In vitro and in vivo evaluation of the efficacies of commercial probiotics and disinfectant against acute hepatopancreatic necrosis disease and luminescent vibriosis in Litopenaeus vannamei.

The bioactivities of two commercially available probiotics and one chemical disinfectant were tested against strains of Vibrio parahaemolyticus (VPAHPND) and V. harveyi. This study aimed to determine shrimp pathogenic Vibrios' in vitro and in vivo sensitivities to commercial probiotics and a chemical disinfectant. The probiotics and disinfectant were tested first in vitro, followed by the in vivo trials. Results showed that upon administration of probiotics either through diet or adding into the tank water, the survivability of shrimp was increased during challenge with VPAHPND and V. harveyi. Also, the disinfectant was tested against the same pathogens and showed positive bactericidal effects at 2500 ppm and 5000 ppm. The present findings suggest that adding probiotics to the rearing water or the shrimp feeds effectively prevents infection by lowering the load of pathogenic bacteria. In comparison, the effectiveness of the disinfectant (PUR) depends on its appropriate concentration and timing of application. It is not only limited to rearing water but is also applicable for decontaminating pond liners, tanks, and other paraphernalia.

Inducing experimental bacterial chondronecrosis with osteomyelitis lameness in broiler chickens using aerosol transmission model.

Lameness disease attributed to bacterial chondronecrosis with osteomyelitis in broilers affects production, animal welfare, and food safety in the poultry industry. The disease is characterized by necrotic degeneration of the rapidly growing femora and tibiae due to bacterial translocation from the respiratory or gastrointestinal tracts into the blood circulation, eventually colonizing the growth plate of the long bones. To investigate the etiology, pathogenesis, and intervention measures for BCO, developing an experimental model that reliably induces BCO lameness is of the utmost importance. In the past, we have employed a wire-flooring model and a litter-flooring model administered with a bacterial challenge to investigate strategies for mitigating BCO. However, multiple issues on labor-intensive barn setup and cleanout efforts for the wire-flooring system and concern of direct pathogenic exposure to the broilers for the litter-flooring models rendered these research models less effective. Thus, we investigated a new approach to induce experimental BCO lameness using an aerosol transmission model employing a group of birds reared on wire-flooring pens as a BCO infection source, and the disease is further disseminated through the airborne transmission to other birds reared on litter flooring in the same housing environment. The effectiveness of the aerosol transmission model in inducing BCO lameness was concluded from 4 independent experiments. The cumulative lameness generated from the BCO source group on the wire floors versus negative control treatments on the litter floors from Experiments 1, 2, 3, and 4 were 84% vs. 69.33%, P = 0.09; 54.55% vs. 60%, P = 0.56; 78% vs. 73.50%, P = 0.64; 81% vs. 74.50%, P = 0.11. Overall, the cumulative lameness generated from the wire floors was successfully transmitted to the birds on litter floors without significant statistical differences (P > 0.05). The effectiveness of the aerosol transmission model for experimentally triggering BCO lameness provides a reliable system for evaluating practical intervention strategies for BCO lameness in broilers.

West Nile Virus Infection in a Pekin Duck ( Anas platyrhynchos domesticus ) Developer Flock.

Developer ducks are ducks being reared for breeding. Like breeder candidate chickens, they are raised with appropriate light and feed programs. A commercial Pekin duck (Anas platyrhynchos domesticus) developer flock experienced an extraordinary, elevated mortality event at 6 wk of age. Weekly mortality rate that week was 162 ducklings out of a flock of 6420 (2.5%). Mortality jumped to 988 (15.4%) ducklings the next week. On first elevated mortality, six dead ducks from that flock were submitted for diagnostic investigation at Michigan State University Veterinary Diagnostic Laboratory. Myocarditis, pale striping or diffuse pallor of the epicardium, was grossly evident in five of the six submitted ducklings. All of the ducklings had hydropericardium, three had ascites, and three had congested meninges. Histology confirmed myocarditis with myocardial necrosis. Cerebrum and brainstem had lymphocytic vasculitis with rare neuronal necrosis in affected areas, as well as Purkinje cells in the cerebellum. West Nile virus was confirmed by PCR the day after submittal and by immunohistochemistry soon thereafter.

Reporte de caso- Infección por el virus del Nilo occidental en una parvada en desarrollo de patos Pekin (Anas platyrhynchos domesticus) reproductores. Los patos reproductores en desarrollo son patos que se crían para la reproducción. Al igual que los pollos candidatos para reproducción, se crían con programas de iluminación y alimentación adecuados. Una parvada comercial en desarrollo de pato Pekín (Anas platyrhynchos domesticus) experimentó un evento de mortalidad elevada y extraordinaria a las seis semanas de edad. La tasa de mortalidad semanal de esa semana fue de 162 patitos de una parvada de 6420 (2.5%). La mortalidad se elevó a 988 (15.4%) patitos la semana siguiente. En el primer aumento de mortalidad, seis patos muertos de esa bandada fueron enviados para una investigación de diagnóstico en el Laboratorio de Diagnóstico Veterinario de la Universidad Estatal de Michigan. La miocarditis, caracterizada por rayas pálidas o palidez difusa del epicardio, fue evidente en cinco de los seis patitos presentados. Todos los patitos mostraron hidropericardio, tres tenían ascitis y tres tenían meninges congestionadas. La histología confirmó miocarditis con necrosis miocárdica. El cerebro y el tronco del encéfalo tenían vasculitis linfocítica con rara necrosis neuronal en las áreas afectadas, así como de las células de Purkinje en el cerebelo. El virus del Nilo Occidental se confirmó mediante PCR el día después de la llegada al laboratorio y mediante inmunohistoquímica poco tiempo después.

Determination of the virulence status of Clostridium perfringens strains using a chicken intestinal ligated loop model is important for understanding the pathogenesis of necrotic.

Necrotic enteritis (NE) is a poultry intestinal disease caused by virulent strains of the bacterium Clostridium perfringens (C. perfringens). This anaerobic bacterium produces a wide range of enzymes and toxins in the gut which leads to NE development. It is generally accepted by the poultry veterinarians that netB-positive C. perfringens strains are virulent and netB-negative strains do not cause NE. However, NE pathogenesis remains unclear as contradictory results have been reported. The use of experimental in vivo models is a valuable tool to understand the pathogenesis of a disease. In this study, a chicken ligated loop model was used to determine the virulence status of 79 C. perfringens strains from various geographical locations, sources, and genotype profiles. According to our model and based on histologic lesion scoring, 9 C. perfringens strains were classified as commensal, 35 as virulent, and 34 as highly virulent. The virulence of only 1 C. perfringens strain could not be classified as its lesion score was variable (from <10 to >15). In general, NE lesions were more severe in intestinal loops inoculated with netB-positive C. perfringens strains than those inoculated with netB-negative strains. The prevalence of netB among strains classified as commensal, virulent, and highly virulent was 56% (5/9), 54%, (19/35), and 59% (20/34). These results suggest that NetB is not required to cause NE lesions and that other factors are also involved. The classification of the virulence status of C. perfringens strains should not be based solely on the presence or absence of this toxin. Therefore, the use of an in vivo model is essential to distinguish commensal from virulent strains of C. perfringens.

Early decrease of ionized calcium and static symmetric dimethylarginine concentration in a model of renal tubular necrosis in corn snakes (Pantherophis guttatus).

OBJECTIVE: To determine if plasma concentrations of symmetric dimethylarginine (SDMA), N-acetyl-beta-d-glucosaminidase (NAG), GGT, ALT, AST, lactate, total calcium, and ionized calcium (iCa) and the calcium:phosphorus ratio are clinically relevant biomarkers to detect early stages of tubular lesions in snakes. ANIMALS: 6 adult corn snakes (Pantherophis guttatus). METHODS: Corn snakes were administered 11 injections of gentamicin at 50 mg/kg, SC, q 24 h in an experimental model of induced tubular necrosis. Plasma biochemistry and blood gas analyses were performed at baseline and after the 3rd and 11th injections. Parameters were compared between time points using a paired Wilcoxon test. In 3 individuals, renal biopsies were collected at baseline before starting injections and at the 3rd and 11th injections, while renal tissue samples were procured after euthanasia in all individuals. RESULTS: Renal proximal and distal tubular necrosis and hepatic steatosis were present in all individuals at necropsy. Compared to baseline, decreased blood concentrations of lactate, ionized calcium, and total calcium and a decreased calcium:phosphorus ratio were noted. A significant decrease of lactate and ionized calcium was observed after 3 days. Conversely, no changes in SDMA, NAG, ALT, AST, GGT, and sodium were detected. CLINICAL RELEVANCE: Ionized calcium and lactate concentrations were the earliest parameters to decrease compared to baseline values in this experimental model. While SDMA is a sensitive indicator of renal disease in mammals, this biomarker did not increase in a model of induced acute tubular necrosis in corn snakes.

Concurrent hepatopathy in dogs with gallbladder mucocele: Prevalence, predictors, and impact on long-term outcome.

BACKGROUND: Information is limited regarding the prevalence and importance of hepatic histologic abnormalities in dogs with gallbladder mucocele (GBM). OBJECTIVES: To (a) report prevalence of hepatic histologic abnormalities in dogs with GBM (b) evaluate for association between hepatic abnormalities and outcome in dogs with GBM (c) evaluate whether neutrophil-to-lymphocyte ratio (NLR) differs in dogs with GBM with and without specific hepatic lesions. ANIMALS: Fifty-two dogs with grossly and histologically confirmed GBM. METHODS: Multicenter, retrospective study of dogs with GBM undergoing cholecystectomy with concurrent liver biopsy. Archived histological sections of gallbladder and liver evaluated by investigators blinded to data. Proportions of dogs with each histologic abnormality alive vs deceased at 1, 3, and 12 months post-cholecystectomy compared. Mann-Whitney U performed to determine if NLR differed in dogs with or without selected lesions. RESULTS: 51/52 (98%, 95% CI [89%, 99%]) dogs with GBM had at least 1 hepatic histologic abnormality. Hepatic fibrosis (37/51; 73%, 95% CI [59%, 83%]), biliary hyperplasia (29/52; 56%, 95% CI [42%, 68%]), and portal inflammation (25/52; 48%, 95% CI [35%, 61%]) were most common. The proportion of dogs alive vs dead differed based on the fibrosis score at 1, 3, and 12 (P ≤ .04) months post-cholecystectomy. Dogs with hepatic necrosis (P = .006) and cholangitis/cholangiohepatitis (P = .02) had higher NLRs compared to dogs without these lesions. CONCLUSIONS AND CLINICAL IMPORTANCE: Histologic abnormalities of the liver are common in dogs with GBM. A higher portal fibrosis score might be associated with shortened long-term survival after cholecystectomy for dogs with GBM. An increase in NLR might predict hepatic necrosis and cholangitis/cholangiohepatitis in dogs with GBM.

Outbreak of epizootic hemorrhagic disease in captive reindeer (Rangifer tarandus).

In September 2020, an outbreak of epizootic hemorrhagic disease occurred in captive reindeer (Rangifer tarandus) and was associated with neurological signs and mortality. Four reindeer died or were euthanized after acute illness over a 12-day period. Affected reindeer displayed abnormal behavior, neurologic signs, lethargy, and/or lameness. The most consistent gross finding was dark red streaks throughout the adrenal gland cortices (4/4). One animal had acute hemorrhage involving the subcutis and skeletal muscles over the ventrolateral body wall and back, and abomasal serosa. Histologically, the most common lesions were adrenal gland cortical hemorrhage (4/4) with necrosis (3/4) and lymphoplasmacytic meningoencephalitis with gliosis, glial nodules, satellitosis, and nonsuppurative perivascular cuffing (4/4). The brain lesions were most frequent in the gray matter of the cerebrum, hippocampus, and thalamus but also involved the cerebellum and brainstem. Epizootic hemorrhagic disease virus serotype 6 was detected through PCR and sequencing of the spleen in all cases.

Identification, pathological, and genomic characterization of novel goose reovirus associated with liver necrosis in geese, China.

Since 2021, a novel strain of goose reovirus (GRV) has emerged within the goose farming industry in Guangdong province, China. This particular viral variant is distinguished by the presence of white necrotic foci primarily localized in the liver and spleen, leading to substantial economic losses for the poultry industry. However, the etiology, prevalence and genomic characteristics of the causative agent have not been thoroughly investigated. In this study, we conducted an epidemiological inquiry employing suspected GRV samples collected from May 2021 to September 2022. The macroscopic pathological and histopathological lesions associated with GRV-infected clinical specimens were examined. Moreover, we successfully isolated the GRV strain and elucidated the complete genome sequence of the isolate GD21/88. Through phylogenetic and recombination analysis, we unveiled that the GRV strains represent a novel variant resulting from multiple reassortment events. Specifically, the µNS, λC, and σNS genes of GRV were found to have originated from chicken reovirus, while the σA gene of GRV exhibited a higher degree of similarity with a novel duck reovirus. The remaining genes of GRV were traced back to Muscovy duck reovirus. Collectively, our findings underscore the significance of GRV as a pathogenic agent impacting the goose farming industry. The insights gleaned from this study contribute to a more comprehensive understanding of the epidemiology of GRV in Southern China and shed light on the genetic reassortment events exhibited by the virus.

Effect of temperature on the protective efficacy of a live attenuated vaccine against herpesviral haematopoietic necrosis in goldfish.

The live attenuated vaccine P7-P8 strain against herpesviral haematopoietic necrosis, which is caused by cyprinid herpesvirus 2 (CyHV-2), exhibits high protective efficacy in goldfish at 25°C, the predominant temperature for this disease; however, the effect of water temperature during the vaccination period on efficacy has not been determined. In this study, an in vitro experiment revealed that the vaccine strain grew between 15 and 30°C in the goldfish cell line RyuF-2. Subsequent in vivo efficacy tests were conducted with vaccination temperatures ranging from 15 to 30°C. During the vaccination period, organs were sampled to determine the vaccine growth dynamics. Blood plasma was collected to assess anti-CyHV-2 antibody titres. The protective efficacy of the vaccine at 15, 20, 25, and 30°C after subsequent virulent CyHV-2 challenge resulted in a relative percentage survival of 73.3%, 77.8%, 100%, and 77.8%, respectively, which indicated that the vaccine is effective over this temperature range. The vaccine virus load in the spleen was lowest at 15°C (103.7 DNA copies/mg) and highest at 25°C (106.5 DNA copies/mg). This indicates that the vaccine virus load over 104 DNA copies/mg may elicit sufficient acquired immunity. No significant differences in antibody titre were observed between groups, which suggests that cell-mediated immunity can be fundamentally involved in protection.

Effects of temperature on viral load, inclusion body formation, and host response in Pacific Herring with viral erythrocytic necrosis (VEN).

OBJECTIVE: The primary objective of this study was to determine the effects of temperature on viral erythrocytic necrosis (VEN) progression under controlled conditions. Secondarily, this study was intended to evaluate the combined effects of temperature and VEN on the Pacific Herring Clupea palasii transcriptome. METHODS: The effects of temperature on VEN progression were assessed by waterborne exposure of laboratory-reared, specific-pathogen-free Pacific Herring to tissues homogenates containing erythrocytic necrosis virus (ENV) at 6.9, 9.0, or 13.5°C. RESULT: Exposure of Pacific Herring to ENV resulted in the establishment of infections characterized by high infection prevalence (89%; 40/45) and mean viral loads (5.5 log10 [gene copies/µg genomic DNA]) in kidney tissues at 44 days postexposure. Mean viral loads were significantly higher in fish from the ambient (mean = 9.0°C) and warm (mean = 13.5°C) treatments (6.1-6.2 log10 [gene copies/total genomic DNA]) than in fish from the cool (mean = 6.9°C) treatment (4.3 log10 [gene copies/µg genomic DNA]). Similarly, the peak proportion of diseased fish was directly related to temperature, with cytoplasmic inclusion bodies detected in 21% of fish from the cool treatment, 52% of fish from the ambient treatment, and 60% of fish from the warm treatment. The mean VEN load in each fish (enumerated as the percentage of erythrocytes with cytoplasmic inclusions) at 44 days postexposure increased with temperature from 15% in the cool treatment to 36% in the ambient treatment and 32% in the warm treatment. Transcriptional analysis indicated that the number of differentially expressed genes among ENV-exposed Pacific Herring increased with temperature, time postexposure, and viral load. Correlation network analysis of transcriptomic data showed robust activation of interferon and viral immune responses in the hepatic tissue of infected individuals independent of other experimental variables. CONCLUSION: Results from this controlled laboratory study, combined with previous observations of natural epizootics in wild populations, support the conclusion that temperature is an important disease cofactor for VEN in Pacific Herring.

Analysis of histopathology and changes of major cytokines in the lesions caused by Mycoplasma ovipneumoniae infection.

BACKGROUND: Mycoplasma ovipneumoniae (M. ovipneumoniae) is one of the main pathogens of sheep pneumonia, causing a series of clinical symptoms, such as depression, anorexia, hyperthermia, cough, dyspnea, and tract secretions. In recent years, the prevalence of M. ovipneumoniae pneumonia has become increasingly serious in sheep farms in Ningxia, China, leading to the death of sheep, and causing significant economic losses. In this study, the pathological organs infected by M. ovipneumoniae were collected to observe histopathological change, to determine the tissue localization of M. ovipneumoniae, and to analyze the cytokine changes, which lays a basis for the diagnosis and pathogenesis of M. ovipneumoniae disease. RESULTS: In this study, M. ovipneumoniae was detected in 97 of 105 samples collected from 13 large-scale sheep farms for nucleic acid by PCR. One representative isolate per farm was isolated from 13 farms. The lesions caused by M. ovipneumoniae were mainly in the trachea, bronchus, and lung, including necrosis of tracheal mucosal epithelial cells, disintegration of some epithelial cells, edema of mucosal lamina propria, with inflammatory cell infiltration, cytoplasmic vacuolization of epithelial cells of bronchial mucosa, massive infiltration of inflammatory cells in the alveolar space of lung, necrosis and hyperplasia of alveolar epithelial cells. Immunohistochemical analysis showed that the proportion of M. ovipneumoniae positive area in the lung was the largest, followed by that in the bronchus and trachea. Compared to healthy animals, diseased animals exhibited up-regulated gene expression levels of IL-1ß, IL-6, and NF-κB in the trachea, bronchus, and lungs. In contrast, the expression of IL-10, IL-12, and IFN-γ was primarily limited to the trachea and bronchus. The expression of IL-1ß showed differential patterns across different lung regions, with variations observed among lung lobes. Additionally, other cytokines consistently showed significant up-regulation specifically in the bronchus. CONCLUSIONS: M. ovipneumoniae is primarily found in the lungs of infected individuals. NF-κB, an essential transcription factor, is involved in the regulation of IL-1ß transcription. IL-12 may enhance the cytotoxic function of natural killer cells during M. ovipneumoniae infection. Those findings demonstrate the distinct expression profiles of cytokines in various anatomical sites throughout disease progression, suggesting the potential role of bronchial tissue as a major site of immune response.

Native ApxIIA secreted by Actinobacillus pleuropneumoniae induces apoptosis in porcine alveolar macrophages dependent on concentration and acylation.

Actinobacillus pleuropneumoniae is an important swine respiratory pathogen causing substantial economic losses to the global pig industry. The Apx toxins of A. pleuropneumoniae belong to the RTX toxin family and are major virulence factors. In addition to hemolysis and/or cytotoxicity via pore-forming activity, RTX toxins, such as ApxIA of A. pleuropneumoniae, have been reported to cause other effects on target cells, e.g., apoptosis. A. pleuropneumoniae ApxIIA is expressed by most serotypes and has moderate hemolytic and cytotoxic activities. In this study, porcine alveolar macrophages (3D4/21) were stimulated with different concentrations of purified native ApxIIA from the serotype 7 strain AP76 which only secretes ApxIIA. By observation of nuclear condensation via fluorescent staining and detection of apoptosis and necrosis by flow cytometry, it was found that high and low concentrations of native ApxIIA mainly caused necrosis or apoptosis of 3D4/21 cells, respectively. ApxIIA purified from an AP76 mutant with a deleted acetyltransferase gene (apxIIC) did not induce necrosis nor apoptosis. Western blot analysis using specific antibodies showed that a cleaved caspase 3 and activated capase 9 was detected after treatment of cells with a low concentration of native ApxIIA, while general or specific inhibitors of caspase 3, 8, 9 blocked these effects. ApxIIA-induced apoptosis of macrophages may be a mechanism of A. pleuropneumoniae to escape host immune clearance.

Therapeutic trial of fluvastatin in a cell line xenograft model of canine mammary gland cancer.

The Hippo signalling pathway is involved in breast cancer and canine mammary tumour (CMT). This study sought to evaluate the efficacy of fluvastatin on the Hippo pathway and its main effectors, YAP and TAZ, in vivo in a murine CMT cell line xenograft model. On treatment day 1, mice were divided into four groups: vehicle, fluvastatin, doxorubicin or a combination therapy. Tumour volumes were monitored with callipers and tissues harvested on day 28th of treatment. Histopathological examination of tumour tissues and major organs was performed as well as tumour evaluation of necrosis, apoptosis, cellular proliferation, expression of YAP, TAZ and the mRNA levels of four of their target genes (CTGF, CYR61, ANKRD1 and RHAMM2). Results showed a statistically significant variation in tumour volumes only for the combination therapy and final tumour weight only for the doxorubicin group compared to control. There was no significant difference in tumour necrosis, expression of CC3, ki-67, YAP and TAZ measured by immunohistochemistry and in the mRNA levels of the target genes. Unexpectedly, lung metastases were found in the control group (9) and not in the fluvastatin treated group (7). In addition, mass spectrometry-based quantification of fluvastatin reveals concentrations comparable to levels reported to exert therapeutic effects. This study shows that fluvastatin tumours concentration reached therapeutic levels without having an effect on the hippo pathway or various tumour parameters. Interestingly, only the control group had lung metastases. This study is the first to explore the repurposing of statins for cancer treatment in veterinary medicine.

Porcine ear necrosis: characterization of lesions and associated pathogens.

Porcine ear necrosis (PEN) is characterized by ulcerative lesions of the ear auricle. To investigate that problem, three farms with PEN in nursery pigs were included, and the study aim was to characterize PEN and the potential role of pathogens and mycotoxins. Within each farm, one batch of weaned piglets was included and the prevalence and severity of PEN were monitored for 6-7 weeks. Within each batch, 30 PEN-affected/non-affected animals were randomly selected. Blood samples were taken from these animals, to assess the systemic presence of pathogens and mycotoxins, as well as punch biopsies from the ear auricle for histopathological examination. From 10 animals, scrapings and swabs from the lesions were subjected to nanopore metagenomic sequencing and bacteriological cultivation, respectively. In all three farms, lesions appeared within 3-4 weeks post-weaning. The prevalence at the end of the nursery was 33%, 24%, and 46% for farms A, B, and C, respectively. Most affected pigs had mild to moderate lesions. Blood samples revealed low to very low levels of pathogens and mycotoxins. Different bacteria such as Staphylococcus, Streptococcus, Fusobacterium, Mycoplasma, and Clostridium species were identified by sequencing in the scrapings. The first two pathogens were also most often identified in bacterial cultures. Mycoplasma hyopharyngis was only found in PEN-affected pigs. Histopathological changes were primarily observed in the outer layer of the epidermis. The results suggest that PEN lesions develop by damage to the outer part of the skin e.g. by ear suckling or biting, followed by multiplication of opportunistic pathogens.

Short-term outcome and complications following cutaneous reconstruction using cranial superficial epigastric axial pattern flaps in dogs: six cases (2008-2022).

OBJECTIVES: To investigate short-term outcome and complications following the use of the cranial superficial epigastric axial pattern flap to reconstruct cutaneous defects in dogs. MATERIALS AND METHODS: Medical records from dogs undergoing reconstructive surgery between 2008 and 2022 by means of cranial superficial epigastric axial pattern flap were reviewed. Data on signalment, reason for reconstruction, defect size, flap healing, post-operative complications and need for revision surgery were collected. RESULTS: Six dogs were included in the study. Indications for reconstruction included neoplasia (4/6), skin necrosis due to vehicular trauma (1/6) and dog bite (1/6). Postoperative complications occurred in 50% of the patients and included seroma (1/6), bruising (2/6) and necrosis of the distal portion of the flap (2/6), with two dogs developing concurrent complications. One dog required open wound management and additional surgery. Overall outcome was scored excellent in three, good in two, and fair in one dog. CLINICAL SIGNIFICANCE: Despite the relatively high complication rate, most of the complications were deemed minor and could be managed conservatively. Eventually, all wounds healed completely and only one flap required revision surgery.

Standardization of canine meningioma grading: Validation of new guidelines for reproducible histopathologic criteria.

Canine meningiomas are currently graded using the human grading system. Recently published guidelines have adapted the human grading system for use in dogs. The goal of this study was to validate the new guidelines for canine meningiomas. To evaluate the inter-observer agreement, 5 veterinary surgical pathologists graded 158 canine meningiomas following the human grading system alone or with the new guidelines. The inter-observer agreement for histologic grade and each of the grading criteria (mitotic grade, invasion, spontaneous necrosis, macronucleoli, small cells, hypercellularity, pattern loss and anaplasia) was evaluated using the Fleiss kappa index. The diagnostic accuracy (sensitivity and specificity) was assessed by comparing the diagnoses obtained with the 2 grading systems with a consensus grade (considered the reference classification). The consensus histologic grade was obtained by agreement between 4 experienced veterinary neuropathologists following the guidelines. Compared with the human grading alone, the canine-specific guidelines increased the inter-observer agreement for: histologic grade (κ = 0.52); invasion (κ = 0.67); necrosis (κ = 0.62); small cells (κ = 0.36); pattern loss (κ = 0.49) and anaplasia (κ = 0.55). Mitotic grade agreement remained substantial (κ = 0.63). The guidelines improved the sensitivity in identifying grade 1 (95.6%) and the specificity in identifying grade 2 (96.2%) meningiomas. In conclusion, the new grading guidelines for canine meningiomas are associated with an overall improvement in the inter-observer agreement and higher diagnostic accuracy in diagnosing grade 1 and grade 2 meningiomas.

Glomerulocystic kidney in two red piranhas Pygocentrus nattereri.

Glomerulocystic kidney (GCK) is defined by a dilatation of the Bowman's space (greater than 2 times the normal size) of more than 5% of all glomeruli. Although GCK has been occasionally documented in dogs, cats, and humans with renal failure, in fish, reports of spontaneous GCK are rare. For the present study, 2 captive adult red piranhas Pygocentrus nattereri from a closed population were submitted for post-mortem examination. Clinical history included lethargy, inappetence, dyspnea, and altered buoyancy. Macroscopically, the fish displayed coelomic distension and ascites. The kidneys were markedly enlarged and dark yellow. Histologically, Bowman's space was noticeably dilated, occasionally with atrophic glomerular tufts. Degeneration and necrosis of the tubular epithelium, infiltration, and nephrocalcinosis were also present. To the authors' knowledge, this present study is the first report of spontaneously occurring GCK in red piranhas and freshwater fish in general. Despite being rare, GCK is a condition with the potential to impair the health of fish and mammals, and further studies are needed to shed new light on this condition.

Laser Capture Microdissection, Culture Analysis, and Bacterial Sequencing to Evaluate the Microbiota of Focal Duodenal Necrosis in Egg Layers.

Focal duodenal necrosis (FDN) is a common intestinal disease of table egg layers. In this research we aimed to identify the bacteria commonly found in FDN lesions as seen with histopathological analysis. Fifty-nine ethanol-fixed duodenum samples were collected from egg layers on eight FDN-affected farms, and 42 samples had typical FDN lesions. Excision of bacteria-containing lesions using laser capture microdissection was performed, followed by 16S rRNA gene sequencing of extracted DNA for bacterial identification. Bacterial sequencing analysis revealed no consistent bacterial species identified from samples with FDN. However, analysis of the relative phylum abundance revealed differences in the duodenal microbiota between layers with FDN and healthy birds. There were differences in the abundance of Proteobacteria, Firmicutes, and Actinobacteria between FDN-positive and FDN-negative control samples compatible with intestinal dysbiosis. In addition, 10 duodenal samples with FDN lesions were collected for bacteriological analysis, yielding 47 colonies on tryptone soy agar, MacConkey agar, and blood agar plates. Using 16S rRNA gene PCR, 39/47 (53.8%) colonies were identified as Escherichia coli. PCR for E. coli virulence genes identified 21/39 (53.8%) E. coli isolates as avian pathogenic E. coli-like. PCR analysis for 19 E. coli virulence genes associated with intestinal disease strains including inflammatory bowel disease found 11/39 (28.2%) isolates containing more than 10 of these virulence genes. In conclusion, FDN appears to be a multifactorial inflammatory intestinal disease associated with intestinal dysbiosis, and Gram-negative bacteria including E. coli may contribute to the pathogenesis of this disease.

Microdisección por captura láser, análisis de cultivos y secuenciación bacteriana para evaluar la microbiota de la necrosis duodenal focal en aves de postura de huevo comercial. La necrosis duodenal focal (FDN) es una enfermedad intestinal común en las gallinas de postura de huevo comercial. En esta investigación, el objetivo fue identificar las bacterias que se encuentran comúnmente en las lesiones provocadas por la necrosis duodenal focal tal como se aprecian con el análisis histopatológico. Se recolectaron 59 muestras de duodeno fijadas con etanol de gallinas de postura de ocho granjas afectadas por necrosis duodenal focal, y 42 muestras tenían lesiones típicas de dicha enfermedad. Se realizó la escisión de las lesiones que contenían bacterias mediante microdisección por captura láser, seguida de la secuenciación del gene 16S rRNA del ADN extraído para la identificación bacteriana. El análisis de secuenciación bacteriana no reveló especies bacterianas consistentes identificadas a partir de muestras con necrosis duodenal focal. Sin embargo, el análisis de la abundancia relativa del phylum reveló diferencias en el microbiota duodenal entre gallinas de postura con necrosis duodenal focal y aves sanas. Hubo diferencias en la abundancia de Proteobacteria, Firmicutes y Actinobacteria entre las muestras controles positivas y negativas para la necrosis duodenal focal compatibles con disbiosis intestinal. Además, se recolectaron 10 muestras duodenales con lesiones de la necrosis duodenal focal para análisis bacteriológico, lo que produjo 47 colonias en placas de agar triptona soya, agar MacConkey y agar sangre. Utilizando un método de PCR para el gene 16S rRNA, 39/47 (53.8 %) colonias se identificaron como Escherichia coli. El método de PCR para genes de virulencia de E. coli identificó 21/39 (53.8 %) aislados de E. coli como similares a E. coli patogénica aviar. El análisis de PCR para 19 genes de virulencia de E. coli asociados con cepas que provocan enfermedades intestinales, incluida la enfermedad inflamatoria intestinal, detectó 11/39 (28.2 %) aislados que contenían más de 10 de estos genes de virulencia. En conclusión, la necrosis duodenal focal parece ser una enfermedad intestinal inflamatoria multifactorial asociada con disbiosis intestinal, y las bacterias Gramnegativas, incluida E. coli, pueden contribuir a la patogenia de esta enfermedad.

Cellular Immune Responses in Lymphoid Tissues of Broiler Chickens Experimentally Infected with Necrotic Enteritis-Producing Clostridium perfringens Strains.

Host cellular responses against Clostridium perfringens (CP), the causative agent of necrotic enteritis (NE) in chickens, are poorly understood. In the present study, we first tested the NE-producing ability of seven netB+ CP strains (CP5, CP18, CP26, CP64, CP67, CP68, and NCNE-1), using an experimental infection model of broiler chickens. Evaluation of intestinal gross lesions showed that all the strains, except CP5, were able to produce NE, while CP26 and CP64 strains produced relatively more severe lesions when compared with other groups. Next, cellular responses in the cecal tonsil (CT), bursa of Fabricius, and spleen were evaluated in chickens infected with strains representing variation in the level of virulence, namely, avirulent CP5, virulent CP18, and a relatively more virulent CP26 strain. Immunophenotyping analysis showed that CT or splenic macrophage frequencies were significantly higher in CP18- and CP26-infected chickens compared with uninfected controls, while the frequencies of γδ T-cells and B-cells in the CT of CP26-infected chickens were significantly higher than those in the uninfected, CP5- or CP18-infected groups. The T-cell analysis showed that chickens infected with CP18 and CP26 had a significantly higher number of splenic CD4+ and CD8+ T-cells expressing CD44 and CD28 activation molecules, while CP26-infected chickens also had significantly increased CT frequency of these activated CD4+ and CD8+ T-cells when compared with uninfected or CP5-infected groups. Collectively, our findings suggested that cellular responses, including activation of T-cells, are selectively induced against virulent CP strains and that the NE-producing characteristics of this pathogen may influence the outcome of immunity to NE.

Respuestas inmunes celulares en tejidos linfoides de pollos de engorde infectados experimentalmente con cepas de Clostridium perfringens productoras de enteritis necrótica. Las respuestas celulares del huésped contra Clostridium perfringens (CP), el agente causante de la enteritis necrótica (NE) en pollos, son poco conocidas. En el presente estudio, primero se analizó la capacidad de producción de enteritis necrótica de siete cepas de C. perfringens netB+ (CP5, CP18, CP26, CP64, CP67, CP68 y NCNE-1), utilizando un modelo de infección experimental de pollos de engorde. La evaluación de las lesiones macroscópicas intestinales mostró que todas las cepas, excepto CP5, podían producir enteritis necrótica, mientras que las cepas CP26 y CP64 produjeron lesiones relativamente más severas en comparación con los otros grupos. Posteriormente, se evaluaron las respuestas celulares en las tonsilas cecales (CT), la bolsa de Fabricio y en el bazo de pollos infectados con cepas que representan variaciones en el nivel de virulencia, por ejemplo las cepas CP5 avirulenta, CP18 virulenta y la cepa CP26 relativamente más virulenta. El análisis de inmunofenotipado mostró que las frecuencias de los macrófagos esplénicos o de las tonsilas cecales fueron significativamente más altas en los pollos infectados con las cepas CP18 y CP26 en comparación con los controles no infectados, mientras que las frecuencias de células T γd y células B en tonsilas cecales de los pollos infectados con la cepa CP26 fueron significativamente más altas que las de los pollos de los grupos no infectados, o infectados con las cepas CP5 o CP18. El análisis de células T mostró que los pollos infectados con las cepas CP18 y CP26 tenían un número significativamente mayor de células esplénicas T CD4+ y CD8+ que expresaban moléculas de activación CD44 y CD28, mientras que los pollos infectados con la cepa CP26 también tenían una frecuencia significativamente mayor en las tonsilas cecales de estas células T CD4+ y CD8+ activadas en comparación con grupos no infectados o infectados con la cepa CP5. En conjunto, estos hallazgos sugirieron que las respuestas celulares, incluida la activación de las células T, se inducen selectivamente contra las cepas virulentas de C. perfringens y que las características productoras de enteritis necrótica de este patógeno pueden influir en el resultado de la inmunidad contra la enteritis necrótica.

Gastrointestinal Impaction and Necrotic Enteritis in a Backyard Chicken.

The carcass of a 4-mo-old, female, mixed-breed backyard chicken was submitted for postmortem evaluation and diagnostic workup. The bird was previously presented to a veterinary clinic because of chronic weight loss and loose stool, and was euthanized before submission to the California Animal Health and Food Safety, Turlock lab. On gross examination, the proventriculus, gizzard, and duodenum were markedly distended and impacted with a mixture of fibrous plant material, cereal grain, and litter material. The koilin layer of the gizzard was eroded. There were multifocal to coalescing, 0.2-1-cm diameter white nodules on the serosal surface of the duodenal loop and lesions extended into the distal jejunum. The duodenum had multifocal, transmural, umbilicated, and ulcerated mucosal lesions, which were covered with a white pseudomembrane. Microscopically, there was segmental, transmural necrosis of the intestinal wall with diffuse sloughing of villi epithelium and accumulation of fibrino-hemorrhagic exudate with numerous bacterial colonies in the lumen. The gross and microscopic findings were indicative of gastrointestinal impaction and necrotic enteritis. Proliferation of Clostridium perfringens within the intestine was demonstrated by anaerobic bacterial culture, intestinal gram stains, and immunohistochemistry. The C. perfringens isolate was type F (encoding the gene for alpha toxin -cpa- and for enterotoxin -cpe) by PCR toxinotyping. Overgrowth of C. perfringens was likely exacerbated by the rough fibrous forage and highly fermentable grain diet. To our knowledge, gastrointestinal impaction concurrent with necrotic enteritis has not been described in backyard chickens. In addition, to our knowledge, C. perfringens type F has not been associated with necrotic enteritis in chickens.

Reporte de caso- Impactación gastrointestinal y enteritis necrótica en un pollo de traspatio. Para realizar una evaluación post mortem y estudios de diagnóstico, se recibió un pollo de traspatio muerto, hembra, de raza mixta y de cuatro meses de edad. El ave fue presentada previamente a una clínica veterinaria debido a la pérdida de peso crónica y heces acuosas y fue sacrificada antes de ser enviada al Laboratorio de Salud Animal y Seguridad Alimentaria de California con sede en Turlock. En el examen macroscópico, el proventrículo, la molleja y el duodeno estaban marcadamente distendidos e impactados con una mezcla de material vegetal fibroso, granos de cereal y material de hojarasca. La capa de koilin de la molleja estaba erosionada. Había nódulos blancos de 0.2 a 1 cm de diámetro, multifocales a coalescentes, en la superficie serosa del asa duodenal y las lesiones se extendían hacia el yeyuno distal. El duodeno presentaba lesiones mucosas multifocales, transmurales, umbilicadas y ulceradas, las cuales estaban cubiertas por una pseudomembrana blanca. Microscópicamente, había necrosis transmural segmentaria de la pared intestinal con desprendimiento difuso del epitelio de las vellosidades y acumulación de exudado fibrino-hemorrágico con numerosas colonias bacterianas en el lumen. Los hallazgos macroscópicos y microscópicos fueron indicativos de impactación gastrointestinal y enteritis necrótica. La proliferación de Clostridium perfringens dentro del intestino se demostró mediante cultivo de bacterias anaerobias, tinciones de Gram intestinales e inmunohistoquímica. El aislado de C. perfringens fue tipo F (que codifica el gene de la toxina alfa ­cpa- y de la enterotoxina ­cpe) por tipificación de toxina mediante PCR. El crecimiento excesivo de C. perfringens probablemente fue exacerbado por el forraje áspero y fibroso y la dieta de granos altamente fermentables. Hasta donde se conoce, la impactación gastrointestinal concurrente con enteritis necrótica no se ha descrito en pollos de traspatio. Además, hasta donde se sabe, C. perfringens tipo F no se ha asociado con enteritis necrótica en pollos.