Neospora caninum in cattle: experimental infection with oocysts can result in exogenous transplacental infection, but not endogenous transplacental infection in the subsequent pregnancy

Whilst it is presumed that infection of pregnant cattle with Neospora caninum oocysts can provoke abortion and is the likely cause of epidemic abortion outbreaks, only two previous experiments have involved inoculation of pregnant cows with oocysts (and only one abortion was provoked in 22 pregnancies). Here, we describe the oral oocyst challenge of 18 cows synchronously bred and inoculated precisely at 70 (n = 6), 120 (n = 6) and 210 (n = 6) days in pregnancy with a nominal dose of 40,000 oocysts. Only one abortion occurred (at the 120 days challenge) which could be definitively ascribed to N. caninum and no transplacental infection (TPI) was detected in any of the other 11 calves born in the 70 and 120 day challenge groups. In contrast, 4/5 live calves born to cattle challenged at 210 days were transplacentally infected. When cows which had transplacentally infected their calves in the first pregnancy were rebred, no TPI occurred. The results show that the timing of challenge influences clinical and parasitological outcomes and that cattle in late pregnancy are exquisitely sensitive to oocyst challenge leading to exogenous TPI and congenitally infected calves. However, cattle which were indisputably systemically infected in their first pregnancy did not induce endogenous TPI in their subsequent pregnancy. This confirms previous results with experimental tachyzoite challenge and suggests that post-natal infection does not lead to persisting infections which can recrudesce in pregnancy.     

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE

Identification of differentially expressed proteins during Neospora caninum tachyzoite–bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5‐fold in relative abundance, p<0.05, t‐test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde‐3‐phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.     

Regional distribution of bovine Neospora caninum infection in the German state of Rhineland-Palatinate modelled by Logistic regression

To obtain a rapid overview over the distribution of bovine Neospora caninum-infections in the German state of Rhineland-Palatinate, an ELISA to determine specific bovine antibodies against a p38 surface antigen of N. caninum tachyzoites was modified to examine bulk milk samples from cattle herds. Experimental bulk milk samples were used to demonstrate that the seroprevalence in a group of animals can be estimated with this ELISA. A cut-off was selected for the specific detection of herds having a seroprevalence ≥10%. About 90% of the dairy herds located in Rhineland-Palatinate were examined. An overall prevalence of bulk milk-positive herds of 7.9% (95% confidence interval 7.0–8.9%), respectively, was determined. Major regional differences in the distribution of bulk milk-positive herds were observed. Prevalences were higher in regions with an increased degree of urbanisation. Logistic regression was applied to model the prevalence of bulk milk-positive herds on a district and city level. Variables describing the dog density, mean temperature in July, mean temperature in January and the total yearly precipitation in districts and cities were able to explain most of the observed variability in the regional prevalences. Our results provide evidence that in addition to risk factors related to individual farms also risk factors related to the farm location such as dog density in the surrounding and climate factors are important in the epidemiology of bovine neosporosis.     

Serum antibodies against Toxoplasma gondii and Neospora caninum in southeast Queensland dugongs

The dugong (Dugong dugon) is an herbivorous marine mammal that inhabits tropical inshore waters and thus may be vulnerable to pollutants and terrestrial pathogens as a result of coastal runoff. In this study, serum samples collected from live, wild dugongs (n = 114) in an embayment located on the urbanized southeast Queensland coast of Australia during 2008–2014, were measured for IgG antibody levels specific to Toxoplasma gondii and Neospora caninum. An ELISA used to measure T. gondii tachyzoite antibodies indicated a non-Gaussian distribution of antibody level, with five dugongs identified as high outliers. Mean levels of antibodies specific for T. gondii in dugongs sampled in 2014 were significantly higher than in 2010 (p = .006) and 2011 (p = .009) with an elevation in mean antibody levels after a major 2011 flood event relative to antibody levels prior to the flood (p < .0001). A competitive ELISA to detect N. caninum antibody indicated a normal distribution of antibody with no high outliers. Mean antibody level for N. caninum was highest in 2012 and declined significantly in 2014 (p = .004). This is the first survey of antibodies directed against T. gondii and N. caninum in dugongs and suggests future health monitoring of this species.     

Oocysts of Neospora caninum, Hammondia heydorni, Toxoplasma gondii and Hammondia hammondi in faeces collected from dogs in Germany

Faecal samples of 24,089 dogs were examined coproscopically in two veterinary laboratories in Germany between March 2001 and October 2004. In 47 dogs, oocysts of 9–14 μm size were found. Their morphology was similar to those of Hammondia heydorni and Neospora caninum. Samples of 28 of these dogs were further examined by inoculation into gerbils: seven isolates induced a specific antibody response against antigens of N. caninum NC-1 tachyzoites. This response suggests that the isolates contained N. caninum. In addition to H. heydorni (12 times isolated), Toxoplasma gondii occysts (twice) and Hammondia hammondi oocysts (twice) were observed in dog faeces. The latter findings suggest that coprophagia with a subsequent intestinal passage by dogs plays a role in the dissemination of coccidian parasites for which cats are definitive hosts. Five of the seven N. caninum (NC-GER2, NC-GER3, NC-GER4, NC-GER5, NC-GER6) and the two T. gondii isolates (TG-dgGER1, TG-dgGER2) were successfully passaged into cell culture and are now available for detailed characterization. In contrast to oocysts of other parasites, N. caninum oocysts were predominantly found between January and April (Fisher exact; P=0.038). In the sera of dogs shedding N. caninum, no reactions against the immunodominant antigens with apparent molecular weights of 19, 29, 30, 33 and 37 kDa of N. caninum tachyzoites were observed 3–5 weeks after shedding. However, the animals recognized a 152-kDa N. caninum antigen. Compared with those identified as H. heydorni, T. gondii or H. hammondi, N. caninum oocyst isolates were significantly smaller in length with the 75th percentiles ≤10.7 μm when measured in concentrated sucrose solution and smaller length–width ratios with the 75th percentiles ≤1.06. It may thus be possible to develop criteria for a preliminary identification of N. caninum in dog faeces based on the oocyst morphology.     

Review of Neospora caninum and neosporosis in animals

Neospora caninum is a coccidian parasite of animals. It is a major pathogen for cattle and dogs and it occasionally causes clinical infections in horses, goats, sheep, and deer. Domestic dogs are the only known definitive hosts for N. caninum. It is one of the most efficiently transmitted parasite of cattle and up to 90% of cattle in some herds are infected. Transplacental transmission is considered the major route of transmission of N. caninum in cattle. Neospora caninum is a major cause of abortion in cattle in many countries. To elicit protective immunity against abortion in cows that already harbor a latent infection is a major problem. This paper reviews information on biology, diagnosis, epidemiology and control of neosporosis in animals.     

Seroprevalence of antibodies to Neospora caninum in dogs and raccoon dogs in Korea

Neospora caninum is an important cause of abortion in cattle, and dogs are its only known definitive host. Its seroprevalence among domestic urban and rural dogs and feral raccoon dogs (Nyctereutes procyonoides koreensis) in Korea was studied by indirect fluorescent antibody test (IFAT) and by the neospora agglutination test (NAT), respectively. Antibodies to N. caninum were found in 8.3% of urban dogs and in 21.6% of dogs at dairy farms. Antibody titers ranged from 1:50 to 1:400. Antibodies to N. caninum were found in six (23%) of 26 raccoon dogs. However, the potential role of raccoon dogs as a source of horizontal transmission of bovine neosporosis needs further investigation. The results of this study suggest that there is a close relationship between N. caninum infection among dairy farm dogs and cattle in Korea. This study reports for the first time upon the seroprevalence of N. caninum infection in raccoon dogs in Korea.     

On the Phospholipid Metabolism of Glial Cell Primary Cultures.

Abstract: Primary cultures were prepared from newborn rat brain. After 16-18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1-[1-³H]alkyl- sn -glycero-3-phosphoethanolamine (1-alkyl-GPE), for 1–20 h. Five main products were formed: 1-alkyl-2-acyl-GPE; 1-alkyl-2-acyksn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC); 1-alkenyl-2-acyl-GPE (ethanolamine plasmalogen); 1-alkenyl-2-acyl-GPC (choline plasmalogen); and 1-alkyl-glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmaiogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl-, 27.5% alkyl-acyl-, and 46.0% alkenyl-acyl- compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1-alkyl-glycerol was a minor reaction.     

Detection of the protozoan Neospora caninum Using in situ Polymerase Chain Reaction

A new method to detect the protozoan Neospora caninum using indirect in situ polymerase chain reaction (PCR) is described. In situ PCR combines the advantages of the extraordinarily high sensitivity and specificity of PCR and the in situ representation of immunohistochemical methods. We describe an indirect in situ PCR, whereby the amplified products were detected using a primed in situ (PRINS) reaction with hapten-labeled nucleotides and visualized using fluorochrome-labeled antibodies. This technique was carried out in both infected cell cultures and formalin fixed, paraffin embedded tissues. Clear signals were obtained in the N. caninum positive samples using in situ PCR, whereas control slides with Toxoplasma gondii infected tissues always yielded negative results.     

Reduction in transplacental transmission of Neospora caninum in outbred mice by vaccination

Infection with the protozoan parasite Neospora caninum is an important cause of abortion in cattle. A major source of infection is transplacental transfer of the parasite from mother to offspring during pregnancy. This study describes investigations on the immunisation of outbred Qs mice before pregnancy with live or a crude lysate of N. caninum (NC-Nowra isolate) to prevent transplacental transfer of a challenge infection administered during pregnancy. Parasites present in the brains of pups from mice challenged with N. caninum (NC-Liverpool) were detected by PCR. Injection of live NC-Nowra tachyzoites before pregnancy dramatically reduced transplacental transfer from 75 to 0.8% in one experiment and from 76 to 8% in a second experiment. Injection of a crude lysate of NC-Nowra tachyzoites reduced transplacental transfer from 67 to 53% in one experiment and from 76 to 63% in a second experiment. Analysis of N. caninum-specific IgG1 and IgG2a antibody levels prior to pregnancy and challenge showed that NC-Nowra lysate induced a response skewed towards IgG1 whereas live parasites induced both IgG1 and IgG2a antibodies. After pregnancy and a challenge infection, a similar IgG1/IgG2a response was seen in all challenged groups. These results provide further positive support for the hypothesis that transplacental transmission of this parasite is preventable by vaccination.     

Detection of IgG antibody against Neospora caninum in cattle in Korea

A total of 492 cattle sera was screened by IgG-ELISA against Neospora caninum (Nc-1 strain and a Korean isolate, KBA-2) and Toxoplasma gondii. Out of 492, 113 sera (23.0%) reacted positively to either Nc-1 or KBA-2 strains of N. caninum. Among the 113 positive sera, 92 sera (81.4%) reacted with antigens of both strains, but 6 sera (5.3%) with Nc-1 and 15 sera (13.3%) with KBA-2 strain only. And with T. gondii antigen, 6 sera (1.2%) were positive but all reacted with N. caninum antigen also. Western blot revealed typical binding pattern according to ELISA values, such that high OD group reacted specifically to the major surface proteins including 43 kDa protein. Seroprevalence of 23.0% indicates that neosporosis seemed to be one of major causes of abortion in cattle. It is suggested here to establish more epidemiological researches nationwide systematically.     

First demonstration of protective immunity against foetopathy in cattle with latent Neospora caninum infection

The parasite Neospora caninum is an important cause of abortion in cattle world-wide. Chronically infected dams transmit the parasite transplacentally and infected foetuses may be aborted or born chronically infected but clinically normal. Chronically infected cows repeatedly transmit the parasite to foetuses in several pregnancies and some may abort more than once suggesting that the immune response in these cattle is compromised during pregnancy. To investigate the nature of the immune response in chronically infected cattle, five naturally, chronically infected cows were challenged with N. caninum tachyzoites at 10 weeks of gestation. No foetopathy occurred and all five delivered live calves at full-term. In four naive pregnant cows challenged at the same time, all four foetuses died within 3-5 weeks of challenge. Of the five live calves born to the chronically infected challenged cows, three were transplacentally infected with N. caninum. The kinetics of the maternal anti-N. caninum antibody responses during gestation suggested that these transplacental infections were not the result of the superimposed challenge, but the result of the recrudescence of the maternal chronic infection-which occurred concurrently in non-challenged, chronically infected pregnant controls. These data provide the first experimental evidence that protective immunity occurs in neosporosis. They also suggest that whilst immunity to a pre-existing infection will protect against an exogenous challenge, this protective immunity will not prevent transplacental infection. This implies that a subtle form of concomitant immunity exists in chronically infected cattle and has important implications for vaccine development.     

Histochemical Method for Detecting Nitric Oxide Synthase Activity in Cell Cultures

NADPH diaphorase histochemistry has been used extensively for detecting nitric oxide synthase (NOS) activity in various cell types including neuronal cell bodies, vascular endothelium, cells of the immune system and epithelial cells. The use of the diaphorase technique in cell cultures to study the induction of NOS has not been investigated. In this paper we report the use of diaphorase histochemistry as a good marker for the detection of NOS activity in cultured cells. This technique can be used in conjunction with other established techniques to determine the presence and activity of NOS in cultured cells.     

NO对金丝桃悬浮细胞生长及金丝桃素生物合成的促进作用研究

一氧化氮 (NO)是近年来发现的一种新型植物信号分子。以硝普钠 (Sodiumnitroprusside ,SNP)为一氧化氮 (NO)的供体 ,研究外源NO对金丝桃悬浮细胞生长及金丝桃素生物合成的影响。试验结果表明 ,金丝桃悬浮细胞在含 0 5和 15 0mmol LSNP的培养基中培养 2 0d后 ,细胞的干重分别为对照组的 140%和50% ;细胞中金丝桃素的含量分别为对照组的 98%和210%。试验结果表明 ,低浓度SNP处理有利于金丝桃悬浮细胞生长 ,而高浓度SNP可以促进金丝桃素的合成。在细胞培养初期 (0d)加入 0.5mmol LSNP并在指数生长后期 (14d)加入15.0mmol LSNP的金丝桃悬浮细胞在培养 2.5d后 ,细胞的干重和金丝桃素的含量分别为对照组的1.4和1.8倍 ,金丝桃素的产量达15.2mg/L ,比对照高3.2倍。SNP对金丝桃悬浮细胞生长及金丝桃素含量的影响可以被NO专一性淬灭剂CPITO(2-4-carboxyphenyl-4 ,4 ,5 ,5-tetramethylimidazoline-1-oxyl-3-oxide)所抑制,说明SNP是通过其分解产物NO影响细胞生长和金丝桃素的合成。试验结果同时表明,在15.0mmol/L的SNP处理下,金丝桃悬浮细胞中的苯丙氨酸解氨酶(PAL)的活性显著升高,推测NO可能通过触发金丝桃悬浮细胞的防卫反应,激活了细胞中金丝桃素的生物合成途径。     

Inhibition of nitric oxide synthase (NOS) underlies aluminum-induced inhibition of root elongation in Hibiscus moscheutos

Aluminum (Al) is toxic to plants when solubilized into Al(3+) in acidic soils, and becomes a major factor limiting plant growth. However, the primary cause for Al toxicity remains unknown. Nitric oxide (NO) is an important signaling molecule modulating numerous physiological processes in plants. Here, we investigated the role of NO in Al toxicity to Hibiscus moscheutos. Exposure of H. moscheutos to Al(3+) led to a rapid inhibition of root elongation, and the inhibitory effect was alleviated by NO donor sodium nitroprusside (SNP). NO scavenger and inhibitors of NO synthase (NOS) and nitrate reductase had a similar inhibitory effect on root elongation. The inhibition of root elongation by these treatments was ameliorated by SNP. Aluminum inhibited activity of NOS and reduced endogenous NO concentrations. The alleviation of inhibition of root elongation induced by Al, NO scavenger and NOS inhibitor was correlated with endogenous NO concentrations in root apical cells, suggesting that reduction of endogenous NO concentrations resulting from inhibition of NOS activity could underpin Al-induced arrest of root elongation in H. moscheutos.     

Activation of High-Affinity Uptake of Glutamate by Phorbol Esters in Primary Glial Cell Cultures

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C, on high-affinity Na(+)-dependent glutamate transport were investigated in primary cultures of neurons and glial cells from rat brain cortex. Incubation of glial cells with TPA led to concentration- and time-dependent increases in the glutamate transport that could be completely suppressed by the addition of the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. The TPA effects could be mimicked by oleoylacetylglycerol and by the diacylglycerol kinase inhibitor R59022. The effects of TPA were potentiated by the Ca2+ ionophore A23187. Under the chosen experimental conditions TPA had no effect on glutamate transport in neurons. We conclude that PKC activates the sodium-dependent high-affinity glutamate transport in glial cells and that it has dissimilar effects on neurons and glial cells.     

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity ^1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection ³. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop ^4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.The principle and protocol of this methodology have been described in the literature ^7,8. Additionally, alternate methodologies to isolate primary microglia are well described ^9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia ¹². However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture ^9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.     

Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.     

Obligatory Relationship Between the Sterol Biosynthetic Pathway and DNA Synthesis and Cellular Proliferation in Glial Primary Cultures

Primary cultures of newborn rat brain, which are composed predominantly of astroglia, were used to examine the relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation. Reduction of the fetal calf serum content of the culture medium from 10 to 0.1% (vol/vol) for an interval of 48 h between days 4 and 6 in culture resulted in a quiescent state characterized by inhibition of DNA synthesis and cellular proliferation. When 10% fetal calf serum was returned to the medium for these quiescent cells, within 24 h DNA synthesis increased markedly. Preceding the rise in DNA synthesis was an increase in sterol synthesis, which occurred within 12 h of the return of the quiescent cells to the 10% fetal calf serum. Exposure of the quiescent cells to mevinolin, a specific inhibitor of sterol synthesis at the 3-hydroxy-3-methylglutaryl-CoA reductase step, completely inhibited the increase in DNA synthesis that followed serum repletion. The increase in total protein synthesis that followed serum repletion was not similarly inhibited by mevinolin. When mevinolin was removed after causing the 24-h inhibition of DNA synthesis, the cultured cells underwent active DNA synthesis and proliferation. Thus, inhibition of the sterol biosynthetic pathway resulted in a specific and reversible inhibition of DNA synthesis and glial proliferation in developing glial cells. These findings establish a valuable system for the examination of glial proliferation, i.e., primary glial cultures subjected to serum depletion and subsequent repletion. Moreover, the data establish an obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation in developing glia.