water temperature, a control tank containing mock-injected shedder fish and a tank with PRV-3 exposed fish were included. Samples were collected from all experimental groups every 2nd week post challenge (WPC) up until trial termination at 12 WPC. PRV-3 RNA load measured in heart tissue of cohabitants peaked at 6 WPC for animals maintained at 12 and 18°C, while it reached its peak at 12 WPC in fish maintained at 5°C. In addition to the time shift, significantly more virus was detected at the peak in fish maintained at 5°C compared to 12 and 18°C. In shedders, fish at 12 and 18°C cleared the infection considerably faster than the fish at 5°C: while shedders at 18 and 12°C had cleared most of the virus at 4 and 6 WPC, respectively, high virus load persisted in the shedders at 5°C until 12 WPC. Furthermore, a significant reduction in the hematocrit levels was observed in the cohabitants at 12°C in correlation with the peak in viremia at 6 WPC; no changes in hematocrit was observed at 18°C, while a non-significant reduction (due to large individual variation) trend was observed at cohabitants held at 5°C. Importantly, isg15 expression was positively correlated with PRV-3 virus load in all PRV-3 exposed groups. Immune gene expression analysis showed a distinct gene profile in PRV-3 exposed fish maintained at 5°C compared to 12 and 18°C. The immune markers mostly differentially expressed in the group at 5°C were important antiviral genes including rigi, ifit5 and rsad2 (viperin). In conclusion, these data show that low water temperature allow for significantly higher PRV-3 replication in rainbow trout, and a tendency for more severe heart pathology development in PRV-3 injected fish. Increased viral replication was mirrored by increased expression of important antiviral genes. Despite no mortality being observed in the experimental trial, the data comply with field observations of clinical disease outbreaks during winter and cold months.

Local osteogenic expression of cyclooxygenase-2 and systemic response in porcine models of osteomyelitis.

It is suggested that cyclooxygenase 2 (COX-2) derived prostaglandins contributes to the progressive bone loss seen in osteomyelitis lesions. In the present study we examined the expression of COX-2 in bones from 23 pigs with experimental osteomyelitis. Osteomyelitis was induced with Staphylococcus aureus and groups of animals were euthanized following 6 h, 12 h, 24 h, 2 days, 5 days, 11 days and 15 days, respectively. Expression of COX-2 was evaluated immunohistochemically and combined with characterization of morphological changes in bone tissue. Furthermore, the serum concentrations of alkaline phosphatase and haptoglobin were measured. Extensive COX-2 expression by osteoblasts was present 2 days after inoculation together with many activated osteoclasts. Simultaneously, the serum concentration of alkaline phosphatase decreased whereas the haptoglobin concentration increased. This is the first in vivo study showing an early wave of COX-2 mediated bone resorption during osteomyelitis. Therefore, treatment aiming to reduce the break down of bone tissue directed by the COX-2 pathway might be suggested early in the course of the disease.

Epidemiology of canine glaucoma presented to University of Zurich from 1995 to 2009. Part 1: Congenital and primary glaucoma (4 and 123 cases).

OBJECTIVE: To investigate the epidemiology of canine congenital and primary glaucoma in the cases presented to the University of Zurich, Vetsuisse Faculty (UZH) from 1995 to 2009. METHODS: Information was obtained from the computer database of patients examined by members of the UZH Ophthalmology Service, between January 1995 and August 2009. Congenital and primary glaucoma was diagnosed based on the age of onset, the lack of evidence of any antecedent eye conditions, and/or the presence and severity of iridocorneal angle defects. The data was evaluated for breed, gender and age at presentation. RESULTS: A total of 5984 dogs presented to the UZH Ophthalmology service between 1995 and 2009. Four dogs of different breed were diagnosed with congenital glaucoma and 123 dogs were diagnosed with primary glaucoma. For the primary glaucomas the overall male to female ratio (M:F) was 1:1.41 and the age of onset ranged from 0.12 to 18.3 years with a mean of 7.3 ± 3.6 years. Data suggested a predisposition for primary glaucoma in the Siberian Husky, Magyar Vizsla and Newfoundland from 2004 to 2009. CONCLUSION: The report presents the epidemiology of canine congenital and primary glaucomas presented to the UZH from 1995 to 2009. A previous suspicion of predisposition for primary glaucoma in the Newfoundland dog (n = 6) and the Magyar Vizsla breed (n = 8) was confirmed.

Epidemiology of canine glaucoma presented to University of Zurich from 1995 to 2009. Part 2: secondary glaucoma (217 cases).

OBJECTIVE: To investigate the epidemiology of canine secondary glaucomas in the cases presented to the University of Zurich, Vetsuisse Faculty (UZH) from 1995 to 2009 focusing on possible risk factors for developing secondary glaucoma in this population of dogs. METHODS: Information was obtained from the computer database of patients examined by members of the UZH Ophthalmology Service, between January 1995 and August 2009. Secondary glaucoma was diagnosed based on the presence of antecedent eye conditions. The data was evaluated for breed, gender, age at presentation, and for antecedent eye conditions known to cause glaucoma including anterior uveitis of unknown cause (AU), lens luxation (LL), intraocular surgery (SX), intraocular neoplasia (IN), unspecified trauma to the globe (T), ocular melanosis (OM), hypermature cataract (PY), hyphema (HY), and six other less frequent conditions. RESULTS: A total of 217 dogs were diagnosed with secondary glaucoma from 1995 to 2009. The age of the dogs with secondary glaucoma ranged between 88 days and 19 years (mean 7.7 ± 3.6 years). Data suggested a predisposition for secondary glaucoma in the Cairn Terrier and the Jack Russell Terrier breeds from 2004 to 2009. Common causes of secondary glaucoma from 1995 to 2009 were AU (23.0%), LL (22.6%), SX (13.4%), IN (10.6%), T (8.3%), OM and PY (both 6.9%) and HY (3.23%). CONCLUSION: The report presents the epidemiology of secondary glaucomas presented to UZH from 1995 to 2009. Fourteen risk factors were recorded for secondary glaucoma. This is the first paper documenting OM in the Swiss Cairn Terrier dog population.

A non-traumatic Staphylococcus aureus osteomyelitis model in pigs.

BACKGROUND: The propensity for bacterial localization within bones of juvenile pigs is similar to the situation in humans, where haematogenously based osteomyelitis most commonly occurs in infants and children. In both pigs and humans, Staphylococcus aureus is a dominant cause of pyaemic lesions including osteomyelitis. The aim of the present study was to evaluate the pig as a model for the development of osteomyelitis following haematogenous spread of S. aureus. MATERIALS AND METHODS: Twelve animals were challenged intravenously once or twice with 1x10(8) bacteria/kg body weight and euthanased consecutively from 6 h to 48 h after challenge. Following euthanasia, tissues were sampled from the lungs and bones for histology and immunohistochemical staining of vessels, different inflammatiory cells, apoptosis cells, and S. aureus. RESULTS: Disseminated microabscesses developed within the lungs by 6 h but had disappeared at 48 h. Within the metaphyseal area of bones, microabscesses developed after 12 h and progressed until 48 h after challenge. Within bones, lesions were localized in separate foci from where the infection progressed towards the growth plate, which was in some cases bypassed due to bacterial spread through transphyseal vascular channels. Often, bone lesions resulted in trabecular osteosis, in which apoptotic cells were sometimes present. CONCLUSION: The model revealed a pattern of development and presence of lesions similar to the frequently occurring osteomyelitic lesions, especially in pre-pubertal children following haematogenous spread of S. aureus. Therefore, this model can be reliably applied in studies of this disease with respect to pathophysiology, pathomorphology, impact of strain virulence, and therapy.

Emergence and Spread of Piscine orthoreovirus Genotype 3.

Piscine orthoreovirus (PRV) is a relevant pathogen for salmonid aquaculture worldwide. In 2015, a new genotype of PRV (genotype 3, PRV-3) was discovered in Norway, and in 2017 PRV-3 was detected for first time in Denmark in association with complex disease cases in rainbow trout in recirculating aquaculture systems (RAS). To explore the epidemiology of PRV-3 in Denmark, a surveillance study was conducted in 2017 to 2019. Fifty-three farms, including both flow through and RAS, were screened for PRV-3. Of the farms examined, PRV-3 was detected in thirty-eight (71.7%), with the highest prevalence in grow-out farms. Notably, in Denmark disease outbreaks were only observed in RAS. Additionally, wild Atlantic salmon and brown trout populations were included in the screening, and PRV-3 was not detected in the three years where samples were obtained (2016, 2018, and 2019). Historical samples in the form of archived material at the Danish National Reference Laboratory for Fish Diseases were also tested for the presence of PRV-3, allowing us to establish that the virus has been present in Denmark at least since 1995. Sequence analyses of segment S1 and M2, as well as full genome analyses of selected isolates, did not reveal clear association between genetic makeup in these two segments and virulence in the form of disease outbreaks in the field.

Chronic dietary exposure to environmental organochlorine contaminants induces thyroid gland lesions in Arctic foxes (Vulpes lagopus).

The impact of dietary organochlorine (OC) exposure on thyroid gland pathology was studied in farmed male Arctic foxes (Vulpes lagopus). The exposed group (n=16) was fed a diet based on wild minke whale (Balaenoptera acutorostrata) blubber as a main fat source in order to mimic the exposure to OC cocktails in the Artic environment. This resulted in an exposure of approximately 17 microg Sigma OC/kg day and a Sigma OC residue adipose tissue and liver concentration of 1700 and 4470 ng/gl.w., respectively, after 16 months of exposure. Control foxes (n=13) were fed a diet with pork (Sus scrofa) fat as a main fat source containing significantly lower OC concentrations. The food composition fed to the control and exposed group was standardized for nutrient contents. Four OC-related histopathological changes were found: (1) flat-epithelial-cell true thyroid cysts (TC) characterized by neutral content; (2) remnants of simple squamous epithelial-cell embryonic ducts containing neutral debris (EDN); (3) remnants of stratified squamous epithelial-cell embryonic ducts containing acid mucins often accompanied with debris of leukocyte inflammatory nature (EDM) and (4) disseminated thyroid C-cell hyperplasia (HPC). Of these, the prevalence of TC, EDN and HPC was significantly highest in the exposed group (chi(2) test: all p<0.04). The study shows that the OC mixture in minke whale blubber may cause development of thyroid gland cysts, C-cell hyperplasia and increase the prevalence of cystic remnants of embryonic ducts. The mechanism causing these effects could include endocrine disruption of the hypothalamus-pituitary-thyroid (HPT) axis, a disturbance of the calcium homeostasis/metabolism or energy metabolism or immune suppression. Because concentrations of OCs are higher in wild Arctic foxes, it is likely that these animals could suffer from similar OC-induced thyroid gland pathological and functional changes.

Staphylococcus aureus infected embolic stroke upregulates Orm1 and Cxcl2 in a rat model of septic stroke pathology.

OBJECTIVE: Ischaemic brain lesions and brain abscesses are frequent in both human and animal cases of septic embolic stroke. However, existing models of brain infection do not reflect central aspects of septic embolic stroke. Our aim was to compare septic and non-septic embolic stroke in order to identify gene expressions, inflammatory mediators and brain damage in a rat model. METHODS: We created precisely located focal brain infarcts in a rat model of Staphylococcus aureus infected embolic stroke. To cause septic embolic stroke we used a fibrin-rich embolus with bacteria, while every rat in the control group received a non-infected embolus. 64 rats were randomized to receive sham-surgery, sterile embolic stroke or septic embolic stroke. All groups were compared for brain pathology, mortality, gene expressions and inflammatory mediators using histology and reverse transcription quantitative real-time PCR. RESULTS: Although infarct volumes did not differ, septic embolic stroke caused higher mortality than sterile embolic stroke (p=  0.002). Brain abscesses were observed only in the septic group. Approximately 400-500 fold increases were observed for Orm1 and Cxcl2 respectively (1.00E-08 < p < 1.92E-07) in the septic group compared to the sterile group, and these were the most dramatically regulated genes in septic embolic stroke compared to sterile embolic stroke. CONCLUSIONS: Septic embolic stroke caused brain abscesses, increased mortality and upregulated Orm1 and Cxcl2 gene expressions compared to non-infected embolic stroke. The dramatic Orm1 increase observed in the septic group is unprecedented and suggests a significant biological role of Orm1 during septic neuroinflammation.

A time course study of immunological responses in Trichuris suis infected pigs demonstrates induction of a local type 2 response associated with worm burden.

In order to investigate immunological changes over time in pigs infected with Trichuris suis, we inoculated 40 pigs with 5000 infective T. suis eggs and left 40 pigs as uninfected controls. Equal numbers of pigs from both groups were sacrificed every other week from 1 to 11 weeks p.i. At necropsy tissue samples were collected from all pigs and their worm burdens were determined. In the proximal colon of T. suis-infected pigs infiltration of eosinophils peaked 5 weeks p.i. and mast cell infiltration developed from 5 to 11 weeks p.i. Histological evaluation of the proximal colon revealed that the presence of T. suis was closely associated with intestinal histopathological changes such as crypt hyperplasia, goblet cell hyperplasia and a general hypertrophy of mucosa. The crypt lengths were positively associated with worm burdens. Real-time PCR analysis of genes related to immune function indicate a local increased transcription of genes coding for CCR3, ARG1, MUC5AC, IL-4, IL-5, IL-13, FcepsilonR1alpha, and IL-13Ralpha2 and decreased expression of genes coding for iNOS, TNF-alpha, IL-10, CD3epsilon, CD80, CD86, IL-4Ralpha, IL-13Ralpha1 and CD40 in the proximal colon of pigs infected with T. suis. This local T-helper cell Type 2-like gene-expression pattern indicates that the Type 2 immune response characteristic of helminth infections in both mouse and humans also develops in pigs infected with T. suis. The results from this study expand our knowledge of the immunomodulatory effect of T. suis, a parasite that has proven effective in treating inflammatory bowel disease, when its eggs are administered regularly to patients.

Biocompatibility of electrochemical glucose sensors implanted in the subcutis of pigs.

BACKGROUND: Lowering blood glucose concentration slows or prevents the development of complications in diabetes. One of the tools to control glucose levels is continuous glucose measurements. A promising technique involves measurements from glucose sensors implanted directly in skin/subcutis. However, in vivo bioinstability and drift in sensor signals have been reported after implantation, suggestively caused by the infiltration of inflammatory cells and adhesion of proteins to sensor membranes. The aim of this study was to evaluate the in vivo biocompatibility of two electrochemical glucose sensors after implantation in the skin of pigs. METHODS: In vivo biocompatibility of in-house fabricated electrochemical glucose sensors and a commercially available continuous glucose monitoring system (CGMS, Medtronic MiniMed, Northridge, CA) implanted 1 h, 2 h, 24 h, 3 days, or 7 days was examined by histological and immunohistochemical techniques. RESULTS: The extent of inflammation increased significantly as a function of time. The inflammation ranged from an acute focal fibrinous/suppurative dermatitis to a chronic fibrinous and granulating foreign body dermatitis 7 days after implantation. Immunohistochemical stainings showed that heterophilic granulocytes, macrophages, and fibrinogen/fibrinogen fragments D and E were consistent findings. Infiltration of CD3epsilon-positive T-cells was primarily confined to day 7 of implantation. In addition, the pro-inflammatory cytokines interleukin-1 and tumor necrosis factor-alpha played a role in the reaction to sensors. CONCLUSION: The reported in vivo bioinstability of sensors is likely to be caused by protein and cellular biofouling on the sensor membrane. Furthermore, the consistent finding of fibrinogen and fibrinogen fragments D and E at the sensor-tissue interface seems to play an important role in the pathogenesis as it possibly maintains the inflammation by promoting the recruitment of inflammatory cells to the implantation site.

Biocompatibility of an enzyme-based, electrochemical glucose sensor for short-term implantation in the subcutis.

BACKGROUND: Continuous glucose measurements provide improved glycemic control and may prevent hypoglycemia and long-term complications of diabetes. One of the most promising techniques is the short-term implantation of electrochemical glucose sensors in subcutis. However, the inflammatory reaction to these sensors may lead to bioinstability of sensor measurements. The purpose of the present investigation was to examine factors contributing to the observed subcutaneous inflammatory reaction to an enzyme-based electrochemical glucose sensor for continuous glucose measurements. The sensor biocompatibility was assessed in vitro and in vivo. METHODS: A toxicological assessment was performed on sensor materials and leachables, and the endotoxin content of sensors was determined by a Limulus amoebocyte lysate (LAL) test. Moreover, as a consequence of permanent penetration of the skin by the sensor the role of bacterial migration to the tissue was investigated. In vivo biocompatibility was investigated through histological examination of implanted sensor membranes for 3 days in pigs. Additionally, the effect of needle size and type (normal vs. inserter needle) on tissue trauma at sensor insertion was evaluated, and the healing of subcutis was assessed histologically from 3 to 14 days after removal of sensors. RESULTS: The toxicological assessment and the LAL test showed no concerns in a 3-day implantation scenario, and bacterial migration to the subcutis could not be detected. The histological examination showed that a reduction in needle size reduced the extent of inflammation to very low levels, and that the different sensor membranes showed similar extent and type of inflammation. Additionally, the extent of subcutaneous tissue reaction after removal of sensors declined gradually over time and returned to near-normal levels after 2 weeks. CONCLUSION: The electrochemical enzyme-based glucose sensor for continuous glucose measurements in subcutis is acceptable from a biocompatibility point of view. Reducing the inserter needle in size reduces the trauma induced at sensor implantation to neglible levels. Furthermore, the tissue reaction to the sensor returns to near-normal 2 weeks after the sensor has been removed following a 3-day implantation period.

Distribution of mast cells in relation to Schistosoma japonicum induced lesions in pigs.

The pathogenesis of schistosomiasis japonica has been extensively studied, however only little attention has been paid to the presence and localization of mast cells in relation to Schistosoma japonicum induced lesions. The aim of the present pilot study was to assess the parasitological and pathological responses in S. japonicum infected pigs with emphasis on the description of the distribution of mast cells in relation to lesions in the liver and cecum. Six pigs were exposed to 2,000 cercariae and examined 9 weeks post-infection. Three unexposed pigs of the same age served as helminth free controls. All infected pigs developed granulomatous hepatitis and typhlitis. In the liver, the degree of mast cell infiltration was higher in the infected pigs compared to the unexposed control group. This distinction could not be shown in the cecum. In both the liver and cecum, a mild to moderate number of mast cells were present within the granulomas. A significant relation was found between infection with S. japonicum and the mast cell infiltration in the liver. Due to their possible association with hepatic fibrosis, it seems as if they have some function in the fibrogenic process and thereby play a dual role in the pathogenesis of S. japonicum. In conclusion, the results show that mast cells are recruited to egg induced lesions in both the liver and the cecum.

Modelling severe Staphylococcus aureus sepsis in conscious pigs: are implications for animal welfare justified?

BACKGROUND: A porcine model of haematogenous Staphylococcus aureus sepsis has previously been established in our research group. In these studies, pigs developed severe sepsis including liver dysfunction during a 48 h study period. As pigs were awake during the study, animal welfare was challenged by the severity of induced disease, which in some cases necessitated humane euthanasia. A pilot study was therefore performed in order to establish the sufficient inoculum concentration and application protocol needed to produce signs of liver dysfunction within limits of our pre-defined humane endpoints. METHODS: Four pigs received 1 × 10(8) cfu/kg BW of S. aureus, and two controls were sham inoculated with saline. A fixed infusion rate of 3 mL/min was used, while the inoculum concentration, i.e., the dose volume, was changed between the pigs. The following dose volumes were used: 10 mL (n = 1), 20 mL (n = 2), and 30 mL (n = 1), corresponding to infusion durations of 3.33, 6.66, and 10 min at dose rates of 3 × 10(7), 1.5 × 10(7), and 1 × 10(7) cfu/min/kg BW, respectively. Blood samples were drawn for complete blood count, clinical chemistry, and inflammatory markers before and every 6 h after inoculation. Prior to euthanasia, a galactose elimination capacity test was performed to assess liver function. Pigs were euthanised 48 h post inoculation for necropsy and histopathological evaluation. RESULTS: While infusion times of 6.66 min, and higher, did not induce liver dysfunction (n = 3), the infusion time of 3.33 min (n = 1) caused alterations in parameters similar to what had been seen in our previous studies, i.e., increasing bilirubin and aspartate aminotransferase, as well as histopathological occurrence of intravascular fibrin split products in the liver. This pig was however euthanised after 30 h, according to humane endpoints. CONCLUSIONS: A usable balance between scientific purpose and animal welfare could not be achieved, and we therefore find it hard to justify further use of this conscious porcine sepsis model. In order to make a model of translational relevance for human sepsis, we suggest that future model versions should use long-term anaesthesia.