Cri-du-Chat Syndrome Cytogenetically Cryptic Recombination Aneusomy of Chromosome 5: Implications in Recurrence Risk Estimation.

Cri-du-chat syndrome is caused by haploinsufficiency of the genes on the distal part of the short arm of chromosome 5, and characteristic features include microcephaly, developmental delays, and a distinctive high-pitched mewing cry. Most cri-du-chat syndrome cases result from a sporadic de novo deletion that is associated with a low recurrence risk. On rare occasions, however, cri-du-chat syndrome with 5p monosomy can be accompanied by 5q trisomy. This combination is virtually always associated with parental large pericentric inversions. Among previously reported cri-du-chat syndrome cases with 5p monosomy accompanied by 5q trisomy, the aneusomy of chromosome 5 in all but one case was cytogenetically visible using G-banding. When an accompanying 5q trisomy is detected, a significant recurrence risk is expected. We here report on a patient with cri-du-chat syndrome phenotype who initially exhibited a normal karyotype on G-banding but in whom molecular analysis using multiplex ligation-dependent probe amplification and array comparative genomic hybridization revealed a 5p deletion accompanied by a 5q duplication. Parental chromosomal testing led to the identification of a very large pericentric inversion, of which breakpoints resided at the terminal regions of 5p15.31 and 5q35.1. This information was vital for counseling the family regarding the significantly high recurrence risk.

Juvenile Muscular Atrophy of a Unilateral Upper Extremity (Hirayama Disease) in a Patient with CHARGE Syndrome.

CHARGE syndrome is an autosomal dominant congenital anomaly syndrome, and the causative gene is CHD7. We report a patient with a CHD7 mutation who presented with juvenile muscular atrophy of a unilateral upper extremity, a presumably heterogeneous condition that is also known as Hirayama disease. This association has not been previously described. Weakness and atrophy of the hands should be carefully examined in patients with CHARGE syndrome, since Hirayama disease might be a possible complication in adolescent patients with this syndrome.

A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions.

Cross-protection between Haemophilus parasuis serovars 2 and 5 was examined in pigs using a bacterin based vaccine, and subsequently the safety and efficacy of a bivalent vaccine were evaluated. Upon intratracheal challenge of a serovar 2 or 5 strain, pigs immunized with a monovalent vaccine were protected against challenge with a homologous serovar strain, but not with a heterologous serovar strain. Immunization with a bivalent vaccine containing both serovars 2 and 5 bacterins conferred protection in pigs against lethal challenge with each of the serovar strains. A total of 86 pigs from two SPF herds were injected with the bivalent vaccine intramuscularly twice at a four-week interval. No adverse reactions following the vaccination were observed. On day 7 after the second vaccination, vaccinated and non-vaccinated control pigs from herd A were transferred to herd B, where Glasser's disease had broken out. Pigs in the control group developed clinical signs of the disease, and 6 of 8 (75%) pigs died until slaughter, in contrast with only 4 of 46 (9%) pigs in the vaccinated group. In herd C, where there was no outbreak of Glasser's disease, complement fixation antibody titer was raised only in the vaccinated group. A challenge experiment on days 20 and 79 after the second vaccination showed that only the vaccinated pigs were protected. From these findings, the safety and efficacy of the bivalent vaccine were confirmed under laboratory and field conditions.

Intranasally inoculated Mycoplasma hyorhinis causes eustachitis in pigs.

Specific-pathogen-free pigs were experimentally inoculated with Mycoplasma hyorhinis, Pasteurella multocida, or both bacterial isolates to evaluate the role of these bacteria in the pathogenesis of otitis media. Six pigs were inoculated intranasally with 4.4 X 10(8) colony-forming units (CFU) of M. hyorhinis. Twenty-one days later, three of these six pigs were inoculated intranasally with 5.0 X 10(8) CFU of P. multocida. Three additional pigs were also inoculated intranasally at the time with P. multocida alone. Two pigs served as uninoculated controls. Seven days later, all pigs were euthanatized. Histologically, subacute inflammation was found in 10 auditory tubes of six pigs and two tympanic cavities of two pigs inoculated with M. hyorhinis. Immunohistochemically, M. hyorhinis antigens were detected on the luminal surface of eight of 10 inflamed auditory tubes, and ultrastructural examination confirmed mycoplasmal organisms in two pigs. M. hyorhinis was isolated from the inflamed tympanic cavities of two pigs. None of the pigs inoculated only with P. multocida had otitis, and P. multocida was not isolated from the tympanic cavity. These findings indicate that M. hyorhinis can cause eustachitis but rarely otitis media in specific-pathogen-free pigs.

Induction of temporary otitis media in specific-pathogen-free pigs by intratympanic inoculation of Mycoplasma hyorhinis.

OBJECTIVE: To examine whether Mycoplasma hyorhinis inoculated into the tympanic cavity can cause otitis media in pigs. ANIMALS: 17- or 22-day-old specific-pathogen-free pigs. PROCEDURE: Histologic and bacteriologic examinations were performed on specimens from the tympanic cavity and auditory tube at 0, 7, 14, and 25 days after intratympanic inoculation of M hyorhinis (auditory tube cloning strain 14). RESULTS: In M hyorhinis-inoculated pigs, mild to moderate inflammation of the auditory tube and tympanic cavity first appeared at postinoculation day (PID) 7. In pigs euthanatized at PID 14, the degree of inflammation was aggravated. Immunohistochemical analysis revealed M hyorhinis antigens on the luminal surface of the auditory tube and tympanic cavity. By PID 25, lesions had lessened. By use of transmission and scanning electron microscopic examinations, mycoplasmal organisms were identified among the cilia in the auditory tubes at PID 14 but not at PID 25. Results of bacteriologic examination indicated that 10(4) to 10(6) color-changing units of M hyorhinis were isolated from the tympanic cavity at PID 0. Variable numbers of M hyorhinis were isolated at PID 7 and 14, and numbers were decreased at PID 25. CONCLUSIONS: M hyorhinis inoculated into the tympanic cavity can cause a self-limiting otitis media in SPF pigs.

An avian pathogenic Escherichia coli strain produces a hemolysin, the expression of which is dependent on cyclic AMP receptor protein gene function.

An avian pathogenic Escherichia coli strain M1000 showed a clear zone of erythrocyte lysis on sheep blood agar plates. The hemolytic activity was not detected in the culture supernatant nor was any DNA sequence homologous to the E. coli alpha-hemolysin gene detected in the chromosome or plasmid DNA of the strain, indicating that the observed hemolysis was different from alpha-type. To identify the genetic determinant responsible for the hemolysis, we performed random Tn5 insertional mutagenesis and obtained one mutant, named M5005, that totally lacked the hemolytic activity. Cloning and nucleotide sequencing of the region flanking the transposon insertion site in the M5005 chromosome revealed that the transposon was inserted within an open reading frame of the cyclic AMP receptor protein (CRP) gene, which is involved in one of the global regulatory networks of gene expression in E. coli. Nucleotide sequence analysis of the intact crp gene of the strain M1000 showed that the CRP protein of M1000 is 99% identical to that of K-12. Introduction of the intact crp gene on a low copy plasmid into the mutant M5005 restored the hemolytic phenotype, confirming that the mutation site in M5005 was in the crp gene. CRP plays a central role in catabolite repression, the phenomenon by which the synthesis of many enzymes required to metabolize various sugars is repressed in the presence of glucose. When the hemolytic activity of E. coli M1000 grown in the presence of glucose was examined, the hemolysis was totally impaired. These results indicate that the avian pathogenic E. coli strain M1000 produces a hemolysin the expression of which is dependent on crp gene function.

Natural case of salpingitis apparently caused by Mycoplasma gallisepticum in chickens.

A natural case of salpingitis, apparently caused by Mycoplasma gallisepticum (MG), in layer chickens is described. In the flock from which the chickens originated, there was a 3 to 4% drop in egg production per month around 250 days old. The production was reduced 70% at 400 days of age, which was 77% of the predisease level. Salpingitis was characterized by marked thickening of the oviductal mucosa due to epithelial hyperplasia and marked lymphoplasmacytic infiltration. Colonization of MG on the epithelial surface was evidenced by electron microscopy and immunohistochemistry using rabbit hyperimmune serum against the S6 strain of MG. Antibodies to MG were detected in all the chickens examined..

Ribotyping of Mycoplasma gallisepticum strains with a 16S ribosomal RNA gene probe.

Ribotyping with a 16S ribosomal RNA gene (rDNA) probe was applied to 25 Mycoplasma gallisepticum strains composed of nine originally isolated in the US and UK, and 16 field isolates from Japan. Four distinct ribotypes were identified among the M. gallisepticum strains on the basis of sizes of the bands produced by digesting genomic DNA with restriction enzymes HindIII, EcoRI, BglII and EcoRV. Three ribotypes were recognized among the 16 Japanese isolates. The original and vaccine F strains were classified as the same ribotype, although they produced different banding patterns with BglII, suggesting that the vaccine strain may be a mutant of the original. Other avian mycoplasma species produced banding patterns different from each other and from M. gallisepticum strains. Characterization of M. gallisepticum strains by ribotyping with a 16S rDNA probe may benefit surveillance and epidemiological investigations on M. gallisepticum infection.

Demonstration of Mycoplasma hyorhinis as a possible primary pathogen for porcine otitis media.

A study of the pathology of the ear was performed on 479 pigs ranging in age from 1 day to 1 year. Histologically, 364 (76.0%) of 479 pigs were affected with otitis. Eustachitis was the most common and preceded an inflammation of other sites of the ear, and an acute eustachitis occurred from as early as 1 week of life. Immunohistochemical examination of frozen cryostat sections revealed Mycoplasma hyorhinis (Mhr) antigens on the luminal surface of the eustachian epithelia in 14 (50.0%) of 28 piglets examined. All the pigs positive for Mhr had an acute eustachitis. Ultrastructural examination on the auditory tubes with positive immunostaining disclosed many mycoplasmas among the cilia. Mhr was isolated from the auditory tubes and tympanic cavities of 19 (67.9%) and 16 (57.1%) of 28 piglets examined, respectively. Porcine otitis media may be caused primarily by Mhr infection in the auditory tube.

Occurrence of keratoconjunctivitis apparently caused by Mycoplasma gallisepticum in layer chickens.

Natural cases of keratoconjunctivitis, apparently caused by Mycoplasma gallisepticum (MG), in layer chickens are described. The disease occurred in a commercial flock consisting of 36,000 pullets (Babcock), first appearing around 30 days of age. Clinically, affected chickens showed unilateral or bilateral swelling of the facial skin and the eyelids, increased lacrimation, congestion of conjunctival vessels, and respiratory rales. Some of the severely affected chickens closed their eyes. The morbidity reached 27.8%, and it was estimated that approximately 10% died from reduced feed intake due to impaired vision. Ten 70-day-old chickens with clinical diseases were examined for lesions. There was acute to subacute keratoconjunctivitis in all of the chickens, and some exhibited laryngitis. Adherence of mycoplasma organisms to epithelial cells of the conjunctiva, cornea, and larynx was frequently observed. These organisms had an ultrastructure characteristic of MG and showed a positive reaction with rabbit polyclonal antibodies against the S6 strain of MG by immunohistochemical analysis. MG was isolated from tissue homogenates of the eyelids and tracheas of the affected chickens. Many of the chickens had atrophic bursae, and infectious bursal disease virus antigens were detected in necrotic bursal follicles by immunostaining. Therefore, immunosuppression due to infectious bursal disease was implicated in the pathogenesis of keratoconjunctivitis in the present cases.

Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by PCR.

A PCR assay was developed for the differentiation of toxigenic Pasteurella multocida subsp. multocida strains, the major etiologic agent for progressive atrophic rhinitis in pigs, from nontoxigenic strains. The PCR targeted a toxA gene encoding a 143-kDa dermonecrotic toxin that is considered to be the central etiologic factor in progressive atrophic rhinitis. toxA fragments were amplified from toxigenic P. multocida isolates but not from nontoxigenic isolates or other bacteria isolated from pigs. The sensitivity of the reaction was as low as 10 pg of chromosomal DNA from a toxigenic strain. The results obtained by PCR of the DNAs of 187 field isolates of P. multocida were consistent with those obtained by the guinea pig skin test and Western blot (immunoblot) analysis. Restriction fragment analysis of the PCR-amplified fragments from 67 field isolates and comparison of the DNA sequences of fragments from capsular serotype A and D strains suggest that the PCR-amplified region, which is considered to encode the major immunologic determinants of the toxin, would be the same among P. multocida strains. The PCR that we describe should be useful for the diagnosis and the etiologic survey of progressive atrophic rhinitis.

Isolation of Mycoplasma hyorhinis and Mycoplasma arginini from the ears of pigs with otitis media.

Mycoplasmas were isolated from the middle ears and nasal cavities of clinically ill, young pigs that were slaughtered due to an unfavourable prognosis. The isolation rates of Mycoplasma hyorhinis were 27 of 43 (62.8 per cent) from the auditory tube, 22 of 43 (51.2 per cent) from the tympanic cavity, and 15 of 25 (60.0 per cent) from the nasal cavity. M arginini was also recovered at rates of 11 of 43 (25.6 per cent) from the auditory tube, 7 of 43 (16.3 per cent) from the tympanic cavity, and 9 of 25 (36.0 per cent) from the nasal cavity. Dual infections with M hyorhinis and M arginini occurred in these sites in some cases. M hyorhinis was isolated from 16 (80.0 per cent) of 20 pigs with otitis media diagnosed histologically, and from two (25.0 per cent) of eight pigs without this condition. The isolation rate was significantly different (P < 0.05) between the groups, suggesting either that the organism triggers development of the disease of the ear or that the agent(s) or factors responsible for otitis media in the pigs created favourable conditions at this site for colonisation by M hyorhinis.

DNA sequence analysis of an allelic variant of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxIA) from serotype 10.

The structural gene encoding Actinobacillus pleuropneumoniae-RTX-toxin I (ApxIA), one of the major hemolytic and cytolytic toxins of the organism, was cloned from serotype 10. The nucleotide sequence data showed that the gene from serotype 10 was 98% identical to that from serotype 1 at both DNA and amino acid levels. The sequence difference was found to localize at the 3' terminal region of the gene. We then analyzed the 3' terminal region of the apxIA gene from other serotypes, 5a, 5b, 9 and 11, using polymerase chain reaction-amplified DNA fragments. Results of DNA sequence indicated that apxIA gene can be divided into the original form including serotypes 1, 9 and 11, and the allelic variant including serotypes 5a, 5b and 10. These gene products, ApxIA proteins, appear to have different second structures at the carboxyl terminal proximal region.

hlyIA-like sequence present in a genetic locus with low homology to appA in Actinobacillus pleuropneumoniae serotype 10.

Using the appA gene encoding Actinobacillus pleuropneumoniae-RTX-toxin II (ApxII) as a probe, Southern hybridization demonstrated the presence of appA-sequence in all 12 serotypes except serotype 10. Serotype 10 strains had a genetic locus with low homology to appA (tentatively designated Lha). DNA sequence analysis revealed that Lha contained a part of hlyIA gene encoding a 105-kDa hemolysin ApxI. Southern hybridization demonstrated the presence of Lha (hlyIA) sequence not only in serotype 10 but also in serotypes 1, 5a, 5b, 9, and 11, which had strong hemolytic activity with sheep red blood cells and were highly virulent for mice. These results suggest that ApxI may be associated with the virulence phenotype of the organism.

Seroepidemiology of mycoplasmal pneumonia of swine in Japan as surveyed by an enzyme-linked immunosorbent assay.

Antibodies to Mycoplasma hyopneumoniae were surveyed by enzyme-linked immunosorbent assay (ELISA) on serum, colostrum and milk samples collected from sows and on sera of growing/finishing pigs in Japan. Only one of 196 specific-pathogen-free sows induced a low ELISA value, while 72% of 411 sows from conventional herds were seropositive. A seropositive rate in the conventional sows gradually decreased with an increase in farrowing frequency or with age. In 3267 growing/finishing pigs ranging in age from one to six months, a seropositive rate increased remarkably from the age of 4 months onwards, reaching the maximum at the age of 6 months. A survey conducted on 42 conventional farms revealed that the highest seroconversion occurred when pigs were 4 months of age. The level of maternal antibodies was proportional to that of the dam's colostral antibodies. After maternal antibodies waned, active immunity in newborn piglets from dams with high colostral antibodies appeared earlier and higher than that in piglets from dams with low colostral antibodies. In 950 slaughter pigs, there was a correlation between seropositiveness and the presence of pneumonic lesions, but the ELISA value did not correlate with the degree of the lesions. Pigs that were raised under unfavorable environmental conditions developed pneumonic lesions more frequently than pigs rearing under better conditions, regardless of their immune status. These results suggested that M. hyopneumoniae and some secondary respiratory pathogens may have been involved in the development of these pneumonias.

Comparison of immunity induced with a Mycoplasma gallisepticum bacterin between high- and low-responder lines of chickens.

Chickens of the high-responder line GSP and low-responder line BM-C, which had been known to have different antibody responses to Mycoplasma gallisepticum (MG) antigen, were immunized by intramuscular injection and by subsequent intratracheal instillation of MG bacterin. They were then challenged with the pathogenic strain SAS of MG. The preventive effects of local antibodies detectable in the trachea, saliva, and lacrima were compared between the two lines of chickens. The local antibody responses, as determined by an enzyme-linked immunosorbent assay, were higher in line GSP than in line BM-C before and after challenge. Following challenge, chickens of both lines were protected from colonization by MG and the development of respiratory lesions. The degree of protection in line GSP was higher than that of line BM-C. The results suggest that local antibodies may play an important role in the host defense mechanism to respiratory MG infection.

Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma.

Pathogenicity for chickens of six strains of Mycoplasma gallisepticum isolated from various birds.

Pathogenicity of the type strain and five field strains of Mycoplasma gallisepticum isolated from various avian hosts was evaluated by chicken inoculation. Only two field strains isolated from chickens were highly pathogenic for the chicken respiratory tract.