Colonic inflammation involves the changes in chemical coding of not only sensory but also enteric and sympathetic prevertebral neurons innervating inflamed target tissue. Since the sympathetic chain ganglia neurons (SChG) affect colonic function, the purpose of our study was to determine the precise location of the SChG neurons innervating the porcine descending colon, define their chemical coding and answer the question of whether paraformaldehyde-induced colitis in female pigs is able to change the chemical phenotype of the perikarya. Using the Fast Blue (FB) retrograde tracing, the greatest concentration of the (FB+) perikarya was revealed in L3 ganglia. Applying double immunohistochemistry, we detected a strongly catecholaminergic character and in majority neuropeptide Y (NPY)-positive staining of the colon-projecting FB+ neurons both in the control and the inflamed animals. Furthermore, colonic inflammation caused the significant increase in density of galanin- (P<0.001), somatostatin- (P<0.005), leu5-enkephalin- (P<0.05), substance P- (P<0.005), and calcitonin gene-related peptide- (P<0.005) immunoreactive (-IR) nerve fibers surrounding the FB+ perikarya, while the density of nitric oxide synthase-IR and NPY-IR fibers remained unaffected compared to the control animals. The increase in visible immunofluorescent detection of neuropeptide-containing fibers could possibly be caused by enhanced demand and release of neuropeptides that modulate intestinal immune response and regulate the gastrointestinal activity through inflammatory mediators.
Aquaporin-4 (AQP4) is an isoform of membrane water channel aquaporins. It is most abundant in the brain, and is believed to be expressed by astrocytes and ependymal cells. We have reported previously that rat pituitary glands express some aquaporin subtypes, including AQP4, but the precise distribution of AQP4 in the pituitary gland is not known. The present work investigated whether AQP4 immunoreactivity exists in various pituitary cell types in adult rats, using double immunofluorescent staining and confocal microscopy. In the adenohypophysis, co-labeling for S-100 protein indicated that folliculo-stellate (FS) cells and marginal layer cells in Rathke's residual pouch have extensive labeling for AQP4. Some AQP4-expressing cells also contained glial fibrillary acidic protein (GFAP) in parenchymal tissue of the anterior lobe, and cytokeratin in marginal layer cells. AQP4 was not coexpressed in any hormone-producing pituitary cells, however. In the neurohypophysis, AQP4 was expressed in some pituicytes, which have been identified as GFAP-positive. These results suggest that AQP4, which is expressed in FS cells, in marginal layer cells and in pituicytes, but not in hormone-producing cells, is important in water transfer within the pituitary gland; astrocytes and ependymal cells do the same in the brain.
Placental complement has the potential to induce autologous embryo injury. We have previously found a significant elevation of adipsin, an activating factor of the alternative complement pathway, in mouse placentas from spontaneous abortions. The present study was aimed to evaluate regulation of the alternative complement pathway in placentas of mouse spontaneous abortions. Protein was purified from mouse placentas at normal and abortion implantation sites on day 14.5 of pregnancy. The activity of the alternative complement pathway was slightly intensified following addition of protein from abortion placentas. Western blotting revealed that Crry was clearly present in the placentas from abortions. Thus, the complement regulating system through Crry is functional to restrict alternative complement activity in abortion placentas.
Avian infectious bronchitis viruses (IBVs) isolated from commercial layer flocks kept in Ibaraki Prefecture in 2009 were genetically and serologically characterized. Reverse transcription-PCR coupled with direct nucleotide sequencing and GenBank BLAST database analysis of the hypervariable region of the S1 subunit of the virus spike gene showed that these isolates are genetically very different from the previously known IBV genotypes in Japan. Furthermore, none of the antisera used in this study neutralized the index isolate (JP/Ibaraki/168-1/2009) in virus neutralization tests. These results suggest that the isolates are a novel IBV genotype in Japan (designated JP-IV).
A 13-year-old mixed-breed dog showing ascites, anorexia and anemia was found to have leukocytosis with marked eosinophilia, splenomegaly and hepatomegaly. The dog died 4 days after initial presentation and was diagnosed with splenic high-grade B-cell lymphoma at necropsy. Remarkable infiltrations of eosinophils were observed in spleen and liver tissues. The eosinophilia and infiltration of eosinophils into the lesions could have been associated with B-cell lymphoma because causes other than lymphoma were excluded. This is the first report of eosinophilia and eosinophilic infiltrations into neoplastic lesions in a dog with high-grade B-cell lymphoma.
In this study, we investigated the expression of immunoglobulin A (IgA) in porcine salivary gland and its relationship with restraint stress in pigs. IgA was expressed in plasma cells in pig salivary gland, as confirmed by immunohistochemical staining. IgA was also detected in pig saliva itself by ELISA, and salivary IgA levels were increased by a restraint stress. Moreover, there was a circadian rhythm of IgA over the course of a day. These results are the first evidence of IgA expression related to stress in the pig saliva and may make IgA useful as a non-invasive biological marker to evaluate acute stress condition in the pigs.
We investigated the anti-inflammatory effects of ethyl pyruvate (EP) on LPS-stimulated canine PBMCs in vitro. We found that EP treatment inhibited the mRNA expressions of proinflammatory cytokines (TNF-α and IL-6), but induced mRNA expression of anti-inflammatory cytokines (IL-10). ELISA measurements revealed that EP also effectively downregulates the LPS-induced increase in proinflammatory cytokine release, while upregulating anti-inflammatory cytokine release. These data indicate that EP could be an effective anti-inflammatory agent in dogs.
We measured the plasma D-dimer (DD) concentration in 205 dogs. Simultaneously, fibrinogen/fibrin degradation products (FDPs) concentration, platelet (PLT) count, prothrombin time (PT), activated partial thromboplastin time (APTT), and plasma concentrations of fibrinogen (Fib) were measured in the same dogs. From these results, we were able to divide the animals into four groups: control (healthy dogs, n=18), pre-disseminated intravascular coagulation (preDIC) (n=20), disseminated intravascular coagulation (DIC) (n=21), and other (n=146). Significant differences in plasma DD concentration were found among the four groups: control, 0.45 ± 0.46 μg/ml (reference range, 0-1.37); preDIC, 5.0 ± 4.8 μg/ml; DIC, 16.3 ± 12.2 μg/ml; and other, 1.5 ± 2.7 μg/ml. A weak positive correlation (r=0.62) was found between FDPs and DD concentrations in the DIC group. As a DIC diagnostic test, the PLT/APTT/FDPs/DD combination had the highest accuracy of 100%, with a sensitivity of 73% and a specificity of 97%. We propose the use of FDPs and DD concentrations as part of the DIC diagnostic test panel, with DD and FDPs to provide accurate diagnosis.
Surfactant protein D (SP-D) is a pattern recognition molecule that has an important role in pulmonary host defense. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for bovine SP-D and determined the concentration of SP-D in bronchoalveolar lavage fluid (BALF) from calves. Bovine SP-D was purified from BALF using a mannose-Shepharose 6B column. The obtained 44 kDa protein was identified as bovine SP-D by N-terminal amino acid sequence analysis and SDS-PAGE analysis. The peptides corresponding to bovine SP-D amino acid residues SDTRKEGT, which have little homology across bovine serum collectins, were synthesized and used to raise an antibody in rabbits. The obtained antibody was specific for bovine SP-D and did not react with collectins in serum. The anti-bovine SP-D antibody was purified and an ELISA system was developed. The detection range of this assay was 4-125 ng/ml, and the intra-assay and inter-assay coefficients of variation were 5.6 and 9.7%, respectively. The concentrations of SP-D in BALF collected from calves experimentally infected with bovine adenovirus type-3 or Mannheimia haemolytica were determined by the ELISA. Elevation of SP-D was found in BALF from inoculated lobes of infected calves compared with those of non-inoculated lobes and those from control animals. These data suggest that the ELISA developed in this study may be available to investigate the physiological role of bovine SP-D in bovine lung.
The objective of this paper was to evaluate the sequence of feline prepro-adrenomedullin (AM) and its tissue distribution and to investigate whether expression of feline AM mRNA increases in association with spontaneous cardiomyopathy. The feline prepro-AM cDNA sequence and deduced amino acids were 564 base pairs and 188 residues, respectively. The cDNA sequences of feline prepro-AM including AM and proadrenomedullin N-terminal 20 peptide showed high homology with those of other mammalian species. The mRNA expression of AM was detectable in various normal tissues. The mRNA levels of AM were elevated in hearts with cardiomyopathy compared with normal hearts. This study suggests that AM has an important role as a neurohumoral factor in cats with spontaneous heart diseases.
A 3-year-old castrated male domestic short-haired cat was presented with nodules on the left nasal wing and the left earflap. Prototheca cells were found after excision biopsy of one of the nodules located on the left earflap. The patient cat was generally in good condition without skin problems. Prototheca wickerhamii was isolated from all 6 masses after they were surgically nucleated. The cat was recovered two months after intervention with no recurrence of skin nodules. This report deals with the first case of feline protothecosis in Japan.
In order to decipher the amino acid (AA) balance in the dairy cattle with left displaced abomasum (LDA), blood samples were obtained from thirty LDA and 12 healthy control cattle. Plasma from LDA cattle exhibited significantly higher free fatty acid and β-hydroxybutyrate, lower glucogenic AA, such as methionine, alanine and serine, and higher ratio of ketogenic AA among blood free AA, such as leucine and lysine. The Fischer ratio, phenylalanine and albumin were similar to control cattle. These results suggested that LDA cattle were in negative energy balance, but AA metabolism and protein synthesis were within a normal range. Lower glucogenic AA was due to the higher gluconeogenesis, and higher ketogenic AA was due to higher ketogenesis.
The ICGN mouse strain is a glomerulosclerosis (GS) model that shows characteristic proteinuria, podocyte morphological abnormalities and increased extracellular matrix accumulation in the glomeruli, which are the final common pathology associated with a variety of kidney diseases leading to end-stage renal failure. Previously, we performed a quantitative trait locus (QTL) analysis to identify the causative genes for GS in ICGN mice and found the deletion mutation of the tensin2 (Tns2) gene that creates both a premature stop codon and dramatically decreases mRNA expression levels within the region of the major QTL (this mutation was designated Tns2nep). The severity of GS varies considerably in humans and other animals, indicating the influence of several genes controlling the disease phenotype. In this study, to identify the modifier/resistant gene(s) for GS, we produced congenic strains carrying the Tns2nep mutation on the C57BL/6J (B6) genetic background and analyzed GS severity. Interestingly, the B6 congenic mice exhibited milder phenotypes than the ICGN strain mice. The results suggest that B6 mice have a modifier(s) of GS resistance. Therefore, identification of the modifier loci in B6 mice will provide important new information regarding gene interactions controlling GS.
Innate cells, such as natural killer (NK) cells and NKT cells, play essential roles as primary effector cells at the interface between the host and parasite until establishment of adaptive immunity. However, the roles of NK and NKT cells in defense against Neospora caninum have not been well clarified. NK and NKT cells were depleted by the treatment with an anti-CD122 (interleukin-2 receptor beta chain) monoclonal antibody (mAb, TM-β1) in vivo. The parasite burden in the brain of mice was promoted by the treatment with anti-CD122 mAb. However, there was no significant difference in the infection rates between controls and the mice treated with anti-asialoGM1 antibody to deplete NK cells. Activation of CD4+ T cells was suppressed in the mice treated with anti-CD122 mAb compared with controls and the mice treated with anti-asialoGM1 antibody. On the other hand, depletion of CD122+ cells or NK cells did not affect the number of activated CD8+ T cells, dendritic cells and B cells following N. caninum infection. These results indicate that CD122+ cells (probably NKT cells) play a crucial role in host defense by activating CD4+ T cells against N. caninum infection.
The genetic diversity of Babesia gibsoni strains worldwide is currently poorly defined. The aim of the present study was to characterize B. gibsoni strains in naturally infected dogs in Taiwan using a combination of polymerase chain reaction (PCR) and sequence analysis of both 18S rDNA and the gene encoding thrombospondin-related adhesive protein (TRAP). Genomic DNA was extracted from 29 parasitemic dogs, and the target genes were separately amplified, sequenced and aligned with corresponding sequences available in GenBank. All 18S rDNA sequences (1,262 bp) amplified from the Taiwanese isolates were identical to each other and had very high similarity (99.9-100%) with previously reported B. gibsoni sequences. These results provide the first molecular evidence showing infection of dogs with B. gibsoni from Taiwan. On the other hand, a phylogenetic analysis based on the deduced amino acid sequence of the TRAP gene demonstrated that the Taiwanese isolates were closely related to strains previously identified from Okinawa Island, Japan, but genetically distinct from strains found on Honshu in Japan and Jeju Island in South Korea. The divergence of TRAP among the geographically dispersed strains examined in this study and others supports the conclusion that this gene is useful for molecular genotyping of B. gibsoni strains.
In order to reveal DNA types of aspermic Fasciola forms in Japan, Fasciola specimens obtained from eight prefectures that had not been previously reported were analyzed for DNA of ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase 1 (ND1) gene. Five combinations in DNA types of both ITS1 and ND1 were revealed from the results of this study and previous studies. The DNA type Fsp2, which is identical to that of F. gigantica in both ITS1 and ND1, was the most predominant in Japan, followed by Fsp1, which is the same DNA type as that of F. hepatica. Fasciola forms with Fsp1 mainly occurred in the northern region of Japan and those with Fsp2 were mainly in the western region. The founder effect related to migration of definitive host and susceptibility of intermediate host snail might play an important role in both geographical distribution and frequency of DNA types in Japanese Fasciola specimens.
Progressive glomerular injury associated with early-onset proteinuria was investigated in male Osborne-Mendel (OM) rats aged 5 to 20 weeks. Age-matched male Fischer 344 (F344) rats were used for comparison. OM rats developed mild hypertension and selective proteinuria (albuminuria) from 5 weeks of age, and non-selective proteinuria from 7 weeks of age. Light microscopy of OM kidney revealed hyaline droplets in the podocyte at 5 weeks of age and vacuolation of podocytes and adhesion of the capillary loop to the Bowman's capsule at 7 weeks of age. Segmental glomerulosclerosis developed in OM rats from 15 weeks of age, and global sclerosis appeared at 20 weeks of age. Desmin, a marker of podocye injury, was expressed in podocytes from 10 weeks of age, and the intensity of expression increased with age. Ultrastructurally, damage to podocytes such as effacement of foot processes, decreasing number of filtration slits, and rearrangement of the actin cytoskeleton were observed from 5 weeks of age in OM rat. Glomerular volume in OM rats increased with age and was consistently higher than in age-matched F344 rats. The number of WT-1-positive podocytes and vimentin-positive podocyte area were lower in OM rats and decreased with age. These findings suggest that glomerulonephropathy in male OM rats is associated with glomerular hypertrophy, progressive podocytopathy, and a reduction in podocyte number and area. Renal injury in OM rats was associated with development of early-onset proteinuria and was more progressive than in age-matched F344 rats.
Pathological findings associated with scuticociliatosis in farmed Japanese flounder in Japan are described. Ten moribund fishes, farmed in Tottori Prefectural Fisheries Experimental Station, showed cutaneous ulcers, darkened skin, fin and tail rot, exophthalmia and alterations in swimming behaviour. Histopathologically, severe epidermal degeneration and necrosis, hyperplasia of branchial epithelium, myositis, myelitis, encephalitis associated with heavy accumulation of scuticociliates in the periorbital cavity and optic nerve fiber were observed. Moreover, masses of ciliates were found to feed on the host tissues such as skeletal muscles, gills and brain, causing severe degenerative changes associated with abundant neutrophilic and lymphocytic infiltration. These findings suggest that the present scuticociliate, Miamiensis avidus, is a highly invasive and destructive pathogen infecting Japanese flounder and capable of developing systemic fatal infection.
Rhabdomyosarcoma (RMS) is a malignant neoplasm derived from mesenchymal tissue with a tendency toward myogenic differentiation associated with the embryogenesis of skeletal muscle. According to the histological features, it can be classified in embryonal, botryoid, alveolar, and pleomorphic, which usually correspond to clinical behavior and prognosis. The spindle cell (SCRMS) variant is a rare subtype of the embryonal RMS and is considered to be less aggressive lesion. The aim of the present paper is to report an unusual case of SCRMS in an 11-month-old male boxer dog diagnosed as extensive SCRMS that affected the frontal region of the skull. Due to the aggressive nature of the lesion and poor clinical prognosis the dog's owners preferred euthanasia as a treatment. A full postmortem examination was carried out. Microscopically, the lesion was composed of a highly cellular proliferation of spindle cells arranged in long and intersecting fascicles. After performing the immunohistochemical studies (HHF-35, smooth muscle actin, desmin and MyoD1), the present case was diagnosed as SCRMS.
The paper describes clinical and pathological features of Alexander's disease (AD)-like disorder in a 1 year and 8 months old French bulldog. Clinically, the dog exhibited megaesophagus, emaciation and weakness without any specific neurological symptoms. The dog died of aspiration pneumonia. On the gross observation of formalin-fixed brain, discolored foci were observed in the white matter of the cerebellum and brain stem. Histologically, numerous Rothenthal fibers and hypertrophic astrocytes were distributed especially in the perivascular, subependymal and subpial area of both the cerebrum and cerebellum. The Rosenthal fibers were intensely immunopositive for GFAP and ubiquitin. Demyelination of the white matter was occasionally found in the brain stem. The present case is likely to be categorized in the adult form of AD, though previous AD-like cases in dogs were in the juvenile form.
Paratuberculosis (Johne's disease) has emerged as one of the most important diseases in cattle. The role of infected bull semen in the spread of infection remains unclear, as the correlation between the amount of excreted Mycobacterium avium subsp. paratuberculosis (semen and feces) and the infection load (blood and tissues) has not been defined. The aim of the present study was to study by culture, and a quantitative real-time polymerase chain reaction, the presence of bacteria in consecutive semen, blood, and fecal samples collected from one infected Piedmont breeding bull during a 380-day period. Five out of seven blood samples and all nine semen samples were positive in the real-time quantitative polymerase chain reaction with 101 to 102 and 102 to 104 copies of IS900/F57 per ml, respectively. In all, there were 9 fecal culture positive samples with too numerous to count colony forming units and positive real-time quantitative polymerase chain reactions ranging from 105 to 107 copies of IS900/F57. After the bull was euthanized, Mycobacterium avium subsp. paratuberculosis was cultured from various parts of the small and large intestines, liver tissue and lymph nodes and from the epididymis and vesicular glands. The results demonstrate a wide extraintestinal distribution of the bacterium and that breeding bulls should be considered a source of paratuberculosis infection due to their contact with other breeding bulls and a high number of heifers and cows through the natural mating process.
In the pregnant bitch, the placenta is a major source of circulating relaxin, but its local expression in the reproductive organs is not clear. This study demonstrated expression of relaxin mRNA in the corpus luteum, uterus, uterine cervix as well as placenta in the pregnant and nonpregnant bitch by reverse transcriptase-polymerase chain reaction (RT-PCR).
Studies of environmental pollution in Africa indicate that toxic metal pollution has reached unprecedented levels over the past decade. Human exposure to toxic metals has become a major health risk on the continent and is the subject of increasing attention from national and international environmentalists. This paper reviews data from the past decade on environmental pollution in Africa and highlights countries where most heavy metal pollutions have been reported. Characteristics of heavy metal pollution in North, West, East and Southern regions of Africa have been described, as have major sources of pollution in the different regions. This review summarizes the sites where most of the heavy metal pollution has been reported in Africa and, where applicable, presents reported levels of pollution in different environmental compartments in the context of internationally acceptable limits. Contaminations in fish and food animals as well as impacts of heavy metal pollution on humans are also described.
This research was conducted to evaluate the effect of naringenin (NAG) on fate and dispositions of deoxynivalenol (DON) in piglets following intravenous (i.v.) administration. Three piglets (Group 1) were pretreated orally with NAG at a dosage of 25 mg/kg bw, once a day for 3 consecutive days, followed by a single i.v. injection of DON at a dosage of 1 mg/kg bw. The other three piglets (Group 2) were intravenously administered with DON at the same dosage. The level of DON in the plasma and various piglets tissues were measured using liquid chromatography/tandem mass spectrometry. The plasma levels of DON were higher in the NAG-untreated piglets than in the NAG-pretreated piglets at each time point. However, the plasma DON concentrations in the piglets pretreated with NAG was lower than those of NAG-untreated piglets. The elimination half-life was longer in the NAG-untreated piglets than in the piglets pretreated with NAG. The initial peak concentration, area under the curve and mean residence time were higher in the NAG-untreated piglets than in the piglets pretreated with NAG. Plasma biomarker enzyme activities were also monitored and the levels of gamma glutamyltranspeptidase, aspartate aminotransferase, alanine aminotransferase, creatine phosphokinase, blood urea nitrogen, and creatinine were considerably lower in the piglets pretreated with NAG than in the NAG-untreated piglets. The toxicokinetic data and blood biochemical parameters indicate that NAG enhances the excretion of DON and reduces the opportunity for damage in piglets. Consequently, its toxicity is greater in NAG-untreated piglets than in piglets pretreated with NAG.