The largest area of the avian telencephalon (Tc) is the subpallium [basal ganglia (BG)], and the pallium (cortex) is a narrow area located at the surface of the Tc. However, recent studies have proposed that most of the area of the avian Tc is the pallium, which corresponds to the cerebral cortex of mammals. This theory is based on neuronal elements with little regard to glial cells, which play important roles in neurogenesis. In the present study, we observed the distribution of glial cells using immunohistochemistry during maturation and discuss the division of the Tc by glial elements. In the early stage, the distribution and morphology of vimentin-positive radial glial cells were different between dorsal and ventral areas when they began to spread their processes toward the pia matter. During the development stage, vimentin-positive long processes divide the pallium and BG by the lamina pallio-subpallialis. Moreover, the pallium was divided into four regions by vimentin and glial fibrillary acidic protein-positive elements in the later stage.

View full abstract

Although MSB1 is the most-commonly used lymphoblastoid cell line for isolation of chicken anemia virus (CAV), some researchers have reported a few biological drawbacks. None of them were supported by the results of the present study. Another four avian (HP1, HP2, BK3 and CU10) and two mammalian (BTL-26 and KO-1) cell lines were investigated for susceptibility to the TK-5803 and AH-9409 strains. Both strains caused CPE on BK3 like MSB1. The mean number of positive cells for each strain in MSB1 and BK3 were not significantly different. The majority of the HP2, CU10 and HP1 cells showed no CPE. The virus titers of both strains were higher in MSB1 and BK3 (10^6.5-7.5 TCID₅₀/0.1 ml) than in HP2, CU10 and HP1 (10^3.5-4.5 TCID₅₀/0.1 ml). BTL-26 and KO-1 were resistant to CAV. BK3 could be used for isolation of CAV.

View full abstract

The purpose of this study was to compare the existence of virulence genes in hemolytic Escherichia coli (HEC) and nonhemolytic E. coli (NHEC) isolated from weaner pigs in Thailand, and to determine their susceptibility to 10 antimicrobial agents. A total of 304 E. coli isolates were obtained from 90 piglets with diarrhea and 110 healthy piglets. Of these, 74 HEC isolates were obtained from 70 pigs with diarrhea, and 4 were obtained from 4 healthy pigs, while 190 and 40 NHEC were recovered from 110 healthy and 20 pigs with diarrhea, respectively. A ten digoxigenin (DIG)-labeled probe system was utilized for detecting genes encoding virulence-associated toxins and proteins in these isolates, and the minimal inhibitory concentration values against 10 antimicrobials were determined by means of the agar dilution technique. In total, 70.3% of the HEC isolates contained an exotoxin gene, lth, estp or stx2e, whereas 2.6% of the NHEC isolates hybridized with a gene probe for estp or stx2e. Over 90% of the isolates were resistant to most agents other than colistin and halquinol. The MIC₉₀ values of the HEC isolates for halquinol and colistin were 4 and 8 times greater than those of the NHEC isolates, respectively. The results represent the first characterization of resistant pathogenic E. coli distributed in the Thai pig industry. Amongst the HEC isolates, there appeared to be an association between the presence of some exotoxin genes, including lth, estp and stx2e, and reduced antimicrobial susceptibility.

View full abstract

The antimicrobial susceptibilities of 72 Brachyspira hyodysenteriae isolates collected from clinical cases of swine dysentery (SD) in 11 prefectures in Japan between 1985 and 2009 were investigated by an agar dilution method using five antimicrobial agents. There is a tendency of Japanese field isolates of B. hyodysenteriae to acquire resistance to the main antimicrobials used in SD treatment such as tiamulin, valnemulin, and efrotomycin. A responsible approach for selection and use of antimicrobial agents is required for SD treatment.

View full abstract

We examined the susceptibility in vitro of 99 isolates of Mycoplasma (M. bovirhinis, M. bovis, M. alkalescens and M. bovoculi) and one of Acholeplasma laidlawii collected in 2008 and 2009 from Japanese homebred cattle and cows imported from Australia to 13 antimicrobial agents. Mycoplasma species isolated from homebred cattle were susceptible to spectinomycin and fluoroquinolones but resistant to macrolides, tetracyclines, thiamphenicol and flumequine. Kanamycin, lincomycin and chloramphenicol maintained intermediate effectiveness against these species. The isolates from the imported cows maintained sufficient susceptibility to all antimicrobials examined except for erythromycin and thiamphenicol. These results showed that four species of Mycoplasma isolated from Japanese homebred cattle had obviously higher drug resistance compared with previous reports, even within this decade.

View full abstract

Hemoplasma (hemotropic mycoplasma) often causes hemolytic anemia in infected cats, especially those with immune suppression. An updated nationwide epidemiological survey of feline hemoplasmosis was conducted in Japan. Blood samples were collected from 1,770 outdoor-accessing cats from March to October 2008. The infections were molecularly detected by PCR analyses, which are able to distinguish Mycoplasma haemofelis (Mhf), `Candidatus M. haemominutum' (CMhm), and `Candidatus M. turicensis' (CMt) infections. Of the 1,770 cats, 468 cases (26.4%) revealed a single- or co-infection of feline hemoplasmas [Mhf alone, 42 cases (2.4%); CMhm alone, 280 cases (15.8%); CMt alone, 48 cases (2.7%); Mhf+CMhm, 28 cases (1.6%); Mhf+CMt, 6 cases (0.3%); CMhm+CMt, 50 cases (2.8%); Mhf+CMhm+CMt, 14 cases (0.8%)]. In addition, male gender, middle to old age, history of fight wounds, and feline immunodeficiency virus infection were shown to be risk factors for hemoplasma infection. Close attention must be paid to the acute onset of disease in feline practice because a prevalence of hemoplasma infection was detected even in clinically healthy cats.

View full abstract

Atypical bovine spongiform encephalopathy (atypical BSE) has recently been identified in several countries including Japan. Most cases of atypical BSE have been reported in cattle older than 8 years of age. To clarify the association between age and occurrence of atypical BSE, we investigated both the physiological properties and amount of cellular prion protein (PrP^C) in brain homogenates from young and aged cattle by enzyme-linked immunosorbent assay and immunoblotting. The amount of PrP^C in the brain homogenates was not significantly different between young and aged cattle, but the amount in the detergent-insoluble fraction in the aged cattle was significantly higher than that of young cattle. Significant differences were observed in neither of the glycosylation forms nor in proteinase K sensitivity in young and aged cattle. Age-related changes included deposition of lipofuscin pigment and a decrease of 33% in proteasome activity in the brains of aged cattle compared to that of young cattle.

View full abstract

The chemokine (C-X-C) receptor 1 (CXCR1) expressed on the neutrophil surfaces interacts primarily with interleukin-8 (IL-8) and has an important role in immune response. Two interesting single nucleotide polymorphisms (SNPs), SNP CXCR1+777G>C and SNP CXCR1-1768T>A, that exhibit an association with subclinical mastitis and milk quality in dairy cattle, respectively, have been reported in the bovine CXCR1 gene. The aim of this study was to demonstrate the presence of the two SNPs in the CXCR1 gene of Japanese Black cattle and examine the association between the SNPs and clinical diseases including intestinal and respiratory diseases in calves. Genotyping of the SNPs in healthy Japanese Black cattle showed that the SNPs were also present in Japanese Black cattle with gene frequencies of 0.37 and 0.15 for the C-type allele in SNP CXCR1+777 and for the A-type allele in SNP CXCR1-1768, respectively. Statistical analysis of the genotype distribution of the SNPs in the bovine CXCR1 gene in healthy and clinical intestinal or respiratory diseased Japanese Black cattle indicated no significant association of the SNPs with clinical diseases in the calves. However, a significant correlation of the number of A alleles in SNP CXCR1-1768 with white blood cell (WBC) and platelet counts was found in the disease group. It is possible that the SNP in the bovine CXCR1 gene plays a role in modulating the hematological profile of WBC and platelet counts.

View full abstract

The effect of down-regulation of survivin expression by small interfering RNA (siRNA) against the canine survivin gene on apoptosis was investigated by transfecting MCM-N1 (a canine malignant oral melanoma cell line) cells with siRNA using cationic liposomes. The siRNA against the canine survivin gene induced an increase in the rate of apoptotic cells and a decrease in the number of viable cells. We also measured sequence-specific down-regulation of survivin expression by reverse transcription-PCR and western blot analysis. The siRNA directed against survivin reduced both mRNA and protein expression in MCM-N1 cells. These findings suggest the importance of survivin in canine melanoma tumors for inducing apoptosis, and reinforce the possibility of using survivin as a putative therapeutic target in canine malignant melanoma tumor.

View full abstract

Severe leukopenia was incidentally found in a newborn Japanese Black calf by blood testing during the clinical trial of an iron dextran drug (day 1). At that time, no clinical problems were observed. On day 15, the calf presented with a high rectal temperature and tachypnea. Treatment with antibiotics and non-steroidal anti-inflammatory drugs did not improve clinical signs. Anemia, melena, and prolonged bleeding were also recorded. Necropsy findings revealed subcutaneous petechial hemorrhage and severe bone marrow aplasia. This is the first confirmed case of pancytopenia and hemorrhage associated with bone marrow aplasia in a Japanese Black calf.

View full abstract

Emotional behavior can be characterized by interactions between genes and environments, especially in the developmental period. However, in mice, the effects of paternal care on offspring's emotional development are not well understood even in cases in which the male mice show stable paternal care to the pups. In this study, we used a reciprocal hybrid model of C57BL/6 and BALB/c mice, which show different parental behaviors and emotional responses, and compared the emotional responses in pups in their adulthood by using the elevated plus-maze test. It was found that inbred male mice showed intensive parental behavior toward their pups, especially as parental contact behavior, and that the licking/grooming behavior of males was dependent on the strain of the female they were paired with. Also, BALB/c pairs showed less parental contact behavior than C57BL/6 pairs. In the elevated plus-maze test, BALB/c males and females showed higher anxiety behavior, and reciprocal hybrid offspring showed intermediate values between C57BL/6 and BALB/c mice. Although no significant difference was found in the parental care that offspring received and anxiety-related behavior between the two reciprocal hybrid strains, BALB/c pairs showed less parental contact behavior toward the pups as compared to C57BL/6 pairs, which would be associated with the higher anxiety-related behavior exhibited in the adulthood of BALB/c strain.

View full abstract

Herbs including Curcuma longa, Houttuynia cordata, Prunus mume and Rubus coreanus have potential immune enhancing and antimicrobial effects. Probiotics also have antibacterial effects, and some are important in regulating the immune system. The aims of the present study were to evaluate the immune enhancing effects of a probiotic fermented four-herb combination (PFH) in broiler chicks and to demonstrate the prophylactic effect of PFH against Salmonella Gallinarum in experimentally infected broiler chicks as an initial step towards the development of feed supplements for promotion of immune activity and disease prevention. Continuous ingestion of PFH markedly increased lysozyme activity in serum and the spleen, peripheral blood mononuclear cell (PBMC) proliferation, the CD4⁺:CD8⁺ T lymphocyte ratio in the spleen and antibody production level in broiler chicks. Conversely, prostaglandin E₂ synthesis in serum and PBMC culture medium was significantly decreased in the PFH-fed chicks compared with the control group in a dose-dependent manner. In the chicks experimentally infected with S. Gallinarum, mortality was delayed in the 2% PFH-fed chicks. Moreover, the survival rates in the 2% PFH-fed group remained the highest among all the trial groups throughout the experimental period. Taken together, these findings suggest that PFH enhances immune activity in broiler chicks and increases survivability against Salmonella Gallinarum in experimentally infected broiler chicks, likely because of potent stimulation of nonspecific immune responses.

View full abstract

This study was carried out to determine the effect of Sophy β-glucan on immunity and growth performance in broilers. One group was treated with 1% β-glucan ad libitum with water and the other group was kept as the control. Vaccination for Infectious Bursal Disease was carried out on days 16 and 21. Blood samples were collected from birds, and antibody titres against IBD were measured. The mean body weight of the β-glucan treated group was significantly (P<0.05) higher than that of the control group. The mean antibody titres measured on days 25, 36 and 42 were significantly (P<0.05) higher in 1% β-glucan treated group than that of the control group, suggesting the presence of immune stimulating effect of β-glucan.

View full abstract

To clarify the significance of fecal trimethylamine, lactate and volatile fatty acids (VFAs) in fermentative development of the hindgut in neonatal calves and the occurrence of diarrhea, 143 fecal samples (47 diarrheic, 93 normal and 3 meconium) were collected from 58 dairy calves fed milk. An additional 20 fecal samples were obtained from 10 weaned calves aged 13 to 18 weeks. Fecal pH, trimethylamine, ammonia, lactate and VFAs were analyzed. Compared with weaned calves, the trimethylamine level was higher in milk-fed calves and was associated with a large number of cases of diarrhea, but it had little relationship with the fecal ammonia level. Feces collected from the youngest (<2 weeks age) calves were more acidic and were associated with a higher lactate concentration. Lactic fermentation and fecal acidity were tremendously accelerated, particularly in diarrhea. Despite large fluctuations, the VFA concentrations were lower in diarrheic than in normal feces. In diarrheic feces at the youngest stage, the lower proportion of n-butyrate in the VFAs was accompanied by the elevated proportion of acetate. However, the fermentative differences between the diarrheic and normal feces were less apparent with advancing age. Thus, the fecal lactate and VFA profiles revealed marked changes with advancing age and suffering from diarrhea. Upon comparison with weaned calves, the trimethylamine level was clearly higher in the milk-fed calves and showed huge elevations in diarrheic cases.

View full abstract

To understand species distribution, trends of antimicrobial susceptibility and prevalence of methicillin resistance in canine staphylococci in Japan, 190 coagulase-positive staphylococci (CoPS) were isolated from dogs with pyoderma in 2 Japanese veterinary referral hospitals. Using a multiplex polymerase chain reaction (M-PCR) method, two CoPS species were identified: 170 Staphylococcus pseudintermedius (89.5%) and 20 S. schleiferi subsp. coagulans isolates (10.5%). In these isolates, susceptibility to 7 antimicrobial agents was determined. Overall, the levels of susceptibility to cefalexin (CEX), amoxicillin/clavulanic acid (CVA/AMPC), minocycline (MINO), ofloxacin (OFLX), norfloxacin (NFLX), lincomycin (LCM) and clindamycin (CLDM) in S. pseudintermedius isolates were 38.2, 52.4, 34.7, 31.2, 34.1, 1.2 and 11.2%, respectively. In S. schleiferi subsp. coagulans isolates, 55% demonstrated susceptibility to CEX, 80% to CVA/AMPC, 70% to MINO, 45% to OFLX or NFLX and 30% to CLDM. None of S. schleiferi subsp. coagulans isolates was susceptible to LCM. To determine the prevalence of methicillin-resistant strains, we used a PCR method, which enabled detection of the fragment of mecA gene in 66.5% (113 of 170) in S. pseudintermedius and 30.0% (6 of 20) in S. schleiferi subsp. coagulans isolates. The frequencies of susceptibility to CEX, CVA/AMPC, OFLX, NFLX and CLDM were significantly lower in methicillin-resistant CoPS than in methicillin-susceptible CoPS isolates. These data suggest a high level of methicillin resistance in staphylococci isolated from dogs with pyoderma in Japan.

View full abstract

Ischemic myelopathy is a disorder of acute onset that is characterized by nonprogressive paresis/plegia. Magnetic resonance imaging (MRI) is particularly helpful in supporting the antemortem diagnosis of ischemic myelopathy. Cats, which were suspected to have ischemic myelopathy between 2005 and 2008, were reviewed retrospectively. The acute onset of nonprogressive and nonpainful myelopathy, the conduct of spine MRI, and the availability of complete medical records were included in the inclusion criteria. The presumptive diagnosis was based on medical history as well as clinical, cerebrospinal fluid, and MRI findings. Six cats met the inclusion criteria based on medical records that had been made for about 3 years, and had relatively good prognoses. The less number of patients with ischemic myelopathy has been reported in cats than in dogs. However, the number of cats with ischemic myelopathy in clinical settings appears to be greater than previously considered.

View full abstract

Cryptosporidium parvum HNJ-1 is widely used as a reference strain in Japan. In the present study, the parasite was subjected for further molecular analysis including transcribed ribosomal region (ITS rRNA), dihydrofolate reductase (DHFR) and surface glycoprotein (GP60) genes. Partial sequence analysis of these genes indicated extensive polymorphism in ITS region compared with relevant sequences of other Cryptosporidium parvum isolates. In addition, this strain was identified as C. parvum IIaA15G2R1 subtype, based on the sequence results of GP60 gene locus.

View full abstract

Recently, excessive losses from Marek's disease (MD) have been noted in adult laying flocks over the age of 40 weeks. We defined these late outbreaks in adult chickens as "late MD", and experimentally reproduced the disease in adult SPF P2 line (50-week-old) or commercial line (74-week-old) chickens inoculated with a virulent strain of Marek's disease virus (MDV). Commercial line chickens were given MDV vaccines (HVT and CVI 988) at hatch. The occurrence of MD was evaluated periodically by the evidence of neurologic signs such as paralysis, torticollis, ataxia, and/or nervous tics, as well as histopathological examination. In P2 line chickens, neurologic signs and MD lymphoma were observed from day 21 onward, and they tended to increase in a time-dependent manner. Meanwhile, in commercial line chickens, only one chicken exhibited MD lymphoma on day 70 post inoculation, but its pathogenesis was questionable. No regression of MD lymphoma was noted in either case. The lesions in the visceral organs, thymus, peripheral nerves, and feather pulps of P2 line chickens were characterized by proliferation of variably sized lymphoid cells. In the feather follicle epithelium, numerous inclusion bodies were noted on day 21 post-inoculation, which tended to decrease afterwards. The morphological findings obtained resembled late MD in field cases. In conclusion, our results demonstrate that adult SPF P2 line chickens are susceptible to virulent MDV, and would be useful for investigation of late MD.

View full abstract

Japanese flounder (Paralichthys olivaceus) were experimentally infected with the highly pathogenic scuticociliate Miamiensis avidus (syn. Philasterides dicentrarachi) using the immersion method to clarify/identify the possible neural routes of entry and possible ways of dissemination of the scuticociliate in the fish body. Scuticociliates were observed on the skin and gills right from day 0-1 post-infection, muscle tissue on day 2 post-infection, reached the brain, and spinal cord on day 3 post-infection, and systemic infection was prominent afterwards. Brain lesions were observed in most of the examined fish from days 3 and 4 post-infection and considered to be the cause of the sudden increase in mortality. Affected fish showed varying degrees of tissue damage including severe epidermal and dermal necrotic lesions, necrotic myositis, encephalitis and myelitis. Whereas, scuticociliates were frequently observed along the optic and/or olfactory nerve in the fish which were accompanied by severe brain lesions but by minimum lesions in the gills and skin, suggesting that in addition to skin and/or gills, neural routes including periorbital and nasal routes may play a role in scuticociliate invasion to the brain. Scuticociliates were also observed in the peripheral nerve fibers in the muscle tissue, cranial and spinal nerves, cranial cavity and in the vertebral canal, suggesting that nerve fibers and/or cerebrospinal fluid circulation may be involved in the spread of the scuticociliate throughout the body in addition to the blood circulation and connective tissue.

View full abstract

Estimation of radical scavenging capacity of lipophilic antioxidants by electron spin resonance (ESR) in vitro is a challenging issue due to their poor solubility in aqueous radical generating and measuring systems. Water-miscible organic solvents are used for this purpose. A novel radical trapping agent, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), that has practical advantages over well-known trapping agents was synthesized. However, no available data for the influence of solvents in an ESR system that uses CYPMPO has been presented. The influences of six water-miscible organic solvents, acetonitrile (AcN), acetone, dimethyl sulfoxide (DMSO), ethanol, polyethylene glycol (PEG), and dimethoxyethane (DME), on ESR responses to Fenton Fe²⁺/H ₂O₂ OH· and hypoxanthine/xanthine oxidase superoxide generation systems in vitro were studied. Reduction of the ESR signal to CYPMPO-OH· adducts by 55.86 ± 5.95 and 83.17 ± 2.50% compared with the control was observed in the presence of AcN and acetone, respectively, at a final concentration of 5% (v/v). AcN of less than 1% had minimal effects. DMSO, ethanol, PEG and DME at 5% (v/v) strongly inhibited the ESR signals and/or caused derangement in the signal patterns. The six water-miscible solvents at 5% (v/v) had no influence on the ESR spectra of CYPMPO-superoxide adducts. From these results, AcN, at less than 1% (v/v), is a useful water-miscible organic solvent for assessing radical scavenging capacities of lipophilic compounds in the CYPMPO-Fenton Fe²⁺/H₂O₂ OH· reaction system in an ESR assay. Any of the solvents used in the present study can be used in a hypoxanthine/xanthine oxidase superoxide generation system.

View full abstract

Organ culture of blood vessels is a useful technique to analyze long-term effects of drugs. Various growth factors are responsible for structural and functional changes during vascular remodeling. We investigated 1) basic contractile characteristics in organ-cultured rat mesenteric arteries (MA) in serum-free condition and 2) long-term effects of fetal bovine serum (FBS). Rat isolated MA with [E (+)] or without [E (-)] endothelium were divided into 1) freshly isolated (fresh), 2) cultured for 3 days without FBS (control) or 3) with 10% FBS (FBS). In E (+) control, maximal contraction by noradrenaline (NA), endothelin (ET)-1 and 5-hydroxytriptamine (5-HT) was similar to that in fresh. In E (-) control, maximal contraction by NA decreased whereas that by ET-1 and 5-HT didn't change from fresh. In E (+) FBS, maximal contraction by NA, ET-1, and 5-HT increased from control. In E (-) FBS, maximal contraction by NA and ET-1 decreased whereas that by 5-HT increased. In E (+) or E (-) control and FBS, sensitivity to NA and ET-1 increased from fresh. In E (+) and E (-) control, sensitivity to 5-HT decreased from fresh, and that in FBS further decreased from control. Three-day organ-cultured rat MA in serum-free condition preserved enough contraction to enable analysis for long-term effects of drug. In FBS, maximal contractions by NA and ET-1 increased in E (+) and decreased in E (-) from control, while those by 5-HT increased in both E (+) and E (-).

View full abstract

Cytochrome P450 2D (CYP2D) is one of the major metabolizing enzymes for drugs in humans and other mammals. Although domestic cats (Felis catus) are the most common companion animal in many countries, the properties of their drug-metabolizing system are largely unknown. We determined the complete cDNA sequence of the CYP2D open reading frame (1,500 bp) with both 5'- and 3'-untranslated regions in domestic cats. Feline CYP2D consists of 500 deduced amino acids and shows high homology with CYP2Ds of primates and rodents, particularly with the canine CYP2D homologue, and was designated as CYP2D6 based on its genomic structure. CYP2D6 transcripts were expressed predominantly in the liver and to a much lesser in the testis, suggesting the role of xenobiotic metabolism in the liver.

View full abstract

Adult rat dorsal root ganglion (DRG) neurons cultured in the presence of 100 ng/ml NGF show spontaneous action potentials and fluctuations in their cytosolic Ca²⁺ concentrations ([Ca²⁺]_i). In the present study, the Ca²⁺ sources of the [Ca²⁺]_i fluctuations and the types of neurons whose excitability was affected by NGF were examined. In the subpopulation of NGF-treated neurons, obvious fluctuations of [Ca²⁺]_i were observed. The [Ca²⁺]_i fluctuations were inhibited by Ca²⁺ removal or inhibitors of voltage-gated Ca²⁺ channels. Regardless of the treatment with NGF, about half of the neurons responded to capsaicin and 10% of the neurons responded to icilin, and almost all icilin-responding neurons also responded to capsaicin. Fluctuations of [Ca²⁺]_i with large amplitudes were observed in 12 out of 131 NGF-treated neurons. Among these 12 neurons, 10 neurons responded to both capsaicin and icilin. The degree of the [Ca²⁺]_i fluctuations in the NGF-treated neurons responding to both capsaicin and icilin was significantly larger than in other neurons. These results suggest that neurons expressing both capsaicin- and icilin-sensitive TRP channels are susceptible to NGF and become hyperexcitable and that Ca²⁺ influx through voltage-gated Ca²⁺ channels is the major source contributing to the [Ca²⁺]_i fluctuations. Since such DRG neurons could play a physiological role as nociceptors, the NGF-induced spontaneous activity of DRG neurons may be the underlying mechanism of neuropathic pain.

View full abstract

The melting temperatures of Mycoplasma wenyonii and `Candidatus Mycoplasma haemobos' should be read as 82.04 ± 0.27°C and 86.98± 0.12°C, respectively.

View full abstract

In the original publication of the article, on page 1519 has been published incorrectly as “serine83l →eucine”. The correction is “serine83 →leucine”.

View full abstract