The Cornell COVID-19 Testing Laboratory: A Model to High-Capacity Testing Hubs for Infectious Disease Emergency Response and Preparedness.

The unprecedented COVID-19 pandemic posed major challenges to local, regional, and global economies and health systems, and fast clinical diagnostic workflows were urgently needed to contain the spread of SARS-CoV-2. Here, we describe the platform and workflow established at the Cornell COVID-19 Testing Laboratory (CCTL) for the high-throughput testing of clinical samples from the university and the surrounding community. This workflow enabled efficient and rapid detection and the successful control of SARS-CoV-2 infection on campus and its surrounding communities. Our cost-effective and fully automated workflow enabled the testing of over 8000 pooled samples per day and provided results for over 2 million samples. The automation of time- and effort-intensive sample processing steps such as accessioning and pooling increased laboratory efficiency. Customized software applications were developed to track and store samples, deconvolute positive pools, track and report results, and for workflow integration from sample receipt to result reporting. Additionally, quality control dashboards and turnaround-time tracking applications were built to monitor assay and laboratory performance. As infectious disease outbreaks pose a constant threat to both human and animal health, the highly effective workflow implemented at CCTL could be modeled to establish regional high-capacity testing hubs for infectious disease preparedness and emergency response.

Viral RNA Load and Infectivity of SARS-CoV-2 in Paired Respiratory and Oral Specimens from Symptomatic, Asymptomatic, or Postsymptomatic Individuals.

In the present study, we assessed the diagnostic sensitivity and determined the viral RNA load and infectivity of SARS-CoV-2 in paired respiratory (nasopharyngeal and anterior nares) and oral samples (saliva and sublingual swab). Samples were collected from 77 individuals of which 75 were diagnosed with COVID-19 and classified as symptomatic (n = 29), asymptomatic (n = 31), or postsymptomatic (n = 15). Specimens were collected at one time point from each individual, between day 1 and 23 after the initial COVID-19 diagnosis, and included self-collected saliva (S), or sublingual (SL) swab, and bilateral anterior nares (AN) swab, followed by health care provider collected nasopharyngeal (NP) swab. Sixty-three specimen sets were tested using five assay/platforms. The diagnostic sensitivity of each assay/platform and specimen type was determined. Of the 63 specimen sets, SARS-CoV-2 was detected in 62 NP specimens, 52 AN specimens, 59 saliva specimens, and 31 SL specimens by at least one platform. Infectious SARS-CoV-2 was isolated from 21 NP, 13 AN, 12 saliva, and one SL specimen out of 50 specimen sets. SARS-CoV-2 isolation was most successful up to 5 days after initial COVID-19 diagnosis using NP specimens from symptomatic patients (16 of 24 positives, 66.67%), followed by specimens from asymptomatic patients (5 of 17 positives, 29.41%), while it was not very successful with specimens from postsymptomatic patients. Benefits of self-collected saliva and AN specimens balance the loss of sensitivity relative to NP specimens. Therefore, saliva and AN specimens are acceptable alternatives for symptomatic SARS-CoV-2 diagnostic testing or surveillance with increased sampling frequency of asymptomatic individuals. IMPORTANCE The dynamics of infection with SARS-CoV-2 have a significant impact on virus infectivity and in the diagnostic sensitivity of molecular and classic virus detection tests. In the present study we determined the diagnostic sensitivity of paired respiratory (nasopharyngeal and anterior nares swabs) and oral secretions (saliva and sublingual swab) and assessed infectious virus shedding patterns by symptomatic, asymptomatic, or postsymptomatic individuals. Understanding the diagnostic performance of these specimens and the patterns of infectious virus shedding in these bodily secretions provides critical information to control COVID-19, and may help to refine guidelines on isolation and quarantine of positive individuals and their close contacts identified through epidemiological investigations.

Does having a cat in your house increase your risk of catching COVID-19?

Due to the zoonotic origin of SARS-Coronavirus 2 (SARS-CoV-2), the potential for its transmission from humans back to animals and the possibility that it might establish ongoing infection pathways in other animal species has been discussed. Cats are highly susceptible to SARS-CoV-2 and were shown experimentally to transmit the virus to other cats. Infection of cats has been widely reported. Domestic cats in COVID-19-positive households could therefore be a part of a human to animal to human transmission pathway. Here, we report the results of a qualitative risk assessment focusing on the potential of cat to human transmission in such settings. The assessment was based on evidence available by October 2021. After the introduction of SARS-CoV-2 to a household by a human, cats may become infected and infected cats may pose an additional infection risk for other members of the household. In order to assess this additional risk qualitatively, expert opinion was elicited within the framework of a modified Delphi procedure. The conclusion was that the additional risk of infection of an additional person in a household associated with keeping a domestic cat is very low to negligible, depending on the intensity of cat-to-human interactions. The separation of cats from humans suffering from SARS-CoV-2 infection should contribute to preventing further transmission.

From People to Panthera: Natural SARS-CoV-2 Infection in Tigers and Lions at the Bronx Zoo.

Despite numerous barriers to transmission, zoonoses are the major cause of emerging infectious diseases in humans. Among these, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and ebolaviruses have killed thousands; the human immunodeficiency virus (HIV) has killed millions. Zoonoses and human-to-animal cross-species transmission are driven by human actions and have important management, conservation, and public health implications. The current SARS-CoV-2 pandemic, which presumably originated from an animal reservoir, has killed more than half a million people around the world and cases continue to rise. In March 2020, New York City was a global epicenter for SARS-CoV-2 infections. During this time, four tigers and three lions at the Bronx Zoo, NY, developed mild, abnormal respiratory signs. We detected SARS-CoV-2 RNA in respiratory secretions and/or feces from all seven animals, live virus in three, and colocalized viral RNA with cellular damage in one. We produced nine whole SARS-CoV-2 genomes from the animals and keepers and identified different SARS-CoV-2 genotypes in the tigers and lions. Epidemiologic and genomic data indicated human-to-tiger transmission. These were the first confirmed cases of natural SARS-CoV-2 animal infections in the United States and the first in nondomestic species in the world. We highlight disease transmission at a nontraditional interface and provide information that contributes to understanding SARS-CoV-2 transmission across species.IMPORTANCE The human-animal-environment interface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important aspect of the coronavirus disease 2019 (COVID-19) pandemic that requires robust One Health-based investigations. Despite this, few reports describe natural infections in animals or directly link them to human infections using genomic data. In the present study, we describe the first cases of natural SARS-CoV-2 infection in tigers and lions in the United States and provide epidemiological and genetic evidence for human-to-animal transmission of the virus. Our data show that tigers and lions were infected with different genotypes of SARS-CoV-2, indicating two independent transmission events to the animals. Importantly, infected animals shed infectious virus in respiratory secretions and feces. A better understanding of the susceptibility of animal species to SARS-CoV-2 may help to elucidate transmission mechanisms and identify potential reservoirs and sources of infection that are important in both animal and human health.

Complete Genome Sequence of SARS-CoV-2 in a Tiger from a U.S. Zoological Collection.

This report describes the identification and characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a Malayan tiger in a U.S. zoo.

Review of Medical Decision Support and Machine-Learning Methods.

Machine-learning methods can assist with the medical decision-making processes at the both the clinical and diagnostic levels. In this article, we first review historical milestones and specific applications of computer-based medical decision support tools in both veterinary and human medicine. Next, we take a mechanistic look at 3 archetypal learning algorithms-naive Bayes, decision trees, and neural network-commonly used to power these medical decision support tools. Last, we focus our discussion on the data sets used to train these algorithms and examine methods for validation, data representation, transformation, and feature selection. From this review, the reader should gain some appreciation for how these decision support tools have and can be used in medicine along with insight on their inner workings.

Identifying free-text features to improve automated classification of structured histopathology reports for feline small intestinal disease.

The histologic evaluation of gastrointestinal (GI) biopsies is the standard for diagnosis of a variety of GI diseases (e.g., inflammatory bowel disease [IBD] and alimentary lymphoma [ALA]). The World Small Animal Veterinary Association (WSAVA) Gastrointestinal International Standardization Group proposed a reporting standard for GI biopsies consisting of a defined set of microscopic features. We compared the machine classification accuracy of free-text microscopic findings with those represented in the WSAVA format with a diagnosis of IBD and ALA. Unstructured free-text duodenal biopsy pathology reports from cats ( n = 60) with a diagnosis of IBD ( n = 20), ALA ( n = 20), or normal ( n = 20) were identified. Biopsy samples from these cases were then scored following the WSAVA guidelines to create a set of structured reports. Three supervised machine-learning algorithms were trained using the structured and then the unstructured reports. Diagnosis classification accuracy for the 3 algorithms was compared using the structured and unstructured reports. Using naive Bayes and neural networks, unstructured information-based models achieved higher diagnostic accuracy (0.90 and 0.88, respectively) compared to the structured information-based models (0.74 and 0.72, respectively). Results suggest that discriminating diagnostic information was lost using current WSAVA microscopic guideline features. Addition of free-text features (number of plasma cells) increased WSAVA auto-classification performance. The methodologies reported in our study represent a way of identifying candidate microscopic features for use in structured histopathology reports.

A review of medical terminology standards and structured reporting.

Much effort has been invested in standardizing medical terminology for representation of medical knowledge, storage in electronic medical records, retrieval, reuse for evidence-based decision making, and for efficient messaging between users. We only focus on those efforts related to the representation of clinical medical knowledge required for capturing diagnoses and findings from a wide range of general to specialty clinical perspectives (e.g., internists to pathologists). Standardized medical terminology and the usage of structured reporting have been shown to improve the usage of medical information in secondary activities, such as research, public health, and case studies. The impact of standardization and structured reporting is not limited to secondary activities; standardization has been shown to have a direct impact on patient healthcare.

Nonimaging polygonal mirrors achieving uniform irradiance distributions on concentrating photovoltaic cells.

We introduce a design methodology for nonimaging, single-reflection mirrors with polygonal inlet apertures that generate a uniform irradiance distribution on a polygonal outlet aperture, enabling a multitude of applications within the domain of concentrated photovoltaics. Notably, we present single-mirror concentrators of square and hexagonal perimeter that achieve very high irradiance uniformity on a square receiver at concentrations ranging from 100 to 1000 suns. These optical designs can be assembled in compound concentrators with maximized active area fraction by leveraging tessellation. More advanced multi-mirror concentrators, where each mirror individually illuminates the whole area of the receiver, allow for improved performance while permitting greater flexibility for the concentrator shape and robustness against partial shading of the inlet aperture.

Evaluation of supervised machine-learning algorithms to distinguish between inflammatory bowel disease and alimentary lymphoma in cats.

Inflammatory bowel disease (IBD) and alimentary lymphoma (ALA) are common gastrointestinal diseases in cats. The very similar clinical signs and histopathologic features of these diseases make the distinction between them diagnostically challenging. We tested the use of supervised machine-learning algorithms to differentiate between the 2 diseases using data generated from noninvasive diagnostic tests. Three prediction models were developed using 3 machine-learning algorithms: naive Bayes, decision trees, and artificial neural networks. The models were trained and tested on data from complete blood count (CBC) and serum chemistry (SC) results for the following 3 groups of client-owned cats: normal, inflammatory bowel disease (IBD), or alimentary lymphoma (ALA). Naive Bayes and artificial neural networks achieved higher classification accuracy (sensitivities of 70.8% and 69.2%, respectively) than the decision tree algorithm (63%, p < 0.0001). The areas under the receiver-operating characteristic curve for classifying cases into the 3 categories was 83% by naive Bayes, 79% by decision tree, and 82% by artificial neural networks. Prediction models using machine learning provided a method for distinguishing between ALA-IBD, ALA-normal, and IBD-normal. The naive Bayes and artificial neural networks classifiers used 10 and 4 of the CBC and SC variables, respectively, to outperform the C4.5 decision tree, which used 5 CBC and SC variables in classifying cats into the 3 classes. These models can provide another noninvasive diagnostic tool to assist clinicians with differentiating between IBD and ALA, and between diseased and nondiseased cats.

Risk factors and sources of foodborne hepatitis E virus infection in the United States.

Hepatitis E virus (HEV) is an important human pathogen with pigs and other species serving as natural animal reservoirs. Ample evidence documents sporadic cases of hepatitis E acquired via consumption of undercooked meat. Chronic hepatitis E cases in immunosuppressed individuals are mostly caused by zoonotic HEV of swine origin. We report here the identification of genotype 3 HEV from non-liver commercial pork from local grocery stores in southwest Virginia, and association of HEV seropositivity to the consumption of undercooked meat in healthy young adults at a university in the United States. These results raise concerns about foodborne HEV transmission in the United States. J. Med. Virol. 88:1641-1645, 2016. © 2016 Wiley Periodicals, Inc.

Preharvest Salmonella Detection for Evaluation of Fresh Ground Poultry Product Contamination.

Salmonella is an important economic and public health concern for the poultry industry. Fresh ground product has been linked with multiple salmonellosis outbreaks in humans. Exposure can be controlled by proper handling and preparation by consumers; however, the industry desires to minimize carriage levels in the final product. A substantial obstacle in reducing product contamination stems from limitations in diagnostic methodologies. Detection of Salmonella contamination currently requires extended incubation periods, and by the time test results are available, the fresh product has reached retail shelves. The goal of this study was to develop a preharvest diagnostic protocol for the evaluation of ground product contamination. The turkey processing plant where this research was conducted had previously established Salmonella screening (BAX system) of ground product, thus providing an opportunity for preharvest sample comparison. Drag swabs were collected from live-haul trailers entering the processing plant over a 12-month period. The swabs were added to modified buffered peptone water and incubated at 40°C. After incubation for 6 h or overnight, samples were tested for the presence of Salmonella with the DNAble assay and related to ground turkey samples from corresponding lots. The linear relationship for the percentage of Salmonella-positive live-haul trailers was significant for both the 6-h (slope = 1.02, R(2) = 0.96, and P < 0.0001) and overnight (slope = 0.35, R(2) = 0.93, and P = 0.0015) incubations, with the percentage of Salmonella-positive ground turkey samples. These data indicate that preharvest screening provides a meaningful evaluation of product contamination. Additionally, the 6-h incubation protocol is rapid enough to allow for product mitigation and could potentially aid in the reduction of future salmonellosis outbreaks.

Making a Difference in Malawi and Zambia Through Health Education and Public Health Best Practices.

A 2-year U.S. Department of State-funded project* was conducted with the purpose of engaging health professionals from Malawi and Zambia who are actively involved in health care and health education for marginalized populations to develop, implement, and evaluate health education and public health interventions/programs. Twenty-six health professionals from Malawi and Zambia, referred to as Global Health Fellows, participated in the 2-year program, of which the main training component was conducted in the United States. Fellows were exposed to health education and public health best practices and developed an action plan to address a health problem of concern in their respective communities/countries. After completion of the program, Fellows received $300 to implement their action plans. Teams of Americans involved in the training program participated in follow-up visits to Malawi and Zambia to observe real-time progress on Fellows' respective action plans. The project was successful in creating an educational experience focused on health education best practices as well as implementation of action plans to address selected health problems in Malawi and Zambia.

Shiga toxin-producing Escherichia coli distribution and characterization in a pasture-based cow-calf production system.

Shiga toxin-producing Escherichia coli (STEC) strains are commonly found in cattle gastrointestinal tracts. In this study, prevalence and distribution of E. coli virulence genes (stx1, stx2, hlyA, and eaeA) were assessed in a cow-calf pasture-based production system. Angus cows (n = 90) and their calves (n = 90) were kept in three on-farm locations, and fecal samples were collected at three consecutive times (July, August, and September 2011). After enrichment of samples, stx1, stx2, eaeA, and hlyA were amplified and detected with a multiplex PCR (mPCR) assay. Fecal samples positive for stx genes were obtained from 93.3% (84 of 90) of dams and 95.6% (86 of 90) of calves at one or more sampling times. Age class (dam or calf), spatial distribution of cattle (farm locations B, H, K), and sampling time influenced prevalence and distribution of virulence genes in the herd. From 293 stx-positive fecal samples, 744 E. coli colonies were isolated. Virulence patterns of isolates were determined through mPCR assay: stx1 was present in 41.9% (312 of 744) of the isolates, stx2 in 6.5% (48 of 744), eaeA in 4.2% (31 of 744), and hlyA in 2.4% (18 of 744). Prevalence of non-O157 STEC was high among the isolates: 33.8% (112 of 331) were STEC O121, 3.6% (12 of 331) were STEC O103, and 1.8% (6 of 331) were STEC O113. One isolate (0.3%) was identified as STEC O157. Repetitive element sequence-based PCR (rep-PCR) fingerprinting was used to study genetic diversity of stx-positive E. coli isolates. Overall, rep-PCR fingerprints were highly similar, supporting the hypothesis that strains are transmitted between animals but not necessarily from a dam to its calf. Highly similar STEC isolates were obtained at each sampling time, but isolates obtained from dams were more diverse than those from calves, suggesting that strain differences in transference may exist. Understanding the transfer of E. coli from environmental and animal sources to calves may aid in developing intervention strategies to reduce E. coli colonization of young cattle.

A study of the level and dynamics of Eimeria populations in naturally infected, grazing beef cattle at various stages of production in the Mid-Atlantic USA.

There is little information available on the species dynamics of eimerian parasites in grazing cattle in the central Appalachian region of the United States. Therefore, the objective of this study was to describe the level of infection and species dynamics of Eimeria spp. in grazing beef cattle of various age groups over the course of a year in the central Appalachian region. Rectal fecal samples were collected from male and female calves (n=72) monthly from May through October 2005, heifers only (n=36) monthly from November 2005 to April 2006, and cows (n=72) in May, July, and September, 2005. Eimeria spp. oocysts were seen in 399 of 414 (96%) fecal samples collected from the calves from May through October. Fecal oocysts counts (FOC) in the calves were lower (P<0.05) in May than all other months and no significant differences were detected from June through September. Eimeria spp. oocysts were detected in 198 of 213 (92%) of fecal samples collected from the 36 replacement heifers monthly from November to April and monthly mean FOC did not differ during this time period. The prevalence of oocyst shedding increased to 100% in calves in September and remained near 100% in the replacement heifers during the sampling period. Eimeria spp. oocysts were also detected in 150 of 200 (75%) samples collected in May, July, and September from the cows and mean FOC did not differ significantly over the sampling period. Eimeria spp. composition was dominated by Eimeria bovis in fecal samples collected from calves, replacement heifers and cows. Mixed Eimeria spp. infections were, however, common in all groups and 13 Eimeria spp. oocysts were identified throughout the sampling period.

Prevalence of cardiomyopathy in apparently healthy cats.

OBJECTIVE: To determine the prevalence of cardiomyopathy and the relationship between cardiomyopathy and heart murmurs in apparently healthy cats. DESIGN: Cross-sectional study. ANIMALS: 103 privately owned, apparently healthy domestic cats. PROCEDURES: Cats were physically and echocardiographically examined by 2 investigators independently. Left ventricular wall thickness was determined via 2-dimensional echocardiography in short-axis and long-axis planes. Left ventricular hypertrophy was identified when end-diastolic measurements of the interventricular septum or posterior wall were > or = 6 mm. Cats with left ventricular hypertrophy but without left ventricular dilatation were considered to have hypertrophic cardiomyopathy (HCM). The associations between heart murmurs and Doppler echocardiographic velocity profiles indicative of dynamic ventricular outflow tract obstruction were evaluated. RESULTS: Heart murmurs were detected in 16 (15.5%; 95% confidence interval, 9.2% to 24.0%) cats; of these, 5 had cardiomyopathy. Cardiomyopathy was also identified in 16 (15.5%; 95% confidence interval, 9.2% to 24.0%) cats; 15 had HCM, and 1 had arrhythmogenic right ventricular cardiomyopathy. Of the cats with HCM, 11 had segmental left ventricular hypertrophy, 3 had diffuse left ventricular hypertrophy, and 1 had borderline left ventricular hypertrophy with marked systolic anterior motion of the mitral valve. Sensitivity and specificity of auscultatory detection of a heart murmur for diagnosing cardiomyopathy were 31% and 87%, respectively. Echocardiographic evidence of late systolic acceleration within ventricular outflow tracts was associated with the existence of a heart murmur. CONCLUSIONS AND CLINICAL RELEVANCE: Cardiomyopathy was common in the healthy cats evaluated in this study. In apparently healthy cats, detection of a heart murmur is not a reliable indicator of cardiomyopathy.

Effect of body position on intraocular pressure in dogs without glaucoma.

OBJECTIVE: To determine the effects of body position on intraocular pressure (IOP) in dogs without glaucoma. ANIMALS: 24 healthy dogs with no evidence of glaucoma. PROCEDURES: Dogs underwent ophthalmic examinations to ensure that no IOP-affecting ocular diseases were present. Each dog was sequentially placed in dorsal recumbency, sternal recumbency, and sitting position. For each of the 3 positions, IOP in the right eye was measured by use of an applanation tonometer immediately after positioning (0 minutes) and after 3 and 5 minutes had elapsed. The initial body position was randomly assigned; each position followed the other positions an equal number of times, and IOP measurements were initiated immediately after moving from one body position to the next. Proparacaine hydrochloride (0.5%) was applied to the right eye immediately prior to IOP measurements. RESULTS: Intraocular pressure was affected by body position. During the 5-minute examination, IOP decreased significantly in dogs that were dorsally recumbent or sitting but did not change significantly in dogs that were sternally recumbent. For the 3 positions, overall mean IOP differed significantly at each time point (0, 3, and 5 minutes). Mean IOP in dorsal recumbency was significantly higher than that in sternal recumbency at 0 and at 3 minutes; although the former was also higher than that in sitting position at 3 minutes, that difference was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Body position affects IOP in dogs. When IOP is measured in dogs, body position should be recorded and consistent among repeat evaluations.

Characteristics of diagnostic tests used in the 2002 low-pathogenicity avian influenza H7N2 outbreak in Virginia.

An outbreak of low-pathogenicity avian influenza (LPAI) H7N2 occurred in 2002 in the Shenandoah Valley, a high-density poultry production region in Virginia. Infected flocks were identified through a combination of observation of clinical signs and laboratory diagnostic tests designed to detect avian influenza (AI) antibodies, virus, or H7-specific RNA. In this report, fitness for purpose of 3 virus/RNA detection assays used during the outbreak was examined: 1) antigen capture enzyme immunoassay (AC-EIA), 2) real-time reverse transcription polymerase chain reaction (RRT-PCR), and 3) virus isolation (VI). Results from testing 762 turkey and 2,216 chicken tracheal swab pooled specimens were analyzed to determine diagnostic sensitivities and specificities of these tests under field conditions using Bayesian techniques for validation of diagnostic tests in the absence of a "gold standard." Diagnostic sensitivities (with 95% probability intervals) in turkeys of AC-EIA and RRT-PCR, in reference to VI, were 65.9 (50.6; 81.3)% and 85.1 (71.9; 95.7)% and of VI 92.9 (78.0; 98.8)% in reference to AC-EIA or 88.7 (76.0; 97.2)% in reference to RRT-PCR; in chickens, diagnostic sensitivities were 75.1 (45.6; 94.2)%, 86.3 (65.9; 97.1)%, and 86.2 (65.8; 97.1)% or 86.3 (66.4; 97.2)%, respectively. Specificities were 99.1 (97.9; 99.8)%, 98.9 (98.0; 99.5)%, and 98.6 (97.4; 99.4)% or 98.8 (97.8; 99.5)% in turkeys and between 99.25% and 99.27% with probability intervals of approximately +/-0.4% for all tests in chickens. Simultaneous use of AC-EIA and RRT-PCR contributed significantly to the rapid control of the outbreak, but the AI RRT-PCR assay with >85% sensitivity and approximately 99% specificity, combined with relatively low cost and fast turnaround, could be used as the sole diagnostic test in outbreaks of LPAI.

The effect of weaning method on coccidial infections in beef calves.

Weaning is often cited as a stressful event that can precipitate clinical coccidiosis in cattle. Recently, two-stage weaning methods have been investigated as a means to reduce stress in calves. The objective of this study was to investigate the effect of weaning method on fecal oocyst count (FOC) and Eimeria spp. composition. A group of 108 beef calves (190-240 days of age) were allocated to three treatments on day -7: a fenceline weaned group where calves could see and hear their dams, but were physically separated from them by a fence (FL), a noseclipped group where suckling was prevented (NC), and a group left with their dams to be traditionally weaned (TW) by abrupt separation on day 0. On day 0, noseclips were removed from the NC calves and all groups were completely separated from their dams. The steers (n=50) were transported 172 km to another farm, and heifers (n=58) were taken to distant fields on the same farm. Rectal fecal samples were collected from all calves on days -7, 0, and 14. Steers were also sampled on days 7, 28, and 42. A group of steers (n=24) from each of the treatments previously described were fitted with pedometers and intensively observed on days 1-4. The Modified McMaster's test and Wisconsin sugar flotation test were used for quantification and identification of Eimeria spp. oocysts. Clinical coccidiosis was not observed during the study. Fecal oocyst counts did not differ between treatment groups on any sampling day. Differences between steer and heifer FOC were not detected. Treatment had no effect on species composition, but time effects were detected in the steers. The mean percentage of E. bovis was lower (P<0.05) on days 28 and 42 than all other sampling days. The decrease in the percentage of E. bovis was accompanied by an increase (P<0.05) in the percentage of E. canadensis on days 28 and 42. Behavioral observation showed that TW steers exhibited higher levels of stress related behavior (P<0.05) following complete separation, although this was not reflected in FOC.

Estrogen up-regulates inducible nitric oxide synthase, nitric oxide, and cyclooxygenase-2 in splenocytes activated with T cell stimulants: role of interferon-gamma.

Estrogen is implicated in many autoimmune diseases and is a robust immunomodulator. For example, it regulates interferon (IFN)-gamma, a cytokine believed to up-regulate inducible nitric oxide synthase (iNOS). A notable gap in the literature is a lack of information on the regulation of nitric oxide in immune tissues by estrogen. We now show that activation of splenocytes with T cell stimulants [concanavalin-A (Con-A) or anti-CD3 antibodies] results in copious release of nitric oxide in splenocyte cultures from estrogen-treated but not placebo-treated mice. Moreover, even a low dose of T cell stimulants induced nitric oxide in splenocytes from estrogen-treated, but not placebo-treated, mice. Con-A-activated splenocytes from estrogen-treated mice also have up-regulated iNOS mRNA, iNOS protein, and cyclooxygenase-2 (a nitric oxide-regulated downstream proinflammatory protein) when compared with controls. Our studies suggest that the induction of nitric oxide by activated splenocytes from estrogen-treated mice is mediated in part by IFNgamma. First, blocking costimulatory signals mediated through interactions of CD28 and B7 molecules by CTLA-4Ig markedly decreased not only IFNgamma but also nitric oxide. Second, estrogen treatment of IFNgamma-knockout (IFNgamma(-)/(-)) mice did not induce iNOS protein or nitric oxide. Finally, in vitro addition of recombinant IFNgamma to Con-A-activated splenocytes from IFNgamma((-)/(-)) mice induced iNOS protein primarily in estrogen-treated mice. Overall, this is the first report to show that estrogen treatment up-regulates IFNgamma-inducible-iNOS gene expression, iNOS protein, nitric oxide, and cyclooxygenase-2 as an indirect consequence of activation of T cells. These findings may have wide implications to immunity and inflammatory disorders including female-predominant autoimmune diseases.