Sera-controlled preadipocyte growth in culture: an ontogeny study with sera from lean and obese pigs

Genetically lean and obese swine were used to investigate the control of preadipocyte growth in culture by porcine serum. Sera were collected from fetuses from obese and lean strains at 70, 90 and 110 d of gestation. Postnatal serum samples were collected from both lines of pigs at 23 to 27 kg. Rat preadipocytes were isolated and grown in culture. Preadipocyte and stromal-vascular cell proliferation was greater in cultures grown in sera obtained postnatally than in cultures grown in sera from fetuses. Sera from lean and obese fetuses were equipotent in promoting cell proliferation. Glycerol-phosphate dehydrogenase (GPDH) activity was higher in cultures fed serum from obese pigs and fetuses than in cultures fed serum from lean pigs and fetuses. Cultures grown in serum from obese fetuses and pigs had soluble protein levels similar to cultures grown with serum from lean pigs and fetuses. These results demonstrate that serum from genetically obese swine, in the pre-obese (fetal) and obese (postnatal) state, caused increased adipogenic activity in adipocytes in culture.     

Variation in clinical and parasitological traits in Pietrain and Meishan pigs infected with Sarcocystis miescheriana

Future prophylaxis needs new concepts, including natural disease resistance of hosts against infectious agents. Genomic approaches to detect and improve disease resistance in farm animals and the molecular mechanisms involved in host-parasite interactions depend to a high degree on the trait differences between founder breeds, i.e. on the animal model. The present study evaluates differences in susceptibility/resistance against Sarcocystis miescheriana in the European Pietrain (PI) and the Chinese Meishan (ME) pig breeds, based on 25 individuals, infected orally with 5x10(4) sporocysts of S. miescheriana. Significant differences appeared in clinical, serological, haematological and parasitological findings. The major discriminating period post infection (p.i.) was between days 42 and 45. Severity of signs was negatively correlated with specific immunoglobulin titres during the first 3 weeks p.i. and positively with the load of bradyzoites in muscle tissues of the pigs. Loads of bradyzoites in muscle tissues were 20 times higher in PI than in ME. Sarcocystis-specific differences between the two breeds were in the range of 1-2 standard deviations. The study lays the foundation for further experiments to analyse chromosomal regions, candidate genes, and thus the molecular basis of Sarcocystis susceptibility/resistance as a model for host-parasite interaction in protozoan infectious disease.     

Fetal hypophysectomy causes a decrease in preadipocyte growth and insulin like growth factor-1 in pigs

Fetal pigs in one uterine horn of each of five gilts were hypophysectomized (HX) in utero by electrical cauterization at 72-74 days of gestation and sera collected at 110 days of gestation. Sera from HX fetuses had lower levels of insulin-like growth factor-1 compared to control littermates (P less than .05). Sera were tested for their effects on primary cultures of stromal-vascular cells from adipose tissue. The soluble protein concentration/dish was lower when pig cells were cultured in sera from HX fetuses compared to sera from control fetuses (P less than .01). Sera from HX fetuses inadequately supported growth of stromal-vascular cells so subsequent experiments utilized pooled sera from normal and HX adult pigs. Sera from HX and control fetuses were mixed with sera from the two adult pools and tested for incorporation of tritiated thymidine into rat preadipocytes and the appearance of adipocytes (determined histochemically) in pig stromal-vascular cultures. In cultures fed sera from HX fetuses there was a lower (P less than .05) number of pig fat cells/culture and a lower level (P less than .06) of preadipocyte proliferation in rat cell cultures when compared to control fetal sera. Fetal pig serum contains factors (adipogenic) which promote the proliferation and differentiation of adipocytes in culture. Serum from HX fetuses has a lower level of adipogenic factors.     

Differential molecular identification of Taeniid spp. and Sarcocystis spp. cysts isolated from infected pigs and cattle

In the present work, the species-specific identification of Taeniid spp. cysticerci and sarcocystis cysts isolated from infected pigs and cattle was achieved by PCR. In particular: (i) multiplex-PCR derived from HDP2 DNA fragment, specific for Taenia saginata/Taenia solium; (ii) PCRs and PCR-RFLPs of the rDNA internal transcribed spacers 1 and 2 (ITS1 and ITS2) for the differential diagnosis of taeniids; (iii) PCR derived from the 18S rRNA gene and sequencing, specific for Sarcoystis spp. The combined application of these three PCR protocols provided an unequivocally specific diagnosis of T. saginata, T. solium, T. hydatigena, Sarcocystis hominis and Sarcocystis suihominis, and may have practical application in the identification of calcified degenerating or morphologically dubious cysts, for example in the slaughter house situation or in human biopsy samples.     

Sarcocystis neurona and Sarcocystis falcatula: monitoring of schizogony in cell culture using fluorescent nuclear labeling

The nuclei of merozoites of Sarcocystis neurona and Sarcocystis falcatula were labeled with the fluorescent marker Syto21. It was shown that the marker would label the parasites and that they would retain the marker throughout schizogony. Thus, there was sufficient marker in the daughter merozoites to make them easily visible with fluorescence microscopy. This technique will be helpful in studying the developmental biology of these parasites in vitro.     

Hematologic values in calves infected with Sarcocystis cruzi

Calves were inoculated with 2 X 10(5) Sarcocystis cruzi sporocysts. Red cell mass decreased dramatically between Days 21 and 35 post-infection and plasma volume increased concurrently, so that blood volume did not change significantly. Mild reticulocytosis and increased pyrimidine 5' nucleotidase activity in erythrocytes occurred between Days 35 and 42. Antiglobulin tests with anti-bovine IgG, IgM and C3 were negative, with the exception of a positive test for C3 in 1 of 6 infected calves.     

Development of sarcocysts of Sarcocystis levinei in water buffalo infected with sporocysts from dogs

Half a million sporocysts of Sarcocystis levinei obtained from experimentally infected dogs were fed to a buffalo calf, and sarcocysts of this species were recovered from its oesophageal muscles when the animal was killed on the 62nd day of inoculation, thus establishing a buffalo-dog-buffalo cycle.     

Sheep experimentally infected with Sarcocystis from dogs. II. Abortion and disease in ewes.

This is the first study of Sarcocystis-induced abortion in sheep. Eleven pregnant ewes were experimentally inoculated with 50,000, 100,000, or 500,000 Sarcocystis ovicanis sporocytis from dogs. Eight ewes either aborted, died, or became moribund before term; they produced 15 fetuses, 11 of normal appearance and 4 necrotic. No evidence of intrauterine transmission was obtained. All infected ewes became anemic, inappetent, and lost weight. Ewes inoculated with the greatest numbers of sporocysts exhibited the most striking signs of acute illness. At necropsy of acutely ill ewes the heart was the most severely affected organ, appearing nearly black as a result of hemorrhagic pancarditis. Less hemorrhage was seen in the kidney, liver, spleen, and skeletal muscles. Microscopically, schizonts were found in capillary endothelial cells of most organs 24 to 33 days after inoculation. Ewes surviving the acute illness appeared generally unthrifty and exhibited the additional signs of wool breaking, and nervous disturbances. At postmortem, the heart and kidneys of these ewes were moderately hemorrhagic, and the adrenal glands and mesenteric lymph nodes were enlarged. Microscopically, sarcocysts were found in the heart, diaphragm esophagus, tongue, skeletal and eye muscles, cerebellum, and cerebrum.     

Development of Isospora suis from pigs in primary porcine and bovine cell cultures

Sporozoites of Isospora suis penetrated and developed by endodyogeny in primary porcine kidney (PPK) and primary fetal bovine kidney (PFBK) cell cultures. Motile merozoites and binucleate Type I meronts were observed in both types of cultured cells. Multinucleate Type II meronts developed in PPK cell cultures only. These multinucleate meronts were always found singly, were nonmotile and did not form merozoites.     

Prevalence of Sarcocystis cysts in pigs and sheep in Spain

Samples of serum and diaphragm muscle were collected from 100 pigs, and serum samples and oesophagi were collected from 100 sheep. The diaphragm muscle and oesophageal tissues were examined for the presence of macroscopic and microscopic Sarcocystis cysts by compression between trichinoscope plates as well as by tissue digestion with pepsin solution. The sera were examined by the indirect haemagglutination test (IHA), using antigens from Sarcocystis gigantea. With these methods, 95% of the sheep and 43% of the pigs were found to be infected with Sarcocystis sp.     

Heart failure in a pig chronically infected with Sarcocystis miescheriana--a case report

A pig infected with 2 x 10(5) sporocysts of Sarcocystis miescheriana which had survived the acute phase of the disease from 12 dpi until 17 dpi retarded in growth and finally died at 60 dpi. From gross pathological examination heart failure was assumed as the cause of death. Histopathologically severe Myocarditis eosinophilica fibrosa was diagnosed. The sections through the heart muscle contained numerous degenerating and some intact sarcocysts.     

Class-specific antibody responses in pigs following immunization and challenge with sporocysts of Sarcocystis miescheriana

Sixteen pigs were each immunized by oral inoculation with 1000 sporocysts of Sarcocystis miescheriana at 8 weeks of age. Four equal groups were then challenged with 3 million sporocysts per animal at 40, 80, 120 or 160 days post-immunization (dpi). Host antibody responses were monitored using the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent-antibody test (IFAT). When using class-specific ELISAs, dramatic increases in specific IgM-antibodies were observed at 21 dpi and increasing levels of IgG-antibodies were detected at 34 dpi. IgM-antibody titres dropped relatively quickly to insignificant levels, whereas IgG-antibody titres persisted at high levels until the end of the experiment. Following challenge, elevated levels of IgM-antibodies were detected, whereas IgG-antibody titres remained unchanged. The dynamics of the antibody titres detected by the IgG-IFAT closely corresponded to those detected by the IgG-ELISA. Despite the presence of specific antibodies at the time of challenge and the continued production of IgM-antibodies after challenge, the protective immunity decreased after 40 dpi and had disappeared by 120 dpi. Furthermore, none of the techniques used were suitable for the detection of specific antibodies during the early phase of acute sarcocystosis around 12 days after challenge.     

Plasma and tissue concentrations and molecular forms of somatostatin in calves infected with Sarcocystis cruzi

The effects of parasitic infection on plasma and tissue content of immunoreactive somatostatin (SRIF) were studied in 4-mo old male calves inoculated with the protozoan Sarcocystis cruzi. Because feed intake significantly decreased (70%) in infected calves around day 28 postinfection (pi), concomitant with the asexual replication of S. cruzi and outward expression of clinical signs, the relative contributions of infection and associated reduction in nutrition on plasma SRIF were evaluated. Treatment groups were: noninfected ad libitum fed (C), infected (250,000 S. cruzi oocysts per os) ad libitum fed (I) and noninfected calves pairfed to the level of intake of each infected calf (PF). Mean plasma concentrations of SRIF (pg/ml) on day 30 pi were: C, 224 ± 22; I, 742 ± 150; PF, 246 ± 31 (effect of infection P<.05). In another study, SRIF was measured in plasma and in pancreatic, duodenal, jejunal and ileal tissue extracts from normal and S. cruzi infected calves. Plasma and tissue samples were collected on day 42 pi. Mean plasma SRIF were 2.5 times higher in infected than control calves. Plasma insulin and insulin-like growth factor 1 was lower in infected v control calves (P<.02). Plasma glucagon was similar between groups. Duodenal (P<.05) and jejunal (P<.02) SRIF content was higher in infected than control calves. Chromatography of tissue extracts on Sephadex G-50 revealed that the increase in SRIF was accounted for, in part, by molecular forms larger than cyclic SRIF-14. Data suggest that peripheral SRIF is increased in calves during S. cruzi infection. The increase in SRIF is not solely related to plane of nutrition. Altered levels of gut SRIF(s) may be associated with perturbed metabolic regulation in parasitized animals through direct effects on the gut.     

Lymphoproliferative responses in pigs infected with Mycobacterium avium

Naturally infected cases of swine mycobacteriosis were divided into two groups, localized infection (LI) and disseminated infection (DI). Lymphoproliferative response (LPR) was then examined to estimate their immunological states. Both control and LI groups showed strong response to Concanavalin A (Con A) and phytohemagglutinin (PHA) in the LPR, and lymphocytes recovered from the LI responded well to purified protein derived from M. avium (PPD). On the other hand, the DI group showed weak response to both Con A and PHA, despite their strong response to PPD stimulation. These data suggest that the low LPR to Con A and PHA observed in the DI groups was probably not due to the general unresponsiveness of T-cells.     

In vitro culture and synchronous release of Sarcocystis neurona merozoites from host cells

The growth of Sarcocystis neurona, isolate UCD1, in continuous culture was examined in 10 cell lines to identify growth conditions and methods for the preparation of parasites free of gross host cell contamination for molecular studies. The unpredictable, slow release of merozoites in most cell lines prompted development of a method to synchronously release the parasites from infected host cells. The calcium ionophore A23187 at a concentration of 1 microM was found to release intracellular merozoites with a 40 min treatment at 37 degrees C. The release of merozoites en masse from attached host cells allowed for the rapid collection of relatively pure parasites from the culture supernatant. This release of merozoites occurred in five different host cell lines. The ionophore-released parasites were highly infectious for host cells and appeared to be morphologically identical to naturally released merozoites, except that the treated merozoites had an increased number of micronemes when examined by electron microscopy. The ionophore did not enhance the release of sporozoites from sporocysts, but freezing in the presence of 5% DMSO released sporozoites that were infectious to bovine monocytes in in vitro culture.     

Optimization of in vitro conditions for bovine subcutaneous and intramuscular preadipocyte differentiation

The objective of these experiments was to develop an in vitro cell culture system for differentiation of bovine preadipocytes, which will permit examination of differences in differentiation between intramuscular (i.m.) and subcutaneous (s.c.) bovine preadipocytes. Stromal-vascular cells from bovine i.m. and s.c. adipose depots were isolated and cultured. Clonally derived s.c. preadipocytes were used to determine the ability of insulin, bovine serum lipids, octanoate, acetic acid, dexamethasone (DEX), and troglitazone (TRO) to elicit differentiation of these cells when added to serum-free medium. Addition of 10 and 20 microL/mL of a commercially available serum lipids supplement to low-glucose Dulbecco's modified Eagle's medium containing 280 nM insulin increased glycerol-3-phosphate dehydrogenase (GPDH) activity (P < 0.01). Inclusion of 1.25 to 10 microM TRO to medium containing 280 nM insulin and 20 microL/ mL serum lipids supplement also increased GPDH activity (P < 0.001) compared with 0 microM TRO. The combination of 280 nM insulin, 1 mM octanoate, and 10 mM acetic acid, with 48 h exposure to 0.25 microM DEX caused morphological differentiation in a small number of cells but did not stimulate GPDH activity (P = 0.99). When used together, 280 nM insulin, 20 microL/mL of serum lipids supplement, 40 microM TRO, and 0.25 microM DEX stimulated differentiation compared with the aforementioned treatment (P < 0.001). Omission of TRO or insulin from this medium reduced GPDH activity by 68% (P < 0.001), whereas removal of DEX tended to reduce GPDH activity (P = 0.06). Preadipocytes from s.c. (n = 3) and i.m. (n = 2) adipose tissues of 3 steers were used to determine the effects of TRO on differentiation using the established conditions. Forty to sixty microM TRO enhanced differentiation compared with 0 microM TRO (P < 0.02) in both depots. No depot differences in response to TRO were detected (P = 0.32). These data demonstrate that bovine preadipocytes are capable of differentiation in response to combinations of insulin, serum lipids, DEX, and TRO. Although TRO enhanced differentiation of bovine preadipocytes, no differential effects of TRO on the differentiation of s.c. and i.m. cells were detected.