Histomorphometric lung density evaluation of Immulina treatment using a murine influenza pneumonia model.

Histomorphometric lung density measurements were used to evaluate the effects of Immulina on mouse pneumonia. Mice were intra-nasally exposed to H1N1 influenza virus at a dose of 5 × 104 PFU/50 µL/mouse. Lung density was measured using the NIH ImageJ software program. Density values were compared to semiquantitative pneumonia severity scores. Lung photomicrographs were evaluated at 25-×, 40-× and 400-× magnification. The study included viral inoculated controls (IC) and non-inoculated controls (NC) and mice either treated or not treated with Immulina. Three doses of Immulina were included (25, 50 or 100 mg/kg) and administered using 3 protocols: prophylactic treatment (P), prodromal treatment (PD) and therapeutic treatment (TH) (note that in most of the evaluations of the data for the three treatment protocols were combined). Groups of mice were evaluated on days 3, 5, 7, 10 and 15 following exposure. The occurrence of "digital pneumonia" (DP) was defined as a density measurement above the 95% confidence limit of the corresponding NC values. A significant reduction in the occurrence of DP with Immulina treatment at the higher doses compared to IC was seen as early as day 3 and persisted up to day 15. There were also statistically significant dose-variable reductions in lung density in response to Immulina. The study suggests early administration of Immulina (P or PD protocols) may enhance resistance against influenza-induced viral pneumonia. A moderate correlation between pneumonia severity scores and lung density was observed for the 25-× and 40-× images (R = 0.56 and 0.53 respectively), and a strong correlation (R = 0.68) for 400-× images.

Disarticulation-Associated Femoral Head Separation in Clinically Normal Broilers: Histologic Documentation of Underlying and Predisposing Cartilage Abnormalities.

Routine and quantitative histologic studies on femoral head separation (FHS) associated with coxofemoral joint disarticulation at necropsy were conducted on 125 femoral heads collected from 21- to 50-day-old clinically normal broilers. The study compared groups demonstrating grossly detached femoral heads (DFHs) with those having attached femoral heads (AFHs). Marked microscopic lesions compatible with osteochondrosis (OCD) consistently occurred along the separation surface in the DFH population. The histologic changes consisted of cartilage degeneration and necrosis sometimes forming small clefts or microfractures. Hemorrhage and less frequent inflammatory cells were often present along the separation surfaces. Small foci of OCD in the femur occurred in the AFH group with lesser frequency and severity. The histologic changes were mainly found within the proximal proliferative zone of the physis near the epiphyseal junction. Histomorphometry disclosed significant quantitative reductions in chondrocyte density with increased pyknosis occurring adjacent to the separation site and to a lesser extent in deeper regions of the growth plate for the DFH compared with AFH. Measurements made along the separation surface of the percentage length occupied by osteochondrotic defects and actual separated cartilage disclosed significant differences between evaluation groups. However, determinations of vascular canal areas present within two or more regions of the growth plate revealed a slight and significant increased area for DFH compared with AFH. Severity scores for the occurrence of microthrombi within the growth plate showed no difference between the groups. The pathogenesis of FHS in broilers is related to defective cartilage production or degeneration resulting in increased fragility. This contrasts with the proposed pathogenesis of OCD in mammals, which involves ischemic necrosis due to underlying vascular defects. The results for the FHS-disarticulation model also differ from those reported for glucorticoid-induced femoral head necrosis in broilers. The FHS-associated lesions occurred without histologic evidence of bacterial chondritis or osteomyelitis.

Separación de la cabeza femoral asociada a la desarticulación en pollos de engorde clínicamente normales: documentación histológica de anomalías del cartílago subyacentes y predisponentes. Estudios histológicos de rutina y cuantitativos sobre la separación de la cabeza femoral (FHS) asociados con la desarticulación de la articulación coxofemoral durante la necropsia se realizaron en 125 cabezas femorales recolectadas de pollos de engorde clínicamente normales de 21 a 50 días de edad. El estudio comparó los grupos que demostraron cabezas femorales separadas (DFH) con los que tenían cabezas femorales unidas (AFH). Lesiones microscópicas marcadas compatibles con osteocondrosis (OCD) ocurrieron consistentemente a lo largo de la superficie de separación en la población de aves con cabezas femorales separadas. Los cambios histológicos consistieron en degeneración del cartílago y necrosis, formando a veces pequeñas hendiduras o microfracturas. La hemorragia y la presencia menos frecuente de células inflamatorias estaban presentes a lo largo de las superficies de separación. Se produjeron con menor frecuencia y gravedad, focos pequeños de osteocondrosis en el fémur en el grupo de aves con cabezas femorales unidas. Los cambios histológicos se encontraron principalmente en la zona proliferativa proximal de la fisis cerca de la unión epifisaria. La histomorfometría reveló reducciones cuantitativas significativas en la densidad de condrocitos con un aumento de la picnosis que ocurrió adyacente al sitio de separación y en menor medida en regiones más profundas de la placa de crecimiento en las aves con cabezas femorales separadas en comparación con las aves con cabezas femorales unidas. Las mediciones realizadas a lo largo de la superficie de separación del porcentaje de longitud ocupada por defectos osteocondróticos y con cartílago separado revelaron diferencias significativas entre los grupos de evaluación. Sin embargo, las determinaciones de las áreas del canal vascular presentes dentro de dos o más regiones de la placa de crecimiento revelaron un aumento leve y significativo del área para las aves con cabezas femorales separadas en comparación con las aves con cabezas femorales unidas. Las puntuaciones de severidad para la aparición de microtrombos dentro de la placa de crecimiento no mostraron diferencias entre los grupos. La patogenia de la separación de la cabeza femoral en pollos de engorde se relaciona con la producción o degeneración de cartílago defectuoso que resulta en un aumento de la fragilidad. Esto contrasta con la patogénesis propuesta para la osteocondrosis en mamíferos, que involucra necrosis isquémica debido a defectos vasculares subyacentes. Los resultados para el modelo de desarticulación-separación de la cabeza femoral también difieren de los reportados para la necrosis de la cabeza femoral inducida por glucorticoides en pollos de engorde. Las lesiones asociadas con la separación de la cabeza femoral ocurrieron sin evidencia histológica de condritis bacteriana u osteomielitis.

Morphometric Documentation of a High Prevalence of Left Ventricular Dilated Cardiomyopathy in Both Clinically Normal and Cyanotic Mature Commercial Broiler Breeder Roosters with Comparisons to Market-Age Broilers.

Previous studies documented the common occurrence of transitory cyanosis and echocardiographic aortic insufficiency in mature commercial broiler breeder roosters. During further investigations, we observed a high prevalence of hearts exhibiting extensive dilation of the left ventricle chamber compatible with dilated left ventricular cardiomyopathy present in both cyanotic and normal subpopulations. We conducted quantitative studies focused on documentation of cardiac ventricle parameters by using simple gross morphometric methods performed on formalin-fixed hearts obtained from both clinically normal roosters and those exhibiting variable transitory cyanosis, echocardiographic aortic insufficiency, or both. A high prevalence of often dramatic left ventricular dilation reflected in enlarged left ventricular chamber areas and elevated left ventricle-to-total ventricle area ratios was morphometrically documented. However, no statistically significant differences in the occurrence of ventricular abnormalities were observed between normal and cyanotic roosters. Age-associated changes were also demonstrated by comparative morphometric studies on hearts from normal market-age broilers (average age of 7 wk) and those of mature roosters (average age of 42 wk). Elevation in both left and right ventricular weight-to-total heart weight ratios dramatically increased with aging. In addition, values (average ± SD) for the left ventricle chamber area-to-total ventricle area ratios increased from 3.2 ± 2.0% in broilers up to 10.0 ± 8.8% in roosters. None of the normal broilers studied demonstrated left ventricular volume ratios above 10%, whereas 33% of the roosters had left ventricular volume ratios above 10%, including 13% with ratios of 20% or higher. However, the left ventricle wall area-to-body weight ratios were much closer for the two age groups (0.85 ± 0.18 cm(2)/kg in broilers and 0.79 ± 0.13 cm(2)/kg in roosters). Also, the standard right ventricle-to-total ventricle weight ratio (RV/TV) went from 0.18 ± 0.04 in broilers to 0.25 ± 0.12 in roosters, whereas the left ventricle-to-total ventricle weight ratios were similar for the two age groups (0.74 ± 0.12 and 0.75 ± 0.08 in broilers and roosters, respectively). Our results for RV/TV in normal broilers were similar to the reported values for normal market-age broilers. In contrast, 36% of the roosters had RV/TV above values reported for broilers considered reflective of right ventricular hypertrophy due to pulmonary hypertension, whereas 4% had values above the reported threshold for broilers dying with ascites (ratios greater than 0.0249 and 0.299, respectively). However, ascites was not observed for any of the roosters. Although essentially all cardiac morphometric parameters demonstrated statistically significant correlations with the age-class group comparisons, significance could not be documented for comparisons between cardiomorphometrics and the subjective occurrences of transitory cyanosis or echocardiographic aortic insufficiency.

Failed detection of Bovine viral diarrhea virus 2 subgenotype a (BVDV-2a) by direct fluorescent antibody test on tissue samples due to reduced reactivity of field isolates to raw anti-BVDV antibody.

Bovine viral diarrhea virus 1 (BVDV-1) is associated with mild or subclinical infections, whereas BVDV-2 is frequently implicated in outbreaks of severe thrombocytopenia and acute fatal disease. In the present study, the carcass of a beef breed cow and tissue samples of a beef calf were received for laboratory diagnosis. Both animals exhibited severe clinical signs compatible with thrombocytopenia or hemorrhagic syndrome. Direct fluorescent antibody test (DFAT) failed to detect BVDV antigen in the tissue specimens of both cases. However, immunohistochemistry (IHC) revealed the presence of BVDV antigen in oral and esophageal mucosa and Peyer patches of the beef breed cow. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) detected BVDV-2 in selected tissues of both animals. Subsequently, BVDV was isolated from both cases and subjected to genetic and serologic characterizations. Mutations in the 5'-untranslated genomic region (5'-UTR) primer and probe binding sites and the E2 gene were associated with reduced efficiency of an established real-time RT-PCR assay and amino acid alterations in the E2 glycoprotein, respectively. Both viral isolates were classified by real-time RT-PCR and phylogenetic analysis as BVDV-2 subgenotype a. Unlike BVDV reference strains Singer and 125c, the isolates cross-reacted with anti-BVDV-1 and anti-BVDV-2 reference sera, indicating antigenic variations in field isolates. The isolates also showed reduced reactivity to porcine anti-BVDV antiserum (the raw serum used to produce BVDV DFA conjugate). In summary, data from the present investigation indicated that genetic and antigenic variations affected the performance of detection assays, especially DFAT, highlighting the need for regular evaluation and modification of BVDV tests.

Factors influencing diagnostic sample submission by food animal veterinarians in Mississippi.

A focus group was organised to gather information and opinions from food animal veterinarians in Mississippi regarding sample submission to diagnostic laboratories. The research found that a range of factors influence the veterinarian's decision regarding whether samples will be submitted to a diagnostic laboratory, with the cost of diagnostics as the key influence. The veterinarians believed that the relationship they had with diagnostic laboratories was important in the protection of public health, but they thought that their role in disease surveillance was under-utilised. More attention needs to be directed towards strengthening veterinary surveillance at ground level to ensure that emergent diseases are detected effectively by a partnership approach between veterinary practitioners in the field and diagnosticians in diagnostic laboratories. This partnership is a vital component of the 'One Health' concept for the protection of both animal and human health. This study demonstrates that qualitative social science methodologies, such as focus groups, can usefully be applied to topics of relevance to veterinary public health.

Diagnosis of Deerpox virus infection in a white-tailed deer (Odocoileus virginianus) fawn.

A 3-month-old fawn from a group of 12 captive white-tailed deer (Odocoileus virginianus) displaying cutaneous lesions was presented to the Mississippi Veterinary Research & Diagnostic Laboratory for necropsy. Postmortem examination identified multiple discrete, round, alopecic, flat, proliferative dermal lesions scattered along the skin of the lips, muzzle, pinna, ventral thorax, medial limbs, and most notably the abdomen. Multiple ulcers were present on the commissures of the lips, dorsal surface of the tongue, and left caudal buccal surface of the oral cavity. The abdomen was filled with fibrinopurulent exudate and ruminal contents. Multiple to coalescing transmural ulcers were identified in the rumen. Histopathological evaluation of the skin revealed markedly thickened epidermis and focal areas of superficial dermal fibrosis, intracytoplasmic, eosinophilic inclusions in swollen keratinocytes and lymphocytic and plasmacytic perivascular dermatitis. The rumen ulcers were surrounded with necrotic cellular debris mixed with fibrin, bacteria, hemorrhages, and a collection of mixed inflammatory cells. Some swollen ruminal mucosal epithelia had eosinophilic intracytoplasmic inclusions. Poxvirus was isolated from the skin and rumen tissue specimens. Electron microscopy detected viral particles with poxvirus morphology. Polymerase chain reaction assays detected A21, a gene conserved within family Poxviridae, in the skin and rumen tissues. Phylogenic analysis of the A21 sequences indicated that the viral isolate (M10-9055) was closely related to known members of genus Cervidpoxvirus. In conclusion, findings indicate that Deerpox virus can produce extensive lesions in white-tailed deer.

Induction of CXC chemokine messenger-RNA expression in chicken oviduct epithelial cells by Salmonella enterica serovar enteritidis via the type three secretion system-1.

The messenger-RNA (mRNA) expression of selected cytokines and chemokines in primary chicken oviduct epithelial cells (COEC) was determined following in vitro infections with wild-type or type three secretion system (T3SS)-mutant Salmonella enterica serovar Enteritidis (SE) strains. All SE strains examined in this study elicited the expression of proinflammatory immune mediators including inducible nitric oxide synthase (iNOS), CXCLi1 (K60), CXCLi2 (IL-8), CCLi3 (K203), and CCLi4 (MIP-1beta). SE also triggered the expression of an anti-inflammatory cytokine, IL-10, but repressed TGF-beta3 transcription. Both T3SS-1 (sipA and sipB) and T3SS-2 (pipB and ssaV) mutants showed reduced capacity, compared to the wild-type SE, to stimulate iNOS mRNA expression in COEC. T3SS-1 (sipA and sipB) mutants were significantly impaired in their ability to induce the expression of CXCLi1 and CXCLi2. T3SS-2 mutants displayed a wild-type phenotype in terms of modulating the expression of chemokines and cytokines in COEC. The expression of iNOS, but not CXC chemokines, correlated with the number of intracellular bacteria in COEC. Genetic complementation of the sipA mutation restored a wild-type phenotype. Thus, SE induction of CXCLi1 and CXCLi2 was sipA-dependent. These results provide enhanced insights into the complex interplay between local host innate immune system and bacterial virulence factors.

PCR detection of pathogenic Leptospira genomospecies targeting putative transcriptional regulator genes.

The genus Leptospira comprises multiple genomospecies that demonstrate varied pathogenic potential. The availability of rapid and precise diagnostic procedures to differentiate pathogenic from nonpathogenic Leptospira spp. is therefore essential to prevent an otherwise easily treatable malaise from developing into a life-threatening disease. In this report, we conducted an investigation on the diagnostic potential of Leptospira genes encoding putative transcriptional regulators. While PCR primers derived from transcriptional regulator gene la1137 recognized all 24 pathogenic Leptospira strains representing seven species, those from la1937, la3231, la3825, and la4130 detected 19 of the 24 Leptospira strains. However, none of these primers reacted with four nonpathogenic Leptospira species or other common bacteria. The putative transcriptional regulator genes la1137, la1937, la3231, la3825, and la4130 are present in pathogenic Leptospira strains, making them potential targets for diagnostic applications. Further characterization of these genes and their proteins may help elucidate the molecular mechanisms of leptospiral virulence and pathogenicity and pave the way for potential development of novel control strategies against leptospirosis.

Uterine neoplasia in 13 cats.

Thirteen uterine tumors were diagnosed in 13 cats and accounted for 0.29% of all feline neoplasms received during a 9.6-year period. Age at diagnosis ranged from 3 to 16 years; median 9 years. Six were Domestic Shorthair cats, and 7 were purebred cats of 5 different breeds. Eight adenocarcinomas and 1 mixed Müllerian tumor (adenosarcoma) comprised the endometrial tumors. Myometrial tumors included 3 leiomyomas and 1 leiomyosarcoma. One of the adenocarcinomas developed in the uterine stump of an ovariohysterectomized cat; the other cats were sexually intact. Concurrent mammary adenocarcinoma was diagnosed in 1 cat with uterine adenocarcinoma and in another with uterine leiomyoma. Tumors were discovered during elective ovariohysterectomy in 2 cats, but at least 3 others had experienced reproductive problems (infertility or pyometra). Five cats presented for abdominal or pelvic masses. Endometrial adenocarcinomas were positive immunohistochemically for cytokeratins and negative for smooth muscle actin (SMA): 1 of 6 cats was positive for vimentin and 4 of 8 were positive for estrogen receptor-alpha (ER alpha). Adenosarcoma stromal cells were positive for vimentin and ER alpha but negative for cytokeratins and SMA. Smooth muscle tumors were positive for vimentin and SMA and negative for cytokeratins. Leiomyomas, but not the leiomyosarcomas, were positive for ER alpha. Adenocarcinomas in 4 cats had metastasized by the time of ovariohysterectomy. Two other cats were euthanized 5 months after ovariohysterectomy; at least one of these cats had developed an abdominal mass that was not examined histologically. Only 2 cats with endometrial adenocarcinoma had disease-free intervals longer than 5 months after surgery. Metastasis was not detected in any mesenchymal tumor; however, these cats were either euthanized on discovery of the tumor or the tumor was first detected at necropsy.

Immunohistochemical analysis of matrix metalloproteinase-1, -3, and -13 in naturally occurring cartilaginous tumors of dogs.

OBJECTIVE: To determine immunoreactivity of matrix metalloproteinase (MMP)-1, -3, and -13 in cartilaginous tumors of dogs, correlate expression of MMP with histologic grade of tumors and clinical outcome of dogs, and compare MMP immunoreactivity between chondrosarcomas and chondromas. SAMPLE POPULATION: Formalin-fixed, paraffin-embedded tissues obtained from samples of naturally occurring chondrosarcomas (n = 31) and chondromas (8) of dogs that were submitted to our veterinary medical diagnostic laboratory. PROCEDURE: Histologic sections from each sample were stained with H&E and monoclonal antibody to MMP-1, -3, and -13 by use of an avidin-peroxidase immunohistochemical technique. For each section, histologic grade (I, II, or III) and immunohistochemical expression (0, 1, 2, or 3) were evaluated. Clinical outcome was obtained from medical records or interviews with referring veterinarians and scored as a good outcome, moderate outcome, or poor outcome. Correlations among variables and differences between chondrosarcomas and chondromas were analyzed. RESULTS: Samples from chondrosarcomas had significantly higher immunoreactivity of MMP-1 and -13, compared with immunoreactivity in samples from chondromas. In chondrosarcomas, a significant positive correlation (r, 0.386) was found between MMP-1 and -13 immunoreactivities, and a significant negative correlation (r, -0.390) was detected between MMP-3 and -13 immunoreactivities. CONCLUSIONS AND CLINICAL RELEVANCE: A significant increase in expression of collagenases (MMP-1 and -13) in chondrosarcomas, compared with expression in chondromas, suggests that collagenases may play an important role in tumor progression, and possibly metastasis, in chondrosarcomas of dogs.