Effects of milk replacer powder added to pasteurized whole milk over different durations on dairy calves fed ground starter diet with alfalfa hay.

Adding milk replacer powder (MRP) to whole milk during the entire preweaning period can increase growth but raises concern because of low starter feed intake and slumps in average daily gain (ADG) at weaning and postweaning. In the current study, effects of adding MRP to pasteurized whole milk (PWM) during d 10-41 or d 10-59 of age were investigated in comparison with PWM. Calves [24 females and 21 males; 39.8 ± 1.85 kg body weight (BW)] were randomly allocated to 1 of 3 treatments: 1) conventional protocol, 5 L/d PWM from d 3-56, and 2.5 L/d from d 57-59 of age (CONV; TS intake = 31.9 kg), 2) short duration of adding MRP to PWM protocol, 5 L/d PWM from d 3-9, 5 L/d PWM + MRP (18% TS) from d 10-41, 5 L/d PWM from d 42-56, and 2.5 L/d PWM from d 57-59 (SD; TS intake = 42.3 kg), 3) long duration of adding MRP to PWM protocol, 5 L/d PWM from d 3-9, 5 L/d PWM + MRP from d 10-56, 2.5 L/d PWM + MRP form d 57-59 (LD; TS intake = 47.7 kg). The osmolality of PWM and PWM + MRP was 278 and 519 mOsm/L, respectively. Calves were weaned on d 60, and the study terminated on d 75. There was a treatment × time interaction for starter intake, where intake was greater for CONV than other treatments from d 14-41 and was greater in CONV than LD during d 42-48 and d 56-62 of age. Final BW was lower in CONV calves than LD calves. Weaning BW and overall hip height were lower in CONV calves than other treatments. The CONV calves had lower ADG at d 14-27 and 35-41 and SD calves had lower ADG at d 42-48 than other treatments. Calves fed CONV treatment had lower ruminal acetate and greater propionate than SD calves during preweaning. Calves fed LD treatment had lower total volatile fatty acids and tended to have greater ruminal pH than other treatments. Calves fed CONV had greater neutrophils and neutrophils/lymphocytes ratio and lower lymphocytes than other treatments. Glucose concentration was greater for LD versus other treatments at d 56, and lower for SD versus other treatments at d 70 of study. Insulin concentration and homeostatic model assessment of insulin resistance index were greater in LD compared with other treatments during preweaning but were not different postweaning. Serum BHB was greater in CONV than other treatments. Albumin was greater for CONV versus other treatments at d 56, however, it was greater in LD-fed calves at d 70 of study. Results indicate that feeding a PWM + MRP to the calves during the entire preweaning period resulted in lower starter feed intake around weaning, but overall starter intake was similar with a greater final BW and fewer health related issues throughout the study. Shifting a PWM + MRP to the conventional whole milk at d 40 of age decreased the ADG of calves.

In vitro antifungal activities of Actinomyces species isolated from soil samples against Trichophyton mentagrophytes.

BACKGROUND AND PURPOSE: Cutaneous infections arise from a homogeneous group of keratinophilic fungi, known as dermatophytes. Since these pathogenic dermatophytes are eukaryotes in nature, use of chemical antifungal agents for treatment may affect the host tissue cells. In this study, we aimed to evaluate the antifungal activity of Actinomyces species against Trichophyton mentagrophytes (abbreviated as T. mentagrophytes). The isolates were obtained from soil samples and identified by polymerase chain reaction (PCR) technique. MATERIAL AND METHODS: In total, 100 strains of Actinomyces species were isolated from soil samples in order to determine their antagonistic activities against T. mentagrophytes in Kerman, Iran. The electron microscopic study of these isolates was performed, based on the physiological properties of these antagonists (e.g., lipase, amylase, protease and chitinase), using relevant protocols. The isolates were identified using gene 16S rDNA via PCR technique. RESULTS: Streptomyces flavogriseus, Streptomyces zaomyceticus strain xsd08149 and Streptomyces rochei were isolated and exhibited the most significant antagonistic activities against T. mentagrophytes. Images were obtained by an electron microscope and some spores, mycelia and morphology of spore chains were identified. Molecular, morphological and biochemical characteristics of these isolates were studied, using the internal 16S rDNA gene. Active isolates of Streptomyces sequence were compared to GenBank sequences. According to nucleotide analysis, isolate D5 had maximum similarity to Streptomyces flavogriseus (99%). CONCLUSION: The findings of this study showed that Streptomyces isolates from soil samples could exert antifungal effects on T. mentagrophytes.

Emergence of cutaneous leishmaniasis in a border area at south-east of Iran: an epidemiological survey.

BACKGROUND & OBJECTIVES: Cutaneous leishmaniasis (CL) has been recently emerged in new foci, posing a public health problem. Increasing cases of CL have been reported during recent years from a border area between Iran and Pakistan, a previously non-endemic area. The present study was designed for epidemiological and parasitological characterization of the disease for the first time in this area. METHODS: A total of 3100 individuals from the city of Mirjaveh and its four rural districts were randomly selected and surveyed from March 2005 to February 2006. Microscopic examination, in vitro culture, mouse inoculations and species-specific kDNA-PCR assay were carried out for Leishmania detection and species identification. RESULTS: CL was endemic in an important rural district of Mirjaveh, presenting active lesions and scars in 6.6 and 9.5%, respectively. The highest rates of both active lesions and scars were found in the age group of 10 years or under with significant differences (p < 0.05) comparing to the older age groups. No association between genders and the rate of leishmaniasis was observed (p > 0.05). The most affected location was upper limb, 39.2% of ulcers and 41.7% of scars. Inoculation of the clinical isolates on Balb/c mice, led to the development of ulcers in the animals, implying that the causative parasite is Leishmania major. The PCR amplification also generated amplicons specific to L. major. CONCLUSION: It can be concluded that Mirjaveh is an endemic region of cutaneous leishmaniasis as a new focus due to the recent emergence in this border area of south-east of Iran with a major contribution of L. major, as the causative parasite species.

High rate of detection of mixed infections of Plasmodium vivax and Plasmodium falciparum in South-East of Iran, using nested PCR.

Sistan and Baluchestan province, South-East of Iran, has been reported as an endemic area of malaria [Sadrizadeh B. Malaria in the world, in the eastern Mediterranean region and in Iran: Review article. WHO/EMRO Report 2001: 1-13.]. The main objective of this research was to perform rapid and correct diagnoses of malaria infection. Blood specimens were collected from 140 suspected volunteers. The Giemsa-stained slides examination and nested PCR for amplification of the Plasmodium small subunit ribosomal genes (ssrRNA) were utilized. The results demonstrated 118 out of 140 cases (84.3%) positive for malaria parasites, including 60.7%, 20.7% and 2.9% as having Plasmodium vivax (P.v), Plasmodium falciparum (P.f) and mixed infections (P.v+P.f), respectively by microscopy. The nested PCR detected malaria parasites in 134 samples (94.3%), consisting of 51.4% P.v, 12.6% P.f and 29.3% mixed infections. The PCR analysis detected 37 cases of mixed infections more than that of the routine microscopy. These results suggested that there are a considerable number of cases with mixed infections in the study area that mainly remain undiagnosed by microscopy. It is also concluded that the nested PCR is a suitable complement to microscopy for accurate specific diagnosis of malaria species in field.

Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation.

Ectopic expression of telomerase results in an immortal phenotype in various types of normal cells, including primary human fibroblasts. In addition to its role in telomere lengthening, telomerase has now been found to have various functions, including the control of DNA repair, chromatin modification, and the control of expression of genes involved in cell cycle regulation. The investigations on the long-term effects of telomerase expression in normal human fibroblast highlighted that these cells show low frequencies of chromosomal aberrations. In this paper, we describe the karyotypic stability of human fibroblasts immortalized by expression of hTERT. The ectopic overexpression of telomerase is associated with unusual spontaneous as well as radiation-induced chromosome stability. In addition, we found that irradiation did not enhance plasmid integration in cells expressing hTERT, as has been reported for other cell types. Long-term studies illustrated that human fibroblasts immortalized by telomerase show an unusual stability for chromosomes and for plasmid integration sites, both with and without exposure to ionizing radiation. These results confirm a role for telomerase in genome stabilisation by a telomere-independent mechanism and point to the possibility for utilizing hTERT-immortalized normal human cells for the study of gene targeting.

Large-mutation spectra induced at hemizygous loci by low-LET radiation: evidence for intrachromosomal proximity effects.

A mathematical model is used to analyze mutant spectra for large mutations induced by low-LET radiation. The model equations are based mainly on two-break misrejoining that leads to deletions or translocations. It is assumed, as a working hypothesis, that the initial damage induced by low-LET radiation is located randomly in the genome. Specifically, we analyzed data for two hemizygous loci: CD59- mutants, mainly very large-scale deletions (>3 Mbp), in human-hamster hybrid cells, and data from the literature on those HPRT- mutants which involve at least deletion of the whole gene, and often of additional flanking markers (approximately 50-kbp to approximately 4.4-Mbp deletions). For five data sets, we estimated f, the probability that two given breaks on the same chromosome will misrejoin to make a deletion, as a function of the separation between the breaks. We found that f is larger for nearby breaks than for breaks that are more widely separated; i.e., there is a "proximity effect". For acute irradiation, the values of f determined from the data are consistent with the corresponding break misrejoining parameters found previously in quantitative modeling of chromosome aberrations. The value of f was somewhat smaller for protracted irradiation than for acute irradiation at a given total dose; i.e., the mutation data show a decrease that was smaller than expected for dose protraction by fractionation or low dose rate.

Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11.

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Löbrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

The relationship between spontaneous telomere loss and chromosome instability in a human tumor cell line.

Chromosome instability plays an important role in cancer by promoting the alterations in the genome required for tumor cell progression. The loss of telomeres that protect the ends of chromosomes and prevent chromosome fusion has been proposed as one mechanism for chromosome instability in cancer cells, however, there is little direct evidence to support this hypothesis. To investigate the relationship between spontaneous telomere loss and chromosome instability in human cancer cells, clones of the EJ-30 tumor cell line were isolated in which a herpes simplex virus thymidine kinase (HSV-tk) gene was integrated immediately adjacent to a telomere. Selection for HSV-tk-deficient cells with ganciclovir demonstrated a high rate of loss of the end these "marked" chromosomes (10-4 events/cell per generation). DNA sequence and cytogenetic analysis suggests that the loss of function of the HSV-tk gene most often involves telomere loss, sister chromatid fusion, and prolonged periods of chromosome instability. In some HSV-tk-deficient cells, telomeric repeat sequences were added on to the end of the truncated HSV-tk gene at a new location, whereas in others, no telomere was detected on the end of the marked chromosome. These results suggest that spontaneous telomere loss is a mechanism for chromosome instability in human cancer cells.

The use of human repetitive DNA to target selectable markers into only the human chromosome of a human-hamster hybrid cell line (AL)

We used the plasmid BLUR-8 that contains an 800-base pair (bp) sequence of human repetitive Alu DNA in a cotransfection protocol to target the plasmids pSV2neo or EBO-pcD-leu-2 (hygro) into a single site of the sole human chromosome, number 11, of a Chinese hamster-human hybrid cell line (AL). The neo and hygro plasmids confer resistance to the antibiotics G418 and hygromycin, respectively. Of the 33 cotransfected clones with single-site insertions, 1/13 without BLUR-8 and 6/20 with BLUR-8 were only in human chromosome 11. A frequency of insertion of 1/13 is not different than expected by chance (rho = 0.3512). On the other hand, the probability that 6/20 insertions, as seen with BLUR-8, occurred by chance is low (rho = 0.0003). We suggest that the human DNA sequences contained in BLUR-8 targeted insertions into only the human chromosome.