Development of a genomics-based PCR assay for detection of Mycoplasma pneumoniae in a large outbreak in New York State.

A genomics-based PCR method was developed and used to test specimens from patients involved in a large outbreak of Mycoplasma pneumoniae in a closed religious community in New York State. New P1 adhesin gene primers were designed to bind to 9 of 10 target sequences in the repetitive-element sequences obtained from the whole genome sequence of M. pneumoniae. This PCR method had a sensitivity of 0.006 CFU and a specificity of 100% for M. pneumoniae. The PCR was validated by testing a subset of patient samples by culture and comparing the results to those obtained by PCR. Of the initial 280 samples tested, 73 were positive by PCR and 22 were positive by culture. All samples positive by culture were also positive by PCR. Follow-up testing of selected patients 3 to 6 weeks after antibiotic treatment revealed that eight samples remained positive by PCR and that three samples remained positive by culture. Additionally, no nonspecific PCR inhibition was detected as a result of the specimen type, transport medium, or sample preparation methodology. The study demonstrates that the PCR described here is a rapid, sensitive, and specific method for the identification of M. pneumoniae and was helpful for the detection and monitoring of the outbreak.

Myelin deficiency in female rats due to a mutation in the PLP gene.

Myelin deficiency (md) in female rats due to a mutation in the X-linked proteolipid protein (PLP) gene is caused by X-chromosome monosomy. Cytogenetic analysis revealed a single X karyotype [41,X(md/0)]. An immunocytochemical, electron microscopic, and biochemical study was performed on male and female md rats. The central nervous system (CNS) of the female md rat [41,X(md/0)] revealed the same total lack of PLP as the CNS of the affected male littermate [42,XY(md/Y)]. Immunocytochemistry for myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP) revealed "islands" of myelin sheath-like reaction product in both. Electron microscopy showed great paucity of compact myelin sheaths in 41,X(md/0) and 42,XY(md/Y). Reduced levels of MPB, MAG, and CNP were confirmed for both sexes but MAG and CNP were substantially higher in 41,X(md/0). Sexual differentiation of the brain may account for the observed differences since normal female reproductive organs are present in the md female rat.

Axonal regrowth in the amyelinated optic nerve of the myelin-deficient rat: ultrastructural observations and effects of ganglioside administration.

It has been postulated that myelin degradation products may inhibit regrowth of mammalian central axons and that central nervous system (CNS) myelin and oligodendrocytes may constitute a "nonpermissive substrate" for axonal growth. To address these issues, we utilized an X-linked rat mutant, myelin-deficient or md. In the optic nerve of this mutant, 40 days and more postnatally, normal myelin is absent and oligodendrocytes are few (Dentinger et al. Brain Res. 344:255-266, 1985). Twenty-eight days before sacrifice, we operated on four groups of 50-day-old md rats and age-matched normal littermates according to the following protocols: 1) unilateral intraorbital optic nerve crush; 2) beginning within 1 hour of nerve crush, daily intraperitoneal injection of GM1 ganglioside (20 mg/kg) dissolved in phosphate-buffered saline (PBS); 3) daily intraperitoneal injection of PBS alone, also begun within 1 hour of nerve crush; 4) severance of the optic nerve immediately behind the papilla 16 or 21 days after the primary crush lesions. Additionally, normal and md rats were killed 4 and 14 days after unilateral optic nerve injury. Nerves of unoperated md rats and their normal littermates were also processed. In the operated animals that did not receive GM1, ultrastructural analysis 4, 14, and 28 days after lesioning revealed that md optic nerves contained significantly greater numbers of regenerating axons, including growth cones and varicosities, than nerves of normal rats. Notably, 28 days postoperatively, (group 1), regenerating axons were still abundant in md nerve, whereas, in nerves of normally myelinated littermates, axonal numbers were diminished markedly. Regenerating optic axons of both md and normally myelinated rats were oriented by linear astrocytic arrays and often were enclosed by astrocytic cytoplasm. In normal littermates, GM1 administration (group 2) induced a significant increase in the number of axons within the operative lesion. Paradoxically, GM1 inhibited the ordinarily robust regeneration of md axons. PBS-injected md and normal rats (group 3) showed no significant differences from noninjected, operated animals. Severance of the nerve at the papilla (group 4) 7-12 days before sacrifice confirmed the origination of axonal regrowth by retinal ganglion cells. The data provide in vivo support for a role of myelin breakdown products or the secretory products of oligodendroglia in the inhibition of regenerative axonal sprouting within mammalian CNS.

Comparative immunocytochemistry of Pelizaeus-Merzbacher disease, the jimpy mouse, and the myelin-deficient rat.

Patients with Pelizaeus-Merzbacher disease (PM), hemizygous mice with the jimpy mutation (jp/Y), and hemizygous rats with X-linked myelin deficiency (md/Y) share a profound lack of proteolipid protein (PLP) in their central nervous systems (CNS). The peripheral nervous system is normal. These X-linked disorders are associated with or actually caused by the lack of normal oligodendrocytes. Vibratome sections of brain were incubated with antisera to myelin basic protein (MBP), myelin-associated glycoprotein (MAG), 2':3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) (EC 3.1.4.37), PLP, a synthetic PLP-peptide, glial fibrillary acidic protein (GFAP), and transferrin. Reaction product was developed by sequential incubation with biotinylated second antibodies, the avidin-biotin-peroxidase complex (ABC), and diaminobenzidine (DAB) plus hydrogen peroxide as chromogenic substrates. In PM, jp/Y and md/Y, islands of myelin-like structures were revealed by antisera to MBP, MAG, and CNP. Reaction product after application of anti-PLP was absent. Reaction product after anti-PLP-peptide was restricted to infrequent bizarre cells possibly representing abnormal oligodendroglia. The lack of oligodendrocytes in jp/Y and md/Y could also be confirmed by immunocytochemistry for transferrin.

Regional variation in the levels of transferrin in the CNS of normal and myelin-deficient rats.

Transferrin (Tf), the iron mobilization protein, is synthesized mainly in the liver. Recently, both Tf and a mRNA for Tf have been demonstrated in oligodendrocytes in the rat brain. The present study used a biochemical assay for determining the levels of Tf in various brain regions of normal rats compared with the level of those obtained from rats with a genetic mutation characterized by an almost complete failure to develop myelin. In myelin-deficient (md) rats, no Tf-positive oligodendrocytes were seen immunohistochemically in the gray or white matter of the CNS. Quantitatively, levels of Tf throughout the CNS of the md rat were decreased to approximately 5% of the normal values despite a normal hepatic synthetic rate. In the normal rat brain, the cerebellum contained the highest concentration of Tf, followed by the pons, the cerebral cortex, and the caudate-putamen, with the latter two sites being similar. Regional variation in the amount of Tf was in general agreement with published reports on the variation of iron and Tf receptor levels in the CNS. Immunohistochemical examination with antiserum to galactocerebroside (a myelin-specific lipid) was used for extending biochemical reports that glycolipid-synthesizing enzymes are deficient in md rats. No immunostaining in the md rat was observed following immunoreaction for galactocerebroside, whereas white matter oligodendrocytes were intensely marked in the normal rat. Robust astrogliosis was present in both the gray and white matter of the md rats. It is not known at present whether the ability to accumulate Tf is necessary for oligodendrocytic survival or if Tf accumulation is more directly related to myelinogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities of central axons in a dysmyelinative rat mutant.

The absence of normal myelin from the CNS of the dysmyelinative rat mutant, md, is associated with axonal abnormalities including organelle-poor and organelle-rich spheroids (OPS and ORS, respectively), wrinkling of the axolemma, persistence of glycogen aggregates and vacuoles in cerebellar mossy fiber terminals, and coalescence of synaptic vesicles in terminal boutons of the nucleus interpositus. OPS have a special predilection for medullary pyramid and the axons of Purkinje cells and further differ from ORS in their possession of nematosomes and in their lack of neurofilaments, microtubules, and degenerating mitochondria. Purkinje cells of md fail to increase in size after 30 days postnatal age and, unlike these neurons in normal neonatal rats, may have massed or dispersed granules of cytoplasmic glycogen which persist for at least 86 days postnatally. Morphometric study of axons of medullary pyramid and cervical corticospinal tract at 19-43 days of age shows a shift in frequency to axons of smaller size in md, as compared to age-matched controls, except that approximately 1% of md axons are larger than any encountered in controls. Finally, the pyramidal axons of md at 43 days of age have a significantly larger area of axoplasm occupied by mitochondria than obtains for the control condition. We conclude that the described abnormalities are secondary to the lack of a myelin investment and/or the loss of oligodendrocytes.

Glial and axonal development in optic nerve of myelin deficient rat mutant.

Development of glial cell lines and axons is reported for the optic nerve of the myelin deficient rat mutant, md, 3-46 days postnatally. In mutants, optic nerves do not increase in area after 16 days of age whereas, in normal rats, they enlarge through 46 days of postnatal life. The density of glial cells, determined in cross-sections, is similar in md and normal littermates through 19 days postnatally. Thereafter, glial densities are greater in the mutant. Nonetheless, total glial counts are reduced in md as compared to the normal, because cross-sectional areas and lengths of mutant nerve 30-46 days after birth are smaller than those of age-matched, normal littermates. Differential counts of glial cells, made by ultrastructural criteria, show that md optic nerves contain abnormal, vacuolated, immature oligodendroglia from the third postnatal day. Furthermore, oligodendrocytes are reduced in number in older mutants; they constitute 1% of optic nerve neuroglia at 46 days. Astrocytic numbers are increased in relative, not in absolute, terms from 19 days, and microglial numbers are greater than normal in the oldest mutants. Reactive microglia, containing large cytoplasmic lipid droplets, constitute 4-8% of the glia of md nerve 19-46 days postnatally. Mean axonal areas are similar in normal rats and mutants at 19 and 43-46 days of age. However, mitochondrial density is greater in md axons 19 days after birth and mean areas of axonal mitochondria are significantly larger in 43-46 day mutants than in age-matched, normal littermates. Additionally, the percent area of axoplasm occupied by mitochondria is increased in md at both 19 and 43-46 days of age. The myelination defect in md appears to be due primarily to an oligodendroglial abnormality which precedes the normal age of onset of myelination. Astrocytic and microglial changes are secondary. Axonal enlargement proceeds normally over 46 days of postnatal life. Overall, the data do not provide definitive support for an axonal basis for the myelination defect, although measurable differences in axonal mitochondria between mutants and normals are demonstrable and qualitative abnormalities do occur in the axons of the mutant.

2',3'-cyclic nucleotide 3'-phosphodiesterase and 5'-nucleotidase in the central nervous system of a myelin-deficient rat mutant.

2',3'-Cyclic nucleotide 3'-phosphodiesterase activity was examined in brains and spinal cords of normal and myelin-deficient Wistar rats. While the activity in normal brains increased from 0.2 mumol/min/mg protein (units) at 6-10 days to 3.5 units at 25 days of postnatal age, the activity in the myelin-deficient rat remained at 0.2-0.3 units over the same period. In spinal cord, the normal activities were 5.7 and 10.9 units at 12 and 20 days, respectively, whereas they declined in the myelin-deficient rat from 1.06 to 0.79 units for the same age points. 5'-Nucleotidase activities in brain and spinal cord were normal in the myelin deficient rat at both ages.

Developmental changes in glycolipid synthesizing enzymes in the brain of a myelin-deficient mutant Wistar rat.

Changes in the activities of UDP-galactose:ceramide galactosyltransferase (CGalT, EC 2.4.1.45), UDP-glucose:ceramide glucosyltransferase (CGlcT, EC 2.4.1.80) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS): galactosylceramide 3'-sulfotransferase (EC 2.8.2.11) over the myelinating period between 12 and 25 days were studied in the brains of control and myelin-deficient rats. Although the activity of galactosyltransferase with ceramides containing hydroxy fatty acids quadrupled in normal male littermates between 14 and 20 days, hardly any increase was observed in the mutant and the activity was less than 10% of control above 20 days of age. With normal fatty acid containing ceramides as acceptors, the activity decreased from 83% of the control at 12 days to approximately 30% after 20 days. Sulfotransferase activity also did not show the normal increase during the 3rd week of life and declined from 60% to 22%. Glucosyltransferase and lysosomal hydrolases in brain and ceramide galactosyltransferase in sciatic nerves appeared to be normal. These results suggest close similarities to the jimpy mutant mouse in which myelin deficiency is also inherited as an x-linked recessive trait.

Ultrastructure of the central nervous system in a myelin deficient rat.

Myelin deficiency (md) is a new mutant in the Wistar rat caused by an X-linked recessive lethal gene. One-half of the male offspring develop tremor and ataxia at 10-12 days of age and seizures at 16-21 days. Usually, the animals die 24-28 days postnatally unless survival is prolonged by anticonvulsants. Light microscopic examination of the C.N.S. shows a complete lack of myelin. The P.N.S. is normally myelinated, however. Frontal cortex, corpus callosum, optic nerves, cerebellum and spinal cord were studied routinely in affected animals aged 3-46 days. Abnormal males were identified three days after birth by the absence of myelinated axons from the ventral funiculus of the cervical cord. In mutants aged 3-16 days, axons had the usual ultrastructural features but were either entirely non-myelinated or, rarely, were invested by poorly organized, non-compacted, myelin-like loops of membranes, 2 to 4 in number. In mutants aged 17-20 days, axonal swellings appeared. These increased in number with longer survival times and contained large numbers of microtubules, neurofilaments, mitochondria and dense bodies. Normal C.N.S. myelin was not observed at any age. Two types of abnormal glial cell occur in md. The first, present in white matter at three days of age, is an abnormal oligodendrocyte. The cytoplasm contains dilatation of the rough-surfaced endoplasmic reticulum and the nuclear envelope is widened. A second cell-type, conspicuous by 10 days, has an electron-dense nucleus with prominently clumped chromatin and large cytoplasmic lipid droplets. This second cell type is believed to be a microgliacyte. The number of cytologically-normal oligodendrocytes decreases as mutants age while hypertrophied, filament-rich astrocytes occur in increasing numbers. The myelin defect in md C.N.S. is probably due to an abnormality of oligodendrocytes. Axonal alterations are probably secondary. Myelin deficiency resembles the murine mutant, Jimpy (jp), although ultrastructural changes in oligodendrocytes appear to be dis-similar and md, in contrast to jp, contains no normal-appearing C.N.S. myelin.

Lipid class analysis of the central nervous system of myelin-deficient Wistar rats.

Brains and spinal cords of myelin-deficient (md) and littermate control rats were analyzed serially for myelin lipids during the period from 13 to 32 days of age. The glycolipids of md rat brains were severely reduced and remained so during the period of study; brain cholesterol and phospholipids increased moderately but never reached the values for control brains. Deficiency of all three lipid classes was marked in the spinal cord and did not change with age. Among the glycolipids of md rats, deficiency was more severe in cerebrosides than sulfatides. The pronounced reduction of cerebrosides in the early stages of myelination suggests that abnormal synthesis of these glycolipids may be the most important biochemical anomaly responsible for myelin deficiency.--Csiza, C.K. Lipid class analysis of the central nervous system of myelin-deficient Wistar rats.

Myelin deficiency (md): a neurologic mutant in the Wistar rat.

Myelin deficiency (md), a newly discovered neurologic mutation in the Wistar rat, is transmitted by an X-linked, recessive lethal gene. Male rats are affected, and the first symptom is a head tremor recognizable at 12 to 15 days of age. The tremors become generalized within a few days and disappear when the animal is at rest. In the later stages, from 17 to 21 days of age, the slightest disturbance will precipitate a generalized seizure. Pups die within 30 days after birth. The only gross postmortem change is a gray color of the spinal cord instead of its normally white appearance. Microscopic findings reveal total lack of myelin formation at all levels of the central nervous system.

Apparent acaridal dermatitis in a C57BL/6 Nya mouse colony.

Dermatitis in a breeding colony of black (C57BL/6 Nya) mice was characterized by intense pruritus leading to self-mutilation and death. The cause appeared to be infestation with the mite, Myobia musculi. Ulceration of the skin resulted in exudation of serum proteins and exposure of the animals' immune defenses to microorganisms and irritants, with subsequent hyperplasia of regional lymph nodes and spleen. Affected mice had an increase in sessile plasma cells, serum immunoglobulin concentration, and percent of circulating granulocytes. The mites were eliminated by treatment with a dichlorvos-ronnel combination in liquid form, which was dispensed onto the bedding with an automatic syringe. The incidence of dermatitis was reduced to zero. Our experience with M musculi in two other strains of mice, both white, indicates that pathogenicity of this mite for mice varies according to the strain, sex, age, and individual differences in sensitivity of the mice as well as the mating ratios employed.

Characterization and serotyping of three feline reovirus isolates.

Three feline virus isolates were shown to be members of the reovirus group by their growth characteristics in cell cultures, physicochemical properties, and appearance under an electron microscope. Their close serological relationship to, or identity with, human reovirus type III was revealed by hemagglutination, hemagglutination inhibition, serum neutralization, and gel diffusion tests. One feline isolate (636) was pathogenic for suckling mice.

Pathogenesis of Feline Panleukopenia Virus in Susceptible Newborn Kittens II. Pathology and Immunofluorescence.

Twenty-three susceptible newborn kittens were inoculated with feline panleukopenia virus on the day of birth and were sacrificed from 18 hr to 43 days postinoculation (DPI). Macroscopic lesions included thymic atrophy in animals examined at 4 to 14 DPI and cerebellar hypoplasia and degeneration in animals examined at 22 to 43 DPI. Clinical signs of ataxia were not observed in the four kittens with cerebellar lesions sacrificed at 22 to 43 days of age. Intranuclear inclusions were present in a variety of cell types in the organs examined from kittens that died or were sacrificed at 4 to 14 DPI. The most severely infected and depleted tissues were the thymus, spleen, mesenteric lymph nodes, and the cerebellum, whereas the bladder, testes, ovaries, and uterus were the least susceptible to panleukopenia virus infection. Specific fluorescence was demonstrated with panleukopenia antiglobulin conjugate in various cell types in tissues from 2 to 22 DPI and only in cerebellar Purkinje cells of kittens sacrificed at 29, 36, and 43 DPI. The virus replicated in the cells of all layers of blood vessels (endothelial, muscular, and connective tissue cells), suggesting that this is the route of dissemination of the agent throughout the body.

Pathogenesis of feline panleukopenia virus in susceptible newborn kittens I. Clinical signs, hematology, serology, and virology.

Inoculation of susceptible newborn kittens with a large dose of panleukopenia virus caused subclinical infection in 19 of 23 cases. All infected kittens developed severe and prolonged leukopenia. Cell-free virus was present in the blood from 1 to 7 postinoculation days. The virus spread to all organs, regardless of the route of inoculation. The thymus, spleen, mesenteric lymph nodes, and the cerebellum were the most severely infected organs. Kittens responded to virus infection by production of specific antibodies, first detectable in the circulatory system 6 to 8 days after infection. Antibody production preceded recovery from leukopenia by 3 days.