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BIOTECHNOLOGICAL INVENTION OF CALOXINS - A NOVEL CLASS OF ALLOSTERIC INHIBITORS SPECIFIC FOR PLASMA MEMBRANE CALCIUM PUMP ISOFORMSSzewczyk, Maria Magdalena 10 1900 (has links)
<p>This work used biotechnology to invent new caloxins - allosteric peptide inhibitors of plasma membrane Ca<sup>2+ </sup>pumps (PMCA) needed to understand the Ca<sup>2+ </sup>signalling in coronary artery.</p> <p>PMCA are encoded by genes PMCA1-4. Defects in PMCA expression have been associated with several pathologies. The major objectives of my thesis were to determine the expression of PMCA isoforms in the smooth muscle and the endothelium of coronary artery and to invent high affinity and specificity caloxins for the isoforms present in these tissues.</p> <p>In Aim 1 it was determined that the total PMCA protein and activity was much greater in smooth muscle than in endothelium. Both tissues expressed only PMCA1 and PMCA4, with PMCA4 > PMCA1 in smooth muscle and PMCA1 > PMCA4 in endothelium. Therefore, the search for PMCA1 and 4 selective caloxins using phage display technique was conducted.</p> <p>Aim 2 was to invent PMCA1 selective inhibitors. Caloxin 1b3 was invented as the first known PMCA1 selective inhibitor. It inhibited PMCA1 Ca<sup>2+</sup>-Mg<sup>2+</sup>-ATPase with higher affinity than PMCA2, 3 or 4. Aims 1 and 2 were consistent with the greater potency of caloxin 1b3 than a known PMCA4 selective caloxin 1b1 in increasing cytosolic Ca<sup>2+</sup> concentration in endothelial cells.</p> <p>Aim 3 was to obtain ultrahigh selectivity and affinity PMCA4 bidentate inhibitor using the previously invented PMCA4 selective caloxins 1c2 and 1b2. In the first step the affinity of caloxin 1b2 was improved by limited mutagenesis to obtain caloxin 1c4. Caloxin 1c4 had 5-6 times higher affinity than caloxin 1b2 for inhibiting PMCA4 activity. Optimization of the bidentate caloxins from caloxin 1c2 and 1c4 was also attempted.</p> <p>The novel caloxins may aid in elucidating the role of PMCA1 and PMCA4 in the physiology and pathophysiology of coronary artery and other tissues.</p> / Doctor of Philosophy (PhD)
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Antidepressant use during pregnancy: Determining the impact on the gut serotonergic system in the offspringLaw, Harriet 11 1900 (has links)
Approximately 10% of pregnant women take antidepressants. Prenatal exposure to selective serotonin reuptake inhibitors (SSRIs), a class of antidepressants, has been shown to alter serotonergic signaling in the brain. However, the effects of SSRIs on peripheral serotonin (5HT) synthesis and/or signaling have largely been ignored. Serotonin in the gut is critical for intestinal function and dysregulation of this pathway is associated with intestinal disease. Therefore, the goal of this study was to determine the effects of perinatal exposure to the SSRI fluoxetine (Prozac®) on intestinal health in the offspring. Dams were given vehicle or fluoxetine hydrochloride (FLX 10 mg/kg/d; N=15) for 2 weeks prior to mating until weaning. We assessed markers of serotonergic signaling, inflammation, and composition of the gut microbiota in the offspring. Male offspring of fluoxetine-treated dams had significantly elevated serum levels of 5-HT and decreased expression of the 5HT2A receptor and MAO. In female offspring there was no effect of SSRI exposure to alter any components of serotonergic signaling. Although we did not find any evidence of increased inflammation following fluoxetine exposure, there were significant alterations in the composition of the gut microbiota in the exposed offspring.
Male offspring of SSRIs-exposed mothers had changes in key components of the gut serotonergic system in association with elevated levels of serum 5-HT and alterations in the gut microbiota in adulthood. The impact of these changes on intestinal health and the reasons for the sex specific effects remain to be determined. / Thesis / Master of Science in Medical Sciences (MSMS)
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Methods in organosilane assemblyBo, Yingjian January 2012 (has links)
Dialkylsilanediols are a novel class of non-hydrolyzable analogues of the tetrahedral intermediate of amide hydrolysis, shown to be good inhibitors of HIV-1 protease, angiotensin converting enzyme (ACE), and thermolysin. An impediment to utilization of these silanediol structures, however, has been the methods for their assembly. This research describes the reductive lithiation of hydridosilanes and alkoxysilanes, and the use of the resulting silyl anions to develop efficient methods to synthesize silanediol precursors. In the first part of research, lithiation of hydridosilanes was studied. As part of this study, a simple 1H NMR method was developed for monitoring and analyzing the progress of lithiation. In addition, this method was converted to a titration for silyllithium reagents using BHT as an internal standard. Silanediols 107 and 177 are analogues of a potent chymase inhibitor, NK-3201 (82). In the second part, diphenylsilanes 108 and 170, precursors to silanediols 107 and 177, were synthesized using addition of silyllithium to sulfinimine 113 as a key step. In the third part, lithiation of alkoxysilanes was studied. (Si,O)-Dianions, generated from lithiation of silane alcohol 175 or 2,2-diphenyl-1-oxa-2-silacyclopentane (225), were reacted with a wide variety of electrophiles to give potentially useful silicon-containing building blocks. Addition of the (Si,O)-dianion 284 to sulfinimines gave silanediol inhibitor precursors with full control of stereochemistry. In the last part, a new method featuring 1,1-diphenyl-2-azaallyllithium chemistry were utilized to synthesize a series of protected α-amino silanes 323, 329 - 331. / Chemistry
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Engineering α-1 Proteinase Inhibitor to Target Neutrophil Serine Proteinase PR3Al-Arnawoot, Ahmed January 2020 (has links)
Activated neutrophils release a neutrophil serine proteinase (NSP) called Proteinase 3 (PR3). In granulomatosis with polyangiitis (GPA), an autoimmune vasculitis, enhanced PR3 release results in endothelial damage. Serine proteinase inhibitors (serpins) such as α-1 proteinase inhibitor (API) inhibit NSPs through the serpin’s reactive center loop (RCL). However, API is known to bind PR3 with a low specificity, compared to its main inhibitory target Human Neutrophil Elastase (HNE). The current treatment for GPA is immunosuppression, which leaves patients immunocompromised. Thus, the overall aim of this study was to engineer an API variant with a higher specificity to PR3 than HNE, which could serve as a possible novel therapeutic strategy for GPA.
We created an API expression library, hypervariable at RCL residues A355-I356-P357-M358-S359, and expressed it in a T7 bacteriophage display system. This phage library was then biopanned for PR3 binding. Two conditions were used for each round of biopanning: experimental, with PR3, and the negative control, without PR3. The library was biopanned for a total of five consecutive rounds, with the product of one screen serving as the starting material for the next. A bacterial mass lysate screen was also employed to further probe the library with PR3.
The phage-display and bacterial lysate screens resulted in the selection of two novel variants API-DA (D357/A358) and API-N (N359). Serpin-proteinase gel complexing assays indicated that API-N formed complex with PR3 similar to API-WT (wild-type), while API-DA was mainly cleaved as a substrate. There was no significant difference between the second order rate constants of API-N and API-WT reactions with PR3. Rate constants for API-DA binding to PR3 or for API-HNE reactions were not completed due to novel coronavirus (COVID-19) restrictions. However, this project successfully demonstrated the ability to screen a hypervariable API phage library with PR3, yielding two new novel API variants. / Thesis / Master of Science in Medical Sciences (MSMS) / When harmful substances enter our body such as bacteria or viruses, we have ways of protecting ourselves from them. One of those ways is through a cell called the neutrophil. This is an immune cell that can release “fighting tools” into our blood to combat the harm. Some of these tools are called proteins. One of those proteins is Proteinase 3. However, sometimes our neutrophils can be activated without the presence of viruses or bacteria by products made in our bodies called autoantibodies. When this happens, too many of the “fighting tool” Proteinase 3 is released leading to damage to the tubes or vessels that our blood flows through. This project aimed to find a new possible way to stop these extra fighting tools from doing harm to our body. We did this by creating a library of different proteins that can stop Proteinase 3 once it is released by the neutrophil.
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Estudio computacional del receptor GABA_A α1ß2γ2 y su interacción con moléculas de interés biológico en el sitio de unión de benzodiazepinasAmundarain, María Julia 15 March 2019 (has links)
Los receptores GABA_A son canales iónicos activados por ligandos y funcionan como los principales mediadores de la inhibición en el sistema nervioso central de mamíferos. Están
formados por cinco subunidades formando un poro central conductor de iones. Cada combinación de subunidades presenta una función y localización determinada, de las cuales el
subtipo α1ß2γ2 es el más abundante en el ser humano. Los receptores GABA_A intervienen
en una miríada de procesos neurológicos y su desregulación genera las denominadas canalopatías. Por lo cual, el estudio de estos sistemas es indispensable para el desarrollo de fármacos
y de tratamientos para mejorar la calidad de vida.
En este trabajo de tesis se propone el estudio in silico del receptor GABA_A α1ß2γ2
mediante el empleo de técnicas de bioinformática y biofísica computacional, que incluyen
simulaciones de docking molecular, dinámica molecular y técnicas de muestreo avanzado.
Se desarrolló un modelo por homología del receptor empleando el receptor GABA_A homopent
mero de subunidades ß3. El modelo fue validado a través de un cuidadoso análisis
de su estereoquímica y su estabilidad mediante simulaciones de dinámica molecular. A continuación, se realizó un exhaustivo análisis de la unión de compuestos a dos sitios de unión
en el dominio extracelular del modelo: el sitio ortostérico (donde se unen los ligandos que
actúan directamente sobre la activación del canal) y el sitio de unión de gran afinidad de
las benzodiazepinas (moduladores alostéricos). Los modos de unión encontrados fueron contrastados
con información experimental disponible y se halló muy buena concordancia. El
trabajo finalizó con el primer estudio computacional sobre la interacción putativa entre este
receptor y la proteína DBI y fragmentos peptídicos derivados de su digestión. Este análisis
permitió elaborar, por primera vez, una hipótesis respecto a los residuos involucrados en la
interacción. / GABA_A receptors are pentameric ligand-gated ion channels which act as the main mediators
of inhibitory signalling in the central nervous system of mammals. They are formed by
five subunits arranged around a central ion-conducting pore. Each combination of subunits
has a specific function and localization, the α1ß2γ2
subtype being the most abundant in homo
sapiens. These receptors intervene in a myriad of neurological processes and their disregulation
cause several channelopathies. Although they are very complex systems, their study is
fundamental for the development of new drugs and therapies aimed at improving life quality.
In this thesis we performed an in silico study of the α1ß2γ2 GABA_A receptor through the
use of bioinformatics and computational biophysics tools, which include molecular docking,
molecular dynamics and enhanced sampling techniques.
A homology model was developed using the structure of the GABAA_A ß3 homopentamer.
The model was validated through a thorough analysis of its stereochemistry and its stability
was evaluated from molecular dynamics simulations. Moreover, an exhaustive evaluation of
the binding modes of ligands to two extracellular sites was performed: the orthosteric site
(ligands which act directly on the activation of the channel) and the high affinity binding
site for benzodiazepines (allosteric modulators). The comparison of the binding modes to
available experimental information showed great agreement. Finally, a computational study
was carried out for the first time regarding the putative interaction of this receptor with
DBI and its peptide fragments. This study allowed the formulation of the first hypotheses
regarding the aminoacids involved in the interaction.
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Biological and Synthetic Studies of Mitochondrial Respiratory Chain Inhibitors / ミトコンドリア呼吸鎖阻害剤に関する生物および合成化学的研究Tsuji, Atsuhito 23 March 2023 (has links)
京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24555号 / 薬科博第172号 / 新制||薬科||19(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 小野 正博, 教授 掛谷 秀昭 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM
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Design and Synthesis of Orally Bioavailable Sphingosine Kinase 2 Selective InhibitorsSibley, Christopher David 16 July 2020 (has links)
In humans, mammals, and perhaps all vertebrates, sphingolipids exist as a family of cellular signaling molecules and have been shown to be involved in a wide range of biological processes ranging from proliferation to apoptosis. As such, sphingolipid signaling has garnered the attention of numerous researchers as an attractive candidate for pharmacological manipulation. The synthetic pathway of one prominent sphingolipid, sphingosine 1-phosphate (S1P), has been implicated in a variety of disease states such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Formation of S1P is facilitated from the ATP dependent phosphorylation of sphingosine (Sph) through its generative enzyme's sphingosine kinase 1 and 2 (SphK1 and SphK2). Inhibition of SphK1 and SphK2 results in the manipulation of S1P levels, which has been shown to be therapeutic in various animal models of disease. While there are multiple examples of potent SphK1-selective and dual SphK1/2 inhibitors, SphK2-selective inhibitors are scarce.
Herein, we describe the design, synthesis and biological testing of SphK2-selective inhibitors. We first describe the discovery that introducing a trifluoromethyl group onto the internal aryl ring of our inhibitor scaffold led to superior selectivity and potency towards SphK2. We demonstrate that the trifluoromethyl moiety is interacting with a previously unknown side cavity in the substrate binding site of SphK2 that is unique and could be exploited in the design of SphK2-selective inhibitors. The synthesis of 21 derivatives with various substituents spanning off the internal aryl ring was completed, therefore characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site. This work led to the development of the most potent SphK2-selective inhibitor known at the time. We then describe the transformation of our SphK2-selective inhibitors into an orally bioavailable drug. We explain how the guanidine functionality on our inhibitor scaffold hinders our compounds from being orally bioavailable. Consequently, a library of 24 derivatives with various modifications to the guanidine functionality was synthesized and evaluated for improved orally bioavailability. Highlighted in this work is the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832), which displays a hSphK2 Ki of 82 nM and 122-fold selectivity for SphK2. Chemical modification and in vivo assessment of 3.14 (SLS1081832) prodrugs was explored. / Doctor of Philosophy / In humans, sphingosine 1-phosphate (S1P) is a signaling molecule that is generated through an ATP dependent reaction of sphingosine (Sph) via sphingosine kinase 1 and 2 (SphK1 and SphK2). Furthermore, S1P has been shown to be implicated in various diseases such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Inhibition of SphK1 and SphK2 has been shown to be therapeutic towards the symptoms of these diseases. Therefore, in order to alleviate these disorders, the concentrations of S1P must be controlled through pharmacological inhibition of SphK1 and SphK2. There are multiple reported examples of potent SphK1-selective and dual SphK1/2 inhibitors; however, SphK2-selective inhibitors are scarce. This work describes the synthesis and biological assessment of 21 compounds for their effectiveness in selectively targeting and inhibiting SphK2. The work led to the discovery of a previously unrecognized side cavity in the binding pocket of SphK2 that enhances inhibitor potency and selectivity towards SphK2. Furthermore, studies characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site led to the development of the most potent SphK2- selective inhibitor known at the time. Building on this work, we next focused on the transformation of our SphK2-selective inhibitors into a drug that could be administered orally. We describe the synthesis of 24 compounds with various modifications to one portion of our scaffold and their effect on improved orally bioavailability. This work led to the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832).
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Quantification of Fungicide Resistance in Cercospora sojina Populations and Development of a Fungicide Application Decision Aid for Soybean in the Mid-Atlantic U.S.Zhou, Tian 09 October 2019 (has links)
Soybean is an important source of protein in animal feed, and growing demand for meat consumption worldwide has led to increased soybean production. Over 120 million metric tons of soybean were harvested in the United States in 2018, approximately one-third of the world production. In the Mid-Atlantic region, soybean is one of the most valuable field crops. Major foliar diseases that reduce soybean yield in the Mid-Atlantic region are frogeye leaf spot (FLS) and Cercospora leaf blight. In addition to crop rotation and host resistance, foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage these soybean foliar diseases. Yield benefits of foliar fungicides have been inconsistent and this may be the result of low disease pressure, unfavorable environmental conditions for disease development, or the presence of fungal pathogen populations that have developed resistance to fungicides. The objectives of this research were 1) to develop a pyrosequencing-based assay to rapidly quantify QoI resistance frequencies in Cercospora sojina, the causal agent of FLS, 2) to examine the effects of fungicide application timings, disease pressure, and environmental factors on soybean yield, and 3) to develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. using a threshold decision rule. A pyrosequencing assay targeting the G143A mutation was designed, and a Virginia survey of C. sojina populations indicated that the G143A mutation conferring QoI resistance is widespread. In small plot fungicide application timing experiments, five weekly fungicide applications starting at beginning pod (R3) resulted in the greatest yield, but for single fungicide applications, R3 or 1 week after R3 resulted in the greatest yields. There was positive relationship between the cumulative number of disease favorable days (mean daily temperature 20-30°C and ≥ 10 hours of relative humidity >90%) from planting to R3 and disease severity at the full pod stage (r = 0.97, P = <0.01). Higher disease severity was associated with greater yield loss (r2 =0.53, P = 0.10) suggesting foliar fungicide applications are more likely to have yield benefits as the number of disease favorable days prior to R3 increase. A disease favorable-days threshold (FDT) using the environmental parameters indicated above was evaluated in on-farm experiments throughout Virginia, Maryland, and Delaware. Based on decision rules, FDT = 8 three weeks prior to R3 was the best predictor of a yield benefit with an R3 fungicide application. The decision aid was also able to correctly predict when a fungicide application would not be profitable ≥90% of the time. This weather-based decision aid along with monitoring of fungicide resistance development within the region will provide soybean growers in the Mid-Atlantic U.S. with tools to maximize yields and profitability. / Doctor of Philosophy / Soybean is the third most valuable field crop in the world, ranked only behind rice and wheat in value. Over 98% of the soybean crop is used for animal feed due to its high protein content. The United States is the largest soybean producer in the world, responsible for one-third of global production. Soybean is the top cash crop in the Mid-Atlantic region. Foliar fungal diseases can reduce the soybean yield by causing lesions on the leaves that reduce photosynthesis and cause premature defoliation. Frogeye leaf spot (FLS) caused by Cercospora sojina is a major yield reducing soybean foliar diseases in the Mid-Atlantic region. Foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage the disease. However, fungicide efficacy has been inconsistent. Inconsistencies may be due to low disease pressure, improper application timing, or fungicide resistance. The purpose of this research was to investigate the fungicide efficacy inconsistencies and to develop management tools to improve yield and maximize profitability. Our objectives were to 1) develop a molecular assay to quantify frequencies of the mutation conferring fungicide resistance in Virginia populations of C. sojina, 2) examine the effects of fungicide application timings, disease severity, and weather on soybean yield, and 3) develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. The C. sojina fungicide resistance mutation was widespread in Virginia, but overall frequencies were relatively low compared to findings from Midwest and Southern states. In fungicide timing experiments, beginning pod (R3) applications resulted in the most consistent yield benefits, and disease severity and yield loss increased as the number of weather-based disease favorable days prior to R3 increased. We used data from on-farm experiments in Virginia, Maryland, and Delaware to develop a weather-based disease favorable-days threshold that increased the probability that a fungicide application at R3 would have a yield benefit in soybean. The results of our research have led improved fungal disease management recommendations for soybean in the Mid-Atlantic that will maximize yields and profitability.
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Characterization of Corynespora cassiicola resistance to the quinone outside inhibitor fungicides, elucidation of fitness parameters, and defining alternative fungicide product strategies in Mississippi soybeanWang, Xiaopeng 13 May 2022 (has links) (PDF)
Target spot, caused by Corynespora cassiicola, is a common lower canopy disease of soybean in the southern United States. Given the recent resurgence of target spot and increasing reports of resistance to the quinone outside inhibitor (QoI) fungicide class within C. cassiicola, a survey of C. cassiicola from the Mississippi soybean production system was initiated in 2019 to determine the nature of its resistance mechanisms. A total of 819 monoconidial isolates were collected from 228 geographic field locations in 75 Mississippi counties. The molecular mechanism of resistance was determined using a PCR-RFLP analysis by comparing nucleotide sequences in the cytochrome b gene. The percentage of isolates containing the G143A substitution increased from 71.3% in 2016 to 93.5% in 2021. In all, 85.8% of the C. cassiicola isolates carried the G143A substitution. The EC50 values of QoI-resistant and -sensitive isolates to azoxystrobin varied significantly with QoI-sensitive isolates exhibiting lower EC50 values than QoI-resistant isolates. Moreover, results of fitness evaluations indicated that QoI-resistant isolates are more competitive than QoI-sensitive isolates and there were no fitness costs associated with QoI resistance in C. cassiicola. Additionally, the sensitivity of six C. cassiicola isolates to eight fungicide active ingredients in four fungicide classes were evaluated. Results indicated that three succinate dehydrogenase inhibitors benzovindiflupyr, fluxapyroxad, and pydiflumetofen were the most effective in inhibiting mycelial growth regardless of isolate phenotype followed by the methyl benzimidazole carbamate thiophanate-methyl, two demethylation inhibitors (DMI) difenoconazole and flutriafol, the QoI pyraclostrobin, and the DMI prothioconazole. Furthermore, the efficacy of seven commercial fungicides on target spot was evaluated in the greenhouse and field. Pydiflumetofen + difenoconazole, fluxapyroxad + pyraclostrobin, and thiophanate-methyl delayed disease progress and protected soybean yield, which indicated their effectiveness in managing target spot. Pydiflumetofen + difenoconazole also significantly reduced defoliation. Notably, fungicides applied at R3 were more effective in reducing disease severity and defoliation than additional growth stage timings. The current study revealed a reduction in C. cassiicola sensitivity to QoI fungicides and a shift to QoI-resistant populations exhibiting fitness advantages. Our findings provide pertinent information for growers as to which fungicides should be recommended to manage target spot.
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Evaluation of false positive results in microbial inhibitor tests for screening antibiotics in goat milkRomero Rueda, Tamara 31 March 2015 (has links)
Tesis por compendio / Goat milk is primarily destined for the production of fermented products, in particular
cheese. Therefore, the control of antibiotic residues in milk is of great importance, since
these could have negative repercussions on technological properties of the milk as well
as on the health of consumers.
In milk quality control programs, microbial inhibitor tests are widely applied to detect
antibiotics during the screening stage. However, tests are non-specific and may be
affected by substances other than antimicrobials which could inhibit the growth of the
test micro-organism, causing false positive results.
The aim of this thesis was to evaluate the interference, related to the presence of
different contaminants in goat milk, on the response of microbial inhibitor tests
commonly used in Spain to detect antibiotics (BRT MRL, Delvotest SP-NT MCS and
Eclipse 100 tests). The influence of the physicochemical characteristics of goat milk on
the false positive outcomes in microbial screening tests was also investigated.
The suitability of microbial inhibitor tests for screening antibiotics in colostrum
secretions was studied by analysing antibiotic-free colostrum and milk samples from
forty-three Murciano-Granadina goats, collected every 12 hours during the first week
post-partum. Microbial inhibitor tests were not suitable for the analysis of goat
colostrum because they presented a high percentage of doubtful and positive results
(up 37.2% in the 36 hours after partum).
To evaluate the effect of caprine colostrum on the microbial test response,
antimicrobial-free goat milk spiked with different concentrations of colostrum was
analysed to calculate the inhibitory concentrations producing 5% of positive results.
The highest interferences were obtained for the addition of colostrum from 12 to 24
hours post-partum and the colostrum concentrations producing 5% positive results
were between 5.1 and 34.6%. The BRT MRL was the test the most affected.
In another study, the interference of detergents and disinfectants used for the cleaning
of milking equipment and milk storage tanks of dairy farms was investigated.
Antimicrobial-free goat milk was spiked with eight concentrations of different cleaning
products (5 acid, 5 alkaline, 5 domestic washing-up liquids, and 1 disinfectant) and
analysed using microbial screening tests. The presence of acid detergent and
disinfectant based on sodium hypochlorite in goat milk did not affect the microbial test
response. However, alkaline detergents at concentrations ≥ 1 ml/l could lead to false
positive results in microbial inhibitor tests (up to 16.7%) and from 4 ml/l on 100%
positive results were obtained. Regarding the products used for home use, and those
used on farms and small size dairies, washing-up liquid containing sodium laureth
sulphate and ethanol had the greatest effects on microbial inhibitor tests, even starting
from a relatively low concentration (1 ml/l). On the other hand, the presence of a
relatively low concentration of detergents in goat milk (0.5 ml/l) slightly modified the
detection capability of the microbial inhibitor tests for amoxicillin, ampicillin,
benzylpenicillin, and cloxacillin, although the detection of these drugs at MRL (safe
level) was not compromised.
Antiparasitic agent residues in goat milk could be another possible cause of false
positive results in microbial screening tests. An in vitro study to evaluate the effect of
seven parasiticides commonly used in dairy goats was carried out. Further two studies,
where albendazole and ivermectin were applied to two groups of dairy goats in
lactation were performed. It should be noted that the parasiticide ivermectin is banned
for the treatment of animals producing milk for human consumption, although its
inclusion in this study was considered interesting to understand the potential effect of
their residues in milk, in the event the practice was performed illegally.
In the in vitro study, raw antibiotic-free milk from goats was spiked individually with eight
different concentrations of albendazole, closantel, diclazuril, febendazole, levamisole,
diazinon, and ivermectin. The microbial inhibitor test results showed a great variability
according to the test and the drug under study. Of the tests considered, the BRT MRL
test was the most sensitive to antiparasitic agents, with the lowest concentrations of
antiparasitic agent causing 5, 10, and 50% of positive results. Generally, closantel and
diazinon were the antiparasitic agents that produced higher interferences in all tests,
since low concentrations already resulted in positive results, while only higher
concentrations of diclazuril and ivermectin showed an inhibitory effect.
To evaluate the effect of albendazole residues on the microbial inhibitor test response,
eighteen healthy Murciano-Granadina goats in mid-lactation were treated with a single
oral administration of the commercially available albendazole registered for dairy sheep
(7.5 mg/kg b.w. of active compound) with a withdrawal period of 4 days for milk
production in ovine. Albendazole and its metabolite residues in goat milk after under
cascade treatment were not detected above MRL from the third day post-administration.
However, a high occurrence of non-compliant results was obtained for the BRT MRL test
during the first six days after treatment, suggesting that factors related to the
albendazole application other than the drug concentration are able to affect the microbial
inhibitor test response in some cases.
Regarding the ivermectin study, twenty-eight Murciano-Granadina goats infested with
Sarcoptes scabiei var. caprae were treated with a subcutaneous injection of ivermectin
(200 μg/kg b.w.), with a second dose applied seven days after the first treatment. Drug
residues in goat milk were recorded during the first fifteen days of the experiment with
concentrations ranging from 8.13 to 24.25 ng/ml. In addition, all the microbial screening
tests seem to be affected by the ivermectin treatment, with BRT MRL the most affected
(20%) compared with Delvotest SP-NT MCS and Eclipse 100 (6.6 and 5.7%,
respectively). These positive results cannot be associated with the ivermectin
concentration in goat milk, as the concentrations measured were lower than the
inhibitory concentrations as reported in a previous in vitro study for these microbial
tests. Thus, as suggested by some authors, interferences could be related to changes
or alterations caused by the application of the parasiticide agent or by the parasitic
disease itself, which could affect the immune response of the animals favouring the
presence of inhibitory substances in milk.
The study of the effect of the goat milk composition on the specificity (rate of false
positive results) of microbial inhibitor tests for screening antibiotics was also
considered. Thus, individual goat milk samples (n=200) were analysed by microbial
inhibitor tests using both visual and instrumental classification of the test results. The
highest specificity values were obtained for the instrumental interpretation of the test
results (94-99% vs 90-96%) due to the occurrence of samples with intermediate
colorations (green-yellow, yellow-blue) making the visual classification more difficult
and subjective. A relation was found between positive results in BRT MRL and Eclipse
100 tests and an elevated fat content in the goat milk. Positive outcomes in Eclipse 100
were associated with the butyric acid concentration in the milk. Further, the Delvotest
SP-NT MCS test response was affected by elevated pH values, high lactoferrrin and
myristoleic acid concentrations in the goat milk. This percentage of positive results
could be minimized by a pre-treatment prior to microbial inhibitor test analysis, such as
fat removal by centrifugation (3,100 g for 10 min at 4 ºC) and/or heating (80 ºC for 10
min).
Undoubtedly, improvements on the specificity of the microbial inhibitor tests for
screening antibiotics in goat milk are desirable to avoid the destruction of milk
compliant for human due to the occurrence of false positive results. The related
financial losses affect farmers and dairies. However, it should be noted that the
presence of contaminants in goat milk could be avoided by applying good farming
practices designed to ensure that milk is obtained from healthy animals under proper
hygienic conditions so ensuring the food safety of goat milk and related dairy products. / Romero Rueda, T. (2015). Evaluation of false positive results in microbial inhibitor tests for screening antibiotics in goat milk [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48552 / Compendio
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