Monitoring udder health on routinely collected census data: Evaluating the short- to mid-term consequences of implementing selective dry cow treatment.

In 2013, the preventive use of antimicrobials in Dutch livestock was prohibited, including a ban on the blanket application of antimicrobial dry cow treatment (BDCT). Since then, selective dry cow treatment (SDCT) has become the standard approach. In this study, we aimed to determine the effect of the ban on BDCT and the extent of the subsequent adoption of SDCT on antimicrobial usage (AMU) and udder health on Dutch dairy farms. In the Dutch cattle health surveillance system, AMU for dry cow treatment (AMUDCT), AMU for intramammary treatment at any point in time (AMUIMM), and udder health indicators are routinely and continuously monitored. This provided the opportunity to study associations among SDCT, udder health, and AMU on census data of approximately 17,000 dairy herds, with about 1.67 million cows in total (>2 yr old) at one moment in time in the period from 2013 until 2017. Six udder health parameters were evaluated using multivariable population-averaged generalized estimating equation models. The year in which the ban on BDCT was introduced (2013) was compared with the period thereafter (2014-2017). Additionally, AMUIMM and AMUDCT were included as independent variables to evaluate whether the extent to which SDCT was implemented on the herd level was associated with udder health. Demographic parameters were included as potential confounders. Since the ban on BDCT, overall declines of 63% in AMUDCT and 15% in AMUIMM were observed. The raw data show an improvement in 5 out of 6 evaluated udder health parameters between 2013 and 2017. Nevertheless, the multivariable model results showed that the period since the ban on BDCT was associated with a small but significant increase in the percentage of cows with high somatic cell count (HSCC) and new HSCC (+0.41% and +0.06%, respectively). Additionally, the probability of belonging to the group of herds with more than 25% of primiparous cows having HSCC during the start of lactation increased slightly, associated with the period after which BDCT was banned (odds ratio = 1.08). The probability of belonging to the group of herds with more than 25% cows having a persistent HSCC during the dry period was not affected and bulk milk somatic cell count showed a slight but significant reduction. The only udder health parameter that notably worsened during the study period was the probability of belonging to the group of herds with more than 25% of multiparous cows with a new HSCC after the dry period, during the start of lactation (odds ratio = 1.23). In herds where the farmer decided not to apply any dry cow therapy (≈20% of all herds), all udder health parameters were poorer compared with herds in which dry cow therapy was applied to some extent. The ban on BDCT and implementation of SDCT in the Netherlands was associated with a considerable reduction in AMU without a major impairment in udder health at the national level. Although negative effects of changed dry cow management were observed in some herds, we conclude that SDCT can be introduced without substantial negative effects on udder health.

The economic impact of drying off cows with a dry-off facilitator (cabergoline) compared with 2 methods of gradual cessation of lactation for European dairy farms.

An abrupt method to dry off cows has disadvantages and is considered inappropriate for current dairy cows due to welfare issues and risks for intramammary infections (IMI). A gradual cessation of lactation (by feeding or milking frequency reduction) has been the generally recommended method for drying off cows to prevent these adverse effects. However, a new alternative to the gradual approach is to abruptly stop milking at the same time as using cabergoline (CAB), a prolactin inhibitor. The aim of the study was to compare the net costs of 3 different methods of drying off cows [gradual reduction in feed (referred to as gradual feeding), gradual reduction in milking frequency (referred to as gradual milking), and abrupt cessation of milking with CAB]. A stochastic Monte Carlo simulation model, at cow level, was developed to calculate the net costs of applying these methods. All inputs for the model were based on literature information, authors' expertise, and expert knowledge. The net costs were determined by only including costs and benefits, which varied between the 3 methods. The model simulated a cow from 7 d before the day of drying off until the end of the next lactation. The likelihood of whether a cow was leaking milk early in the dry period was determined. Subsequently, it was determined whether or not the cow will get an IMI during the dry period, where the probability of getting an IMI was higher for cows leaking milk than for cows not leaking milk. If the IMI was not cured during the dry period, the cow had an IMI at calving. Also, milk production and feed requirements were modeled, and labor for applying the drying off method was included. For all methods, the net costs were calculated as the sum of costs for feed during the gradual feed reduction period, costs for applying the gradual-milking method, and the IMI costs during the dry period and lactation, minus the milk revenues during the transition from lactation to the dry period. Under default conditions, the average net cost of abrupt cessation of milking with CAB was €49.6/cow. The data showed that 90% of the net costs ranged from -€13.7 to €307.8/cow. The average net costs for gradual feeding and gradual milking were €99.1 and €71.5/cow, respectively. In conclusion, abrupt cessation of milking with CAB saved €49.5 and €21.9/cow on average compared with gradual feeding and gradual milking, respectively. This difference was mainly due to more milk returns and lower labor and IMI costs during lactation.

Assessment of acoustic pulse therapy (APT), a non-antibiotic treatment for dairy cows with clinical and subclinical mastitis.

Clinical and subclinical mastitis affects 30% of cows and is regarded as the most significant economic burden on the dairy farm reducing milk yield and quality and increasing culling rate. A proprietary Acoustic Pulse Therapy (APT) device was developed specifically for treating dairy cows. The APT device was designed to produce deep penetrating acoustic pulses that are distributed over a large treated area at a therapeutic level. This paper presents findings from a clinical assessment of this technology for the treatment of dairy cows with subclinical and clinical mastitis. In subclinical mastitis, a group of 116 cows from 3 herds were identified with subclinical intramammary infection and enrolled in the study; 78 cows were assigned to the treatment group and 38 cows to the control group. Significant differences (P<0.001) were found where 70.5% of the cows in the treatment group returned to normal milk production, compared with only 18.4% of the control group. Daily milk yields of the treated cows increased significantly (P<0.05) and the percentage of cows with log somatic cell count under 5.6 cells/mL was significantly higher (P<0.001). Milk of the infected quarters appeared normal with lactose greater than 4.8%, but this difference was not significant. Of the treated cows with identified bacteria, 52.6% of the quarters were cured, while in the control group only 25.0% (P<0.001). Specifically, all cows identified with Escherichia coli in the treatment group were cured, with 66.6% cured with no intervention in the control. Spontaneous cure of glands infected with coagulase negative staphylococci (CNS) and Streptococci was low while treatment successfully increased the cure of CNS from 13.3% to 53.8% and that of Streptococci from 18.2% to 36.4%. Of the 4 cows identified with Staphylococcus aureus, 3 were cured. The clinical mastitis study group included 29 infected cows that were submitted either to a gold standard antibiotic treatment subgroup of 16 cows (n = 16) or to an APT treatment subgroup of 13 cows (n = 13). A cure of 18.7% was shown for the antibiotic treatment, of which logSCC returned to <5.6 cell/mL and 56.2% were culled. A cure of 76.9% was shown for the APT treatment with only one cow culled (7.7%).

Carcinogenicity assessment of baricitinib in Tg.rasH2 mice and Sprague-Dawley (Crl:CD) rats.

Baricitinib is a potent and selective Janus kinase (JAK)1 and JAK2 inhibitor, and is approved for the treatment of moderately to severely active RA in adults in Europe, Japan, and other countries. This study evaluated the carcinogenic potential of baricitinib in Tg. rasH2 mice and Sprague-Dawley (Crl:CD) rats. Baricitinib was administered daily by oral gavage to Crl:CD rats for up to 94 weeks (dose levels of 0, 1, 3, or 8 mg/kg for males and 0, 3, 8, or 25 mg/kg for females) and to Tg. rasH2 mice for 26 weeks (dose levels of 0, 15, 40, or 300 mg/kg for males and 0, 10, 30, or 150 mg/kg for females). Baricitinib was well tolerated with no incidence of compound-related neoplasms at any dose levels in rats and mice. In mice, non-neoplastic events observed were bone marrow hypocellularity and increased adipocytes. In rats, baricitinib administration was associated with a dose-dependent increase in survival, with a decreased incidence of neoplasm (hematopoietic and mammary), potentially secondary to drug-related decreased weight gain. The incidence of proliferative changes such as neoplastic and hyperplastic lesions in the mammary glands of females and in the livers of males and females also decreased. In conclusion, baricitinib is not considered to be carcinogenic.

Economic optimization of selective dry cow treatment.

The objective of this study was to develop a mathematical model to identify a scenario with the lowest costs for mastitis associated with the dry period while restricting the percentage of cows to be dried off with dry cow antimicrobials. Costs of clinical and subclinical mastitis as well as antimicrobial use were quantified. Based on data from a large field trial, a linear programming model was built with the goal to minimize the costs associated with antimicrobial use at drying off. To enable calculations on minimizing costs of dry cow treatment on herd-level by drying-off decisions in an "average" herd, we created an example herd. Cows were projected on 3 different types of herds, based on bulk tank somatic cell count, and were categorized in groups based on parity and somatic cell count from the last test recording before drying-off. Economically optimal use of antimicrobials was determined while restricting the maximum percentage of cows dried off with antimicrobials from 100 to 0%. This restriction reveals the relationship between the maximum percentage of cows dried off with antibiotics and the economic consequences. A sensitivity analysis was performed to evaluate the effect of variation in the most important input variables, with the effect of dry cow antimicrobials resulting in a lower or higher percentage of clinical and subclinical mastitis depending on being dried off with or without dry cow antimicrobials, respectively, and the milk price. From an economic perspective, blanket dry cow treatment seems not to be the optimal approach of dry cow therapy, although differences between approaches were small. With lower bulk tank somatic cell counts, more dry cow antimicrobials can be omitted without economic consequences. The economic impact of reducing the percentage of clinical mastitis was found to be much larger than reducing the bulk tank somatic cell count. The optimal percentage of cows to be dried off with antimicrobials depends on the udder health situation, expressed as the bulk tank somatic cell count and the incidence of clinical mastitis. For all evaluated types of herds, selective dry cow treatment was economically more beneficial than blanket dry cow treatment. Economic profits of selective dry cow treatment are greater if bulk tank somatic cell count and clinical mastitis incidence are lower. Economics is not an argument against reduction of dry cow antimicrobials by applying selective dry cow treatment.

Doxorubicin-induced female reproductive toxicity: an assessment of ovarian follicular apoptosis, cyclicity and reproductive tissue histology in Wistar rats.

Doxorubicin is a widely used chemotherapeutic agent for various cancers, particularly for the female breast cancer patients. Although the rate of young female cancer patients is increasing every year, conversely the lack of knowledge of adverse effects of doxorubicin on female reproductive system insisted us to assess the toxic effects of doxorubicin on the female reproductive tissue histoarchitecture, cyclicity, and mammary glands in Wistar rats. The rats were divided into two groups depending on the treatment period, i.e., 24 h and 28 d and further subdivided into three subgroups and administered with doxorubicin at 3 mg/kg bw (subgroup I), 6 mg/kg bw (subgroup II), and equal volume of normal saline (subgroup III) intraperitoneally once during the whole treatment period. We observed a significantly altered estrous cycle with a prolonged diestrous and short proestrous in higher dose group and dose-dependent significant changes in the uteri and mammary gland histoarchitecture in 28 days treated rats as compared to control. Moreover, the micronuclei and chromosomal aberration frequency were increased significantly in both treatment groups. A significant increase in follicular atresia in ovaries of the 28 days treated rats was observed. The immunohistochemical analysis of ovarian tissues showed an increased p53 and caspase 3 expression and apoptosis in primordial follicles of treated rats. The results suggest that though doxorubicin is a potential chemotherapeutic drug for many tumors, but the risk of adverse effects on the female reproductive system is there even at low doses.

Combining web-based tools for transparent evaluation of data for risk assessment: developmental effects of bisphenol A on the mammary gland as a case study.

Different tools have been developed that facilitate systematic and transparent evaluation and handling of toxicity data in the risk assessment process. The present paper sets out to explore the combined use of two web-based tools for study evaluation and identification of reliable data relevant to health risk assessment. For this purpose, a case study was performed using in vivo toxicity studies investigating low-dose effects of bisphenol A on mammary gland development. The reliability of the mammary gland studies was evaluated using the Science in Risk Assessment and Policy (SciRAP) criteria for toxicity studies. The Health Assessment Workspace Collaborative (HAWC) was used for characterizing and visualizing the mammary gland data in terms of type of effects investigated and reported, and the distribution of these effects within the dose interval. It was then investigated whether there was any relationship between study reliability and the type of effects reported and/or their distribution in the dose interval. The combination of the SciRAP and HAWC tools allowed for transparent evaluation and visualization of the studies investigating developmental effects of BPA on the mammary gland. The use of these tools showed that there were no apparent differences in the type of effects and their distribution in the dose interval between the five studies assessed as most reliable and the whole data set. Combining the SciRAP and HAWC tools was found to be a useful approach for evaluating in vivo toxicity studies and identifying reliable and sensitive information relevant to regulatory risk assessment of chemicals. Copyright © 2016 John Wiley & Sons, Ltd.

Failure and preventive costs of mastitis on Dutch dairy farms.

Mastitis is an important disease from an economic perspective, but most cost assessments of mastitis include only the direct costs associated with the disease (e.g., production losses, culling, and treatment), which we call failure costs (FC). However, farmers also invest time and money in controlling mastitis, and these preventive costs (PC) also need to be taken into account. To estimate the total costs of mastitis, we estimated both FC and PC. We combined multiple test-day milk records from 108 Dutch dairy farms with information on applied mastitis prevention measures and farmers' registration of clinical mastitis for individual dairy cows. The aim was to estimate the total costs of mastitis and to give insight into variations between farms. We estimated the average total costs of mastitis to be €240/lactating cow per year, in which FC contributed €120/lactating cow per year and PC contributed another €120/lactating cow per year. Milk production losses, discarded milk, and culling were the main contributors to FC, at €32, €20, and €20/lactating cow per year, respectively. Labor costs were the main contributor to PC, next to consumables and investments, at €82, €34, and €4/lactating cow per year, respectively. The variation between farmers was substantial, and some farmers faced both high FC and PC. This variation may have been due to structural differences between farms, different mastitis-causing pathogens, the time at which preventive action is initiated, stockmanship, or missing measures in PC estimates. We estimated the minimum FC to be €34 per lactating cow per yr. All farmers initiated some preventive action to control or reduce mastitis, indicating that farmers will always have mastitis-related costs, because mastitis will never be fully eradicated from a farm. Insights into both the PC and FC of a specific farm will allow veterinary advisors and farmers to assess whether current udder health strategies are appropriate or whether there is room for improvement from an economic perspective.

Effect of different scenarios for selective dry-cow therapy on udder health, antimicrobial usage, and economics.

The goal of dry-cow therapy (DCT) is to reduce the prevalence of intramammary infections (IMI) by eliminating existing IMI at drying off and preventing new IMI from occurring during the dry period. Due to public health concerns, however, preventive use of antimicrobials has become questionable. In this study, we evaluated the effects of 8 scenarios for selecting animals for DCT, taking into account variation in parity and cow-level somatic cell count (SCC) at drying off. The aim of this study was to evaluate udder health, antimicrobial usage, and economics at the herd level when using different scenarios for selecting cows for DCT. To enable calculation and comparison of the effects of different scenarios to select cows for DCT in an "average" herd, we created an example herd, with a virtual herd size of 100 dairy cows to be calving during a year. Udder health, antimicrobial usage, and economics were evaluated during the dry period and the first 100 d in lactation, the period during which the greatest effect of DCT is expected. This leads to an estimated 13,551 cow-days at risk during a year in a 100-cow dairy herd. In addition to a blanket DCT (BDCT) scenario, we developed 7 scenarios to select cows for DCT based on SCC. The scenarios covered a range of possible approaches to select low-SCC cows for DCT, all based on cow-level SCC thresholds on the last milk recording before drying off. The incidence rate of clinical mastitis in the example herd varied from 11.6 to 14.5 cases of clinical mastitis per 10,000 cow-days at risk in the different scenarios, and the prevalence of subclinical mastitis varied from 38.8% in scenario 1 (BDCT) to 48.3% in scenario 8. Total antimicrobial usage for DCT and clinical mastitis treatment varied over the scenarios from 1.27 (scenario 8) to 3.15 animal daily dosages (BDCT), leading to a maximum reduction in antimicrobial usage of 60% for scenario 8 compared with BDCT. The total costs for each of the scenarios showed little variation, varying from €4,893 for scenario 5 to €5,383 for scenario 8. The effect of selective DCT compared with BDCT on udder health, antimicrobial usage, and economics is influenced by the SCC criteria used to select cows for DCT. Scenario 2 resulted in the lowest increases in clinical and subclinical mastitis compared with BDCT. The greatest reduction in antimicrobial usage was achieved under scenario 8. From an economic perspective, lowest costs were achieved with scenario 5. Drying off dairy cows with antimicrobials has an effect on udder health, antimicrobial usage, and economics.

Quantitative Assessment of Mouse Mammary Gland Morphology Using Automated Digital Image Processing and TEB Detection.

The assessment of rodent mammary gland morphology is largely used to study the molecular mechanisms driving breast development and to analyze the impact of various endocrine disruptors with putative pathological implications. In this work, we propose a methodology relying on fully automated digital image analysis methods including image processing and quantification of the whole ductal tree and of the terminal end buds as well. It allows to accurately and objectively measure both growth parameters and fine morphological glandular structures. Mammary gland elongation was characterized by 2 parameters: the length and the epithelial area of the ductal tree. Ductal tree fine structures were characterized by: 1) branch end-point density, 2) branching density, and 3) branch length distribution. The proposed methodology was compared with quantification methods classically used in the literature. This procedure can be transposed to several software and thus largely used by scientists studying rodent mammary gland morphology.

Short communication: an in vitro assessment of the antibacterial activity of plant-derived oils.

Nonantibiotic treatments for mastitis are needed in organic dairy herds. Plant-derived oils may be useful but efficacy and potential mechanisms of action of such oils in mastitis therapy have not been well documented. The objective of the current study was to evaluate the antibacterial activity of the plant-derived oil components of Phyto-Mast (Bovinity Health LLC, Narvon, PA), an herbal intramammary product, against 3 mastitis-causing pathogens: Staphylococcus aureus, Staphylococcus chromogenes, and Streptococcus uberis. Plant-derived oils evaluated were Thymus vulgaris (thyme), Gaultheria procumbens (wintergreen), Glycyrrhiza uralensis (Chinese licorice), Angelica sinensis, and Angelica dahurica. Broth dilution testing according to standard protocol was performed using ultrapasteurized whole milk instead of broth. Controls included milk only (negative control), milk + bacteria (positive control), and milk + bacteria + penicillin-streptomycin (antibiotic control, at 1 and 5% concentrations). Essential oil of thyme was tested by itself and not in combination with other oils because of its known antibacterial activity. The other plant-derived oils were tested alone and in combination for a total of 15 treatments, each replicated 3 times and tested at 0.5, 1, 2, and 4% to simulate concentrations potentially achievable in the milk within the pre-dry-off udder quarter. Thyme oil at concentrations ≥2% completely inhibited bacterial growth in all replications. Other plant-derived oils tested alone or in various combinations were not consistently antibacterial and did not show typical dose-response effects. Only thyme essential oil had consistent antibacterial activity against the 3 mastitis-causing organisms tested in vitro. Further evaluation of physiological effects of thyme oil in various preparations on mammary tissue is recommended to determine potential suitability for mastitis therapy.

Assessment of the proliferative capacity of the flavanones 8-prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and naringenin in MCF-7 cells and the rat mammary gland.

8-Prenylnaringenin (8-PN) and naringenin (Nar) are phytoestrogens found in food items and nutritional supplements, while 6-(1.1-dimethylallyl)naringenin (6-DMAN) is a component of an African plant. Besides their assumed beneficial effects they may promote mammary and endometrial cancer. We therefore assessed their proliferative and estrogenic potential on the mammary gland in vitro and in vivo. In competitive estrogen receptor (ER) ligand binding assays 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα and had the strongest mitotic effect on MCF-7 cells among the test substances. In a three day exposure in young adult ovariectomized female rats 15 mg/kg 8-PN had the highest capacity to increase the number of terminal end buds (TEB) in the mammary gland and stimulated expression of proliferation markers in epithelial ductal cells, followed by 6-DMAN and Nar, but overall their capacity to stimulate proliferation was weak in comparison to 17ß-Estradiol (E2).

Assessment of ABCG2-mediated transport of xenobiotics across the blood-milk barrier of dairy animals using a new MDCKII in vitro model.

The ATP-binding cassette (ABC) efflux transporter ABCG2 represents the main route for active secretion of drugs and toxins across the blood-milk barrier, thereby producing a potential health risk for dairy consumers through formation of relevant residues in milk. However, no suitable in vitro model is as yet available to systematically investigate ABCG2-mediated transport of xenobiotics into milk of dairy animals. We recently cloned ABCG2 from the lactating mammary gland of dairy cows (bABCG2) and goats (cABCG2). Thus, the objective of this study was to generate a suitable blood-milk barrier in vitro model using polarized MDCKII monolayers stably expressing mammary bABCG2 or cABCG2. ABCG2 protein was localized by confocal microscopy to the apical and lateral plasma membrane of polarized MDCKII cells. Intact barrier function of MDCKII-bABCG2 and MDCKII-cABCG2 monolayers was confirmed by determination of cell permeability of transcellular marker propranolol and paracellular marker atenolol which was ≤1 %. In flux assays, ABCG2 substrate 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) showed preferential basolateral to apical (B > A) transport in ABCG2-MDCKII cells. This apically directed PhIP transport was significantly inhibited by ABCG2 inhibitor fumitremorgin C (FTC) or the flavonoid equol. PhIP B > A transport in MDCKII-bABCG2 monolayers was additionally decreased by ABCG2 inhibitor Ko143. The fluoroquinolone antibiotic enrofloxacin was identified as a substrate of ruminant mammary ABCG2. The analgesic drug sodium salicylate was shown to be substrate of bABCG2 but not of cABCG2. Thus, the generated mammary ABCG2-expressing MDCKII cells represent a valuable tool to study active secretion of drugs and toxins into milk.

Quantitative assessment of mammary gland development in female Long Evans rats following in utero exposure to atrazine.

In this study, we quantified the effects of in utero exposure to the herbicide atrazine on subsequent mammary gland development. Atrazine was administered to pregnant female Long Evans rats from gestation days 13-19 at doses of 0, 6.5, 50, or 100 mg/kg/day. A pair-fed control group was yoked to the high-dose atrazine-treated group. Litter size was standardized to 10 pups on postnatal day (PND) 4. Whole mounts of the left fourth mammary gland and histologic sections of the right fourth gland were obtained from a subgroup of offspring on PND1, 21, 33, on day of vaginal opening (VO), or around PND65 at diestrus. A blinded, quantitative analysis of key morphological features in mammary gland whole mounts (ductal elongation, ductal network area, epithelial area, terminal end bud [TEB] incidence, and epithelial density) as well as epithelial proliferation within different parenchymal structures was conducted. There was no effect of atrazine exposure on any of the measures of mammary gland development at the maternal dose of 6.5 mg/kg/day. On PND1, ductal elongation was increased by approximately 20% (p < 0.05) in the female offspring born to dams exposed to 50 and 100 mg/kg/day atrazine, coincident with decreased epithelial proliferation in the 100 mg/kg/day group at this age. These differences were not present on PND21, or thereafter. An increased incidence of TEB in the mammary glands from females that were born to both the pair-fed and 50 mg/kg/day-treated dams at the time of VO indicated that this response was a specific result of maternal caloric restriction. Collectively, these data indicate that maternal atrazine exposure has no long-term effects on mammary gland development in female offspring beyond a transitory response to high doses at PND1.

Current assessment of the effects of environmental chemicals on the mammary gland in guideline rodent studies by the U.S. Environmental Protection Agency (U.S. EPA), Organisation for Economic Co-operation and Development (OECD), and National Toxicology Program (NTP).

BACKGROUND: Evaluation of the structural and/or functional integrity of the mammary gland (MG) across life stages is integral to the assessment of developmental, reproductive, and carcinogenic risk for environmental chemicals. OBJECTIVES: In this commentary I characterize MG assessment recommended in U.S. Environmental Protection Agency, Organisation for Economic Co-operation and Development, and National Toxicology Program guideline toxicology study protocols and identify any information gaps for the evaluation of MG development, structure, and function. DISCUSSION: Several data gaps, issues, and challenges were identified. Current guidelines that include a lactation phase do not provide specific recommendations to record observations on maternal or offspring lactation or nursing behavior. In guideline studies, the assessment of MG toxicity often relies upon indirect, nonspecific, or surrogate end points, and information that could be useful in the interpretation of these data (e.g., mode of action or toxicokinetics) is often unavailable. Most guideline studies designed to assess general organ toxicity do not expose test animals during sensitive stages of MG development; histopathological evaluation of the developing MG is not routinely conducted; and evaluation of MG tissue for both sexes is inconsistently recommended. CONCLUSIONS: I propose the following general recommendations to enhance MG assessment in guideline toxicology studies: a) inclusion of more specific criteria for the evaluation of MG end points in guideline language, b) inclusion of histopathological evaluation of MG development (using whole-mount techniques) in existing or new guideline protocols that include offspring with perinatal and/or pubertal treatment, c) incorporation of perinatal exposures into rodent subchronic and carcinogenicity assays, and d) expansion of the histopathological evaluation of male MG tissue.

Assessment of bovine mammary chemokine gene expression in response to lipopolysaccharide, lipotechoic acid + peptidoglycan, and CpG oligodeoxynucleotide 2135.

During intramammary infections pathogen associated molecular patterns (PAMPs) induce an inflammatory response, recognized clinically as mastitis. Recognition of PAMPs by mammary cells leads to the production of the pro-inflammatory cytokines, TNF-alpha and IL-1beta. These cytokines augment the secretion of various chemokines that are responsible for directing the host cellular immune response, and consequently the outcome of infection. Previous research has shown that gram-negative and gram-positive bacteria elicit different types of innate immune responses. The purpose of this study, therefore, was to characterize the expression of various chemokine genes in bovine mammary gland explants in response to lipopolysaccharide (LPS), peptidoglycan (PTG) combined with lipotechoic acid (LTA), and CpG oligodeoxynucleotide (CpG-ODN) 2135 representing gram-negative bacteria, gram-positive bacteria, and bacterial DNA, respectively, to determine if these PAMPs induce different chemokine gene expression patterns. Explants from 3 Holstein cows were cultured with 10 microg/mL of LPS, LTA + PTG, or CpG-ODN 2135 for 6 and 24 h. Total RNA was extracted and the expression of CXCL8, MCP-1, MCP-2, MCP-3, MIP1-alpha, and RANTES genes was measured by real-time polymerase chain reaction (RT-PCR). Lipopolysaccharide significantly induced MCP-1, MCP-2, and MCP-3 expression, and slightly increased CXCL8 gene expression. The combined PAMPs, LTA + PTG, on the other hand, significantly induced MCP-1 gene expression, and slightly increased MCP-3 expression. No significant expression differences for any of the chemokine genes were observed in explants stimulated with CpG-ODN 2135. These results demonstrate that PAMPs associated with different mastitis-causing pathogens induce chemokine-specific gene expression patterns that may contribute to different innate immune responses to bacteria.

Assessment of DNA adducts and the frequency of 6-thioguanine resistant T-lymphocytes in F344 rats fed 2,4-toluenediamine or implanted with a toluenediisocyanate-containing polyester polyurethane foam.

Toluenediamines have been of toxicological concern because of their industrial use as intermediates in polyurethane synthesis and because of the potential of their release from degradation of the Microthane polyesterurethane covering of some breast implants. In this study, we have assessed the extent of DNA damage in rats treated with a carcinogenic toluenediamine isomer, 2,4-toluenediamine (2,4-TDA), under conditions that result in tumor induction, and in rats implanted with Microthane polyesterurethane foam. Time and dose-dependent formation of adducts was observed in DNA from the liver and mammary gland of rats fed 10, 40, 80 and 180 ppm 2,4-TDA for up to 6 weeks. In assays conducted 1 to 32 weeks after the start of treatment, no adducts were detected in the DNA of T-lymphocytes isolated from the spleens of animals fed 40 or 180 ppm 2,4-TDA, nor was there an increase in mutations at the hprt locus in these lymphocytes. In rats fed 40 or 180 ppm, 2,4-TDA for 6 weeks, adducts were detectable in DNA isolated from liver and mammary gland for 26 to 43 weeks after termination of the treatment. No DNA damage, as assessed by both DNA adduct measurement and induction of T-lymphocyte hprt mutations, was observed in rats up to 42 weeks after receiving subcutaneous implants of polyesterurethane foam (67 or 267 mg/kg). Although 2,4-TDA is clearly capable of damaging DNA, the results of this study are consistent with the conclusion that Microthane foam-containing implants present a minimal risk of genotoxicity through release and subsequent metabolic activation of 2,4-TDA. The study also indicates that DNA adduct formation and mutation induction in lymphocytes are inadequate biomonitors for measuring exposure to toluenediamines.

Carcinogenicity assessment of lonidamine by dietary administration to Sprague-Dawley rats.

Following chronic dietary administration of 20, 60 and 180 mg/kg per day of lonidamine for 2 years to groups of Sprague-Dawley rats, treatment-related non-tumour findings seen microscopically included the following: atrophy of the testis with associated changes in epididymis and pituitary at all dosages; neuropathy in the sciatic nerve accompanied by skeletal muscle atrophy which was dose-related, particularly in male animals. Neither the incidence of tumour-bearing animals, nor the spectrum of tumours seen, was significantly changed. In the females given 180 mg/kg per day an overall reduction in tumour incidence was noted, which was reflected in a significant reduction (P < 0.001) in mammary tumours.

Assessment of bovine mammary gland macrophage oxidative burst activity in a chemiluminescence assay.

A major bactericidal mechanism of neutrophils and macrophages is the generation of toxic oxygen-free radicals upon phagocytosis of microbes. Studies were conducted to assess the oxidative metabolism of bovine mammary gland macrophages. Bovine mammary gland macrophages were challenge exposed with a variety of phagocytic stimuli in an in vitro, luminol-assisted chemiluminescence assay. A measurable oxidative burst was observed when macrophages were challenge exposed with heat-aggregated bovine immunoglobulin, opsonified zymosan, and nonosponified zymosan. Addition of superoxide dismutase decreased mammary gland macrophage chemiluminescence in a dose-dependent manner. Brucella abortus, when opsonified with antiserum, lacteal antibody, or normal serum, produced an oxidative event, whereas nonopsonified B abortus did not. When challenge exposed with phagocytic stimuli, mammary gland macrophages produced an oxidative burst similar to that produced by other phagocytes for which an oxidative event is known to be bactericidal.