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PubMed1996

Characteristics of major structural protein coding gene and leader-body sequence in subgenomic mRNA of porcine reproductive and respiratory syndrome virus isolated in Japan.

Nucleotide sequence, 1713nt in length, of porcine reproductive and respiratory syndrome virus (PRRSV) isolated in Japan was determined. The sequence encompassed 3 overlapping open reading frames (ORFs), ORF5 to ORF7. These ORFs encodes major structural proteins of PRRSV. The deduced amino acid sequence of each ORF showed higher than 87.5% identity with an American isolate, and lower (54.6 to 80.5%) identity with an European isolate. This result supported a previous report about antigenic characteristics of the EDRD-1 strain. Leader-body junction sequence in subgenomic mRNA of the EDRD-1 strain was determined by sequencing cDNA clones of subgenomic RNAs. A common sequence motif of 5 nucleotide, represented by UA(A/G)CC, was identified as the junction sequence.

Amino Acid Sequence↗
PubMed1992

[Effect of various adamantane compounds on the reproduction of Sindbis virus. Isolation and properties of a resistant strain].

Rimantadine and its structural analogs, i. e. amide of 1-adamantane carboxylic acid (AACA) and 1-adamantane acetic acid amide, were shown to be able to inhibit reproduction of Sindbis virus in culture Vero cells. AACA had the maximum antiviral activity. Subcultures of the initial sensitive population of Sindbis virus in the presence of AACA led to formation of mutants resistant to AACA as well as to rimantadine, adamantane acetic acid amide and ammonium chloride. The Sindbis virus population was heterogenous in sensitivity to AACA, which was evident from isolation of separate clones with various levels of sensitivity to the above mentioned compounds from the population. It was found that reproduction of the AACA sensitive and resistant strains of Sindbis virus differed: the latent period of the resistant strain was 2 hours longer than that of the sensitive strain. The same effect was observed in the comparative study on synthesis of the virus-specific RNA.

Animals↗
PubMed2001

Analysis of open reading frame 5 in Japanese porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism.

For the epidemiological survey of porcine reproductive and respiratory syndrome virus (PRRSV) isolated in Japan, the open reading frame (ORF) 5 gene of 37 field isolates in Chiba prefecture from 1991 to 1999 were analyzed by restriction fragment length polymorphism (RFLP). Reverse tanscription-polymerase chain reaction (RT-PCR) amplifying the ORF 5 gene detected 35 field isolates except for 2 isolates. RFLP analysis with MluI, HincII, SacII, HaeIII and MspI demonstrated that 35 field isolates were divided into 14 distinct codes and 34 isolates were distinguished from VR2332-derived modified live PRRSV vaccine, indicating the existence of genetic diversity in PRRSV field isolates in Japan. Only one strain 98-4A had an RFLP pattern identical to the vaccine strain. Nine out of the 35 field isolates (25.7%) had the RFLP code 1-3-2-1-1 and these patterns occurred throughout the examination period. This suggests that these isolates are the prevailing strain of PRRS in Chiba prefecture. RFLP with 5 enzymes demonstrated that various strains existed in the same year, and that there were different codes on the same farm according to the year. These observations indicate that there is a genetic variation among field isolates in very limited regions and some viruses possess the ability to cause their own genomic substitutions within a herd in a short time.

Animals↗
PubMed1997

Isolation and identification of porcine reproductive and respiratory syndrome virus in cell cultures.

Three strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in porcine lung macrophage (PLM) cultures from three swine herds. This has been the first successful isolation of PRRSV in the Czech Republic and the strains received the designations CAPM V-501, CAPM V-502 and CAPM V-503, respectively. All the three isolates in PLM were identified by immunofluorescence and immunoperoxidase tests and the strain CAPM V-502 also by electron microscopy using the ultrathin section technique. The strain CAPM V-502 has been adapted to the cell line MARC-145. Viral RNA in PLM cultures infected with any of the isolated PRRSV strains was demonstrated by RT-PCR targeted to the more conserved ORF 7 genomic region encoding the nucleocapsid protein. The assessment of PCR products in agarose gel revealed a uniform size of 394 bp in all the three isolates and the European prototype strain Lelystad used as positive control.

Animals↗
PubMed2000

A method for the dispersal and characterization of leukocytes from the human female reproductive tract.

PROBLEM: The isolation of human female reproductive tract (RT) cells that maintain viability and are representative of the entire population is essential for a thorough evaluation of mucosal immunity in the reproductive tract mucosa. Here, we describe the isolation of RT cells in high yields and with high viability from the Fallopian tube, uterine endometrium, endocervix, ectocervix and vagina. METHOD OF STUDY: This cell dispersion method uses an enzyme cocktail composed of pancreatin, hyaluronidase, and collagenase (PHC), and employs a 250-microm mesh screen to facilitate cell dispersion. RESULTS: The yields of cells isolated per gram of tissue in the presence of this PHC cocktail were compared and found to be strikingly higher relative to the yields obtained with other enzyme cocktails or in the absence of enzymes. Flow cytometry was used to characterize leukocyte subsets isolated from uterine endometrium in the presence of the various enzyme cocktails. The common leukocyte antigen marker CD45, pan T-cell marker CD3, monocyte/macrophage marker CD14 and B-cell marker CD19 were retained after exposure to the PHC cocktail of enzymes. The expression of CD8 and CD4 was lost after exposure to added enzymes but regained after culture overnight. CONCLUSION: These studies demonstrate the feasibility of using enzymatic digestion for the isolation of whole populations of Fallopian tube, endometrial, cervical and vaginal cells, including leukocyte subsets in high yields, and provide a foundation for investigating mucosal immune cell function in the human female RT.

CD4 Antigens↗
PubMed1997

Classification of Arcobacter species isolated from aborted pig fetuses and sows with reproductive problems in Brazil.

Seventeen field isolates of Arcobacter species were recovered in Brazil from aborted porcine fetal livers (n = 3), kidneys (n = 2), and thoracic fluid (n = 1). Arcobacter species were also recovered from uterine and oviductal tissues (n = 5) and a placenta from sows with reproductive problems. These isolates were initially presumed to be Arcobacter cryaerophilus on the basis of aerobic growth at 30 degree C, indoxyl acetate hydrolysis, catalase and oxidase reactions, growth on MacConkey agar, sensitivity to 3.5% sodium chloride, and susceptibility to nalidixic acid (40 mg/ml). The isolates were confirmed as Arcobacter using polymerase chain reaction, and were classified as A. cryaerophilus 1A (24%), A. cryaerophilus 1B (71%), and A. butzleri (6%) using restriction fragment length polymorphism.

Abortion, Veterinary↗
PubMed2001

Stability and reproductive fitness of Schistosoma mansoni isolates with decreased sensitivity to praziquantel.

These studies are focused on schistosomes derived from human infections not cured by three successive doses of praziquantel that also produced infections in mice that were significantly more difficult to cure than infections with control worms. Half (three of six) of these isolates retained their decreased response to praziquantel after multiple passages through the life-cycle in the absence of therapeutic pressure. Two of the isolates, including the one initially least sensitive to praziquantel; reverted, to a sensitivity not significantly different from controls. For example, the EE6 isolate initially required 680 mg/kg praziquantel to affect a 50% reduction in worm load in murine infections, but after only six passages through the life cycle over 5 years this was reduced to 113 mg/kg, not different from control infections. The stability of some of the isolates and the reversion of others indicates that the biological or genetic factors conferring decreased praziquantel response varies among the isolates. The three isolates that retained decreased sensitivity to praziquantel all showed compromises in reproductive fitness in the laboratory, expressed most frequently as a decreased cercarial production from snails infected with those isolates compared to controls. For example, the total cercarial production of snails infected with the EE10 isolate was only 57% that of controls. The reversion of some of the isolates to a praziquantel sensitive state and the decreased reproductive fitness of those that did not revert suggest that there is some biological cost associated with the relative praziquantel insensitivity of these worms, which could help limit the impact of such isolates in the field. Infections with the less sensitive isolates also produced significantly less circulating schistosomal antigen in mice, suggesting that a decrease in the host immune response elicited by these worms could be one of the factors contributing to the diminished praziquantel efficacy.

Animals↗
PubMed1998

Comparative sequence analysis of open reading frames 2 to 7 of the modified live vaccine virus and other North American isolates of the porcine reproductive and respiratory syndrome virus.

To elucidate changes associated with the attenuated virulence in a modified live porcine reproductive and respiratory syndrome (PRRS) vaccine (Boehringer Ingelheim Animal Health, St. Joseph, MO), derived from an American prototype ATCC virus VR-2332, nucleotide sequence of 3' genome covering open reading frames (ORFs) 2 to 7 coding regions from the vaccine virus was determined by RT-PCR with two overlapping fragments. Comparisons showed 98 base changes (94 substitutions, 3 deletions, and 1 addition) out of 3318 nucleotides between the vaccine virus and its parental virus. There were 15, 26, 17, 29, 9, and 6 base substitutions in ORFs 2, 3, 4, 5, 6, and 7, respectively, resulting in 5, 13, 8, 13, 2, and 3 amino acid (a.a.) substitutions in their deduced proteins, respectively. Most of these a.a. substitutions were also present in 17 known virulent/wild type PRRS virus isolates from North America. However, there were 1, 4, 1, and 1 unique a.a. substitutions in the vaccine virus ORFs 2, 3, 4, and 5 deduced proteins, respectively. These unique amino substitutions may be responsible for the attenuated virulence in the vaccine virus.

Amino Acid Sequence↗
PubMed1999

Antigenic and genetic variations of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome virus isolates.

The antigenic variability of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome (PRRS) virus was characterized with a panel of 24 monoclonal antibodies (MAbs) raised against the American PRRS virus isolate ISU-P. Five continuous epitopes designated EpORF7-A through E were revealed by the reactivity pattern of these MAbs with 67 American field isolates, two modified-live vaccine viruses, and the European Lelystad virus as determined by the indirect immnofluorescence assay and Western immunoblotting and confirmed by additivity and blocking enzyme-linked immunosorbent assays. The reactivity pattern of isolates in the IFA permitted their subdivision into 4 American antigenic groups which represented 84.1, 11.6, 2.9 and 1.4% of viruses tested. The antigenic variation among isolates was correlated to single, group specific nucleotide substitutions and mediated by a combination of at least 4 of the 5 epitopes. EpORF7-A was conserved in all American isolates and the Lelystad virus which constituted a separate antigenic group. Consequently, monoclonal antibodies specific for EpORF7-A may prove useful as the antigenic basis for a universal diagnostic test for the PRRS virus. EpORF7-C, D and E were only present in the American isolates tested.

Amino Acid Sequence↗
PubMed2006

Classification of Actinobacillus spp isolates from horses involved in mare reproductive loss syndrome.

OBJECTIVE: To identify Actinobacillus spp isolates recovered from fetuses and pericardial fluid from horses affected with mare reproductive loss syndrome (MRLS) and determine whether these bacterial species are the same as those isolated from clinically normal horses. SAMPLE POPULATION: Isolates of actinobacilli recovered from 18 horses with pericarditis and 109 fetuses aborted by mares affected by MRLS. Procedures-Actinobacillus spp isolates were identified to the level of species or subspecies by use of conventional phenotypic tests and biochemical and enzyme test kits. The 16S rRNA gene from selected isolates was amplified, purified, and sequenced. Sequence data were compared with sequence data for actinobacilli in GenBank. RESULTS: Of the 109 isolates obtained from fetuses, 14 were Actinobacillus equuli subsp equuli, 65 were A equuli subsp haemolyticus, 28 were Bisgaard taxon 10-like bacterium, and 2 were Actinobacillus genomospecies 1. Of the 18 isolates from horses with pericarditis, 4 were A equuli subsp equuli, 13 were A equuli subsp haemolyticus, and 1 was Bisgaard taxon 10-like bacterium. Comparisons with published data and GenBank data revealed that the isolates recovered from horses with MRLS were the same as those isolated from the oral cavity or alimentary tract of healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE: Actinobacillus spp isolates recovered from fetuses and pericardial fluid samples of horses affected by MRLS in 2001 to 2003 were identical to Actinobacillus spp found in the oral cavity and alimentary tracts of healthy horses.

Aborted Fetus↗
PubMed2006

An ultrasonographic method to study reproductive seasonality in ewes isolated from rams.

In studies concerning the seasonality in sheep, the effect of the teaser-ram, which can by itself induce oestrus as well as the development of ovary function bodies, has not been considered. In this study, a method is developed to determine ovarian activity during the year while excluding a possible influence of the ram. The study was carried out on 10 German Blackhead Mutton ewes (GBM) and 10 German Mutton Merino ewes (GMM) and lasted for 14 months. The ewes were kept without contact with any ram, and once a week a transrectal ultrasonography was conducted to determine ovary function bodies (corpora lutea, follicles) and a blood sample was taken to analyse progesterone (P4) concentration. In both breeds, the number of ovulating ewes, the ovulation rate (OR), the size of the corpora lutea (CL) and P4-concentrations showed significant seasonal fluctuations. During the main breeding period (September-February), the OR was higher (2.0 +/- 0.4) than during the rest of the year (1.6 +/- 0.5). Outside the main breeding season (March-August), approximately 40% GBM ewes and only approximately 15% GMM ewes produced CL. The cycle and the related seasonal changes in the P4-concentration coincide with different ultrasonically diagnosed CL (developing or regressing, insufficient, no CL). Between the volume of fully developed CL and P4-concentration, correlations of 0.57 (GMM) and 0.45 (GBM), respectively, were found. On average, P4-concentration in September to February was by approximately 1.60-1.66 nmol/l in the GMM and by approximately 1.80-1.86 nmol/l in the GBM higher than in spring and summer. Considering the OR, the P4-values corresponded with the CL volumes, i.e. in the months from August to February the CL values were significantly larger than in the months March to August. The ultrasonic ovarian diagnosis is a useful method to determine the ram-uninfluenced seasonality of reproduction in sheep.

Animals↗
PubMedDate not supplied

[Changes in reproductive function during chronic isolation and increases in the population density of rats with different typologic behavioral characteristics].

It has been shown on white outbred female rats that animals with a strong balanced mobile nervous system and active-search behaviour had the greatest stress resistence against the action of chronic isolation and increase of population density. Weak non-balanced excitable animals with non-directed motor restlessness had a minimal resistence against stress.

Animals↗
PubMed1996

Morphological-cytochemical and molecular genetic analyses of mitochondria in isolated human oocytes in the reproductive age.

Molecular genetic, cytochemical and morphometric analyses have been performed on isolated oocytes from 41 women (27-39 years of age) in order to detect mutations of mitochondrial DNA (mtDNA), defects of the respiratory chain (ubiquinone-cytochrome-c-oxidoreductase = complex III; cytochrome-c-oxidase = complex IV) and alterations of mitochondrial volume during cellular ageing. Morphometric analyses showed an increase in mitochondrial numerical density with age from the mean values of 7.36 per micron2 and 6.97 per micron3 up to 30 years to 10.74 per micron2 and 11.66 per micron3 in the age group 31-40 years (P < 0.001). Similarly, an increase in the mitochondrial profile area from 0.074 per micron2 in the age group < 30 years to 0.101 per micron2 was noted in the fourth decade. The mitochondrial volume fraction was also significantly increased in the elder age group. Neither point mutations of mtDNA (nucleotide pairs 3243, 8344) nor the common deletion (4977 bp, nucleotide pairs 8482-13460) could be detected. In parallel, ultra- and immunocytochemical studies of the complexes III-IV failed to reveal functional defects. In conclusion there is an age-related increase in the volume fraction of the mitochondria which might reflect subtle changes in the oxidative phosphorylation capacity, but is not linked to mutations of mtDNA or functional defects of the respiratory chain enzymes in mature human oocytes from women of reproductive age.

Adult↗
PubMed1990

Isolation of Gardnerella vaginalis from the reproductive tract of four mares.

A gram-variable pleomorphic bacillus was isolated from the reproductive tracts of 4 mares during routine prebreeding soundness examinations. Using a commercial bacterial identification system, these organisms were identified as Streptococcus acidominimus. However, colonial and Gram-staining characteristics did not support this identification. Subsequent testing indicated the organism was similar to Gardnerella vaginalis. Additional growth and biochemical analysis performed in our laboratory and at the Michigan Department of Public Health and by the Center for Disease Control, Atlanta, Georgia, confirmed the identification of G. vaginalis.

Animals↗
PubMed1996

Serological property and replication in cell cultures of porcine reproductive and respiratory syndrome viruses isolated in Japan.

The serological property and replication in swine alveolar macrophages (SAM) and MARC-145 cell cultures of 35 porcine reproductive and respiratory syndrome (PRRS) viruses isolated in SAM were investigated. All the isolates were reacted almost equally with antisera against three Japanese isolates including EDRD-1 strain (American type) in immunoperoxidase monolayer assay (IPMA), but weakly or did not react with antiserum against Lelystad virus (European type) indicating that the Japanese isolates are more closely related to an American type virus. Twenty two of 35 isolates replicated with CPE both in SAM and MARC-145 cells, whereas remaining 13 isolates did not show CPE in MARC-145 cells. The antigenicity of the isolates did not relate to the virus origin and the replication in the cell cultures.

Animals↗