We previously reported changes in gene expression in mammary tissue from non-inflamed mammary glands adjacent to an inflamed gland challenged with lipopolysaccharide (LPS). We determined if changes in the expression of selected genes in non-inflamed glands would be replicated in RNA isolated from milk fat. Cows were milked twice daily prior to experiment. Per cow, one mammary gland (QLPS) was randomly assigned to receive an intramammary infusion of 50 µg LPS immediately after morning milking on d-0. The ipsilateral (QI) and contralateral (QC) mammary glands adjacent to QLPS remained untreated. Quarter foremilk samples from all mammary glands were collected on d-1 and d-0 for milk composition and isolation of RNA for quantification of selected genes via quantitative polymerase chain reaction. Symptoms of clinical mastitis developed only in QLPS and were apparent within 3 h post-challenge. In QI and QC, lactose percentages were lower at 12 h post-challenge compared to d-1, but milk fat and protein contents were not different. For gene expression, 7 of 13 selected genes were differentially regulated in non-inflamed glands. In QI but not QC, LALBA expression was lower at 12 h post-challenge than on d-1. One gene of interest, LPIN1, was significantly upregulated in QI and QC but downregulated in QLPS at 12 h post-challenge. Five additional immune or stress-related genes were significantly upregulated in QLPS and, to a lesser but significant degree, in QI and QC compared to d-1. Notably, expression of two immune genes (NFKBIA, PTX3) was significantly greater in QI than QC despite QI having a numerically lower somatic cell count. Minor changes in the composition of milk secreted by non-inflamed mammary glands were linked to several immune and stress responses in those glands. Further, individual non-inflamed mammary glands responded differently depending on their position relative to the mastitic gland.

The present study investigated the effect of Bacillus subtilis DSM 29784 (Ba) and enzymes (xylanase and β-glucanases; Enz), alone or in combination (BE) as antibiotic replacements, on the growth performance, digestive enzyme activity, immune response and the intestinal barrier of broiler chickens. In total, 1200 1-d-old broilers were randomly assigned to five dietary treatments, each with six replicate pens of forty birds for 63 d as follows: (a) basal diet (control), supplemented with (b) 1 × 109 colony-forming units (cfu)/kg Ba, (c) 300 mg/kg Enz, (d) 1 × 109 cfu/kg Ba and 300 mg/kg Enz and (e) 250 mg/kg enramycin (ER). Ba, Enz and BE, similar to ER, decreased the feed conversion rate, maintained intestinal integrity with a higher villus height:crypt depth ratio and increased the numbers of goblet cells. The BE group exhibited higher expression of claudin-1 and mucin 2 than the other four groups. BE supplementation significantly increased the α-diversity and β-diversity of the intestinal microbiota and markedly enhanced lipase activity in the duodenal mucosa. Serum endotoxin was significantly decreased in the BE group. Compared with those in the control group, increased superoxide dismutase and glutathione peroxidase activities were observed in the jejunal mucosa of the Ba and BE groups, respectively. In conclusion, the results suggested that dietary treatment with Ba, Enz or BE has beneficial effects on growth performance and anti-oxidative capacity, and BE had better effects than Ba or Enz alone on digestive enzyme activity and the intestinal microbiota. Ba or Enz could be used as an alternative to antibiotics for broiler chickens.

We report seven cases of probable endotoxin poisoning linked to contaminated compounded glutathione. Five of the cases were using the infusions for treatment of Lyme disease highlighting the risks of using compounded sterile preparations for unapproved indications, especially if the quality of source products cannot be assured.

Subacute ruminal acidosis (SARA) is a prevalent metabolic disorder in dairy cows known to elicit local and systemic immune responses. We recently showed that cows experiencing SARA and challenged intramammarily with lipopolysaccharide (LPS) experienced stronger metabolic disturbances compared with cows without SARA. Therefore, we hypothesized that cows experiencing SARA have a modulated innate immune response and impaired plasma metabolome compared with healthy cows when experiencing an acute mastitis challenge. A total of 18 Simmental cows were subjected either to a Control (CON, n=6) or SARA (n=12) feeding regimen, receiving either 40% or 60% concentrates for 30 days. Thereafter, six SARA (SARA-LPS) and the CON (CON-LPS) cows were intramammarily challenged with 50 µg LPS from Escherichia coli (O26 : B6), while the remaining six SARA cows (SARA-PLA) received a placebo. Blood and milk samples were analyzed for acute phase proteins and a targeted ESI-LC-MS/MS-based metabolomics approach was performed in blood samples 24 h after the LPS challenge. The LPS infusion caused a strong increase in immune response variables, with a higher concentration of milk amyloid A 48 h after the LPS challenge in SARA-LPS compared with CON-LPS cows. Cows receiving the LPS infusion had a lower plasma concentration of several amino acids and lysophosphatidylcholines but without differences in SARA cows and healthy cows. In conclusion, our results revealed that an intramammary LPS infusion increased acute phase proteins and modulated the blood metabolome. While no systemic differences between SARA and healthy cows were observed, cows experiencing SARA showed a higher concentration of an acute phase protein at the local level of the mammary gland. Further research is required to elucidate the underlying mechanisms and to evaluate its clinical significance for udder health.

Excessive rainfall and dam failures resulted in floodwater contaminating our public water supply. The endotoxin risk in the contaminated water created challenges in recovery of sterile processing for our surgical equipment. Recovery plans should include a potable water source and a method to connect it to the required location. We share our solution of plumbing our sterile processing equipment to tanker-transported potable water sources. (Disaster Med Public Health Preparedness. 2018; 12: 415–418)

The gastrointestinal tract is a large reservoir of both Gram positive and negative bacteria, of which the Gram negative bacteria act as a source of lipopolysaccharide (LPS). Luminal LPS, commonly known as endotoxin, can enter systemic circulation via paracellular and transcellular permeation. It has been shown that endotoxin exposure can increase intestinal paracellular permeability and alter intestinal structure and function, resulting in impaired absorption and utilisation of nutrients with negative impact on both poultry heath and growth. Acute exposure to large amounts of endotoxin suppresses feed intake in chickens and activation of the innate immune system. Substantial evidence suggests that endotoxin is recognised via toll-like receptor (TLR)-4, located on the surface of immune cells, which activates kinases to enhance the transcription of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α) and interleukin (IL)-1 and 6 which mediate the inhibitory effect of endotoxin on the intestinal nutrient absorption. Inflammation can divert energy and nutrients away from growth supp and into supporting the immune system responses, leading to growth suppression and lowered feed efficiency. In addition, sustained exposure to endotoxin may elicit tolerance, which can make animals refractory against secondary Gram-negative infections. In this review, the effects of endotoxin on intestinal barrier functions and the subsequent negative impacts on the production of chickens are summarised. Overall, this review combines the current knowledge of the chicken's intestinal responses to Gram negative bacterial infection, illustrating the importance of gut health in defence against bacterial infections and improving poultry health, growth and welfare.

Evidence from animal and human studies has associated gut microbiota, increased translocation of lipopolysaccharide (LPS) and reduced intestinal integrity (II) with the inflammatory state that occurs in obesity and type 2 diabetes mellitus (T2DM). Consumption of Ca may favour body weight reduction and glycaemic control, but its influence on II and gut microbiota is not well understood. Considering the impact of metabolic diseases on public health and the role of Ca on the pathophysiology of these diseases, this review critically discusses possible mechanisms by which high-Ca diets could affect gut microbiota and II. Published studies from 1993 to 2015 about this topic were searched and selected from Medline/PubMed, Scielo and Lilacs databases. High-Ca diets seem to favour the growth of lactobacilli, maintain II (especially in the colon), reduce translocation of LPS and regulate tight-junction gene expression. We conclude that dietary Ca might interfere with gut microbiota and II modulations and it can partly explain the effect of Ca on obesity and T2DM control. However, further research is required to define the supplementation period, the dose and the type of Ca supplement (milk or salt) required for more effective results. As Ca interacts with other components of the diet, these interactions must also be considered in future studies. We believe that more complex mechanisms involving extraintestinal disorders (hormones, cytokines and other biomarkers) also need to be studied.

Activity of locus coeruleus (LC) neurons, the major noradrenergic cell-body group in the brain whose axons give rise to approximately 70% of norepinephrine (NE) in the brain, is believed to play an important role in attention/vigilance, cognitive functions and behavioral disorders, particularly depression. Results described here show that in the rat, intraperitoneal (i.p.) injection of lipopolysaccharide (LPS, a bacterial endotoxin) causes long-lasting changes in electrophysiological activity of LC neurons that are mediated by interleukin-1 (IL-1) acting locally in the LC region. First, it was found that IL-1, when microinjected into the LC region or stimulated/expressed in that brain region, increased activity of LC neurons. The only exception to this was that a very low dose of microinjected IL-1 (5 pg) decreased LC activity, which could be blocked by an antagonist to corticotropin-releasing hormone (CRH), thus suggesting that the decrease was due to IL-1 stimulation of CRH release. All of these effects could be blocked by injection and/or infusion of IL-1 receptor antagonist (IL-1RA) specifically into the LC region. Next, intraperitoneal (i.p.) injection of a low dose of LPS(10 µg/kg or 100 ng/kg) was also found to increase LC activity. The excitation of LC produced by 10 µg/kg i.p. LPS increased progressively for at least 1 week, with LC neurons firing at more than twice their normal rate at 1 week after the i.p. LPS injection. Alteration of LC activity lasted for 3 weeks after a single i.p. injection of 10 µg/kg LPS. The effects of i.p. LPS on LC activity at any time after i.p. injection could be blocked by a brief microinfusion of IL-1RA into the LC region, thereby indicating that changes in LC activity seen after the i.p. LPS were caused by IL-1 acting in the LC region. Finally, i.p. injection of peptidoglycan, representing gram-positive bacteria, and polyinsinic-polycytidylic acid [poly(I):(C)], representing viral infection, also caused increases in LC activity, and the effects of peptidoglycan [but not those of poly(I):(C)] were blocked by microinfusion of IL-1RA into LC. These findings suggest that bacterial infections can give rise to prolonged changes in brain activity through cytokine action in brain.

Adverse uterine environments caused by maternal stress (such as bacterial endotoxin) can alter programming of the fetal hypothalamic–pituitary–adrenal axis (HPAA) rendering offspring susceptible to various adulthood diseases. Thus, protection against this type of stress may be critical for ensuring offspring health. The present study was designed to determine if maternal supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFAs) during pregnancy helps to protect against stress-induced fetal programming. Briefly, 53 ewes were fed a diet supplemented with fishmeal (FM) or soybean meal (SM) from day 100 of gestation (gd100) through lactation. On gd135, half the ewes from each dietary group were challenged with either 1.2 μg/kg Escherichia coli lipopolysaccharide (LPS) endotoxin, or saline as the control. The offspring’s cortisol response to weaning stress was assessed 50 days postpartum by measuring serum cortisol concentrations 0, 6 and 24 h post weaning. Twenty-four hours post-weaning, lambs were subjected to an adrenocorticotropic hormone (ACTH) challenge (0.5 μg/kg) and serum cortisol concentrations were measured 0, 0.25, 0.5, 1 and 2 h post injection. At 5.5 months of age, offspring were also challenged with 400 ng/kg of LPS, and serum cortisol concentrations were measured 0, 2, 4 and 6 h post challenge. Interestingly, female offspring born to FM+LPS mothers had a greater cortisol response to weaning and endotoxin challenge compared with the other treatments, while female offspring born to SM+LPS mothers had a faster cortisol response to the ACTH stressor. Additionally, males born to FM+LPS mothers had a greater cortisol response to the ACTH challenge than the other treatments. Overall, FM supplementation during gestation combined with LPS challenge alters HPAA responsiveness of the offspring into adulthood.

Low somatic cell count (SCC) is a reliable indicator of high-quality milk free of pathogenic microorganisms. Thus, an important goal in dairy practice is to produce milk with low SCC. Selection for cows with low SCC can sometimes lead to extremely low SCC in single quarters. The cells in milk are, however, predominantly immune cells with important immune functions. To investigate the mammary immune competence of quarters with very low SCC, healthy udder quarters of cows with normal SCC of (40–100)×103 cells/ml and very low SCC of <20×103 cells/ml were challenged with lipopolysaccharide (LPS) from Escherichia coli. In the first experiment, SCC and cell viability after a challenge with 50 ng of LPS/quarter was investigated. In the second experiment, tumour necrosis factor α (TNF-α) concentration and lactate dehydrogenase (LDH) activity in milk, and mRNA expression of various innate immune factors in milk cells were measured after a challenge with 100 μg LPS/quarter. LPS challenge induced an increase of SCC. SCC levels reached were higher in quarters with normal SCC and maximum SCC was reached 1 h earlier than in very low SCC quarters. The increase of TNF-α concentrations in milk in response to LPS challenge was lower in quarters with very low SCC than in quarters with normal SCC. The viability of cells and the LDH activity in milk increased in response to LPS challenge, however, without a difference between the groups. The mRNA expression of IL-1β and IL-8 was increased in milk cells at 12 h after LPS challenge, whereas that of TNF-α and lactoferrin was not increased at the measured time points (12, 24 and 36 h after LPS challenge). No differences of mRNA expression of measured immune factors between normal and very low SCC samples were detected. The study showed that udder quarters with very low SCC responded with a less marked increase of SCC compared with quarters with normal SCC. This difference corresponded with simultaneously lower TNF-α concentrations in milk. However, the immune competence of the cells themselves based on mRNA expression of TNF-α, IL-8, IL-1β, and lactoferrin, did not differ. The results may indicate that very low SCC can impair the immune competence of udder quarters, because the immune response in udder quarters with lower SCC is less efficient as fewer cells contribute to the production of immunoregulators.

Lactoferrin (Lf) is a molecule naturally present in bovine milk that affects the availability and transport systems of iron. Lf also binds endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria and modulates the immunological response. In the present study, concentrations of bovine Lf (bLf) and citrate in milk were determined in early (EL) and late (LL) lactating dairy cows, using an experimentally induced endotoxin mastitis model and a crossover design. Nine clinically healthy Finnish Ayrshire cows were challenged twice with 100 μg endotoxin infused into one udder quarter. Milk samples were collected from the challenged and control quarters of each cow before and after endotoxin infusion during 3 d, and bLf and citrate concentrations were measured. In all cows, clinical signs of mastitis were seen at both times of challenge, but the response was more severe in EL than in LL. Concentration of bLf in the milk started to rise approximately 8 h after endotoxin infusion and was still higher than normal on the third day, especially in the late-lactating cows. In milk of the LL group, concentrations of bLf were significantly higher than in the EL group. In contrast, concentrations of citrate were higher in milk of the EL cows compared with the LL cows. Concentration of bLf and citrate varied substantially among cows. The molar ratio of citrate to bLf before and after challenge was significantly higher during the EL period. The results of this study partly explain why cows in early lactation are more susceptible to intramammary infections and why mastitis is more severe in them.

Endotoxin levels were measured in the blood and cerebrospinal fluid (CSF) of control individuals and 2 groups of patients with African sleeping sickness. Endotoxin levels were markedly elevated in the blood (infected groups mean endotoxin values 40·2 pg/ml and 53·8 pg/ml, compared to control 11·6 pg/ml, P < 0·0001 for both increases) and CSF (infected groups mean endotoxin values 45·8 pg/ml and 50·1 pg/ml compared to control 6·3 pg/ml, P < 0·0001 for both increases) of the patients. The levels were reduced 6 weeks following different drug treatments in the 2 groups (blood levels to mean 33·8·pg/ml and 28·5 pg/ml; CSF levels to 37·4 pg/ml and 27·0 pg/ml). The blood endotoxin values correlated with the CSF values before treatment (r = 0·74 and 0·57 for the 2 groups; P < 0·0001 for both) and after treatment (r = 0·57 and 0·56 for the 2 groups; P < 0·0001 for both). It is concluded that raised endotoxin equilibrates in the blood and CSF compartments, and may contribute significantly to the pathology of sleeping sickness.

CBA/T6 and DBA/2J mice inoculated with Plasmodium berghei ANKA (PbA) develop cerebral involvement 6–8 days post-inoculation, from which the CBA mice almost invariably die and the DBA mice recover. Dexamethasone (DXM; 80 mg/kg) given to inoculated CBA mice twice, on day 3 and again within 48 h, reduced the cerebral symptoms and prevented death from cerebral malaria. Plasma tumour necrosis factor (TNF) levels, which increased at the time of the cerebral symptoms, were also reduced in these DXM-treated mice. Intravenously administered Evans Blue, a dye which binds to albumin, diffused extensively across the blood-brain barrier only during the period of cerebral symptoms, in proportion to the severity of the cerebral symptoms and the disease. In PbA-infected CBA mice, cerebral symptoms and the amount of Evans Blue diffusing into the brain tissue were both reduced by DXM treatment, but only if the steroid was given on day 3 and again within 48 h. Endotoxin injected intravascularly into PbA-infected DBA mice after day 5 resulted in an exaggeration of cerebral symptoms and death between days 6 and 9. Plasma TNF and the amount of Evans Blue in the brain parenchyma increased above normal levels in these mice. Endotoxin injections had only minor effects on the severity of the cerebral symptoms in PbA-infected CBA mice and did not cause the animals to die sooner.

The objective was to determine the effects of supplementing the diet with fish oil during the peri-partum period on the immune competence and the pathophysiological response to a lipopolysaccharide-induced mastitis challenge. Multiparous Holstein cows (n=30) were completely randomized to one of two treatments at 3 weeks pre-partum. Treatments differed only in the source of supplemental lipid and included either Energy Booster® or fish oil. Treatment diets were fed from −21 d relative to expected date of parturition until 10 d post partum. Treatments were fed as a bolus prior to the a.m. feeding. The dose of lipid during the pre-partum period was 250 g/d, whereas the amount of lipid supplemented post partum was adjusted to the level of intake, approximately 0·92% of the previous day's dry matter intake. Ex-vivo analyses of immune competence were measured including the antimicrobial activity of whole blood against Escherichia coli, Salmonella typhimurium and Candida albicans as well as the production of interferon-γ by peripheral blood mononuclear cultures. At 7 days in milk cows were infused with 100 μg of Esch. coli lipopolysaccharide into one rear quarter. Supplementing fish oil increased plasma concentrations of eicosapentaenoic and docosahexaenoic acids, but had no affect on the proportions of arachidonic acid at calving. Fish oil did not influence the production of interferon-γ or the antimicrobial activity of whole blood against any of the microorganisms. Furthermore, fish oil had no ameliorative effect on either the local or the systemic acute phase response following an intramammary lipopolysaccharide challenge in early lactating Holstein cows. Supplementing fish oil in the diet of peri-partum cows will not protect them from deleterious effects of an excessive acute phase response.

A water purification and sterilization device was tested for its functional capabilities. Challenge water consisting of potable water augmented with bacteria, endotoxin, virus, suspended solids, and dissociable ions (sodium chloride, lead or arsenic salts) was passed through the device. The product water quality attributes were analyzed. The device demonstrated reduction in bacteria of >7 logs, endotoxin was reduced by >4 logs, virus was reduced by >4 logs, and dissociable ions were reduced by >3 logs. The product water of the device met the limits for a range of chemical entities specified by the United States Pharmacopeia and Association for the Advancement of Medical Instrumentation. The product water met the quality attributes of Sterile Water for Injection, USP, Sterile Purified Water, USP, and the Water for Dialysis. The device provides a logistical advantage in reducing the weight of transport of packaged water by 83% and the cube by 67%. It operates manually by gravity and is disposable after a single use. The device provides an effective alternative to the transport and use of packaged sterile water in remote locations by production of sterile water at the pointof-need using available water. It also is capable of producing safe drinking water following the production of clinical waters. This device has been cleared by the US Food and Drug Administration for production of three liters Sterile Purified Water, USP from Environmental Protection Agency (EPA) grade drinking water.