Comparison of polymerase chain reaction and immunohistochemistry for the detection of Mycoplasma pulmonis in paraffin-embedded tissue.

The polymerase chain reaction (PCR) is a reliable method of detecting specific DNA sequences. The purpose of this study was to develop a PCR method of detecting Mycoplasma pulmonis in paraffin-embedded tissue sections and to compare the sensitivity of this method with that of the avidin-biotin complex (ABC) immunohistochemistry technique. We infected mice intranasally with 10(7) M. pulmonis and sacrificed them 28 days later. Using a published oligonucleotide primer sequence for M. pulmonis, we examined 8-microns paraffin sections by PCR for a 710 base-pair segment of the genome. A sequential 8-microns paraffin section was stained for M. pulmonis, using standard ABC immunohistochemistry methods. By PCR and dot blot hybridization, 60 of 62 paraffin-embedded lungs were positive for M. pulmonis, whereas only 17 of 62 lungs were positive, using the ABC method. In this study, we demonstrated that PCR of paraffin-embedded tissues followed by dot blot hybridization of the PCR products was much more sensitive than the ABC method in identifying mouse lungs infected with M. pulmonis.

Comparison of xylazine with tiletamine-zolazepam (Telazol) and xylazine-ketamine anesthesia in rabbits.

Although widely used to provide short term anesthesia, ketamine-xylazine does not always produce satisfactory anesthesia. We compared the efficacy of ketamine-xylazine to tiletamine-zolazepam-xylazine for producing surgical anesthesia in rabbits. Four of six rabbits receiving ketamine-xylazine and all of the 12 animals given tiletamine-zolazepam-xylazine were anesthetized successfully. The mean surgical anesthesia time in the ketamine-xylazine group was 35 +/- 6 minutes as compared to the tiletamine-zolazepam-xylazine group, 72 +/- 8 minutes (p less than 0.05). There was no significant difference in the interval between the injection of the different anesthetic mixtures and the loss of either the righting reflex, the jaw reflex or the toe web pinch reflex. Respiratory rates and arterial oxygen partial pressure were higher in the ketamine-xylazine group (p less than 0.05). However, in both groups arterial blood pressure and arterial PO2 were lowered, while arterial PCO2 was elevated. No nephrotoxicity occurred. Tiletamine-zolazepam-xylazine provides effective surgical anesthesia in rabbits and in many cases may be preferable to conventional ketamine-xylazine regimen.

Detection of Mycoplasma pulmonis in arthritic joints of rats by indirect immunoperoxidase staining.

Neonatal and 8-week-old rats were inoculated with Mycoplasma pulmonis. A portion of the animals developed polyarthritis. Indirect immunoperoxidase staining was used to identify the localization of M. pulmonis within arthritic joints. M. pulmonis antigen was most often observed within cartilage in the neonatal group and in synovial tissue in the 8-week-old group.

Mycoplasma pneumoniae-induced hydrocephalus in hamsters.

Hydrocephalus was induced in neonatal hamsters after intracerebral inoculation of Mycoplasma pneumoniae. Examination of the ependyma from affected animals by electron microscopy did not reveal mycoplasma. However, in an ependymal organ culture system, M. pneumoniae cytadsorbed to ependymal cells.

Effect of estrogen-promoted bacterial infections of the rat uterus on bioassay of mammalian cell growth factor activities in uterine luminal fluid.

Exogenous estradiol treatment of intact or ovariectomized rats causes accumulation of significant volumes of fluid in the uterine horns. In this report, evidence is presented showing the presence of mammalian cell growth factor(s) in uterine luminal fluid (ULF), along with other data showing that the exogenous estradiol treatment needed to cause significant accumulation of the fluid also facilitates the movement of vaginal origin bacteria into the uterine horns. It is shown that microorganisms infect the uteri of 80% or more of rats administered exogenous estradiol, and that the microorganisms are most probably of vaginal origin; procedures such as ligation of the uterine body above the cervix or antibiotic treatment did not suppress the infections. Administration of different doses of exogenous estrogen by either implantation of a single 25-mg estradiol/cholesterol pellet which causes a 20- to 50-fold elevation of estradiol levels above physiological plasma concentrations, or instead, by a Silastic tube delivery method that elevates levels only 2- to 3-fold above the normal range, resulted in equal frequency of uterine infections and in the appearance of infection at the same time after starting treatment. A number of bacterial species are present in the contaminated ULF, and these are the origins of intracellular products which are potent inhibitors of mammalian cell growth; the presence of these bacterial origin inhibitors interferes with the bioassay of the ULF growth factor activity, and hence, impedes the characterization of the growth factor(s) present in luminal fluid. Characterization of the origins of the growth-inhibiting activities showed that Pseudomonas aeruginosa and Proteus mirabilis are the predominant species present in infected uteri and that both produce exotoxin activities which inhibit growth of mammalian cells in culture; Pseudomonas appears to be the greater producer of cytotoxic activity. Evidence is presented that suggests that the well-known Exotoxin A produced by Pseudomonas may be responsible, in part, for the toxic effects of this organism. Other, as yet unidentified, cell growth inhibitors also may be produced by the bacteria found in ULF. Surgical separation of the uterine body from the cervix allows preparation of ULF which contains no bacteria and substantially reduced levels of growth inhibitors to mammalian cell lines.

Localization of Mycoplasma pulmonis in cartilage.

Transmission electron microscopy of articular cartilage in Mycoplasma pulmonis-infected neonatal rats revealed the presence of mycoplasmas within the matrix and lacunae. The mycoplasmas appeared to have a tropism for the chondrocytes and induced lysis of both the chondrocytes and matrix of the cartilage.

Cytadsorption of Mycoplasma pulmonis to rat ependyma.

Mycoplasma pulmonis was inoculated intracerebrally into neonatal rats. Hydrocephalus was induced, and the lateral ventricles and aqueduct were examined by scanning electron microscopy. Mycoplasmas were observed to be cytadsorbed to the ependymal surface.

Pathogenicity of influenza A virus in ependymal organ culture.

Ependymal organ culture was used as a model to study the effect of influenza A virus on the ciliated ependyma of the rat. One-square-milli-meter portions of cerebellum from newborn rats were harvested in roller tubes containing Ham's F-12 medium calf serum, and glutamine. Ciliary activity was monitored by stereomicroscopy and, after vigorous ciliary motion was established, tubes were inoculated with 0.1 ml of an inoculum containing 35 plaque-forming units (PFU) of neurotropic influenza A virus (WSN strain). Explants were examined by scanning electron microscopy (SEM) at 2 and 7 days and by transmission electron microscopy (TEM) at 7 days. When compared to noninfected controls, SEM showed that at 2 days the density of microvilli and cilia was decreased in influenza A virus-infected explants. At 7 days, the ependymal cells were nearly denuded of cilia and microvilli, and macrophage like cells were frequently resting upon the ependymal surface. TEM showed numerous viral particles, both budding from the cells surface and located extracellularly near the cell surface.

A new model of congenital hydrocephalus in the rat.

Hydrocephalic neonates were observed in a small breeding colony of rats. Normal rats from this colony were obtained and brother-sister mated for seven generations. The overall prevalence of hydrocephalics was approximately 23%; however, in one subline, the prevalence approached 50%. Breeding data suggested the trait to be polygenic. Hydrocephalics could be detected at 1-2 days of age, and survived for 4-5 weeks. Dilatation of the ventricles was restricted to the lateral ventricles. No evidence of developmental anomalies was seen within the ventricles. Preliminary evidence suggested that the pathophysiology may be related to poorly developed veins in the periosteal-dural layers and to underdeveloped pia-arachnoid cells. The hydrocephalus was classified as being of the communicating type.

Inner ear disease characterized by rolling in C3H mice.

A neurologic disease characterized by rolling developed in C3H mice. Pseudomonas aeruginosa was isolated from the middle and internal ears or meninges of affected mice. The principal pathologic finding was aggressive, primary, purulent otitis media, with extension to the inner ear, acoustic nerve, and meninges. Stresses that may have contributed to induction of the disease were not delineated; however, acidification of the drinking water resulted in near elimination of the disease.

Pathogenicity of Mycoplasma pulmonis in ependymal organ culture.

Ependymal organ culture was used as a model to study the effect of Mycoplasma pulmonis on the ciliated ependyma of the rat. Reduced ciliary activity or ciliostasis occurred 48 to 72 h after infection. Scanning electron microscopy showed that numerous organisms were associated with the cytoplasmic membrane of host cells beginning at 24 h, and transmission electron microscopy indicated that M. pulmonis cytadsorbs to the cell surface. Lesions observed in the organ cultures were limited to changes at the cell surface. These changes included reduction in microvilli density, matting of cilia into bundles, collapse of the cilia onto the cell surface, deciliation of the ependymal cells, and flattening of the cell surface. The results indicated that the response of the ependymal cell to M. pulmonis is similar to that which occurrs in Mycoplasma-infected tracheal and oviduct organ culture systems.

Mycoplasma-induced hydrocephalus in rats and hamsters.

Mycoplasma pulmonis, a pathogen of the respiratory tract in rats, was inoculated intracerebrally into neonate rats and hamsters to determine if it would induce lesions in the ependyma. Hydrocephalus was induced in 116 of 120 rats and in 23 of 28 hamsters. The severity of hydrocephalus was greater in the rats than in the hamsters. Hydrocephalus induction occurred only subsequent to inoculation of viable M. pulmonis. At 2 weeks of age, rats became refractory to induction of hydrocephalus. Light microscopy indicated that the hydrocephalus was communicating without an inflammatory response in the ventricles and meninges. Preliminary electron microscopy revealed that amorphous material covered portions of the ependymal surface and that cilia were sometimes matted together. It was suggested that the hydrocephalus was due to ciliary dysfunction or to an imbalance of cerebrospinal fluid secretion and absorption. This M. pulmonis-induced hydrocephalus may be a useful model for elucidating the pathogenesis of certain types of congenital hydrocephalus in humans.