Labial Vestibular Flap Closure of the Cleft Palate Is Advantageous for Maxillary Development.

Objective: Using labial vestibular flap was performed to close the primary alveolar and hard palate cleft at the second stage of early 2-stage closure surgery for unilateral cleft lip and palate for minimizing the damage to the maxillary periosteum. We analyzed maxillary development to clarify the influence of cleft palate surgery. Design: Retrospective longitudinal study in 5 years after primary palatal closure. Setting: Institutional study Patients: Study subjects included 214 patients with nonsyndromic complete unilateral cleft lip and palate who were consecutively treated in our clinic. Main Outcome: We used a 3D dental model scanner to assess maxillary development in patients aged 3 months to 5 years after using either the conventional pushback method (PB) (51 cases) or 2-stage closure (Local palatal flap closure: LF [67 cases] and Labial vestibular flap closure: VF [96 cases]). Results: Comparing the measurement results, the major axis of maxilla, width, intercanine distance, and intermolar distance was significantly larger in the LF group compared to the PB group. After the age of 3, the cleft side of VF group had grown significantly to compare with LF group in width. It was also confirmed that the inserted labial mucosal flap itself grew. Enlargement of the labial mucosal flap was observed at all sites except the canine. Conclusion: Good maxillary growth occurred in the following order: VF groups > LF group > PB group. Poor growth was correlated with the extent of periosteal damage during surgery and the degree of postoperative bone surface exposure.

Maxillary Development and Dental Arch Relationships Following Early Two-Stage Palatoplasty: A Comparative Study.

OBJECTIVE: To examine skeletal morphology and dental arch relationships at 8 years of age following early 2-stage palatoplasty, which consists of soft palate plasty at 1 year of age and hard palate closure at 1.5 years of age, and to compare the results with those of conventional pushback palatoplasty. DESIGN: Retrospective. SETTING: Single institutional study. PATIENTS: Eighty-six patients with nonsyndromic complete unilateral cleft lip and palate (UCLP) were selected. INTERVENTION: The subjects were divided into 2 groups according to the palatoplasty protocols, as follows: 45 patients, who underwent early 2-stage palatoplasty (ETS group), and 41 patients, who underwent 1-stage pushback palatoplasty (PB group). MAIN OUTCOME MEASURES: Skeletal morphology was assessed using lateral cephalometric analysis, and dental arch relationships were examined using the GOSLON yardstick. RESULTS: Cephalometric analysis revealed that the anterior-posterior length of the maxilla, measured by PTM-A and PTM-ANS, both projected to the nasal floor (NF) plane, was longer in the ETS group than in the PB group (PTM-A/NF, p = .04; PTM-ANS/NF, p = .03, unpaired t-test), although no significant difference was observed in SNA (p = .09, unpaired t-test). Upper posterior facial height was shorter in the ETS group than in the PB group (p = .02, unpaired t). Assessments with the GOSLON yardstick showed that the ETS group presented better dental arch relationships than the PB group (p = 0.04, Mann-Whitney's U-test). CONCLUSIONS: The present results suggested that the ETS protocol reduced the negative effects of palatal surgery on facial development and dental arch relationships in patients with complete UCLP at 8 years of age.

Morphological Correlations in Nasolabial Formation After Primary Lip Repair for Unilateral Cleft Lip.

PURPOSE: We aim to reveal postoperative morphological correlations between the nasolabial components in patients with unilateral cleft lip (UCL). The hypothetical correlations are first, a correlation between the vertical height of the cleft-side alar base and the length of the cleft-side red lip, and second, a correlation between the length of the cleft-side red lip and the vertical position of the cleft-side oral commissure. We explain how these morphological balances are controlled by surgery. METHODS: Three-dimensional bilateral measurements of the length of the red lip, vertical height of the nasal alar base, and vertical height of the oral commissure were conducted retrospectively on 31 patients with UCL and palate (complete UCL: 26; incomplete UCL: 5) who underwent primary lip plasty at Osaka Women's and Children's Hospital from 2017 to 2019. RESULTS: We found a statistically significant correlation between the vertical height of the cleft-side alar base and cleft-side red lip length (P = .012, r = 0.45); thus, the longer the red lip, the lower was the nasal alar base. The correlation between the cleft-side red lip length and the vertical height of the cleft-side oral commissure also showed a statistical significance (P = .00074, r = 0.57); thus, the shorter the red lip, the higher was the oral commissure. CONCLUSIONS: The present results provided objective evidence showing basic morphological relationships between the postoperative nasolabial features of patients with UCL. The results lead to a reasonable approach to define the proposed peak of the Cupid's bow, an unsettled major controversy in cleft lip surgery.

Occlusion at 5 Years of Age Following Hard Palate Closure With Vestibular Flap.

OBJECTIVE: This study aims to assess occlusal relationships and frequency of oronasal fistula at 5 years of age following 2 hard palate closure techniques and to compare results. DESIGN: Retrospective longitudinal study. SETTING: Institutional study. PATIENTS: Study patients included 57 patients with nonsyndromic complete unilateral cleft lip and palate who were consecutively treated. All patients underwent our early 2-stage protocol for palatoplasty, which consisted of soft palate plasty at 1 year of age and hard palate closure at 1.5 years of age. Twenty-nine patients underwent hard palate closure using vestibular flap (VF group) technique (2009-2011) and 28 patients underwent conventional hard palate closure with local palatal flap (LPF group) technique (2006-2008). MAIN OUTCOME MEASURES: Occlusal relationships were assessed with 5-year-olds' index, and frequency of oronasal fistula was investigated. RESULTS: Average 5-year-olds' index scores for VF and LPF groups were 3.11 and 3.57, respectively (P < .001). Oronasal fistula occurred in approximately 7% of patients in the VF group and in 18% of patients in the LPF group. CONCLUSION: Hard palate closure with VF technique may provide better occlusal relationships at 5 years of age than does conventional local closure with the LPF.

Characterization of Highly Pathogenic Avian Influenza Virus A(H5N6), Japan, November 2016.

Highly pathogenic avian influenza viruses (HPAIVs) A(H5N6) were concurrently introduced into several distant regions of Japan in November 2016. These viruses were classified into the genetic clade 2.3.4.4c and were genetically closely related to H5N6 HPAIVs recently isolated in South Korea and China. In addition, these HPAIVs showed further antigenic drift.

Characterization of clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses from wild birds possessing atypical hemagglutinin polybasic cleavage sites.

Since 2014, clade 2.3.4.4 H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have been distributed worldwide. These viruses, which were reported to be highly virulent in chickens by intravenous inoculation, have a consensus HPAI motif PLRERRRKR at the HA cleavage site. However, two-clade 2.3.4.4 H5N8 viruses which we isolated from wild migratory birds in late 2014 in Japan possessed atypical HA cleavage sequences. A swan isolate, Tottori/C6, had a novel polybasic cleavage sequence, PLGERRRKR, and another isolate from a dead mandarin duck, Gifu/01, had a heterogeneous mixture of consensus PLRERRRKR and variant PLRERRRRKR sequences. The polybasic HA cleavage site is the prime virulence determinant of AIVs. Therefore, in the present study, we examined the pathogenicity of these H5N8 isolates in chickens by intravenous inoculation. When 106 EID50 of these viruses were intravenously inoculated into chickens, the mean death time associated with Tottori/C6 was substantially longer (>6.1 days) than that associated with Gifu/01 (2.5 days). These viruses had comparable abilities to replicate in tissue culture cells in the presence and absence of exogenous trypsin, but the growth of Tottori/C6 was hampered. These results indicate that the novel cleavage motif of Tottori/C6 did not directly affect the infectivity of the virus, but Tottori/C6 caused attenuated pathogenicity in chickens because of hampered replication efficiency. It is important to test for the emergence of diversified HPAIVs, because introduction of HPAIVs with a lower virulence like Tottori/C6 might hinder early detection of affected birds in poultry farms.

Cross-protective potential of anti-nucleoprotein human monoclonal antibodies against lethal influenza A virus infection.

The nucleoprotein (NP) possesses regions that are highly conserved among influenza A viruses, and has therefore been one of the target viral proteins for development of a universal influenza vaccine. It has been expected that human or humanized antibodies will be made available for the prophylaxis, pre-emptive and acute treatment of viral infection. However, it is still unclear whether anti-NP human antibody can confer protection against influenza virus infection. In this study, we generated transgenic mice expressing anti-NP human mAbs derived from lymphocytes of a patient infected with H5N1 highly pathogenic avian influenza (HPAI) virus, and experimental infections were conducted to examine antiviral effects of the anti-NP antibodies against H5N1 HPAI viral infections with a high fatality rate in mammals. Transgenic mouse lines expressing the anti-NP human mAbs at more than 1 mg ml-1 showed marked resistance to H5N1 virus infections. In addition, resistance to infection with an H1N1 subtype that shows strong pathogenicity to mice was also confirmed. Although the anti-NP mAbs expressed in the transgenic mice did not neutralize the virus, the mAbs could bind to NP located on the surface of infected cells. These results suggested a possibility that the non-neutralizing anti-NP human mAbs could induce indirect antiviral effects, such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. Taken together, these results demonstrated that anti-NP human mAbs play an important role in heterosubtypic protection against lethal influenza virus infections in vivo.

Influence of Different Palatoplasties on the Facial Morphology of Early Mixed Dentition Stage Children With Unilateral Cleft Lip and Palate.

OBJECTIVE: To compare the maxillofacial morphology in the early mixed dentition phase between two patient groups with unilateral cleft lip, alveolus, and palate who underwent different types of palatoplasty. DESIGN: Cross-sectional study. SETTING: An institutional study. PATIENTS AND INTERVENTIONS: Seventy-one patients with unilateral cleft lip and palate (UCLP) treated at Osaka University Dental Hospital, Japan, were divided into two groups: 33 patients were treated by early two-stage palatoplasty by the modified Furlow Technique (ETS), and 38 patients were treated by one-stage Wardill-Kilner push-back palatoplasty (PB). Intergroup comparisons were performed. MAIN OUTCOME MEASURES: The dental, skeletal, and soft-tissue features were evaluated. For intergroup comparisons, Mann-Whitney U test was used for the statistical analyses. RESULTS: No significant differences existed between the ETS and PB groups regarding the skeletal features except for the mandibular ridge height. Meanwhile, the upper central incisor and upper lip of the ETS group showed a more anterior position than those in the PB group. CONCLUSIONS: Early two-stage Furlow palatoplasty leads to more protruded upper lip, providing more anteriorly positioned upper incisors compared with PB, at least at the early mixed dentition stage.

Validity of the combined use of two esthetic rating systems, the infant index and 5-point aesthetic index, for pre- and postsurgical evaluation of cleft lip repair.

OBJECTIVE: The present study was designed to investigate the usefulness of combining two different ordinal scaling indices, infant index (I-I) and 5-point aesthetic index (5-PAI), for the assessment and prediction of esthetic outcome of primary lip repair for patients with unilateral cleft lip. MATERIALS AND METHODS: The nasolabial appearance of the patients was evaluated before primary lip repair and at 5 years of age using cropped facial photographs with frontal and oblique views. The I-I and 5-PAI employ expanded reference photographs and objective esthetic variables for judgment. RESULTS: The inter- and intrarater Kappa values of both I-I and 5-PAI demonstrated good to very good agreement (range: 0.74-0.84 for I-I and 0.62-0.77 for 5-PAI). Furthermore, both the declination of the columella and the deformity of the alar cartilage in I-I showed a correlation with nasal rating score of 5-PAI and were identified as predictable independent parameters (declination of the columella: Rs = 0.37, P = 0.04; deformity of the alar cartilage: Rs = 0.35, P = 0.02). CONCLUSION: The combined use of I-I and 5-PAI with expanded reference photographs and objective variables could be useful for obtaining greater accuracy of the esthetic assessment and predicting postsurgical nasolabial esthetics at infancy.

Genetic comparisons between lentogenic Newcastle disease virus isolated from waterfowl and velogenic variants.

Avirulent Newcastle disease viruses (NDV) harbored by waterfowl have the potential to become virulent after transmission to and circulation within chicken populations. In order to investigate how virulent viruses are selected from an avirulent background, we compared the complete sequences of the avirulent NDV isolate Goose/Alaska/415/91 and its virulent variant strain 9a5b, which was obtained by nine and five passages in the chick air sac and brain, respectively. Seven amino acid substitutions were detected in the M, F, and HN proteins. Two were detected between variants 9a3b and 9a5b (128P to H and 495E to K in HN protein) that were passed through the brain. Pathogenicity determined by the MDT and IVPI tests also differed between 9a3b and 9a5b. These results suggest that in addition to the F cleavage site sequence, these two amino acids in HN protein are also related to the pathogenicity of NDV in chickens.

Susceptibility of two species of wild terrestrial birds to infection with a highly pathogenic avian influenza virus of H5N1 subtype.

The recent epidemic caused by H5N1 highly pathogenic avian influenza (HPAI) viruses has spread over many parts of Asia, Europe and Africa. Wild birds, particularly waterfowl, are considered to play a role in viral dissemination. However, detailed information on whether wild terrestrial birds act as carriers is currently unavailable. To investigate the susceptibility of terrestrial birds to HPAI viruses, two species of wild bird (great reed warbler and pale thrush) that are common in East Asia were infected with H5N1 HPAI virus. The results showed that both species were highly susceptible to the virus. The great reed warbler showed fatal infection with 100% mortality, but the pale thrush survived for longer periods (>8 days) with viral shedding. These findings suggest that there is variation in clinical outcome after infection of wild terrestrial birds, and that some bird species could become subclinical excretors of the H5N1 virus.

Characterization of H5N1 highly pathogenic avian influenza virus isolated from a mountain hawk eagle in Japan.

On January 4, 2007, an emaciated mountain hawk-eagle was found in Kumamoto Prefecture, Japan. Highly pathogenic avian influenza (HPAI) virus subtype H5N1 was isolated from both tracheal and cloacal swabs of the dead bird. On January 13, an outbreak of HPAI, caused by H5N1 strain, occurred in a chicken farm in Miyazaki Prefecture. Within three weeks, three additional outbreaks had occurred (two in Miyazaki Prefecture and one in Okayama Prefecture). To investigate the relationship between the hawk-eagle isolate and chicken isolates, we studied the virus growth, pathogenicity, and phylogenetic information of this hawk-eagle isolate. The highest virus titer was found in the brain (10(7.25 )EID(50)/g), followed by trachea and muscle (10(2.65) and 10(2.50) EID(50)/g, respectively). Sequence analysis at the hemagglutinin (HA) cleavage site of this isolate revealed a typical virulent-type sequence, R-R-R-K-K-R. Phylogenetic analysis demonstrated that the hawk-eagle isolate belongs to Qinghai Lake type virus group. A homology search of the HA gene also showed major similarity (more than 99%) to the Miyazaki and Okayama isolates in 2007 and also Korean isolates in 2006. These results suggest that Qinghai Lake type H5N1 HPAI virus was newly introduced from Asian Continent into Japan, and had already present in natural environment of Kyusyu district in the beginning of January 2007.

Increase in the neuraminidase activity of a nonpathogenic Newcastle disease virus isolate during passaging in chickens.

A pathogenic mutant of the Newcastle disease virus (NDV) was previously generated by passaging a non-pathogenic isolate from wild waterfowl. Velogenic mutant 9a5b (IVPI=2.67) contains three amino acid substitutions (128H, 495K and 573stop) in the hemagglutinin-neuraminidase (HN) protein, as compared with nonpathogenic waterfowl isolate 415/91 strain, and two of these (128H and 495K) were introduced after mesogenic 9a3b (IVPI=1.88). To investigate the role of the HN protein in NDV virulence, the function of HN protein such as neuraminidase (NA), Hemadsorption (HAd) and fusion promotion activities was examined by introducing the point mutations observed in passaged mutants into the HN gene cDNAs. In vitro functional assay using mutant protein expression demonstrated that the 128H substitution markedly increases NA activity and 573stop substitution increase NA and HAd activities. On the other hand, 495K substitution had little effect on any activities. These results indicate that a single amino acid substitution (128P to H) in the NDV HN protein affects the neuraminidase activity and is possibly correlated with the virulence.

Avian influenza virus and paramyxovirus isolation from migratory waterfowl and shorebirds in San-in district of western Japan from 2001 to 2008.

Surveillance of avian influenza virus and paramyxovirus in migratory waterfowl and shorebirds was conducted in the San-in district of western Japan from the winter of 2001 to 2008. From 4,335 fecal samples from wild birds, 41 avian influenza viruses of 12 different HA and NA combinations, including two H5N3 strains, and 13 avian paramyxoviruses were isolated. Phylogenetic analysis of HA genes revealed that H5N3 strains clustered in a different branch from the recent highly pathogenic H5N1 isolates in Japan; however, the introduction of new highly pathogenic avian influenza virus by migratory birds cannot be ignored. Therefore, it is necessary to continue surveillance of these potentially serious pathogens in waterfowl and shorebirds.

Development of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detection of avian influenza viruses in field specimens.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an established gene amplification method for rapid diagnosis of various infectious diseases. In order to detect avian influenza viruses, particularly in field specimens, specific primers targeting the matrix gene were designed. Thirty-four virus samples, including isolates from wild and domestic avian hosts belonging to various geographical areas, were used to confirm the validity of the primers. All samples were confirmed to be positive in less than 1 hr. The RT-LAMP assay was also able to detect avian influenza virus in the various field samples, such as swabs, tissues, and feces. These results indicate that the developed RT-LAMP assay with uniquely designed primers is potentially useful in comprehensive avian influenza surveillance.

Characterization of Escherichia coli strains obtained from layer chickens affected with colibacillosis in a commercial egg-producing farm.

The present study reports colibacillosis of layer chickens in a commercial egg-producing farm in western Japan. Three flocks of chicken at 18-21 weeks of age were affected during the initiation of egg lay. Postmortem examination revealed pericarditis, perihepatitis, airsacculitis, subcutaneous inguinal lesion, and injured cloaca. Escherichia coli was isolated from the lesions of the affected birds. Twenty-two of 26 E. coli isolates (84.6%) obtained from 18 birds in the 3 flocks showed pulsed-field gel electrophoresis (PFGE) patterns that were considered to be closely associated to each other and arbitrarily designated as pattern A. All the 22 isolates with the PFGE pattern A harbored the putative virulence genes, astA, iss, iucD, tsh, and cva/cvi. Additional 2 PFGE patterns (B and C) were also found in E. coli isolates obtained from the affected flocks and had the putative virulence genes in combinations different from those in the pattern A strains. The results suggested that certain E. coli virulence genes and host factors, such as initiation of egg lay may be associated with occurrence of colibacillosis.

Screening of the enterocin genes and antimicrobial activity against pathogenic bacteria in Enterococcus strains obtained from different origins.

Antimicrobial activities of 139 Enterococcus isolates (48 E. faecium and 91 E. faecalis) obtained from canine feces, boiler meat samples, swine feces, wild waterfowl feces, and human feces were examined against respective bacteria, including Streptococcus pyogenes, Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, Salmonella Enteritidis, and Escherichia coli. Bacteriocin (BAC) production assay revealed that the antimicrobial activity against at least one of 6 indicator strains (BAC+ phenotype) was found in 51 (37%) isolates (29 E. faecium and 22 E. faecalis). Twenty-four of 46 isolates positive for at least one of the enterocin structural genes (entA, entB, entL50AB, and cylL) showed a BAC+ phenotype. The existence of other enterocins or nonenterocin factors was implied because the BAC+ phenotype was detected in a total of 27 Enterococcus isolates that had none of the enterocin genes tested. The antimicrobial activity against Gram-negative strains (Salmonella Enteritidis and E. coli) was detected in the 6 Enterococcus isolates that had either the entA, entB, entL50AB or cylL genes. Moreover, the proportion of the antimicrobial activity against L. monocytogenes among the cylL-positive E. faecalis isolates showing beta-hemolysis (10/16) was significantly (p<0.01) higher than among those lacking beta-hemolysis (2/15). The results suggested that certain characteristics are likely to be associated with the antimicrobial activity against specific organisms.

Strong antiviral activity of heated and hydrated dolomite--preliminary investigation.

Heated and hydrated naturally occurring dolomite showed very strong antiviral activity. Infectivity of avian and human influenza, avian infectious bronchitis (coronavirus), Newcastle disease (paramyxovirus) and avian laryngotracheitis (herpesvirus) viruses dropped at least 1,000 fold following contact with the dolomite for five minutes at 4 degrees C. Dolomite is expected to be useful to inhibit the incidence of emerging and re-emerging infectious diseases.

Inhibitory effects of Enterococcus strains obtained from a probiotic product on in vitro growth of Salmonella enterica serovar enteritidis strain IFO3313.

Enterococcus faecium and Enterococcus gallinarum strains were isolated from a commercial probiotic product and the effects of these strains on the growth of Salmonella enterica serovar Enteritidis strain IFO3313 were investigated. Viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. faecium were significantly (P < 0.05) lower than those in pure cultures after 6, 8, and 24 h when the cultures were incubated in heart infusion broth at 37 and 41 degrees C. Significant differences in viable cell counts of Salmonella Enteritidis in mixed cultures with the probiotic product isolate of E. gallinarum and those in pure cultures were also observed after 8 and 24 h at 37 and 41degrees C. Similar observations were shown in mixed cultures of Salmonella Enteritidis with the reference strains of E. faecium GIFU8355 and E. gallinarum ATCC 49573. Significant differences in viable cell counts of these enterococcal strains were not shown among pure and mixed cultures with Salmonella Enteritidis. The pH values in pure and mixed cultures were 7.0 or 7.5 throughout the experiments. E. faecium strains were found to harbor the genes encoding enterocins A and B and showed inhibitory zones with a diameter of 4 to 6 mm against growth of Salmonella Enteritidis in the enterocin production assays. However, the E. gallinarum strains possessed neither of the enterocin genes tested and exhibited no inhibition zone in the enterocin production assays. These results indicated that enterococcal strains exhibit inhibitory effects on the growth of Salmonella Enteritidis and these effects were due to both enterocin and nonenterocin factors.

Multiplication and motility of Salmonella enterica serovars enteritidis, infantis, and montevideo in in vitro contamination models of eggs.

The invasive ability of Salmonella enterica serovars Enteritidis, Infantis, and Montevideo in eggs was examined. Strains of these serovars originating from egg contents, laying chicken houses, and human patients were experimentally inoculated (0.1-ml dose containing 78 to 178 cells) onto the vitelline membrane of eggs collected from specific-pathogen-free chickens and incubated at 25 degrees C. The test strains were detected in 25 of 138 yolk contents by day 6, indicating the penetration of Salmonella organisms through the vitelline membrane. There were no significant differences in overall rates of penetration between serovars. The organisms were also detected in the albumen from 125 of 138 eggs tested by day 6. Growth to more than 10(6) CFU/ml was observed in 48 of the 125 albumen samples. An inoculum of 1000 Salmonella cells was added to 15 ml of albumen at the edge of a petri plate. A 10-mm-diameter cylindrical well, the bottom of which was sealed with a polycarbonate membrane with 3.0-microm pores, was filled with egg yolk and placed into the albumen at the center of the dish, which was maintained at 25 degrees C. Experiments were performed in triplicate with each strain. Salmonella organisms in all the albumen samples were detected by day 11. However, motility of the organisms toward the yolk was observed in only two dishes inoculated with the Salmonella Enteritidis strain from a human patient and in one dish inoculated with the Salmonella Infantis strain from liquid egg. The albumen samples obtained from the dishes inoculated with the Salmonella Enteritidis strain had high numbers of bacteria (>10(8) CFU/ml). The present study suggests that Salmonella organisms in egg albumen are unlikely to actively move toward the yolk, although depositionon or near the vitelline membrane can be advantageous for proliferation.