Meta-analysis of apparent ruminal synthesis and postruminal flow of B vitamins in dairy cows.

As milk production has significantly increased over the past decade(s), existing estimates of the B-vitamin needs of the modern dairy cow are currently being reconsidered, as suboptimal B-vitamin supply may affect metabolic efficiency. At the same time, however, "true" (i.e., biologically active forms, excluding nonfunctional analogs) B-vitamin supply also cannot be adequately estimated by dietary intake, as the rumen microbiota has been shown to play a significant role in synthesis and utilization of B vitamins. Given their complex impact on the metabolism of dairy cows, incorporating these key nutrients into the next generation of mathematical models could help to better predict animal production and performance. Therefore, the purpose of this study was to generate hypotheses of regulation in the absence of supplemental B vitamins by creating empirical models, through a meta-analysis, to describe true B-vitamin supply to the cow (postruminal flow, PRF) and apparent ruminal synthesis (ARS). The database used for this meta-analysis consisted of 340 individual cow observations from 15 studies with 16 experiments, where diet and postruminal digesta samples were (post hoc) analyzed for content of B vitamins (B1, B2, B3, B6, B9, B12). Equations of univariate and multivariate linear form were considered. Models describing ARS considered dry matter intake (DMI, kg/d), B-vitamin dietary concentration [mg/kg of dry matter (DM)] and rumen-level variables such as rumen digestible neutral detergent fiber (NDF) and starch (g/kg of DM), total volatile fatty acids (VFA, mM), acetate, propionate, butyrate, and valerate molar proportions (% of VFA), mean pH, and fractional rates of degradation of NDF and starch (%/h). Models describing PRF considered dietary-level driving variables such as DMI, B-vitamin dietary concentration (mg/kg of DM), starch and crude protein (g/kg of DM) and forage NDF (g/kg of DM). Equations developed were required to contain all significant slope parameters and contained no significant collinearity between driving variables. Concordance correlation coefficient was used to evaluate the models on the developmental data set due to data scarcity. Overall, modeling ARS yielded better-performing models compared with modeling PRF, and DMI was included in all prediction equations as a scalar variable. The B-vitamin dietary concentration had a negative effect on the ARS of B1, B2, B3, and B6 but increased the PRF of B2 and B9. The rumen digestible NDF concentration had a negative effect on the ARS of B2, B3, and B6, whereas rumen digestible starch concentration had a negative effect on the ARS of B1 and a positive effect on the ARS of B9. In the best prediction models, the dietary starch increased PRF of B1, B2, and B9 but decreased PRF of B12. The equations developed may be used to better understand the effect of diet and ruminal environment on the true supply of B vitamins to the dairy cow and stimulate the development of better-defined requirements in the future.

Analysis of BMAA enantiomers in cycads, cyanobacteria, and mammals: in vivo formation and toxicity of D-BMAA.

Chronic dietary exposure to the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA) triggers neuropathology in non-human primates, providing support for the theory that BMAA causes a fatal neurodegenerative illness among the indigenous Chamorro people of Guam. However, since there are two stereoisomers of BMAA, it is important to know if both can occur in nature, and if so, what role they might play in disease causation. As a first step, we analysed both BMAA enantiomers in cyanobacteria, cycads, and in mammals orally dosed with L-BMAA, to determine if enantiomeric changes could occur in vivo. BMAA in cyanobacteria and cycads was found only as the L-enantiomer. However, while the L-enantiomer in mammals was little changed after digestion, we detected a small pool of D-BMAA in the liver (12.5%) of mice and in the blood plasma of vervets (3.6%). Chiral analysis of cerebrospinal fluid of vervets and hindbrain of mice showed that the free BMAA in the central nervous system was the D-enantiomer. In vitro toxicity investigations with D-BMAA showed toxicity, mediated through AMPA rather than NMDA receptors. These findings raise important considerations concerning the neurotoxicity of BMAA and its relationship to neurodegenerative disease.

Abrupt weaning reduces postweaning growth and is associated with alterations in gastrointestinal markers of development in dairy calves fed an elevated plane of nutrition during the preweaning period.

The benefits of feeding elevated quantities of milk to dairy calves have been well established. However, there is a reluctance to adopt this method of feeding in commercial dairy production because of concerns around growth, health, and ruminal development during weaning. The objective of this study was to characterize the effect of an abrupt (0 d step-down) or gradual (12 d step-down) feeding scheme when calves are fed an elevated plane of nutrition (offered 1.35 kg of milk replacer/d). For this experiment, a total of 54 calves were randomly assigned to an abrupt or a gradual weaning protocol before weaning at 48 d of life. Calves were housed and sampled in individual pens for the duration of the experiment, and milk, starter, and straw intake were measured on a daily basis. Body weight was measured every 6 d, whereas blood, rumen fluid, and fecal samples were collected on d 36 (pre-step-down), 48 (preweaning), and 54 (postweaning) of the experiment. Although the growth rates of the step-down calves were lower from d 37 to weaning (0.62 ± 0.04 vs. 1.01 ± 0.04 kg/d), the postweaning average daily gain was greater compared with the group that was abruptly weaned (0.83 ± 0.06 vs. 0.22 ± 0.06 kg/d). Total ruminal volatile fatty acid was greater in the step-down group on the day of weaning (d 48; 59.80 ± 2.25 vs. 45.01 ± 2.25 mmol), whereas the fecal starch percentage was lower during postweaning compared with the abruptly weaned calves (d 54; 3.31 ± 0.76 vs. 6.34 ± 0.76%). Analysis of the digestive tract of bull calves on d 55 revealed minimal differences between gross anatomy measurements of gut compartments as well as no morphological differences in rumen papillae development, yet the total mass of rumen when full of contents was larger in the step-down calves (7.83 ± 0.78 vs. 6.02 ± 0.78 kg). Under the conditions of this study, the results showcase the benefits of a step-down feeding strategy from an overall energy balance standpoint, due to increased adaptation of the gastrointestinal tract preweaning.

Exogenous essential amino acids stimulate an adaptive unfolded protein response in the mammary glands of lactating cows.

The phosphorylation of mammalian target of rapamycin complex 1 (mTORC1) components and integrated stress response networks in the mammary glands of lactating cows have not accounted for the stimulation of milk protein yield by chronic supplementation with AA or glucose. Faster milk protein synthesis could be a consequence of increased milk protein mRNA per cell, the number of ribosomes per cell, the secretory capacity of cells, or the mammary cell number. To investigate these 4 possibilities using a translational and transcriptional approach, we performed protein and gene expression analyses of mammary and longissimus dorsi tissue collected from lactating dairy cows after 5 d of abomasal infusion with saline or 844 or 1,126 g/d of an essential AA (EAA) mixture, with and without 1,000 g/d glucose. Infusion with EAA increased milk protein yield but did not affect the phosphorylation of mTORC1-related proteins in the mammary gland. In skeletal muscle, phosphorylation of 4EBP1 (eIF4E-binding protein 1) increased in response to both EAA and glucose, and phosphorylated S6K1 (70-kDa ribosomal protein S6 kinase) increased with glucose. In response to EAA, mammary mRNA expression of the marker genes for milk proteins, ribosome biogenesis, and cell proliferation were not upregulated. Instead, reciprocal regulation of 2 arms of the unfolded protein response occurred. Infusion of EAA for 5 d activated XBP1 (X-box binding protein 1) mRNA, encoding a transcription factor for endoplasmic reticulum biogenesis, and it decreased the mRNA expression of genes encoding pro-apoptotic protein CHOP (C/EBP homologous protein) and downstream GADD34 (growth arrest and DNA damage-inducible 34). These findings implicate non-stress-related, adaptive capabilities of the unfolded protein response in the long-term nutritional regulation of milk protein yield in lactating dairy cows.

Glucose supplementation stimulates peripheral branched-chain amino acid catabolism in lactating dairy cows during essential amino acid infusions.

To determine how glucose modulates protein synthesis when essential AA are in abundant supply, 5 early-lactation, rumen-fistulated Holstein dairy cows were fed a diet containing 6.95 MJ/kg of net energy for lactation and 12.4% crude protein and abomasally infused for 5 d with saline, 844 or 1,126 g/d of a complete essential AA mix, with and without the inclusion of 1,000 g/d of glucose, in a 5×5 Latin square design. Infusion of essential AA increased milk yield by 4.1 kg/d, milk protein by 256 g/d, milk fat by 95 g/d, and milk urea nitrogen by 70% compared with saline, with no differences between the level of essential AA infusion. The addition of glucose to essential AA infusate did not stimulate milk protein yield or concentration, but reduced milk urea nitrogen by 17% and decreased milk fat yield. Arterial concentrations of total essential AA increased 3- to 4-fold, mammary clearance decreased 61%, and mammary uptake of essential AA increased 65% in response to essential AA infusion. Arterial branched-chain AA concentrations declined 29% in response to glucose and mammary clearance increased 48%, but mammary AA uptake was unchanged. Essential AA infusion increased plasma 3-methylhistidine by 50% and reduced muscle branched-chain α-keto acid dehydrogenase kinase abundance by 14%, indicating stimulation of muscle protein turnover and branched-chain AA catabolism, respectively. Glucose had no further effect on muscle branched-chain α-keto acid dehydrogenase kinase abundance but decreased mRNA expression of branched chain aminotransferase 1. Lack of further increases in plasma 3-methylhistidine or greater stimulation of muscle branched-chain AA catabolism indicates that muscle protein degradation was unchanged with glucose but that accretion may have been stimulated. The decrease in circulating branched-chain AA concentrations and nitrogen excretion in response to glucose suggests that surplus essential AA were redirected to peripheral, extra-mammary tissues.

The influence of age and weaning on permeability of the gastrointestinal tract in Holstein bull calves.

Fourteen Holstein bull calves were used in a randomized complete block design to investigate the effect of calf age and weaning on permeability of the gastrointestinal tract (GIT). Calves were randomly assigned to 1 of 2 treatments: (1) a weaning protocol that was initiated on d 35; WN; n=7), or (2) a control treatment where calves were not weaned (CON; n=7). Calves were bottle-fed milk replacer (150 g/L), in 3 equal portions/d targeting 15% of their body weight (BW) in liquid milk intake [approximately 21.1g/kg of BW/d, dry matter (DM) basis]. On d 35, the amount of milk replacer offered to WN calves was reduced to 7.5% of BW for 7 d before calves were weaned on d 42. On d 14, 28, and 42, calves were orally dosed with 500 mL of Cr-EDTA (179 mM Cr-EDTA solution) and housed in a metabolism crate to enable total urine collection and determination of total urinary Cr recovery as an indicator of total-tract permeability. On d 44, calves were killed and tissues from the rumen, omasum, duodenum, jejunum, ileum, cecum, and proximal and distal colon were collected, rinsed, and transported in buffer solution (pH 7.4 at 38.5°C). Tissues were incubated in Ussing chambers under short-circuit conditions with buffer solutions designed to mimic the mucosal and serosal energy source that would be available in vivo (glucose for tissues from the small intestine and short-chain fatty acids for tissues that would be exposed to fermentation; rumen, omasum, and large intestinal tissues). The serosal to mucosal flux of (14)C-mannitol and (3)H-inulin was measured for each region. Although we detected treatment × period interactions for BW and starter intake, dietary treatments did not differ within a week. Overall, the time that ruminal pH was <5.5 was less before weaning than after weaning. We observed a differential response for the appearance of Cr in urine for WN and CON calves, where the appearance of Cr (mg/48 h) in urine decreased for both treatments from d 14 to 28, but increased from d 28 to 42 for WN, whereas Cr appearance continued to decrease for CON. The flux of mannitol and inulin did not differ between treatments but did differ among region of the GIT, with rumen, duodenum, and jejunum having the greatest permeability. These data suggest that permeability of the GIT decreases with age but weaning may disrupt this process. The rumen, duodenum, and jejunum appear to be the regions with greatest permeability.

Essential amino acid infusions stimulate mammary expression of eukaryotic initiation factor 2Bε but milk protein yield is not increased during an imbalance.

Essential amino acid (EAA) deficiencies and imbalances were created in lactating cows by using an infusion subtraction protocol to explore effects on milk protein yield and activity state of regulators of mRNA translation in the mammary glands. Six lactating cows on a diet of 11.2% protein were infused abomasally for 5d with saline, 563g/d of a complete EAA mix, or EAA without His, Met, Phe, or Trp in a 6×6 Latin square design. Infusion of complete and imbalanced EAA solutions increased mammalian target of rapamycin (mTOR) signaling in the mammary glands, as evidenced by higher ribosomal S6 kinase 1 (S6K1) phosphorylation compared with saline infusion. Total S6K1 abundance was decreased by imbalanced AA infusions. Except for the mixture lacking Phe, infusion of EAA, whether imbalanced or not, increased abundance of total eukaryotic initiation factor 2Bε (eIF2Bε). A correlation of 0.33 between phosphorylation state of S6K1 and total eIF2Bε abundance suggests that an mTOR-mediated upregulation of eIF2Bε translation occurred. Despite increased mTOR/eIF2Bε signaling, milk protein yields increased only with the complete EAA mixture compared with saline. Low plasma concentrations of His, Met, and Phe during their respective imbalances likely interfered with protein synthesis. Total abundance and phosphorylation state of eukaryotic initiation factor 2α were not responsible for the interference. Further study of eIF2Bε as a regulator of milk protein yield is warranted.

Technical note: Three-dimensional imaging of rumen tissue for morphometric analysis using micro-computed tomography.

Rumen development in calves has been evaluated by measuring papillae length, width, and density using microscopy for over 50 yr. Although common in the literature, disadvantages to this method exist, such as large variations in rumen papillae size and shape, small numbers of total papillae being measured, and the time required. The objective of this study was to develop a more effective technique for assessing rumen papillae using micro-computed tomography (micro-CT) and to compare this technique with microscopy. Rumen tissue was collected from the ventral sac of 20 postweaned bull calves at 55 d of age, immediately fixed in 10% neutral buffered formalin for 48 h, and stored in 70% ethanol at 4°C before the contrast enhancement. After evaluation of contrast-enhancement protocols, it was determined that mercury chloride provided the most pronounced contrast for accurate micro-CT imaging based on relative density of the papillae. A 1-cm(2) tissue section from the ventral sac of all bull calves was tensioned on a rapid prototyped curved plastic holder and imaged at 4 5 µm resolution for 56 min using a GE Locus Explore micro-CT (General Electric, Milwaukee, WI). MicroView V2.2 software (General Electric) was used to create a 3-dimensional virtual model of the entire sample. The length and width of papillae were measured 3-dimensionally and compared with measurements of papillae under the light microscope taken from the same region. The length and width measurements using micro-CT (2.47 ± 0.12 and 0.55 ± 0.01 mm) compared with light microscope (2.96 ± 0.03 and 0.86 ± 0.01 mm) were significantly smaller. The difference may reflect a more accurate determination in the base of the rumen tissue with micro-CT or the specificity of mercury chloride to bind only to intact rumen tissue. The mean number of papillae per centimeter squared viewed using micro-CT was 128.5 ± 33.9 with a total surface area of 681.8 ± 112.4 mm(2) and volume of 156 mm(3) per sample. Micro-CT data demonstrated that surface area and volume are positively associated and that papillae length was negatively associated with papillae per centimeter squared and positively associated with total volume of tissue section. This study represents the first time that micro-CT has been being used to assess morphology of rumen tissue. Micro-CT has the potential to improve the accuracy and efficiency of rumen tissue measurements; however, more standardization of each factor involved in tissue preparation, imaging, and location of papillae measurements is required.

Historical stocking data and 19th century DNA reveal human-induced changes to native diversity and distribution of cutthroat trout.

Many species are threatened with extinction and efforts are underway worldwide to restore imperilled species to their native ranges. Restoration requires knowledge of species' historical diversity and distribution. For some species, many populations were extirpated or individuals moved beyond their native range before native diversity and distribution were documented, resulting in a lack of accurate information for establishing restoration goals. Moreover, traditional taxonomic assessments often failed to accurately capture phylogenetic diversity. We illustrate a general approach for estimating regional native diversity and distribution for cutthroat trout in the Southern Rocky Mountains. We assembled a large archive of historical records documenting human-mediated change in the distribution of cutthroat trout (Oncorhynchus clarkii) and combined these data with phylogenetic analysis of 19th century samples from museums collected prior to trout stocking activities and contemporary DNA samples. Our study of the trout in the Southern Rocky Mountains uncovered six divergent lineages, two of which went extinct, probably in the early 20th century. A third lineage, previously declared extinct, was discovered surviving in a single stream outside of its native range. Comparison of the historical and modern distributions with stocking records revealed that the current distribution of trout largely reflects intensive stocking early in the late 19th and early 20th century from two phylogenetically and geographically distinct sources. Our documentation of recent extinctions, undescribed lineages, errors in taxonomy and dramatic range changes induced by human movement of fish underscores the importance of the historical record when developing and implementing conservation plans for threatened and endangered species.

HIV infection induces changes in CD4+ T-cell phenotype and depletions within the CD4+ T-cell repertoire that are not immediately restored by antiviral or immune-based therapies.

Changes in CD4+ T-cell surface marker phenotype and antigen receptor (TCR) repertoire were examined during the course of HIV infection and following therapy. A preferential decline in naive CD4+ T cells was noted as disease progressed. Following protease inhibitor therapy, naive CD4+ T cells increased only if they were present before initiation of therapy. Disruptions of the CD4+ TCR repertoire were most prevalent in patients with the lowest CD4+ T-cell counts. Antiviral or IL-12 therapy-induced increases in CD4+ T-cell counts led to only minor changes in previously disrupted repertoires. Thus, CD4+ T-cell death mediated by HIV-1 infection may result in a preferential decline in the number of naive CD4+ T cells and disruptions of the CD4+ T-cell repertoire that are not immediately corrected by antiviral or immune-based therapies.

Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy.

The size of the pool of resting CD4+ T cells containing replication-competent HIV in the blood of patients receiving intermittent interleukin (IL)-2 plus highly active anti-retroviral therapy (HAART) was significantly lower than that of patients receiving HAART alone. Virus could not be isolated from the peripheral blood CD4+ T cells in three patients receiving IL-2 plus HAART, despite the fact that large numbers of resting CD4+ T cells were cultured. Lymph node biopsies were done in two of these three patients and virus could not be isolated. These results indicate that the intermittent administration of IL-2 with continuous HAART may lead to a substantial reduction in the pool of resting CD4+ T cells that contain replication-competent HIV.

Peripheral expansion of pre-existing mature T cells is an important means of CD4+ T-cell regeneration HIV-infected adults.

The CD4+ T-cell pool in HIV-infected patients is in a constant state of flux as CD4+ T cells are infected and destroyed by HIV and new cells take their place. To study T-cell survival, we adoptively transferred peripheral blood lymphocytes transduced with the neomycin phosphotransferase gene between syngeneic twin pairs discordant for HIV infection. A stable fraction of marked CD4+ T cells persisted in the circulation for four to eighteen weeks after transfer in all patients. After this time there was a precipitous decline in marked cells in three of the patients. At approximately six months, marked cells were in lymphoid tissues in proportions comparable to those found in peripheral blood. In two patients, the proportion of total signal for the transgene (found by PCR analysis) in the CD4/CD45RA+ T-cell population relative to the CD4/CD45RO+ population increased in the weeks after cell infusion. These findings indicate that genetically-marked CD4+ T cells persist in vivo for weeks to months and that the CD4+ T-cell pool in adults is maintained mostly by the division of mature T cells rather than by differentiation of prethymic stem cells. Thus, after elements of the T-cell repertoire are lost through HIV infection, they may be difficult to replace.

Water administration of the medium-chain fatty acid caprylic acid produced variable efficacy against enteric Campylobacter colonization in broilers.

Campylobacter is one of the most common causes of foodborne illness, and poultry are considered a primary source of Campylobacter infections. Caprylic acid, an 8-carbon fatty acid, has been shown in previous studies to reduce enteric cecal Campylobacter concentrations in poultry when administered in the feed. For greater ease of application for producers, a water-soluble form of caprylic acid, sodium octanoate, was evaluated for efficacy against enteric Campylobacter. The first trial consisted of 70 birds in 7 groups (n = 10 chicks/group): an untreated control and 6 other groups that were challenged with Campylobacter at d 3 and that received 0, 0.175, 0.35, 0.7, 1.4, or 2.8% water-soluble caprylic acid in water 3 d before necropsy at d 14. The second trial consisted of 80 birds in 8 groups (n = 10 chicks/group): an untreated negative control and 7 other groups, all of which were challenged with Campylobacter at d 3 and received 0, 0.044, 0.088, 0.175, 0.35, 0.7, or 1.4% water-soluble caprylic acid for 3 d before necropsy at d 14. In trial 1, only the 0.175% dose caused a reduction in cecal Campylobacter counts in comparison with the positive control (approximately a 3-log reduction). In trial 2, no treatment reduced Campylobacter counts compared with the positive control. Unlike the efficacy of caprylic acid in feed, treatment with caprylic acid in water had an inconsistent effect on intestinal Campylobacter counts.

Cyanotoxin Analysis and Amino Acid Profiles of Cyanobacterial Food Items from Chad.

In some parts of the world, cyanobacteria are used as a food in the human diet, due to their ready availability. Lake Chad, has long been a traditional site for the collection of Arthrospira fusiformis which is dried and processed at the lake into thin wafers called Dihé for later consumption or is transported to market for sale. However, Dihé purchased from markets in Chad has not been analyzed for known cyanobacterial toxins or assessed for total amino acid content. Since BMAA in traditional foodstuffs of the indigenous Chamorro people of Guam causes neurodegenerative illness, it is important that Dihé from Chad be analyzed for this neurotoxin. BMAA and its isomer AEG were not detected in our analyses, but a further isomer DAB was detected as both a free and bound amino acid, with an increase in the free concentration after acid hydrolysis of this fraction. Microcystins were present in 6 samples at up to 20 µg/g according to UPLC-PDA, although their presence could not be confirmed using PCR for known microcystin synthetic genes. Amino acid analysis of the cyanobacterial material from Chad showed the presence of large amounts of canonical amino acids, suggesting that this may supplement indigenous people on low protein diets, although regular monitoring of the foodstuffs for the presence of cyanotoxins should be performed.

Toxin Analysis of Freshwater Cyanobacterial and Marine Harmful Algal Blooms on the West Coast of Florida and Implications for Estuarine Environments.

Recent marine and freshwater algal and cyanobacterial blooms in Florida have increased public concern and awareness of the risks posed by exposure to these organisms. In 2018, Lake Okeechobee and the Caloosahatchee river, on the west coast of Florida, experienced an extended bloom of Microcystis spp. and a bloom of Karenia brevis in the coastal waters of the Gulf of Mexico that coincided in the Fort Myers area. Samples from the Caloosahatchee at Fort Myers into Pine Island Sound and up to Boca Grande were collected by boat. High concentrations of microcystin-LR were detected in the cyanobacterial bloom along with brevetoxins in the marine samples. Furthermore, ß-N-methylamino-L-alanine (BMAA) and isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobuytric acid (DAB) were detected in marine diatoms and dinoflagellates, and cyanobacteria of freshwater origin. High freshwater flows pushed the cyanobacterial bloom to barrier island beaches and Microcystis and microcystins could be detected into the marine environment at a salinity of 41 mS/cm. For comparison, in 2019 collections of Dapis (a new generic segregate from Lyngbya) mats from Sarasota showed high concentrations of BMAA, suggesting the possibility of long-term exposure of residents to BMAA. The findings highlight the potential for multiple, potentially toxic blooms to co-exist and the possible implications for human and animal health.

Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy.

BACKGROUND: Combination antiretroviral therapy suppresses but does not eradicate human immunodeficiency virus type 1 (HIV-1) in infected persons, and low-level viremia can be detected despite years of suppressive antiretroviral therapy. Short-course (28-day) intensification of standard antiretroviral combination therapy is a useful approach to determine whether complete rounds of HIV-1 replication in rapidly cycling cells contribute to persistent viremia. We investigated whether intensification with the integrase inhibitor raltegravir decreases plasma HIV-1 RNA levels in patients receiving suppressive antiretroviral therapy. METHODS: Subjects (n = 10) with long-term HIV-1 suppression receiving combination antiretroviral regimens had their regimens intensified for 4 weeks with raltegravir. Plasma HIV-1 RNA level was determined before, during, and after the 4-week intensification period, using a sensitive assay (limit of detection, 0.2 copies of HIV-1 RNA/mL of plasma). A 4-week intensification course was chosen to investigate potential HIV-1 replication in cells with relatively short (approximately 1-14-day) half-lives. RESULTS: There was no evidence in any subject of a decline in HIV-1 RNA level during the period of raltegravir intensification or of rebound after discontinuation. Median levels of HIV-1 RNA before (0.17 log10 copies/mL), during (0.04 log10 copies/mL), and after (0.04 log10 copies/mL) raltegravir intensification were not significantly different (P > .1 for all comparisons in parametric analyses). High-performance liquid chromatography and mass spectroscopy experiments confirmed that therapeutic levels of raltegravir were achieved in plasma during intensification. CONCLUSIONS: Intensification of antiretroviral therapy with a potent HIV-1 integrase inhibitor did not decrease persistent viremia in subjects receiving suppressive regimens, indicating that rapidly cycling cells infected with HIV-1 were not present. Eradication of HIV-1 from infected persons will require new therapeutic approaches. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00618371.

Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV.

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = -0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.

The natural feed additive caprylic acid decreases Campylobacter jejuni colonization in market-aged broiler chickens.

Campylobacter causes human foodborne illness, and epidemiological evidence indicates poultry and poultry products as a significant source of human infection. Decreasing Campylobacter in the poultry intestinal tract would decrease contamination of poultry products. Caprylic acid is a medium-chain fatty acid reported to be effective in killing a variety of bacterial pathogens, including Campylobacter jejuni, but its effect has not been investigated in the control of C. jejuni in preslaughter market-aged poultry already colonized with this bacterium. The objective of this study was to determine the therapeutic effect of caprylic acid on C. jejuni counts in the cecal contents of 42-d-old chickens. Four trials were conducted. In the first 2 trials, day-of-hatch chicks (n = 60 per trial) were assigned to 6 treatment groups (n = 10 birds per treatment group): positive controls (Campylobacter, no caprylic acid), 0.7 or 1.4% of caprylic acid in feed for the last 3 d of the trial with or without a 12-h feed withdrawal. Treatments were similar for trials 3 and 4 except the doses used were 0.35 or 0.7% caprylic acid supplementation for the last 7 d of the trial. On d 42, ceca were collected and Campylobacter counts determined. The supplementation of caprylic acid at 0.35 and 0.7% consistently decreased (P < 0.05) the colonization of C. jejuni in the chicken ceca compared with positive control treatment. When these treatments were evaluated after a 12-h feed withdrawal period, 0.7% caprylic acid decreased Campylobacter colonization in the 3-d treatment supplementation. Body weight and feed consumption did not differ between the caprylic acid and control groups. The results suggest that therapeutic supplementation of caprylic acid in the feed can effectively decrease Campylobacter in market-aged chickens and may be a potential treatment for decreasing pathogen carriage in poultry.

Campylobacter biofilm phenotype exhibits reduced colonization potential in young chickens and altered in vitro virulence.

In this study, we evaluated the ability of different Campylobacter phenotypes (biofilm versus planktonic) to colonize young poultry. It has been suggested that a persistent Campylobacter biofilm reservoir may be involved in the initial contamination of poultry flocks. Campylobacter jejuni cultured adherent to agar was utilized as the biofilm model and C. jejuni cultured in broth was evaluated as the planktonic model. In 2 independent trials, 1-d-old broiler chicks were given 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). Cecal contents of all birds were evaluated by culturing 12 d after the initial challenge with C. jejuni. In both trials, birds challenged with C. jejuni cultured in broth had approximately 3 to 4 log higher cecal Campylobacter concentration than birds challenged with C. jejuni cultured adherent to agar. Using 2 cell lines (INT 407 and DF1), virulence of C. jejuni cultured in broth versus adherent to agar also was evaluated by challenging monolayers of eukaryotic cells with 1 of 3 treatments: 1) 10(5) cfu.mL(-1) of C. jejuni cultured in broth, 2) 10(5) cfu.mL(-1) of C. jejuni cultured adherent to agar, or 3) no C. jejuni (negative control). The virulence study also showed differences of C. jejuni cultured in broth or agar in attachment and invasion abilities to tissue culture cells, but differences were not as consistent as with the chick colonization study. This study indicates that phenotype may play a role in colonization of chickens and virulence by C. jejuni.

Therapeutic supplementation of caprylic acid in feed reduces Campylobacter jejuni colonization in broiler chicks.

Poultry colonized with Campylobacter species are a significant source of human food-borne illness. The therapeutic use of the medium chain fatty acid caprylic acid consistently reduced enteric C. jejuni colonization in chicks by 3 to 4 logs in three separate trials. These results support caprylic acid's potential to reduce Campylobacter carriage in poultry.