Effect of the topical administration of corticosteroids and tuberculin pre-sensitisation on the diagnosis of tuberculosis in goats.

BACKGROUND: Caprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB control and eradication programmes have traditionally been based on intradermal tuberculin tests and slaughterhouse surveillance. However, this strategy has limitations in terms of sensitivity and specificity. Different factors may affect the performance of the TB diagnostic tests used in goats and, subsequently, the detection of TB-infected animals. In the present study, the effect of two of the factors that may affect the performance of the techniques used to diagnose TB in goats, the topical administration of corticosteroids and a recent pre-sensitisation with tuberculin, was analysed. METHODS: The animals (n = 151) were distributed into three groups: (1) a group topically treated with corticosteroids 48 h after intradermal tuberculin tests (n = 53); (2) a group pre-sensitised with bovine and avian purified protein derivatives (PPDs) 3 days before the intradermal tuberculin test used for TB diagnosis (n = 48); and (3) a control group (n = 50). All the animals were tested using single and comparative intradermal tuberculin (SIT and CIT, respectively) tests, an interferon-gamma release assay (IGRA) and a P22 ELISA. RESULTS: The number of SIT test reactors was significantly lower in the group treated with corticosteroids when compared to the pre-sensitised (p < 0.001) and control (p = 0.036) groups. In contrast, pre-sensitisation with bovine and avian PPDs did not cause a significant reduction in the number of SIT and CIT test reactors compared with the control group. In fact, a higher number of reactors was observed after the prior tuberculin injection in the pre-sensitised group (p > 0.05). No significant effect was observed on IGRA and P22 ELISA due to corticosteroids administration. Nevertheless, a previous PPD injection affected the IGRA performance in some groups. CONCLUSIONS: The application of topical corticosteroid 24 h before reading the SIT and CIT tests can reduce the increase in skin fold thickness and subsequently significantly decrease the number of positive reactors. Corticosteroids used can be detected in hair samples. A previous pre-sensitisation with bovine and avian PPDs does not lead to a significant reduction in the number of intradermal tests reactors. These results are valuable in order to improve diagnosis of caprine TB and detect fraudulent activities in the context of eradication programs.

Evolution of breast milk fatty acids in Spanish mothers after one year of uninterrupted lactation.

The WHO recommends exclusive breastfeeding of infants for the first sixth months of life and advises that it shall continue for up to two years of age or beyond in combination with complementary foods. However, the image of a woman breastfeeding a toddler or a preschooler is unusual in western societies. Exploring the nutritional properties of milk during prolonged lactation can help normalizing prolonged breastfeeding. Human milk fatty acid composition was determined in sixteen lactating mothers practicing prolonged lactation (≥12 months) and sixteen women on their first twelve months of lactation. Breast milk after one year is richer in saturated fatty acids, particularly lauric and myristic, showing a tendency towards lower levels of oleic acid, and higher of arachidonic, α-linolenic and docosahexaenoic acids, in comparison to early milk (< 1 year). The age and body condition of the mother, parity, sex of the baby, and diet influence also the fattyacidome of milk.

Fatty acid composition of breast milk in Galicia (NW Spain): A cross-country comparison.

Based on the premise that the fatty acid composition of human milk can be substantially altered by diet, the current study investigated the fatty acid profile (fattyacidome) of breast milk in Galicia, a small region located in the north-west of Spain and characterized by the Southern European Atlantic Diet (SEAD). A cross-country comparison was also performed to assess worldwide variety and diet impact, reviewing the profiles reported various European, North and South American, Asian and African countries and Australia. Galician human milk appeared similar to the rest of Europe, with some particular features related to the SEAD (dairy, pork, beef and sunflower and olive oils), such as relatively high levels of linoleic acid and lower α-linolenic acid. The results also showed the existence of woman-specific profiles and significant changes over lactation in some fatty acids. Worldwide, the fatty acid profiles were similar, with the clear exception of Asiatic breast milk. The impact of fatty acids on infant health warrants further investigation.

Effects of bovine pregnancy on the fatty acid composition of milk: the significance for humans needs.

Milk from 40 Holstein dairy cows was collected from two different farms in Galicia (Spain). The differences in the fatty acid composition of two groups of cows, 20 pregnant and 20 non-pregnant, was studied to determine whether pregnancy status is a determinant factor that can alter the fatty acid profile of milk. Gas-chromatography (GC) coupled to flame ionisation detection (FID) was used for the determination of the fatty acids. Differences in the milk fatty acids between pregnant and non-pregnant cows were pronounced showing statistically significant differences for some fatty acids and the total saturated and monounsaturated fatty acids. Milk from non-pregnant cows was lower in saturated fatty acids and higher in monounsaturated fatty acids (unlike milk from pregnant cows). The effects of the consumption of bovine milk, particularly milk fat, on human health have been studied in depth and sometimes are associated with negative effects, but milk has also several beneficial characteristics linked to some fatty acids.

Simultaneous determination of sulfonamides, penicillins and coccidiostats in pork by high-performance liquid chromatography-tandem mass spectrometry.

Veterinary drugs are widely and legally used to treat and prevent disease in livestock. However, drugs are also used illegally as growth-promoting agents. To protect the health of consumers, maximum residue limits (MRL) in food of animal origin have been established and are listed in Regulation 37/2010. According to this regulation, more than 300 drugs need to be controlled regularly in laboratories for residues of veterinary drugs. A cost-effective analytical method is very important and explains why the development of multi-residual methods is becoming popular in laboratories. The aim of this work is to describe a simple, rapid and economical high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous identification and quantification of 21 veterinary drugs in pork muscle samples. The sample clean-up procedure is performed with acidified dichloromethane and does not require solid phase extraction. The method is applicable to nine sulfonamides and seven coccidiostats identified within 36 min. Calculated relevant validation parameters such as recoveries (from 72.to 126 %), intra-precision and intermediate precision (relative standard deviation below 40 %) and decision limits (below 7 µg Kg(-1)) were within acceptable range and in compliance with the requirements of Commission Decision 2002/657/EC.

Development of an LC-MS/MS method to quantify sex hormones in bovine milk and influence of pregnancy in their levels.

Hormones work in harmony in the body, and this status must be maintained to avoid metabolic disequilibrium and the subsequent illness. Besides, it has been reported that exogenous steroids (presence in the environment and food products) influence the development of several important illnesses in humans. Endogenous steroid hormones in food of animal origin are unavoidable as they occur naturally in these products. The presence of hormones in food has been connected with several human health problems. Bovine milk contains considerable quantities of hormones and it is of particular concern. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, based on hydroxylamine derivatisation, has been developed and validated for the quantification of six sex hormones in milk [pregnenolone (P5), progesterone (P4), estrone (E1), testosterone (T), androstenedione (A) and dehydroepiandrosterone (DHEA)]. This method has been applied to real raw milk samples and the existence of differences between milk from pregnant and non-pregnant cows has been statistically confirmed. Basing on a revision of existing published data, it could be concluded that maximum daily intakes for hormones are not reached through milk ingestion. Although dairy products are an important source of hormones, other products of animal origin must be considered as well for intake calculations.

Determination of the hormonal growth promoter 17alpha-methyltestosterone in food-producing animals: bovine hair analysis by HPLC-MS/MS.

This paper describes the development, validation and application of a confirmatory method to detect 17alpha-methyltestosterone (MT) in bovine hair, to aid in controlling the administration of this growth promoter in meat-producing animals. After cryogenic grinding, MT was removed from the hair matrix using a single step extraction procedure with acetonitrile. Hydroxylamine derivatisation was used to enhance analyte determination with an electrospray ionisation (ESI) source. Determination was carried out using a triple quadrupole liquid chromatography tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode (MRM). The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC and using deuterated testosterone (T-d(3)) as the internal standard. The decision limit (CCalpha) was 0.07 ng g(-1) and the detection capability (CCbeta) was 0.12 ng g(-1). Repeatability was CV% (7%), within-laboratory reproducibility was CV% (11.0%), and trueness was (87%). Applicability of the method was demonstrated in an animal study. Samples obtained from animal experiments were analyzed and the presence of MT was confirmed.

Quantitative LC-MS/MS method for the sensitive and simultaneous determination of natural hormones in bovine serum.

We have developed a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the simultaneous quantitative analysis of several free forms of steroid hormones in bovine serum [pregnenolone (P(5)), progesterone (P(4)), 17hydroxyP(5), 17hydroxyP(4), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (A), estrone (E(1)), 2, 4 and 16 hydroxyE(1), 2 and 4 methoxyE(1)]. Deuterated analogs were used as internal standards. Serum proteins were eliminated with acetonitrile. Oxime derivatives of steroids were extracted with tert-butylmethylether and analyzed in positive MRM mode. Methodology was validated in accordance with the European Commission Decision 2002/657/EC. Performance characteristics permit the use of this methodology for steroid determination in animal serum samples.

Syntheses of molecularly imprinted polymers: Molecular recognition of cyproheptadine using original print molecules and azatadine as dummy templates.

The use of custom-made polymeric materials with high selectivities as target molecules in solid-phase extraction (SPE), known as molecularly imprinted solid-phase extraction (MISPE), is becoming an increasingly important sample preparation technique. However, the potential risk of leakage of the imprinting molecules during the desorption phase has limited application. The use of a mimicking template, called a dummy molecular imprinting polymer (DMIP), that bears the structure of a related molecule and acts as a putative imprinting molecule may provide a useful solution to this problem. In the current study, cyproheptadine (CPH) and azatadine (AZA) were used as templates in the development of an MIP and DMIP for acrylic acid and methacrylic acid monomers. Our results indicate that DMIPs have equal recognition of CPH, avoiding the problem of leakage of original template during the desorption phase relative to MIPs synthesized in presence of the print molecule CPH. Examination of the surface structure of the two polymer products by SEM shows appreciable differences in structural morphology and function of the monomers employed. These results are well supplemented by data obtained for swelling ratios and solvent uptake. Molecular modelling of CPH and AZA suggests that both substrates are similar in shape and volume.

Prior immunity to Trichinella spiralis prevents (re)occurrence of an explicit stress response in intestines but not in mesenteric lymph nodes, heart and lungs from reinfected rats.

We recently showed that, in our Trichinella spiralis rat model, first exposure, but not re-exposure to infective-stage larvae evoked heat shock responses in 4 test organs. Our work, however, failed to implicate either early complete clearance of challenge muscle larvae (ML), or rapid elimination of newborn larvae (NBL) in the phenomenon noted in reinfected rats. This study clarifies that issue using 2 established facts in T. spiralis biology and anti-T. spiralis immunology. That is, adult worms injure gut cells and immune destruction of NBL requires release of material also toxic to host cells. To approach the above problem we analysed relevant and irrelevant rat organs for increased heat shock protein (HSP) production at 1, 7, 14, 20 and 27 p.i. during first and second infections. Organs examined were intestines, mesenteric lymph nodes (MLN), heart and lungs. Using densitometric analyses of immunoblots, increased HSP expression was detected on day 7 in intestines from both primary and secondary-infected rats albeit that the change in the latter was just short of significant. Interestingly, MLN only exhibited increased HSP levels in the reinfected rat model with increased HSP levels persisting for 1 week. A lasting shock response was detected in reinfected rats; in contrast, first exposure resulted in shock responses being evident in lungs at either day 7 or day 14, only. These findings suggest that (i) in immune rats, a few challenge ML develop into adults, produce NBL which are trapped within MLN, and (ii) that anti-T. spiralis and/or anti-NBL immunity is associated with an, as yet, uncomprehended stress to host's heart tissues.

The true meaning of 'exotic species' as a model for genetically engineered organisms.

The exotic or non-indigenous species model for deliberately introduced genetically engineered organisms (GEOs) has often been misunderstood or misrepresented. Yet proper comparisons of of ecologically competent GEOs to the patterns of adaptation of introduced species have been highly useful among scientists in attempting to determine how to apply biological theory to specific GEO risk issues, and in attempting to define the probabilities and scale of ecological risks with GEOs. In truth, the model predicts that most projects may be environmentally safe, but a significant minority may be very risky. The model includes a history of institutional follies that also should remind workers of the danger of oversimplifying biological issues, and warn against repeating the sorts of professional misjudgements that have too often been made in introducing organisms to new settings. We once expected that the non-indigenous species model would be refined by more analysis of species eruptions, ecological genetics, and the biology of select GEOs themselves, as outlined. But there has been political resistance to the effective regulation of GEOs, and a bureaucratic tendency to focus research agendas on narrow data collection. Thus there has been too little promotion by responsible agencies of studies to provide the broad conceptual base for truly science-based regulation. In its presently unrefined state, the non-indigenous species comparison would overestimate the risks of GEOs if it were (mis)applied to genetically disrupted, ecologically crippled GEOs, but in some cases of wild-type organisms with novel engineered traits, it could greatly underestimate the risks. Further analysis is urgently needed.

The adaptive potential of genetically engineered organisms in nature.

Most genetically engineered organisms are unlikely to pose any threat to the environment because they are already highly selected for survival under restricted conditions. Engineering for new traits in natural or semi-natural populations, however, may entail greater risks. Genetic novelty, i.e. mutation, is an important component of the evolutionary process; a small but significant proportion of natural mutations lead to improved fitness and increased competitiveness. The artificial insertion of a new trait may produce a similar effect, setting an organism on a new and unpredictable evolutionary track. The current challenge is to attain the capacity to identify the small proportion of genetically engineered organisms in which such events might occur.

Changes with age characterize circadian rhythm in telemetered core temperature of stroke-prone rats.

In a stroke-prone (SP) strain of laboratory rats, aging is associated with quantifiable changes of the circadian rhythm in body core temperature: (1) its timing becomes less tight, as revealed by a larger standard error of the acrophase (the peak in the 24-hour cosine function best approximating all data) and (2) the acrophase in old SPs occurs earlier than in young ones--quite apart from (3) a decrease in circadian amplitude reported earlier. These results gain particularly in interest in the context of the recent finding that a large (approximately 90 degrees) acrophase-advance is associated with bilateral lesions of the suprachiasmatic nuclei in inbred (non-SP) Fischer rats. These observations may be of interest to those developing models of aging functions in disorders with blood pressure elevation.

Ecology and evolution of flowering plant dominance.

Birds and mammals are important seed dispersers and their diversification in the Cretaceous may have created niches for many plant specialists on scattered resources. Maintaining sexual recombination through wind pollination in such sparse populations is difficult, and so angiosperms with their sophisticated systems for insect pollination were favored in many critical situations.