Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.     

Engineering Disease Resistant Cattle

Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk. Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis causing organisms. Care will be taken to avoid altering milk’s nutritional and manufacturing properties. Multi-cistronic constructs may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Detection and prevalence of UMP synthase deficiency among dairy cattle

A partial deficiency of UMP synthase has been detected in Holstein dairy cattle. Since affected females secrete milk with elevated concentrations of orotate, milk orotate was used to screen for the condition among 880 cows in 17 randomly selected Holstein herds in Illinois. Mean orotate was 85.0 +/- 1.5 microgram/ml milk and 17 cows had milk orotate in excess of 170 micrograms/ml. Including the latter animals, 42 cows were evaluated for erythrocyte UMP synthase and 15 were found to be partially deficient. Thus, at least 1.7 percent of all cows had the condition; this is a minimal estimate because the initial screen was milk orotate and this may be low, particularly early in lactation. Deficient cows had half the level of UMP synthase as normal, nondeficient cows (1.30 +/- 0.06 vs. 2.79 +/- 0.10 units/ml). The binomial classification of deficient versus normal accounted for 72 percent of the variation noted in UMP synthase. Milk orotate was significantly elevated in deficient cows (337.8 +/- 31.3 micrograms/ml), validating its use as a screening device. Urinary orotate also was higher for deficient cows (28.4 +/- 5.2 vs. 9.2 +/- 0.8 micrograms/ml) and differentiated the two groups as well as milk orotate. Normal and deficient cows did not differ in milk lactose concentrations. Erythrocyte UMP synthase also was measured in 85 Holstein bulls used for artificial insemination and 6 had low levels of UMP synthase (1.39 +/- 0.19 vs. 2.92 +/- 0.05 units/ml); the binomial classification accounted for 42 percent of the variation. The partially deficient animals identified appear to be heterozygotes for a condition expected to be lethal in the homozygous state.     

Expression of Lysostaphin in Milk of Transgenic Mice Affects the Growth of Neonates

As an initial step towards enhancing mastitis resistance in dairy animals, we generated BLG-Lys transgenic mice that secrete lysostaphin, a potent antistaphylococcal protein, in their milk. In the current study, we continue our assessment of lysostaphin as a suitable antimicrobial protein for mastitis resistance and have investigated mammary gland development and function in three lines of transgenic mice. As the lines were propagated, there was a tendency for fewer BLG-Lys litters to survive to weaning (51% as compared to 90% for nontransgenic lines, p = 0.080). Nontransgenic pups fostered on dams from these three lines exhibited diminished growth rates during the first week of lactation. Rates of gain became comparable to pups on nontransgenic dams at later time points. Initial slow growth also resulted in decreased weaning weights for pups nursed by transgenic dams (15.35±0.27 g) when compared to pups delivered and nursed by nontransgenic dams (18.61 ± 0.61 g; p < 0.001), but the effect was temporary, as similar weights were attained by adulthood. Milk yield at peak lactation was not different between BLG-Lys (0.79 ± 0.33 g) and nontransgenic (0.91 ± 0.38 g; p = 0.166) dams. Histological examination of the transgenic mammary glands during gestation revealed no differences when compared to control glands; however, at early lactational stages, the BLG-Lys glands exhibited less alveolar area than control glands and a delay in lobulo-alveolar maturation. The results clearly demonstrate reduced growth of neonates on BLG-Lys dams; whether the poor pup performance can be attributed to delayed mammary development or the gland development merely reflects reduced suckling stimuli from the pups remains to be determined.Authors Abhijit Mitra and Kathleen S. Hruska contributed equally to this work     

Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands

Staphylococci cause bovine mastitis, with Staphylococcus aureus being responsible for the majority of the mastitis-based losses to the dairy industry (up to $2 billion/annum). Treatment is primarily with antibiotics, which are often ineffective and potentially contribute to resistance development. Bacteriophage endolysins (peptidoglycan hydrolases) present a promising source of alternative antimicrobials. Here we evaluated two fusion proteins consisting of the streptococcal λSA2 endolysin endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin (λSA2-E-Lyso-SH3b) or the staphylococcal phage K endolysin, LysK (λSA2-E-LysK-SH3b). We demonstrate killing of 16 different S. aureus mastitis isolates, including penicillin-resistant strains, by both constructs. At 100 μg/ml in processed cow milk, λSA2-E-Lyso-SH3b and λSA2-E-LysK-SH3b reduced the S. aureus bacterial load by 3 and 1 log units within 3 h, respectively, compared to a buffer control. In contrast to λSA2-E-Lyso-SH3b, however, λSA2-E-LysK-SH3b permitted regrowth of the pathogen after 1 h. In a mouse model of mastitis, infusion of 25 μg of λSA2-E-Lyso-SH3b or λSA2-E-LysK-SH3b into mammary glands reduced S. aureus CFU by 0.63 or 0.81 log units, compared to >2 log for lysostaphin. Both chimeras were synergistic with lysostaphin against S. aureus in plate lysis checkerboard assays. When tested in combination in mice, λSA2-E-LysK-SH3b and lysostaphin (12.5 μg each/gland) caused a 3.36-log decrease in CFU. Furthermore, most protein treatments reduced gland wet weights and intramammary tumor necrosis factor alpha (TNF-α) concentrations, which serve as indicators of inflammation. Overall, our animal model results demonstrate the potential of fusion peptidoglycan hydrolases as antimicrobials for the treatment of S. aureus-induced mastitis.     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Compositional analysis of dairy products derived from clones and cloned transgenic cattle

Cloning technology is an emerging biotechnological tool that could provide commercial opportunities for livestock agriculture. However, the process is very inefficient and the molecular events underlying the technology are poorly understood. The resulting uncertainties are causing concerns regarding the safety of food products derived from cloned livestock. There are similar concerns for livestock produced by biotechnologies which enable the purposeful introduction of genetic modifications. To increase the knowledge about food products from animals generated by these modern biotechnologies, we assessed compositional differences associated with milk and cheese derived from cloned and transgenic cows. Based on gross composition, fatty acid and amino acid profiles and mineral and vitamin contents, milk produced by clones and conventional cattle were essentially similar and consistent with reference values from dairy cows farmed in the same region under similar conditions. Whereas colostrum produced by transgenic cows with additional casein genes had similar IgG secretion levels and kinetics to control cows, milk from the transgenic cows had a distinct yellow appearance, in contrast to the white color of milk from control cows. Processing of milk into cheese resulted in differences in the gross composition and amino acid profiles; 'transgenic' cheese had lower fat and higher salt contents and small but characteristic differences in the amino acid profile compared to control cheese.     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method.

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Effects of recombinant lysostaphin on cytotoxicity and interleukin-8 level in normal human epidermal keratinocytes cell line

The normal human epidermal keratinocyte (NHEK) was used to evaluate the cytotoxicity of recombinant lysostaphin. As determined with the Neutral Red (NR) cytotoxicity assay, the midpoint toxicity value (NR50) after 48 h exposure was 16 ± 0.4 g lysostaphin/l. Lysostaphin cytotoxicity effect is much less than the surface active agent, sodium laurate. However, the NR50 value after 48-h exposure was 1.9 ± 0.02 g/l for S. aureus lysate derived from the bacterial lytic action of lysostaphin. A linear increase in interleukin-8 (IL-8) level in NHEK cells from resting levels of 65 ± 3 pg/ml to peak of 760 ± 15 pg/ml during the first 9 hours was noted for the cells treated with 800 mg lysostaphin/l. S. aureus lysate has the same effect on the induction of IL-8 levels. The induced rises in IL-8 were lysostaphin and S. aureus lysate concentration dependence. © Rapid Science Ltd. 1998     

Evaluation of intrauterine antibiotic treatment of clinical metritis and retained fetal membranes in dairy cows

Retained fetal membranes (RFM) and clinical metritis (CM) are frequently diagnosed disease conditions in dairy cows and considered of major economic impact due to negative effect on reproduction and milk production. The objective of this study was to evaluate the efficacy of i.u. tetracycline for the treatment of RFM and CM in dairy cows. Affected cows were randomly assigned to two groups; treatment group animals received i.u. 5g chlortetracycline twice weekly for 2 wks, and no treatment group. A total of 1416 cows and 804 heifers in 5 herds calved during the study period. CM was diagnosed in 18.6% (inter farm range; 15.2-23.5%) and 30% (19.4-42.3%) of cows and heifers, respectively. RFM was diagnosed in 13.1% (9.4-18.1%) and 9.2% (3.6-13.8%) of cows and heifers, respectively. Conception rates after first insemination were 38.3%, 42.5% and 18% in normal, treated and non-treated CM cows, respectively. Numbers of days open were 140.5, 136.2 and 165.5 in normal, treated and non-treated CM cows, respectively. Based on 305-d corrected milk yield, cows and heifers affected by RFM and CM produced 300-500kg less milk compared with their normal herd mates. Cows treated for CM produced 654kg more milk per 305-d corrected lactation compared to non-treated control cows. Treatment of RFM had no effect on reproductive performance or milk production. In conclusion, i.u. chlortetracycline treatment was proven to prevent the detrimental effect of CM on reproductive performance in heifers and cows and on milk production in cows only.     

Evaluation of intrauterine antibiotic treatment of clinical metritis and retained fetal membranes in dairy cows

Retained fetal membranes (RFM) and clinical metritis (CM) are frequently diagnosed disease conditions in dairy cows and considered of major economic impact due to negative effect on reproduction and milk production. The objective of this study was to evaluate the efficacy of i.u. tetracycline for the treatment of RFM and CM in dairy cows. Affected cows were randomly assigned to two groups; treatment group animals received i.u. 5 g chlortetracycline twice weekly for 2 wks, and no treatment group.A total of 1416 cows and 804 heifers in 5 herds calved during the study period. CM was diagnosed in 18.6% (inter farm range; 15.2–23.5%) and 30% (19.4–42.3%) of cows and heifers, respectively. RFM was diagnosed in 13.1% (9.4–18.1%) and 9.2% (3.6–13.8%) of cows and heifers, respectively. Conception rates after first insemination were 38.3%, 42.5% and 18% in normal, treated and non-treated CM cows, respectively. Numbers of days open were 140.5, 136.2 and 165.5 in normal, treated and non-treated CM cows, respectively. Based on 305-d corrected milk yield, cows and heifers affected by RFM and CM produced 300–500 kg less milk compared with their normal herd mates. Cows treated for CM produced 654 kg more milk per 305-d corrected lactation compared to non-treated control cows. Treatment of RFM had no effect on reproductive performance or milk production. In conclusion, i.u. chlortetracycline treatment was proven to prevent the detrimental effect of CM on reproductive performance in heifers and cows and on milk production in cows only.     

Antimicrobial susceptibility and invasive ability of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from mastitis from dairy backyard systems

Fifteen (15) backyard farms were investigated to determine the antimicrobial susceptibility and invasion ability of S. aureus isolates from cows with subclinical mastitis in México. A total of 106 cows were sampled and 31 S. aureus isolates were recovered. S. aureus isolates were resistant to penicillin class antibiotics and susceptible to gentamicin and cetyltrimethylammonium bromide. STA9 and STA13 isolates were resistant to erythromycin (MIC > 25 microg/ml) and lincomycin (STA13, MIC > 25 microg/ml; STA9, MIC > 100 microg/ml). STA9 isolate harbors the erm(B) and msr(A) genes, whereas STA13 isolate harbors the erm(C) gene. STA9 and STA13 isolates contains the lnu(A) gene. Only 5 isolates (STA11, STA13, STA14, STA15 and STA21) were able to internalize in bovine mammary epithelial cells. These results indicate that S. aureus isolates from dairy backyard farms showed differences in the antimicrobial susceptibility patterns and invasion ability in bovine mammary epithelial cells. This kind of evaluations should be performed in different dairy regions, since resistance patterns and isolate diversity vary on a per-region basis.     

Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10³ and 95.4×10³ CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10⁵ and 622.2×10⁵ cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.     

Use of cow-side progesterone tests to improve reproductive performance of high-producing dairy cows

This study evaluated the effectiveness of the cow-side ELISA milk progesterone test in improving postpartum reproductive performance in the Dordt College dairy herd. Cows that produced more than 18,500 lb of milk per lactation were assigned to the high production group (40 cows), while cows that produced less than 18,500 lb of milk (42 cows) were assigned to the low production group. Twenty-one cows in the high production group and 19 cows in the low production group received no ELISA testing (untreated controls), while the remaining cows in each group were evaluated by ELISA test every 7 d beginning on Day 27 post partum (treated cows). A sequence of 2 high progesterone tests and 1 low test indicated the cows were cycling normally. Cows that had low milk progesterone levels (<5 ng/ml) for 3 consecutive tests were assumed to have follicular cysts and were treated with 2 ml GnRH (Cystorelin, 50 mug/ml). Cows that had 3 consecutive high tests (>5 ng/ml) were assumed to have persistent corpora lutea (CL) and were treated with 5 ml PGF(2)alpha (Lutalyse, 5 mg/ml). In both the high and low production groups, treated cows had higher (P < 0.08) pregnancy rates by Day 210 than the untreated controls (63.2 vs 38.1% and 56.5 vs 42.1%, respectively). The days open were reduced (P < 0.05) for the treated animals by 41.6 d compared with the controls. The treated cows produced a net savings of $70.42 (US) per cow assuming a $3.00 savings/day open.     

Interleukin 2 treatment of Staphylococcus aureus mastitis.

A study was conducted in dairy cows to evaluate the efficacy of recombinant bovine interleukin 2 (rBoIL-2) as an adjunct to antibiotic therapy in Staphylococcus aureus mastitis. In normal, non-mastitic cows, intramammary infusion of rBoIL-2 caused a tenfold increase in somatic cell counts (SCC) in milk. Co-administration of 2 mg of rBoIL-2 and sodium cephapirin in cows with established S. aureus mastitis decreased SCC and shedding of S. aureus compared with values from cows that were given only sodium cephapirin or 10 mg rBoIL-2 with sodium cephapirin. Cows in the 2 mg rBoIL-2 group cleared the infection earlier and at 2 weeks after treatment had not relapsed with staphylococcal mastitis. These data suggest that rBoIL-2 may be useful as an immunotherapeutic agent in controlling mastitis.     

Evaluation of blood and milk oxidative status during early postpartum of dairy cows

In dairy cows, the intensity of metabolic activity, associated with the negative energy balance (NEBAL), is responsible for an increased production of reactive oxygen species (ROS) and, subsequently, for the development of the condition of oxidative stress, which may overwhelm the antioxidant potential of the bovine maternal organism, making it prone to the development of many puerperal dysfunctions, as well as to an alteration of colostrum and milk quality. Given these premises, the aims of this study are to evaluate serum and milk concentrations of ROS and lipoperoxides, vitamins A and E, on the 10th, 12th, 14th and 16th day postpartum of dairy cows, a particularly critical period during which the NEBAL reaches its nadir, and to compare the trends of these parameters in two different bovine breeds. The study was performed in pluriparous Italian Friesian and Brown dairy cows. On the 10th day postpartum, all cows underwent a clinical examination to exclude the presence of alterations; furthermore, on the same day, a milk sample was collected from each cow, in order to perform the somatic cell count (SCC; (CE) N. 853/2004) and to establish which of them had an SCC ⩽400 000/ml or >400 000/ml. In this study, among the 110 cows that were initially selected, the evaluation of these parameters allowed the inclusion of 80 animals, which were divided into four groups of 20 subjects each: Group F and F1: Italian Friesian healthy cows, with SCC ⩽400 000/ml and >400 000/ml, respectively; Group B and B1: Italian Brown healthy cows, with SCC ⩽400 000/ml and >400 000/ml, respectively. On the 10th, 12th, 14th and 16th day postpartum, peripheral blood and milk samples were collected. The results obtained show that in group B1 there were higher concentrations of ROS and milk antioxidants compared with Friesian group cows. This datum let us suppose that even in the presence of higher ROS concentrations the antioxidant status found in group B1 seems to be able to counteract the oxidative damage, which is more likely to develop in these cows.     

中国荷斯坦牛IL8基因遗传多态性与泌乳性状以及体细胞评分的关联

以上海某奶牛场30个公牛家系的610头中国荷斯坦牛为试验材料,采用聚合酶链式反应-单链构象多态性(PCR-SSCP)技术对Interleukin-8(IL8)基因的遗传多态性进行了分析,采用混合动物模型分析了IL8基因突变位点与测定日产奶量、测定日乳脂率、测定日乳蛋白率、305d校正产奶量、305d乳脂量、305d乳蛋白量及测定日体细胞评分7个性状的相关性,寻找可用于生产实际的分子标记。共检测到KK、KA和AA3种基因型,频率分别为0.187、0.451和0.362,等位基因K和A的频率分别为0.412和0.588。该位点突变对测定日产奶量、305d乳蛋白量、305d校正产奶量和305d乳脂量以及体细胞评分影响达到极显著水平(P0.01),对测定日乳蛋白率的影响达到显著水平(P0.05),对测定日乳脂率影响不显著(P0.05)。多重比较表明:KK基因型对测定日产奶量、305d校正产奶量、305d乳蛋白量和305d乳脂量极显著高于AA和KA基因型(P0.01)。KK基因型的体细胞评分(SCS)最小二乘均值极显著低于KA、AA基因型(P0.01)。对于测定日的乳蛋白率AA基因型显著低于KA、KK型(P0.05)。IL8基因遗传突变对中国荷斯坦牛泌乳性状和乳房炎抗性有较大的遗传效应,可用于中国荷斯坦牛的分子标记辅助选择。     

Mathematical approaches to detect low concentrations in progesterone profiles

There is a general need for higher objectivity and accuracy in describing the physiological fertility performance of dairy cows. To develop the alternative meaningful starting points for the selection of genetically superior dairy cows, this study focused on the detection of low progesterone concentrations, which are indicative of estrus events. Three mathematical approaches were used: one based on the exponentially weighted moving average control chart, and two threshold methods, which were developed in-house. Data were collected from one data set that included 97 insemination data of first-lactating Holstein dairy cows, and a second set that included 160 inseminations of primiparous and multiparous Holstein dairy cows. On the basis of these 2 data sets, and using a threshold of 1.2 ng progesterone/ml skimmed milk, the sensitivity of the 3 models was high and ranged between 100% and 93.13%, with an error rate between 4% and 22.17%. The specificity varied between 97.92% and 99.93%. The average concentration levels of true-positive–detected progesterone measures were low and ranged between 0.18 and 0.28 ng progesterone/ml skimmed milk (first data set) and 0.21 to 0.26 ng progesterone/ml skimmed milk (second data set). False-positive–detected low progesterone concentrations during estrus events were closely related to progesterone values around the 1.2 ng progesterone/ml skimmed milk threshold and the detecting rules of the control chart. Thus, we suggest that a threshold of 0.8 ng progesterone/ml skimmed milk is indicative for luteal activity in defatted foremilk. By means of the three methods used, the detection of low progesterone concentrations was possible and it can be assumed that this is a good starting point for further studies (such as interval calculation) in this area.     

Novel SNPs of the Bovine PRLR Gene Associated with Milk Production Traits

The single nucleotide polymorphisms (SNPs) within exon 10 of the prolactin receptor gene (PRLR) were detected in Chinese Holstein cows using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods, and their genetic effects on milk production traits were evaluated in this study. Two newly detected SNPs (g.9206G→A and g.9681C→T) caused amino acid variations E378K and A536V, respectively, which were then preliminarily predicted at the topological level. Statistical results indicated that the two SNPs were significantly associated with milk yields, and cows with the combined genotype GGCC showed superior milk performance. A putative phosphorylation site was identified at residue 378K ([ST]-×-[RK]), which offers a partial explanation for the associations. These results suggest that the two novel SNPs within exon 10 of the PRLR gene associated with milk production traits are useful genetic markers in a selection program for Holstein dairy cattle.     

Peptidoglycan hydrolase fusions maintain their parental specificities

The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiae are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated S. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans. Both fusions display lytic specificity for streptococcal pathogens and S. aureus. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63 degrees C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.