The mammalian olfactory system employs sophisticated mechanisms to detect and recognize an extensive range of smells. In rodents, the olfactory epithelium (OE), situated within the nasal cavity, mainly comprises four defined endoturbinates and several ectoturbinates. Olfactory receptors (ORs) belong to a large family, comprising over 1,000 genes in rodents, which are expressed in olfactory sensory neurons in the OE that detect odor molecules. The rodent OE is divided into four topographically distinct zones, defined by individual OR distribution. However, although the structural complexity and the zonal organization of mammalian OE may contribute to successfully interpreting olfactory information, it remains poorly understood. In this study, we investigated the nasal cavity structure and zonal organization of the OE in goats. Morphological observations revealed that the goat nasal cavity possessed well-developed endoturbinates and ectoturbinates and had a structure similar to that of rodents and sheep, previously reported in other studies. In situ hybridization was used to analyze the expression pattern of ORs, NADPH:quinone oxidoreductase 1, and olfactory cell adhesion molecules as markers of zonal organization in the goat OE. Based on the expression patterns of these genes, we concluded that the goat OE was divided into four zones. The well-developed structure of the nasal cavity and distribution of each OR in the OE were similar to those found in rodents, suggesting that these features were highly conserved between mammals and may have fundamental roles in discriminating among numerous odor molecules in the environment.
The broad-spectrum lytic capability of Salmonella bacteriophages against various Salmonella species was evaluated to determine their potential as an alternative for antibiotics, and the safety and preventive effects of the bacteriophages were assessed on mice and pigs. Four bacteriophage cocktails were prepared using 13 bacteriophages, and the lytic capability of the four bacteriophage cocktails was tested using Salmonella reference strains and field isolates. Bacteriophage cocktail C (SEP-1, SGP-1, STP-1, SS3eP-1, STP-2, SChP-1, SAP-1, SAP-2; ≥109 pfu/ml) showed the best lytic activity against the Salmonella reference strains (100% of 34) and field isolates (92.5% of 107). Fifty mice were then orally inoculated with bacteriophage cocktail C to determine the distribution of bacteriophages in various organs, blood and feces. The effects of bacteriophages on Salmonella infection in weaned pigs (n=15) were also evaluated through an experimental challenge with Salmonella Typhimurium after treatment with bacteriophage cocktail C. All mice exhibited distribution of the bacteriophages in all organs, blood and feces until 15 days post infection (dpi). After 35 dpi, bacteriophages were not detected in any of these specimens. As demonstrated in a pig challenge study, treatment with bacteriophage cocktail C reduced the level of Salmonella shedding in feces. The metagenomic analyses of these pig feces also revealed that bacteriophage treatment decreased the number of species of the Enterobacteriaceae family without significant disturbance to the normal fecal flora. This study showed that bacteriophages effectively controlled Salmonella in a pig challenge model and could be a good alternative for antibiotics to control Salmonella infection.
This study described the occurrence of clinical and subclinical forms of mastitis in 250 cattle from 5 dairy farms around the cities of Santa Rosa and Machala, El Oro Province, Ecuador. Clinical mastitis (CM) was determined based on obvious changes in milk (mild), signs of inflammation in the udder (moderate), and/or generalized clinical symptoms (severe). Subclinical mastitis (SCM) was assessed using the California mastitis test. CM and SCM were detected in 30 (12.0%) and 150 (60%) of the 250 tested cattle, respectively. Prevalence at the udder quarter level was 57.7% (577/1,000), which was higher among forequarters (369/577; 63.9%) than hindquarters. Of the 577 mastitic milk samples subjected to microbiological analysis, 35 were excluded due to contamination and 20 tested negative. Identification of bacterial isolates revealed that 33.3% of the 93 CM samples contained coliforms, 25.8% coagulase-positive staphylococci, 20.4% coagulase-negative staphylococci (CNS), 9.7% streptococci, 7.5% Bacillus spp., and 3.2% Klebsiella spp. Bacterial profiling of the 429 SCM milk samples showed that 55.4% contained CNS, 22.1% Bacillus spp., 9.3% streptococci, and 6.1% coagulase-positive staphylococci. In vitro antibiotic susceptibility testing of the obtained isolates indicated that all were susceptible to amoxicillin, ampicillin, cefotaxime, enrofloxacin, sulfamethoxazole-trimethoprim, gentamicin, and neomycin. No multidrug-resistant strains were observed.
The bacterium Ornithobacterium rhinotracheale is associated with respiratory disease in wild birds and poultry. In this study, the phylogenetic analysis of nine reference strains of O. rhinotracheale belonging to serovars A to I, and eight Mexican isolates belonging to serovar A, was performed. The analysis was extended to include sequences from another 23 strains available in the public domain. The analysis showed that the 40 sequences formed six clusters, I to VI. All eight Mexican field isolates were placed in cluster I. One of the reference strains appears to present genetic diversity not previously recognized and was placed in a new genetic cluster. In conclusion, the phylogenetic analysis of O. rhinotracheale strains, based on the 16S rRNA gene, is a suitable tool for epidemiologic studies.
Fowl cholera caused by Pasteurella multocida has always been a disease of global importance for poultry production. The aim of this study was to obtain more information about the epidemiology of avian P. multocida infection in southwest China and the genetic characteristics of clinical isolates. P. multocida isolates were characterized by biochemical and molecular-biological methods. The distributions of the capsular serogroups, the phenotypic antimicrobial resistance profiles, lipopolysaccharide (LPS) genotyping and the presence of 19 virulence genes were investigated in 45 isolates of P. multocida that were associated with clinical disease in poultry. The genetic diversity of P. multocida strains was performed by 16S rRNA and rpoB gene sequence analysis as well as multilocus sequence typing (MLST). The results showed that most (80.0%) of the P. multocida isolates in this study represented special P. multocida subspecies, and 71.1% of the isolates showed multiple-drug resistance. 45 isolates belonged to capsular types: A (100%) and two LPS genotypes: L1 (95.6%) and L3 (4.4%). MLST revealed two new alleles (pmi77 and gdh57) and one new sequence type (ST342). ST129 types dominated in 45 P. multocida isolates. Isolates belonging to ST129 were with the genes ompH+plpB+ptfA+tonB, whereas ST342 included isolates with fur+hgbA+tonB genes. Population genetic analysis and the MLST results revealed that at least one new ST genotype was present in the avian P. multocida in China. These findings provide novel insights into the epidemiological characteristics of avian P. multocida isolates in southwest China.
Cytotherapy with mesenchymal stem cells (MSCs) has been studied in many species, and often requires in vitro cell expansion to obtain therapeutic doses of stem cells. Because the characteristics of MSCs, such as self-renewal and multi-lineage differentiation, can be altered by long-term culture, it is important to maintain stemness during cultivation. This study assessed the changes in the characteristics of feline adipose tissue-derived (fAT)-MSCs during in vitro passaging. Stem cells isolated from the adipose tissue of donor cats were cultured for seven sub-passages. Proliferation capacity was analyzed by calculating the cell doubling time and by colorimetric assay. Expression of stem cell-specific markers was evaluated by quantitative reverse transcription (qRT)-PCR and immunophenotyping. Expression of adipogenic and osteogenic differentiation markers was also measured by qRT-PCR. Histochemical staining and measurement of β-galactosidase activity were conducted to detect cellular senescence. The cell proliferation rate decreased significantly at passage 5 (P5). Gene expression levels of pluripotency markers (Sox2, Nanog and Klf4) and stem cell surface markers (CD9, CD44, CD90 and CD105) decreased during continuous culture; in most assays, statistically significant changes were observed at P5. The ability of cells to undergo adipogenic or osteogenic differentiation was inversely proportional to the number of passages. The proportion of senescent cells increased with the number of passages. These results suggest that repeated passages alter the proliferation and multipotency of fAT-MSCs. In clinical trials, early-passage cells should be used to achieve the maximum therapeutic effect.
A 6-year 5-month-old spayed female Scottish Fold cat presented with a one-month history of gait abnormalities, increased salivation, and decreased activity. A blood test showed hyperammonemia and increased serum bile acids. Imaging tests revealed multiple shunt vessels indicating acquired portosystemic shunt. Histopathologic analysis of liver biopsy showed features consistent with liver hypoperfusion, such as a barely recognizable portal vein, increased numbers of small arterioles, and diffuse vacuolar degeneration of hepatocytes. These findings supported the diagnosis of primary hypoplasia of the portal vein/microvascular dysplasia, (PHPV/MVD). To our knowledge, this is the first case of feline PHPV/MVD that developed multiple acquired portosystemic shunts and presented with hepatic encephalopathy.
A 12 year-old intact male Pembroke Welsh corgi weighing 10.8 kg was presented for evaluation of a 3-month history of dyspnea, and a 1-week history of exercise intolerance and anorexia. Severe hypoxemia (PaO2 56 mmHg), diffuse lung alveolar infiltration, and severe pulmonary hypertension (PH) (tricuspid regurgitation pressure gradient was 81 mmHg) were identified. A tentative diagnosis of severe PH due to lung disease or pulmonary thromboembolism was made and treated intensively. After 5 days of hospitalization, the dog died despite oxygen supplementation and anticoagulant therapy. This dog was diagnosed as unclassified interstitial lung disease based on histopathological findings.
The objectives of this study were to assess if Clinofibrate (CF) treatment improved lipid metabolism in dogs, and to clarify whether its efficacy is influenced by canine characteristics. We collected medical records of 306 dogs and performed epidemiological analyses. Lipid values of all lipoproteins were significantly decreased by CF medication, especially VLDL triglyceride (TG) concentration (mean reduction rate=54.82%). However, 17.65% of dogs showed drug refractoriness in relation to TG level, and Toy Poodles had a lower CF response than other breeds (OR=5.36, 95% CI=2.07–13.90). Therefore, our study suggests that genetic factors may have an effect on CF response, so genetic studies on lipid metabolism-related genes might be conducted to identify variations in CF efficacy.
Prototheca zopfii is associated with bovine mastitis, which causes a reduction in milk production and secretion of thin, watery milk with white flakes. However, the source of infection and an infection route of mastitis have not been clarified. In this study, we evaluated the prevalence of P. zopfii genotype 2 in fecal samples from Japanese dairies with or without a history of protothecal mastitis in 2017. P. zopfii genotype 2 was detected in 23 of 60 (38%) fecal samples in only the herd with a history of protothecal mastitis. These results suggest that occurrence of bovine protothecal mastitis is related to persistent infection in intestine and the source of infection is feces.
Nonalcoholic steatohepatitis (NASH) is a progressive liver disease, and some patients develop hepatic cirrhosis/carcinoma. Animal models play key roles in the development of new therapies for NASH. In this study, the pharmacological effects of metformin and pioglitazone were investigated in female Spontaneously Diabetic Torii (SDT) fatty rats to verify the utility of this model. The anti-diabetic drugs were administered to SDT fatty rats fed a cholesterol-enriched diet from 4 to 25 weeks, and changes in food intake, body weight, and blood chemistry parameters were evaluated every 4 weeks. The hepatic lipid content, mRNA expression in relation to lipid synthesis, inflammation, and fibrosis, and histopathological analyses were performed at 25 weeks. Pioglitazone improved hyperglycemia, hyperlipidemia, and abnormalities in hepatic parameters. The insulin levels were lower than those in the control rats before 16 weeks. Plasma glucose levels in the metformin-treated rats were lower than those in the control rats, and plasma alanine aminotransferase levels temporarily decreased. The lipid content and some mRNA expression in relation to fibrosis in the liver decreased with pioglitazone treatment, and the mRNA expression of microsomal triglyceride transfer protein increased. Hepatic fibrosis observed in the SDT fatty rats improved with pioglitazone treatment; however, the effect with metformin treatment was partial. These results in both drugs are in line with results in the human study, suggesting that the SDT fatty rat is useful for developing new anti-NASH drugs that show potential to regulate glucose/lipid metabolism.
We evaluated a handling method using tunnels to tame laboratory mice (ICR) in the context of animal welfare and ease of handling. During 1-week acclimation to handling and subsequent 1-week oral administration (once per day), voluntary interaction with the experimenter was much greater in mice handled by a tunnel compared to those picked up by the tail. According to a rating of the ease of handling laboratory mice, a tunnel facilitated mouse handling during acclimation to handling and oral gavage of saline compared to tail handling. In addition, mice handled by a tunnel showed less anxiety than mice handled by the tail in the open field test, but not in elevated plus maze. Calculation of experimental variation in behavioral tests, which were used to mimic pharmacological studies, suggested that mice handled by a tunnel exhibited the tendency of less variation compared to those picked up by the tail, in both groups that were intraperitoneally administered saline as placebo and diazepam as an active drug. Thus, tunnel use could be beneficial for improving animal welfare and facilitated handling of ICR mice in mouse studies.
Meat is a rich source of protein, fatty acids and carbohydrates for human needs. In addition to necessary nutrients, high fat contents in pork increase the tenderness and juiciness of the meat, featuring diverse application in various dishes. This study investigated the transcriptomic profiles of intramuscular adipose tissues in Jinhua and Landrace pigs by employing advanced RNA sequencing. Results showed significant interesting to note that there were significant differences in the expression of genes. 1,632 genes showed significant differential expression, 837 genes were up-regulated and 195 genes were down-regulated. Variations in genes responsible for cell aggregation, extracellular matrix formation, cellular lipid catabolic process, and fatty acid binding strongly supported that both pig breeds feature variable fat and muscle metabolism. Certain differentially expressed genes are included in the pathway of mitogen-activated protein kinase signaling pathway, Ras signaling pathway and insulin pathway. Results from real-time quantitative polymerase chain reaction also validated the differential expression of 17 mRNAs between meats of the two pig breeds. Overall, these findings reveal significant differences in fat and protein metabolism of intramuscular adipose tissues of two pig breeds at the transcriptomic level and suggest diversification at the genetic level between breeds of the same species.
The present study investigated the prevalence of Toxoplasma gondii and other intestinal parasites in cats in the Tokachi subprefecture in Japan. A total of 365 household cats were included in the study, and 353 serum and 351 fecal samples were collected and analyzed. T. gondii IgG antibodies were detected in the sera of 16.14% of cats based on Latex agglutination test and ELISA. For ELISA, T. gondii RH strain tachyzoites lysate and T. gondii SAG2 recombinant protein were used as antigens. Low seropositivity was detected in cats younger than one year and older than 11 years; outdoor and hunter cats showed significantly high seropositivities. Neutering either in male or female cats, but not gender, had a considerable effect on seroprevalence. Toxoplasma gondii oocysts were detected in one fecal sample. The overall parasitic infestation in cats was 12.5%. Other detected parasites included Toxocara species, which showed the highest prevalence of 7.7%, followed by Isospora spp. (2%), Taenia spp. (1.7%), and Ancylostoma spp. (0.9%). Spirometra spp. was detected in only one sample. Outdoor cats comprised 50% of all 44 parasite-infested cats. Although T. gondii oocysts were detected in only one sample, the relatively high seroprevalence of T. gondii indicated that it can pose significant risks to the environment. Our findings highlighted the potential of outdoor cats as a source of T. gondii and other parasites.
Allyl isothiocyanate (AITC), a metabolite of the glucosinolate sinigrin, protects the liver of rats injured by carbon tetrachloride (CCl4). This study evaluated whether AITC reduces hepatic fibrosis in rats repetitively exposed to CCl4. Serum chemistry showed that AITC (doses of 5 and 50 mg) administered to rats exposed to CCl4 significantly reduced the levels of alanine aminotransferase and aspartate aminotransferase activity that were elevated in CCl4-intoxicated rats. The connective tissue in AITC-treated rats was significantly reduced based on Sirius staining. In addition, Kupffer cell activation was significantly reduced in the AITC and CCl4 co-treated groups. Collectively, this study suggests that AITC mitigates hepatic fibrosis in rats repetitively exposed to CCl4 with concurrent regulation of Kupffer cell and monocyte activation.
Four aged retired Chinese native pigs, three females and one male, estimated as over 10-year-old, were subjected to autopsy because of infertility due to aging. Grossly, nodular lesions were found bilaterally in the adrenal medulla of all four pigs. Based on the gross and the histopathological findings, they were diagnosed as either medullary nodular hyperplasia or pheochromocytoma. Immunohistochemically, proliferating cells of all these lesions were immuno-positive for chromogranin-A, indicating adrenal medulla-derived. Ultrastructurally, cytoplasmic neurosecretory granules suggestive of secretion were observed in these proliferating cells. There have been only limited numbers of reports on adrenal medullar proliferative changes including pheochromocytoma in pigs. The present cases will provide a valuable information for the characterization of similar changes in animals and human.
Despite being rarely reported, improved diagnostic and prognostic indicators are necessary for treating malignant melanoma in rabbits. In this study, two cases of primary skin lesions, on the scrotum and on eyelid, with systemic metastases, were examined. The tumors formed intra-dermally by sheet-like proliferation of polymorphic cells, with anisocytosis and varying amount of melanin granules. Tumors had displaced almost 50% of the lung and liver tissue, and tumor metastasis was the cause of early death in both rabbits. Ki-67-positive population was high in both, and it was found to be useful in assessing the outcome and malignancy. In addition, Melan-A, HMB-45, PNL2 and S100 established a useful immunohistochemical panel for the diagnosis of melanocytic tumor in rabbits.
Epithelial-mesenchymal transition (EMT) is an orchestral and functional change in epithelial cells. Many signaling pathways are involved in EMT, and transforming growth factor-beta (TGF-β) is considered to be one of the most important factors in induction of EMT. In this study, we treated the rat intestinal epithelial cell line (IEC-6) with TGF-β1 as a signaling stimulant. Gross analysis of IEC-6 cells showed typical characteristics of epithelial cells such as cuboidal morphology and cell-cell contact, whereas treatment with TGF-β1 (10 ng/ml−1) for 7 days produced robust, spindle-shaped morphology. Immunocytochemistry analysis showed distinct E-cadherin staining in IEC-6 cells, but weak and faint in EMT cells. EMT cells showed positive expression of α-SMA and tenascin-C but IEC-6 cells did not. Quantitative real-time PCR analysis showed that myosin light chain kinase and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17) mRNAs were significantly upregulated in EMT cells. Immunocytochemistry analysis also showed that EMT cells strongly expressed CPI-17 but IEC-6 cells did not. A collagen gel contraction assay revealed that EMT cells had greatly increased contraction compared with control cells. These results suggest that the increased contractile activity induced by TGF-β in EMT cells may be attributable to the upregulation of molecules responsible for myosin phosphorylation/de-phosphorylation.
A 2-year-old, exotic shorthair cat presented with baldness and mild scaling on trunk that was confirmed as Microsporum canis (M. canis) infection by the following methods. Wood’s lamp and trichogram were used to demonstrate fungal elements suggestive of dermatophytosis consistent with M. canis. Dermatophyte test medium (DTM) and polymerase chain reaction (PCR) were used for identification. E-test and broth microdilution test were then utilized to estimate antifungal minimal inhibitory concentrations (MICs) towards ITZ and TRF respectively. The strain was isolated from the patient and revealed TRF MIC >32 µg/ml and ITZ MIC 0.023 µg/ml. Patient was cured of dermatophytosis with systemic ITZ.
Tick-borne encephalitis (TBE) and severe fever with thrombocytopenia syndrome (SFTS) are both tick-borne zoonotic diseases caused by TBE virus (TBEV) and SFTS phlebovirus (SFTSV). In 2016, a second domestic TBE case was reported in Hokkaido, Japan, after an absence of 23 years. We conducted IgG ELISA for TBEV and SFTSV on 314 deer (Cervus nippon yesoensis) serum samples collected from 3 places in Hokkaido. There were 7 seropositive samples for TBEV but none for SFTSV by ELISA. The specificity of the 7 positive samples was confirmed by neutralization tests against TBEV, and 5 sera showed 320 to 640 of 50% focus reduction endpoint titers. Our results provide information about the infectious status of TBEV in wild deer in Hokkaido, Japan.
The objective of this study was to evaluate the efficacy of a non-selective COX inhibitor (tolfenamic acid) and a selective COX-2 inhibitor (robenacoxib) for post-operative pain control in cats. Thirty cats undergoing ovariohysterectomy were randomly divided into three groups: the control (placebo) group, the tolfenamic acid (4 mg/kg/day) group, and the robenacoxib (1 mg/kg/day) group. Non-steroidal anti-inflammatory drugs (NSAIDs) were administered orally 2 hr before anesthesia induction and 24 and 48 hr post-operation. Buccal mucosal bleeding times (BMBTs) were assessed prior to anesthesia induction. Colorado pain scores and composite pain scores were evaluated in a blinded fashion before induction and 2, 8, 24, 30 and 48 hr post-operation. The Colorado pain scores of cats receiving robenacoxib were significantly lower than those of cats in the control group at 30 (P=0.0126) and 48 (P=0.0439) hr post-operation. The composite pain scores of cats from the robenacoxib group were lower than those of cats in the control group at 30 (P=0.0299) and 48 (P=0.0103) hr post-operation. The Colorado pain scores of cats receiving tolfenamic acid were significantly lower than those of cats in the control group at 30 hr (P=0.0186) post-operation. The composite pain scores in cats in the tolfenamic acid group were lower than the scores of cats in the control group at 24 (P=0.0403) and 48 (P=0.0413) hr post-operation. BMBTs remained within normal limits in all groups. Both tolfenamic acid and robenacoxib are useful for post-operative pain control in cats.
A 5-year-old castrated male poodle presented with blindness. Ophthalmic examinations including slit-lamp biomicroscopy, tonometry, ultrasonography, and electroretinography were performed. The anterior lens capsule of the right eye (OD) was totally pigmented, with persistent pupillary membranes (PPMs). Ultrasonography of the same eye showed severe lens atrophy and retinal detachment. Electroretinography revealed flat a- and b-waves in OD, but normal amplitudes in the left eye (OS). No ocular defects were detected in OS except mature cataract. In this case, it was determined that hypermature cataract with PPMs caused both lens-induced-uveitis and total anterior lens capsule pigmentation. This condition needs to be differentiated from absent pupil. Notably, PPMs with total anterior lens capsular pigmentation are extremely rare in dogs.
The aim of this paper is to report two cases of sternal dislocation (SD) in cats and the long-term outcomes with and without surgery. In a cat with poly-traumatized SD (Case 1), mandibular, radial, and ulnar fractures were corrected first, and the SD was allowed to heal without intervention for 14 months. However, normal healing did not occur and sternal instability remained. Therefore, the SD was corrected surgically, and the cat recovered fully within 4 weeks. In a cat with isolated SD (Case 2), surgery was performed, and normal posture and gait were regained after 5 weeks. Furthermore, in both cases, no postoperative complications were observed during follow-up. Therefore, surgical correction of SD in cats is recommended.
The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100 and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system.
In ophthalmological research, the use of zebrafish to investigate visual behaviors has been increasing, but can produce misleading, false-positive results if compounds adversely affect their motor functions or central nervous system. Therefore, histological analysis to identify a target organ is important in zebrafish toxicity assay. We investigated the retinal degeneration in zebrafish, using typical retinal toxicants, mainly sodium iodate and N-methyl-N-nitrosourea (MNU). No histopathological changes were found after sodium iodate exposure at 1.0 mM for 5 or 7 days in the retina of larval, juvenile, and adult zebrafish. There were also no obvious histopathological changes in the retina of adult zebrafish at 0.1 mM, even after 30 days treatment with sodium iodate. In addition, many proliferating cell nuclear antigen-positive cells were found not only in the ciliary marginal zone, but also in the outer nuclear layer, especially in larval and juvenile zebrafish with or without sodium iodate exposure. However, the concentrations of iodine in the blood and the eyeballs of adult zebrafish increased remarkably after the treatment. General retinal damage emerged after MNU exposure at 150 mg/l for 60 min in adult zebrafish, but first pyknotic cells appeared in the inner nuclear layer and the ganglion cell layer. Our findings indicate that zebrafish retina have a different reactivity pattern from mammalian animals against some retinal toxicants, and in them it is difficult to detect histopathological changes.
In 2013, the first case of Taiwan ferret badger rabies virus (RABV-TWFB) infection was reported in Formosan ferret badgers, and two genetic groups of the virus were distinguished through phylogenetic analysis. To detect RABV-TWFB using a sensitive nucleic acid-based method, a quantitative real-time reverse transcription polymerase chain reaction targeting the conserved region of both genetic groups of RABV-TWFB was developed. This method had a limit of detection (LOD) of 40 RNA copies/reaction and detected viral RNA in brain and ear tissue specimens of infected and dead Formosan ferret badgers and mice with 100% sensitivity and specificity. The mean viral RNA load detected in the ear tissue specimens of ferret badgers ranged from 3.89 × 108 to 9.73 × 108 RNA copies/g-organ, which was 111-fold to 2,220-fold lower than the concentration detected in the brain specimens, but 2,000-fold to 5,000-fold higher than the LOD of the assay. This highly sensitive technique does not require facilities or instruments complying with strict biosafety criteria. Furthermore, it is efficient, safe, and labor-saving as only ear specimens need be sampled. Therefore, it is a promising technique for epidemiological screening of Taiwan ferret badger rabies.
Influenza B virus has been known to infect humans and other animals, including seals. Vaccination efficacy varies across seasons. Human monoclonal antibodies (mAbs) can be useful for developing novel vaccines, guided by epitope analysis, and can be used therapeutically. Hybridoma technology has been used to make mAbs. Here we evaluated SPYMEG as a fusion partner cell line for human mAb generation specific to influenza B hemagglutinin (HA). SPYMEG is a human/murine myeloma partner cell line that has previously been used to generate human mAbs that recognize the HA of influenza A and B viruses. Peripheral blood mononuclear cells were obtained from 16 volunteers, previously vaccinated with the 2014–2015 trivalent seasonal influenza vaccine, and were fused with SPYMEG to yield hybridomas. The resulting hybridomas were screened for antigen-specific antibody secretion and cloned by limiting dilution. We obtained 32 stable clones secreting anti-influenza B HA human IgG, although most of these clones were obtained from one volunteer (SeaV-29) who had a robust immune response. We conclude that SPYMEG is a good fusion partner cell line, although cloning by limiting dilution may lead to significant loss of hybridomas.
Enzyme-linked immunosorbent assay (ELISA) performed using extensively purified bacterially expressed bovine prion protein (PrP) shows decreased cross-reactivity. We generated a transduced Madin–Darby bovine kidney (MDBK) cell line continuously expressing glycosylphosphatidylinositol (GPI)-anchorless bovine PrP (designated as MDBK ∆GPI protein) by using a lentiviral expression system. The present study also described the method for purifying bovine PrP through sequential culturing without the need for complex purification protocol. Our results showed that the purified bovine PrP could be used as an immunogen for developing anti-PrP monoclonal antibodies. Together, our results suggest that the new GPI-anchorless bovine PrP and its purification method can be used for performing basic studies for employing a cell-based approach.
This study reports a novel adenovirus that was found circulating in pigeons in China. Nucleotide homology analysis of the hexon gene showed a nucleotide similarity of 79.0 and 70.9% with PiAd-2 variant A and PiAd-1, respectively. Phylogenetic analysis suggested that the identified virus, together with PiAd-2 variant, constitutes a monophyletic group (proposed as Pigeon Aviadenovirus B) in the genus Aviadenovirus. The present study contributes to the understanding of the epidemiology, ecology, and taxonomy of adenoviruses in pigeons.
To trace the prevalence of canine distemper virus (CDV) in diarrhoetic dogs, a total of 201 stool samples were collected in the Heilongjiang province of northeastern China from May 2014 to April 2015. The 201 fecal samples were subjected to the detection of CDV by using RT-PCR targeting the partial N gene, phylogenetic analysis based on the complete H gene, and co-infection analysis. Results indicated that 24.88% (50/201) of the samples were positive for CDV. The fifty CDV samples exhibited an overall co-infection rate of 94% (47/50) with four enteric viruses (82%, 41/50) and five bacteria (72%, 36/50). The positivity rate of CDV exhibited differences among regions, seasons, ages and immunization status. Phylogenetic analysis of the complete H genes (n=6) revealed that the CDV strains identified in our study belonged to the Asia-1 group, and showed genetic diversities. These data provide evidence that there are a number of genetically diverse CDV Asia-1 strains circulating in diarrhoetic dogs in northeastern China; the CDV-affected animals exhibit the high co-infection with other enteric viruses and bacteria.
Brown bears communicate with other individuals using marking behavior. Bipedal back rubbing has been identified as a common marking posture. Oily substances are secreted via enlarged sebaceous glands in the back skin of male bears during the breeding season. However, whether apocrine gland secretions are associated with seasonal changes remains unknown. The present study aimed to identify histological and histochemical changes in the secretory status and the glycocomposition of the apocrine glands in the back skin of male bears in response to changes in seasons and/or reproductive status. The apocrine glands of intact males during the breeding season were significantly larger and more active than those of castrated males during the breeding season and those of intact males during the non-breeding season. Lectin histochemical analyses revealed a more intense reaction to Vicia villosa agglutinin (VVA) in the cytoplasm, mainly Golgi zones of apocrine cells during the breeding season among castrated, compared with intact males. Positive staining for VVA was quite intense and weak in intact males during the non-breeding and breeding seasons, respectively. Ultrastructural analysis revealed VVA positivity in the Golgi zone, especially around secretory granules in apocrine cells. Changes in lectin binding might reflect a change in the secretory system in the apocrine cells. The present histological and histochemical findings of changes in the secretory status and glycocomposition of the apocrine glands according to the season and reproductive status suggest that these glands are important for chemical communication.
Although several Edwardsiella tarda infections have been reported, its pathogenic role in marine mammals has not been investigated at the genome level. We investigated the genome of E. tarda strain KC-Pc-HB1, isolated from the false killer whale (Pseudorca crassidens) found bycaught in South Korea. The obtained genome was similar to that of human pathogenic E. tarda strains, but distinct from other Edwardsiella species. Although type III and VI secretion systems, which are essential for the virulence of other Edwardsiella species, were absent, several virulence-related genes involved in the pathogenesis of E. tarda were found in the genome. These results provide important insights into the E. tarda infecting marine mammals and give valuable information on potential virulence factors in this pathogen.