We conducted an i.n. local tolerance and toxicity study with Xylo_0.05%/Carr_0.12% and Xylo_0.05% compared with the base formulation as control (Table 1). The purpose of this study was to obtain information on the tolerability of Xylo_0.05%/Carr_0.12% in rabbits after repeated i.n. administration over a period of 14 days. Xylo_0.05%/Carr_0.12% was applied three times/day at a dose/application of 70 µg xylometazoline HCl and 168 µg iota-carrageenan; application of Xylo_0.05% delivered the same dose of xylo-metazoline HCl but 0 µg of iota-carrageenan. No mortality occurred in the control or any of the dose groups during the treatment period of this study; a three-time daily administration of neither Xylo_0.05%/Carr_0.12% nor Xylo_0.05% was associated with any clinical symptoms in male or female rabbits. At the application site, the test solutions were well tolerated. Throughout the study, neither erythema nor edema were observed at the nostrils of all animals; there was no effect on body weight or on hematological parameters. No treatment-related effect on clinical biochemistry is assumed after treatment with Xylo_0.05%/Carr_0.12% or Xylo_0.05%. Changes in single parameters, e.g., alanine-aminotransferase, aspartate-aminotransferase or glutamate dehydrogenase, were observed in single animals treated with Xylo_0.05%/Carr_0.12%. However, these changes were not severe and were not considered of toxicological relevance, when comparing to minimum/maximum ranges of the control, historical data and/or pretreatment values. In addition, no correlation with other parameters (e.g., histopathology) was determined for the aforementioned findings. No gross pathological abnormalities or abnormal organ weights of toxicological concern were found in animals treated with Xylo_0.05%/Carr_0.12% or Xylo_0.05%. After treatment with Xylo_0.05%/Carr_0.12%, a slightly higher thymus, epididymides, prostate and ovary weight was found. Slightly but not significantly higher weight of thymus, spleen, ovaries and epididymides was also associated with treatment with Xylo_0.05%. However, relative organ weights did not exceed values of respective historical controls.

In conclusion, Xylo_0.05%/Carr_0.12% was well tolerated at the application site with no occurrence of erythema or edema at the nostrils of all animals or any signs of toxicity at the daily dose tested. Xylo_0.05%/Carr_0.12% did not produce any morphological indicators for toxicity in any of the organs and tissues examined. No test item-related changes on clinical biochemistry parameters and relative organ weights were detected in this study.

Non-communicable diseases with the highest health burdens include cardiovascular diseases, cancer and diabetes. The number of POCT that are commercially available for non-communicable diseases is limited and reflects the challenge of measuring low concentrations of protein biomarkers in a variety of different biological samples, including blood, serum, urine and saliva. The key cancer and cardiac biomarkers, as well as their normal values in blood, are given in Table 1. PSA is the most common tumour marker for prostate cancer. IL-6, an interleukin, is overexpressed in several different cancers including prostate cancer, as well as head and neck squamous cell carcinoma. The interleukins are part of the cytokine family and more generally play an important role in the inflammatory response of diseases such as rheumatoid arthritis, cardiovascular diseases (CVD), diabetes and Alzheimer’s disease. The MMP family are part of the zinc-dependent endopeptidases, where MMP-2 is key in tumour growth, invasion and metastasis and MMP-3 is used to diagnose and monitor diseases such as head and neck squamous cell carcinoma and adrenal tumours. The alpha-fetoprotein, an oncofetal glycoprotein, is the most important for liver cancer tumour marker. CEA, a glycoprotein, is raised with inflammation or tumours in any endodermal tissue, including the gastrointestinal tract, respiratory tract, pancreas and breast, and can be used for diagnosis of lung cancer, ovarian carcinoma and breast cancer. The cancer antigen 125 (CA-125) is used for monitoring ovarian, breast and uterine cancer.

CVDs and coronary heart disease are globally among the leading causes of ill health, invalidity and mortality. Troponin-I is most sensitive for myocardial tissue damage. There are several cardiac biomarkers, these can sometimes require 3-hourly or 6-hourly repeat blood tests, checking for dynamic rise and can be used to rule-out or confirm Acute Coronary Syndrome (ACS). D-dimer blood tests can be used to help rule out suspected Deep Vein Thrombosis (DVT) or Pulmonary Embolism (PE), but it suffers from a high false positive rate that may lead to unnecessary or costly advanced imaging.

Similarly, there are certain Human Leukocyte Antigens (HLA) that are associated with specific autoimmune and immune-mediated diseases, such as reactive arthritis, skin lesions, systemic lupus erythematosus (HLA-DR2), rheumatoid arthritis, diabetes mellitus type 1 (HLA-DR3) and ankylosing spondylitis (HLA-B27). HLAs are also very important in organ transplant rejections, where there may have been inadequate matching. We might include tests for Erythrocyte Sedimentation Rate (ESR), Rheumatoid Factor (RF), Antinuclear Antibody test (ANA) and, then potentially more specific autoantibody tests when investigating chronic or autoimmune diseases. There are also many chronic or autoimmune diseases linked to genetic mutatations, or even rare conditions such as Gorlin Syndrome.

POCT with a higher level of connectivity and data analytics could dramatically change the diagnosis and management of both communicable and non-communicable diseases. Moreover, incorporation of findings from the human genome project with decentralized POCT could dramatically change our understanding of disease epidemiology and population health.

Xylometazoline hydrochloride (HCl) (2-[4-(1,1-dimethylethyl)-2,6-dimethylbenzyl]-4,5-dihydro-1H-imidazole HCl) is a well-established nasal decongestant that belongs to the pharmacotherapeutic group of sympathomimetic drugs and acts selectively on α-adrenergic receptors (alpha-adrenergic agonist).1

Xylometazoline HCl is applied topically to relieve nasal congestion associated with acute or chronic rhinitis, common cold, sinusitis, hay fever or other allergies. It causes vasoconstriction in the nasal submucosa, which is manifested as a collapse of the venous sinusoids. Xylometazoline HCl action is characterized by a fast onset with an effect obtained after 5–10 minutes and lasting for 6–8 hours.2 The efficacy of xylometazoline HCl as a topical nasal decongestant is well proven.3,4 Xylometazoline HCl has been used in the EU in the treatment of nasal congestion caused by rhinitis/sinusitis since 1959. Since then a large number of preparations containing xylometazoline HCl have been approved and marketed in several European countries.

Carrageenan is a high-molecular-weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industries, and is generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA), as reviewed by Cohen and Ito.5 Three main forms of carrageenans are commercially used: iota-, kappa- and lambda-carrageenan. They differ in the degree of sulfation, solubility and gelling properties.6 The antiviral mechanism of carrageenan is based on the interference with viral attachment: carrageenan forms a barrier on the mucosal surface, thereby preventing the interaction between virus and cellular surface. As a result, infection of cells is inhibited. The antiviral effectiveness varies with the type of polymer as well as the virus and the host cells,7–17 as reviewed previously.18–20 We already published that iota-carrageenan is a potent inhibitor of major (intercellular adhesion molecule 1 receptor) and minor group (low-density lipoprotein receptor) human rhinovirus (hRV)21 and influenza virus A replication.22 Furthermore, we demonstrated the broad antiviral efficacy of an iota-carrageenan containing nasal spray against common cold viruses in several randomized, double-blind, parallel-group, placebo-controlled clinical trials.23–25 The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus who were intranasally treated with iota-carrageenan showed a 1.9-day faster recovery from common cold symptoms than placebo-treated patients in the intention-to-treat population.26 Carrageenan is contained in medical device nasal sprays that have been approved and marketed in the EU for 10 years for the supportive treatment of common cold/flu-like illnesses caused by viruses.

The rationale for merging xylometazoline HCl with iota-carrageenan into one product is based on the intent to combine their properties, namely the decongestant efficacy provided by xylometazoline HCl and the effectiveness to reduce viral infection accomplished by iota-carrageenan. The intranasal (i.n.) topical application provides reliable and fast relief from the symptoms of nasal congestion and facilitates breathing during common cold/flu-like illnesses and allergic rhinitis. Furthermore, it facilitates drainage of the secretion during sinusitis and can be used as an adjuvant treatment of mucosal swelling due to otitis media. Additionally, iota-carrageenan can reduce the reproduction and spreading of respiratory viruses. The above described indications frequently involve viral infections which worsen the course of the underlying disesase. Therefore, a nasal spray which combines decongestant and antiviral properties is benefitial for patients when compared to conventional xylometazoline HCl products.

Therefore, a medicinal product containing xylometazoline HCl and iota-carrageenan for the proposed indication and route of administration is advantageous and reasonable from a scientific and a medicinal point of view. In order to evaluate whether the properties of xylometazoline HCl are affected by the presence of iota-carrageenan, a set of experiments was performed.

Neisseria gonorrhoeae uses cellular proteins CXCL10 and IL8 to enhance HIV‐1 transmission across cervical mucosa

All calves had baseline total serum protein (TSP) measurements above 5.5 g/dL, indicating no failure in passive immune transfer [18, 19]. However, 31 calves had TSP measurements above 7.5 g/dL and from these, 23 presented diarrhea at the time of enrolment. Consequently, 35% of the enrolled animals might have presented some degree of dehydration that could alter to some extent the values of TSP. No significant difference (P = 0.94) was found for the proportion of TSP above 7.5 g/dL between control (CTR) and SRB calves (OR = 1.03, 95% CI = 0.43–2.47). Additionally, at enrollment, 43 (49%) calves presented signs of slight disease (diarrhea), which may also explain the high TSP level and possible dehydration. No differences (P = 0.39) were found in the odds (95% CI) of diarrheal disease at enrollment (OR = 1.44 [0.62–3.34]) for CTR calves in comparison with the SRB group.

Rearing healthy calves that maintain adequate growth rates is essential for the success of dairy operations. However, during the first month of life, calves face multiple stressors while the immune system is still developing, resulting in a high susceptibility to digestive diseases.

During the first weeks of life of dairy calves, diarrhea is the most prevalent health disorder, as well as the main cause of death. A recent report in the US indicated that 56.4% of calf mortality was a consequence of diarrhea and animals less than 4 weeks old were the most affected. In 2013, 21% of pre-weaned calves presented diarrhea and 16% of all pre-weaned calves were treated with antimicrobials. Rehydration and antibiotic therapy are common treatments for calves with neonatal diarrhea. However, due to consumer concerns, regulations for the use of antibiotics in food animals are becoming more restrictive. Consequently, research focused on alternatives to the use of antimicrobials, including strategies to prevent disease is required.

Prebiotics are defined as non-digestible feed ingredients that stabilize the intestinal microbiota, stimulating the growth of beneficial bacteria and inhibiting the colonization by pathogens [3, 4]. Prebiotic-probiotic interactions have been shown to improve immune responses [3, 5], contrasting with the action of antibiotics that eliminate and restrict the growth of detrimental and beneficial microorganisms with no distinction.

The use of prebiotics has been studied in young ruminants as a prophylactic strategy to prevent disease and as an alternative to antibiotics and one of the most common products is mannanoligosaccharides (MOS), a derivative of the cell wall of the yeast Saccharomyces cerevisiae. However, the effects of prebiotics on performance, health, and immunity of calves has not been consistent. For example, the supplementation of MOS resulted in a reduction in almost 1 point on the severity of neonatal diarrhea in a 1 to 4 scale and decreased the number of days with high diarrhea scores. Contrary, other studies reported no differences in diarrhea cases after the supplementation prebiotics [8–10] in pre-weaned dairy calves. Furthermore, some studies indicated no differences in weight gain [8, 10, 11], while greater gains were reported by others [6, 12, 13] after a prebiotic supplementation in pre-weaned calves.

Heat stabilized rice bran (SRB) contains prebiotics that have been tested in mice, chickens, pigs, horses, dogs and humans. This is a natural product that has been heat stabilized to prevent rancidity. As other prebiotics used in calf health, SRB is a carbohydrate. However, SRB contains ɣ-Oryzanol (omega 6–9), antioxidants (tocopherols, tocotrienols, polyphenols, phytosterols), vitamin E and B, amino acids (tryptophan, histidine, methionine, cysteine, arginine) and micronutrients (magnesium, calcium, phosphorus, manganese), which may have the potential to enhance the host health, not only through a symbiotic effect with the probiotic bacteria in GI tract. Previous research indicated that this product had positive effects reducing the presentation and duration of diarrhea from human rotavirus and human norovirus in pigs, increasing the production of local and systemic IgA and enhancing the immune system in mice and pigs [14–17]. The effect of SRB has not been previously studied in young ruminants and its potential as a supplement or additive in whole milk of pre-weaned dairy calves has not been explored.

We hypothesized that the addition of SRB in milk of pre-weaned calves would reduce the presentation and severity of neonatal diarrhea, improving the immune response and consequently the overall calf performance. Therefore, our specific objective was to determine the effect of SRB on average daily gain (ADG), fecal IgA concentration, presentation of diseases, time to recovery from disease, and animal removal.

POCT can play an important role in both the diagnosis and management of specific diseases. POCT for the rapid identification of specific pathogens is ongoing. The need for POCT for the early identification and management of sepsis is particularly important since this is difficult to diagnose and the mortality increases by an average of 8% for every undiagnosed hour. In the hospital setting, various tests and investigations are undertaken when suspecting sepsis in both adults and children: these include vital signs checks (observations of temperature, blood pressure, respiratory rate, pulse oximetry oxygen saturation, heart rate and responsiveness). A full set of routine blood tests can include blood count, a chemistry profile including urea and electrolytes, C-Reactive Protein (CRP), and glucose, and a coagulation screen. More than 170 biomarkers have been reported in the literature for the diagnosis of sepsis. However, only the most common biomarkers, such as CRP or procalcitonine, can typically be analysed within centralised hospital laboratories. The testing of the other biomarkers typically requires more specialised laboratories. Other tests can include arterial or venous blood gas samples, urine output recorded and urine sample dipstick test and microbiological culture, aerobic and anaerobic microbiological blood cultures, swab of suspected wound or respiratory tract/gynaecological swabs, chest radiograph.

Some of these can be tested immediately to provide results (at the patient’s bedside), others require sampling of bodily substances/fluids which can either be taken to a local analyser or sent to laboratories (these may be onsite or require further delivery to more specialist laboratories elsewhere offsite). Results may be published to patient’s electronic records as soon as possible. Some of the requested tests may be marked urgent or routine, as necessary. Bedside glucose tests, vital signs temperature probe, pulse oximetry or electronic blood pressure cuffs will often provide immediate results. Arterial or venous blood gas samples can be difficult to acquire if there is difficulty with gaining vessel access (sometimes alternative sampling techniques including femoral stab, or ultrasound-guided sampling is necessary). The local availability of analysers can, however, lead to results within minutes, including true oxygen and carbon dioxide saturations, pH, lactate and Electrolyte levels. Similarly urine samples may be tested with a dipstick for common abnormalities (protein, leukocytes, blood, glucose, nitrates), or urine test for pregnancy, prior to being sent for full investigation in the laboratory. Blood or swab cultures taken will require 5–10 days incubation; growth of any organisms and their sensitivities/resistance to various antibiotics will guide management and assist in identifying appropriate treatment(s). Currently, certain patients found to be positive with or exposed to certain microorganisms (’superbugs’) may need more treatments by nursing staff to improve sanitation or nursing in isolation; examples include patients positive for Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococci (VRE), and Carbapenemase-producing Enterobacteriaceae (CPE), among others.

Sexually transmitted infections (STIs) are among the most common acute conditions worldwide. In 2012, there were 357.4 million new global cases of four common curable STIs: chlamydia (130.9 million cases); gonorrhoea (78.3 million cases); syphilis (5.6 million cases) and trichomoniasis (142.6 million cases). Although STIs are not normally fatal they do represent a significant burden of diseases and they can lead to complications such as pelvic inflammatory disease, ectopic pregnancy and infertility. STIs can increase infectiousness of a susceptibility to HIV and in pregnancy they can cause fetal or neonatal death. Chlamydia is globally the most common bacterial STI and causes reproductive complications in women. The LMICs and LDCs have the majority of global incidents of STIs, but the health systems are less well resourced to manage these. POCT could play an important role in supporting the management of conditions for individual patients and also provide wider control of STIs within developed countries, LMICs and LDCs.

According to the Centers for Disease Control and Prevention (CDC), in the United States alone drug-resistant bacteria cause at least 23.000 deaths and 2 million infections every year. This is therefore a public health problem, requiring innovative POCT approaches that can contribute to helping characterise the development and spread of resistant bacteria. This can have a transformative role in the treatments administered, constituting one step further in the path of personalised medicine. By allowing the rapid detection of infectious pathogens and resistance factors, practitioners can for example reduce unnecessary administration of antibiotics, contributing to alleviate the problem of antibiotic resistance.

Respiratory tract infections (RTIs) are one of the problems that can be caused by a variety of bacterial and viral pathogens. Worldwide, they are the second greatest cause of morbidity and mortality. RTIs are the most common infections for those that are immuno-compromised. There is now also concerns about the number of infections caused by antimicrobial resistant (AMR) bacteria as well as community and hospital acquired infections, for example pneumonia. New lethal viruses and bacteria causing RTIs with epidemic potential have emerged over the last decade. These include severe acute respiratory syndrome coronavirus (SARS-CoV), swine-origin influenza A, multi-drug resistance tuberculosis and multi-drug resistance gram negative bacteria, for which there are very few effective therapy options.

The key challenge for effective treatment of RTIs in a variety of different healthcare settings is for fast, sensitive and specific identification of pathogens as well as antibiotic resistance profiles. Also, it would be beneficial to determine whether a pneumonia was Community-acquired or Hospital-acquired (there are cases where this is still unclear): this can have a large impact on treatment regimen. If Hospital-acquired, then treatment with intravenous antibiotics initially is more beneficial. RTIs are the most common infections encountered within primary care and there is evidence that people presenting with acute uncomplicated RTIs will commonly receive antibiotics despite most RTIs being viral. There is therefore a clear need for POCT that can differentiate RTIs within primary care and reduce unnecessary antibiotic prescribing as part of AMR stewardship.

In many cases, amplification is indispensable for the analysis of nucleic acids. Currently, nucleic acid amplification methods include but are not limited to polymerase chain reaction (PCR) (1), strand-displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling-circle amplification (RCA) and the Qβ replicase reaction. Among these methods, PCR has been the most popular due to its simplicity; however Peltier effect or metal-block-based PCR system are characterized by high thermal mass, large reaction volume and thus slow heating/cooling rates. The PCR speed can be improved by increasing the heat transfer rate or decreasing the thermal mass. With the advent of micro-electro-mechanical-systems (MEMS) technology, the development of miniaturized PCR chips becomes possible (2,3). The miniaturization of PCR devices offers several advantages such as short assay time, low reagent consumption and rapid heating/cooling rates, as well as great potential of integrating multiple processing modules to reduce size and power consumption. The number of publications on PCR chips has grown rapidly recently, and the articles are spread over a large number of journals. The development of PCR microchips has been discussed in recent reviews (2–4). In this article, we will review the latest advances and future trends based on literature published since January 2005. In addition, we will also discuss some practical issues related to the development of PCR chips. As a supplement to this review, the reader may wish to refer to several reviews of general microfluidic technologies (5–9).

The organization of this article is as follows. First, several important topics on the microfluidic PCR chips will be presented. Those topics, which are crucial in the development of PCR chips, include chip substrates and surface treatments, PCR chip architecture, on-chip PCR reaction volume and reaction speed and approaches to eliminating cross-contamination. Then, the temperature and fluidic controls and measurements in PCR chips are discussed, which include thermal insulation, evaporation and gas-bubble formation and measures to counteract these phenomena, semi-invasive or noninvasive temperature and fluidic measurements and numerical simulation of temperature and fluid fields in PCR chips. Finally, product detection methods used in PCR chips, e.g. off-line and on-line detection, are covered, followed by integration of functional components in PCR chips, biological samples used in PCR chips and potential applications of PCR chips, as well as practical issues related to the development of PCR chips.

The current trial demonstrated evidence for the beneficial effect of ZM on ADG and neonatal diarrhea as well as an effect of ZS on diarrhea in dairy calves during the pre-weaning period. It is important to consider these results in the context of the entire pre-weaning and hutch period. On average, after 90 days from birth to hutch exit, placebo-treated bull calves gained 38.88 kg body weight while ZM-treated bull calves gained an additional 1.98 kg (40.86 kg). In contrast, the effect of zinc on weight gain in treated heifers depended on birth weight. Low birth weight heifers treated with ZM gained on average less than a placebo-treated heifer of the same birth weight. In contrast, high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight. The switch in direction of the association between ZM treatment and ADG in heifer calves depending on birth weight suggests a dose-response effect rather than a true sex-specific effect of ZM on ADG. Hence, low birth weight calves (including heifers) may require a lower dose of ZM to mitigate any negative effect of what is otherwise a suitable dose for higher birth weight calves. These findings are in agreement with a previous randomized clinical trial testing the effect of daily oral zinc in diarrheic neonatal Holstein calves which, showed that ZM-treated calves had a numerically, though not significantly increased ADG compared to calves treated with zinc oxide or placebo due to small sample size. In general, our trial findings are in agreement with the large body of human literature supporting the use of oral zinc for the prevention and treatment of diarrhea and impaired growth in children [5, 10, 33].

Zinc supplementation is widely accepted by global health organizations as a vital component of therapy for childhood diarrhea [3, 4], however, recent reviews of the literature demonstrated heterogeneity in study results on the basis of age, baseline zinc status, geographic location, and supplementation regimen [10, 34]. Similar to our findings, a sex-specific response to zinc supplementation has been demonstrated in several human studies. Zinc gluconate administered for diarrhea prevention reduced the incidence of dysentery in treated boys but not girls; when given therapeutically, it reduced diarrhea duration and frequency more dramatically in boys compared to girls. Similarly, zinc sulfate was shown to improve diarrhea outcomes in boys but improved growth rates in girls. Broadly, these differences between male and female responses to zinc supplementation are not understood, though theories regarding differences in immune function and response [13, 35], diarrhea etiology, and nutrient requirements have been proposed. In the current study, ZM-treated bulls demonstrated increased ADG compared to placebo-treated bulls while ZM-treated heifers demonstrated decreased ADG compared to placebo-treated heifers. However, due to a significant interaction between ZM treatment and birth weight, this reduction in ADG in ZM-treated heifers was overcome with increasing birth weight, such that ZM-treated heifers with birth weights above 42 kg experienced increased ADG during the pre-weaning period, compared to placebo-treated heifers with birth weights above 42 kg.

Differences in the growth response to ZM supplementation between bull and heifer calves may have been related to its effect on feed intake. Previous research on the effects of feeding various doses of oral zinc oxide to pre-ruminant dairy calves demonstrated that high levels of oral zinc supplementation resulted in reduced feed intake. In the current trial, oral ZM dose was estimated to be significantly higher in heifers compared to bulls due to the significantly lower birth weight of heifers. Additionally, serum zinc concentrations in ZM-treated heifers were numerically higher than that of bulls, though this difference was not significant, likely due to the small sample size. Perhaps the higher zinc dose in heifers was associated with reduced feed intake, leading to reduced growth, and that this effect was more pronounced for ZM compared to ZS. The fact that ZM-treated heifers with birth weights approaching those of average bull calves (and, therefore, a similar zinc dose to that in bulls) experienced an increase in ADG over placebo-treated heifers similar to that of bull calves partially supports this theory. Although management practices on the study dairy were designed to be identical for both bulls and heifers, it is possible that subtle, unrecognized differences in nutritional and health management may also have contributed to sex-specific differences in weight gain. Nevertheless, future trials are warranted to investigate the potential differences in the dose-response to zinc supplementation between bulls and heifers.

We hypothesized that ADG would be increased in zinc-supplemented calves compared to placebo-supplemented calves due to the potential preventive and therapeutic effects of zinc supplementation on neonatal diarrhea. In other words, calf diarrhea is mitigated by zinc supplementation and, therefore, on the causal pathway between zinc and ADG. However, considering the similarly-reduced hazard of diarrhea and increased hazard of cure from diarrhea in both ZM and ZS treatment groups but a lack of effect of ZS on ADG, it is likely that the effect of ZM on ADG is not solely mediated through its effects on diarrhea. Differences in effectiveness between organic and inorganic formulations also may exist. In fact, the underlying mechanism of action of oral zinc remains unknown. Several theories of the mechanisms of action of zinc in the prevention and treatment of childhood diarrhea exist, including a mucosal-protective role, a diarrhea-induced zinc deficiency, an essential element in cell-mediated immunity, and a modifier of intra-luminal electrolyte secretion and absorption [6, 37–39].

The clinical and practical implications of effects of ZM supplementation on ADG and diarrhea must be considered. Pre-weaned calf diarrhea remains an ongoing issue for the dairy industry. The deleterious effects on calf health and performance and the resulting economic burden create a strong incentive to treat and prevent diarrhea in pre-weaned calves. On large dairy operations like those in California’s Central Valley, small changes in disease incidence and duration as well as animal growth and performance can have profound economic consequences. As a non-antimicrobial product, zinc may become increasingly attractive as antimicrobials in livestock feed are under increased scrutiny and regulation due to concerns about antimicrobial resistance [2, 40]. Prevalence of C. parvum fecal shedding in a random sample of 92 study calves at onset and resolution of diarrhea was significantly higher in calves treated with zinc compared to Placebo-treated calves. In contrast, a previous study where calves that tested positive for C. parvum at the start of diarrhea and were treated with ZM had 16 times higher odds of being fecal ELISA negative at exit compared to the Placebo group (P = 0.08; power = 72.3%). The difference in findings may be due to the differences in the timing of diarrhea across treatment groups. For the current study’s random sample of calves that acquired, survived, and were sampled on the correct days, the mean age of calves on both onset and resolution of diarrhea was higher for ZM and ZS calves compared to placebo-treated calves. Although C. parvum oocyst shedding in infected calves can occur as early as 3 days of age, peak shedding occurs at about 14 days of age. It is possible that the increase in prevalence of C. parvum shedding in ZM and ZS treated groups was due to the increased age of zinc-treated calves compared to placebo-treated calves at resolution of diarrhea. The latter explanation is also supported by our findings that the odds of microbiological cure from C. parvum significantly decreased in older calves, with no significant differences in the odds of cure between treatment groups. In addition, the current testing did not estimate the concentration of C. parvum shedding which may still differ between treatment groups.

Despite the large sample size, the current trial was limited to a single California dairy, which may represent other large dairies but does not reflect all the dairy management systems in California or elsewhere. Additionally, our results show that calves respond to zinc supplementation for diarrhea prevention differently depending on chemical formulation and calf sex. The latter could be due to differences in body weight between bulls and heifers and may point towards the need for sex-specific dosing. Furthermore, the current research did not evaluate the potential economic utility of zinc supplementation. Future studies on more accurate dosing of zinc by calf sex, the practical feasibility of weight-based dosing, and the expected cost-effectiveness of zinc administration as part of the management of pre-weaned dairy calves are warranted. Finally, our clinical trial was performed on a single, large, predominately Holstein, California dairy over a six-month period, which precluded our ability to evaluate differences due to season or breed. Hence, future studies to assess any modifying effect of breed and seasonal differences on the effect of zinc on calf health and weight gain are also needed.

The current double blind, block-randomized placebo controlled clinical trial tested the effect of a prophylactic daily oral zinc supplementation in neonatal Holstein calves. Bull calves treated with ZM had a significantly increased ADG (22 g per day) during the pre-weaning period compared to placebo-treated bulls. In comparison, ZM-treated heifers had significantly lower average daily gain (9 g per day) compared to placebo-treated heifers, although higher ZM doses in low birthweight heifers may explain the lower ADG. Calves treated with either ZM or ZS had significantly lower risks of diarrhea and significantly higher risk of cure from diarrhea over the first 30 days of life compared to placebo-treated calves and hence the current trial demonstrated that zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves. Additionally, zinc improved weight gain differentially in bulls compared to heifers, indicating the need for further research to investigate zinc dosing in calves.

Macroautophagy (here called autophagy) is the subject of many recent articles by virologists. With regard to herpesviruses, interest was galvanized by the insightful observation that the herpes simplex virus 1 (HSV-1) neurovirulence protein ICP34.5 bound to Beclin 1, thereby inhibiting maturation of the autophagosome (1, 2). Because several closely related herpesviruses, including varicella-zoster virus (VZV), do not contain an ICP34.5 gene homolog in their genomes (3), autophagy studies were pursued with this virus to investigate differences from HSV-1. Based on the HSV-1 data, an early hypothesis stated that an autophagic response would impair VZV replication.

One of the initial methods used to detect autophagy was microscopy (4). The autophagosome is characterized by its double-walled outer membrane containing microtubule-associated protein 1 light chain 3B (LC3-II), the lipidated form of LC3-I (5). Because of the diameter of the autophagosome (300 to 1,000 nm) and its immunogenic LC3-II protein, confocal microscopy with a fluorescent-antibody probe is an excellent method by which to visualize and enumerate autophagosomes in VZV-infected cells (6). Using this technology, we documented an abundant autophagy response in the skin vesicles within the exanthem of human subjects with herpes zoster (7). We have reproduced these results both in infected cell cultures and in infected human skin xenografts within the severe combined immunodeficient (SCID) mouse model for varicella (8, 9). More recently, these results have been duplicated in a human skin organ culture model for herpes zoster infection (10). In another set of experiments, we documented autophagic flux in VZV-infected cells (9). In other words, the accumulation of autophagosomes was not caused by a block in the autophagy pathway. These VZV data are supported by papers that report similar autophagy results after infection with the closely related alphaherpesvirus pseudorabies virus (PRV), as well as duck enteritis herpesvirus (11, 12), both of which also lack the herpesvirus ICP34.5 homolog.

Cells exhibit basal levels of autophagy even during herpesvirus infection (13). However, the levels of autophagy induced by VZV infection in both (i) human skin during the VZV disease called herpes zoster (shingles) and (ii) human skin explants in either the skin organ culture model or the severe combined immunodeficient mouse model of VZV infection are far above basal levels (10). Further, we found that inhibition of autophagy diminishes VZV cell-to-cell spread and infectivity (8). We also found evidence that an intact autophagy pathway is required for VZV exocytosis after secondary envelopment (14). Because of these findings, we postulated that treatment of VZV-infected cultures with the antiautophagic flux drug bafilomycin A1 (BAF) would decrease viral titers. BAF is known to interrupt late stages of flux, which include fusion of the autophagosome with both lysosomes and endosomes (15, 16), as well as fusion of an early endosome with a late endosome (17). Although we did find that treatment with BAF diminished VZV titers, the findings by electron microscopy were both unexpected and insightful. We also correlate the inhibitory BAF-related effects during the VZV infectious cycle with published data about the inhibitory effects of brefeldin A and monensin at two other Golgi apparatus locations during PRV and HSV-1 infectious cycles. Thus, this report not only reaffirms the centrality of the Golgi apparatus/trans-Golgi network (TGN) as a platform in the alphaherpesvirus infectious cycle, but also expands the antiherpesviral properties of BAF.

(Portions of this research were carried out as part of honors undergraduate thesis research by J. H. Girsch at the University of Iowa.)

Reference values and ranges of thoracic radiography measurements by age and sex are reported in Tables 2–4. The results from the ANOVA tests examining the effects of age, sex, and age × sex interactions on the thoracic radiographic values are presented in Table 5. Significant effects by age were shown for LL, TD, TBr, CBr, and CR. Significant effects by sex were found for TD, TBr, CBr, CR, and R-HHR. Significant effects by age × sex were observed for TD, TBr, CBr, and CR. Both TD and TBr increased with age in both sexes, and both were significantly higher in males than in females in the group aged 49–60 months (Fig. 3A, 3B). CBr increased with age and was significantly higher in males than in females across all age groups (Fig. 3C). CR declined with age and was significantly higher in males than females across all age groups (Fig. 3D).

Heterosexual transmission is the most common route of HIV‐1 infection in women.1, 2 A key co‐factor in the transmission of HIV‐1 in women is the prior existence of bacterial, viral, and parasitic microbes in the cervix that can alter the cervical environment and thereby influence HIV‐1 transmission.3, 4, 5, 6, 7, 8

Gonorrhoeae caused by Neisseria gonorrhoeae (NG), a gram‐negative diplococci, is one of the most severe and common forms of STI9, 10 that has been shown to increase HIV‐1 acquisition.10, 11, 12 The presence of pro‐inflammatory cytokines in the vaginal fluid of NG‐infected women and some cell line‐based studies with NG led to the speculation that NG‐induced inflammatory cytokines either directly or indirectly could increase HIV‐1 transmission.12, 13, 14, 15, 16 Additional mechanisms of NG‐induced enhanced HIV‐1 transmission that have been suggested include recruitment of increased number of endo‐cervical CD4+ T cells in NG‐infected women providing more targets for HIV‐1,17 activation of CD4+ T cells by NG,18 epithelial tight junction disruption,19 and increased HIV‐1 transcription by NG‐secreted proteins.20

The molecular mechanism by which NG enhances HIV‐1/transmission in the female genital tract is still uncertain. Part of the uncertainty is due to lack of a suitable ex vivo model that mimics in vivo situation. HIV‐1/NG interaction has been mostly studied in in vitro cell culture using CD4+ T cells, endometrial epithelial cells,15, 18, 21, 22 and immortalized cell lines.15, 23 However, these cell systems do not accurately reflect situation that occur in human cervix/vaginal tissue. In addition, we do not know the mechanism of HIV‐1 transmission through the epithelia of the cervical mucosa, especially when epithelia do not express CD4 and CCR5/CXCR4.24, 25, 26 Regardless of how HIV‐1 crosses the epithelium, HIV‐1 exposure to the epithelial layer or epithelial cells has been shown to induce production of cytokines and chemokines which serve as signaling molecules.27, 28 These signaling molecules may play an important role in HIV‐1 transmission by attracting target immune cells to fuel HIV‐1 infection in sub‐mucosa and hence transmission.29, 30

Here we describe use of a primary cervical tissue‐based organ culture model of NG infection that provides the natural cervical tissue architecture observed in cervix of NG‐infected women. Using this organ culture, we showed that NG exposure to cervical tissues induced epithelial membrane ruffling and inflammatory cytokine response, reminiscent of in vivo situation. Furthermore, using this model we have shown that NG induces IL‐1β from cervical epithelium post‐exposure and increases the production of epithelial proteins CXCL10 and IL8, two key proteins that may be responsible for HIV‐1 transmission, suggesting that increase in CXCL10 and IL‐8 production in epithelia may be responsible for NG‐induced enhanced HIV‐1 transmission across cervical mucosa. This study for the first time describes a molecular mechanism of NG‐induced enhancement of HIV‐1 transmission across cervical mucosa.

Foot-and-mouth disease (FMD) is one of the most economically and socially devastating diseases affecting cloven-hoofed animals. The infectious agent, foot-and-mouth disease virus (FMDV), is a member of the Aphthovirus genus of the Picornaviridae family, and contains single-stranded positive-sense RNA genomes of about 8,500 nucleotides. As an antigenically variable virus, FMDV consists of seven serotypes (A, O, C, Asia 1, and South African Territories 1, 2, and 3) and a large number of subtypes. In general, slaughtering FMDV-infected/exposed or FMDV-susceptible animals, restricting animal movement, and, in some cases, vaccinating against FMDV and then slaughtering these animals are used as control measures for potential outbreaks in disease-free areas. Although inactivated FMD vaccines have been available since the early 1900s and new novel vaccines are being continuously developed, they offer little or no cross-protection against various serotypes and subtypes of FMDV. In addition, these vaccines do not provide complete clinical protection until seven days post-vaccination. Therefore, there is a need for developing effective and safe alternative antiviral strategies against FMDV.

Mizoribine, an imidazole nucleoside (Figure 1A), has been used as an immunosuppressive agent for the treatment of renal transplantation, autoimmune diseases, and steroid-resistant nephrotic syndrome in some countries owing to its antiproliferative activity against T and B lymphocytes. This drug could be phosphorylated by adenosine kinase and converted to mizoribine 5′-monophosphate, the active form of mizoribine. It has been demonstrated that mizoribine 5′-monophosphate acts as an inhibitor of inosine 5′-monophosphate dehydrogenase (IMPDH) and guanosine monophosphate synthetase. In addition, mizoribine is known to inhibit replication of some DNA and RNA viruses, such as cytomegalovirus, respiratory syncytial virus, severe acute respiratory syndrome-associated coronavirus (SARS-CoV), bovine viral diarrhea virus (BVDV), vaccinia virus, influenza virus types A and B, and herpesviruses, in combination with acyclovir. However, the antiviral activity of mizoribine against FMDV has not yet been investigated. Hence, in this study, the antiviral effect of mizoribine against FMDV was evaluated in vitro using IBRS-2 cells and confirmed in vivo using suckling mice.

Digital PCR conventionally utilizes sequential limiting dilutions of target DNA, followed by amplification using the polymerase chain reaction (PCR),. As a result, it is possible to quantitate single DNA target molecules. We utilize the digital array, which is a novel nanofluidic biochip, where digital PCR reactions can be performed (Figure 1) by partitioning DNA molecules, instead of diluting them. This chip utilizes integrated channels and valves that partition mixtures of sample and reagents into 765 nanolitre volume reaction chambers. DNA molecules in each mixture are randomly partitioned into the 765 chambers of each panel (the total volume of the PCR mix in each panel: 6 nl×765 = 4.59 µl). The chip is then thermocycled and imaged on Fluidigm's BioMark real-time PCR system and the positive chambers that originally contained 1 or more molecules can be counted by the digital array analysis software (Figure 2).

A randomized clinical trial was performed. The study was approved by the UC Davis Institutional Animal Care and Use Committee. Sample size calculation was performed using methods for calculating sample size for 2 independent groups8 based on a standard deviation of 207 mg/dL for serum IgG concentrations in calves given plasma IV in previous studies4 (alpha = 0.05, power = 0.8, minimal detectable IgG concentration = 196 mg/dL). The required sample size was at least 7 calves in each group. To account for an anticipated dropout of up to 50% because of mortality, 30 calves (15 in each group) were enrolled. Thirty Jersey bull calves from a single farm in Hilmar, California were enrolled. Adult cows on the farm of study were vaccinated annually with a modified live respiratory disease vaccine containing infectious bovine rhinotracheitis, bovine viral diarrhea, parainfluenza‐3, and bovine respiratory syncytial viruses. Additionally, the cows were vaccinated with a multivalent vaccine containing Escherichia coli, rotavirus, and coronavirus during the dry cow period. Jersey bull calves delivered from eutocia and observed births were immediately separated from their dams before nursing colostrum. The calves were randomly assigned using a coin toss to 2 groups. Fifteen calves were assigned to the control group to receive colostrum (CL group) by oroesopheagal tubing, and 15 to the treatment group (PL group) to receive bovine plasma IV. The calves were weighed, identified using ear tags, transported within 36 h after enrollment and housed in individual calf hutches at the UC Davis Beef Research facilities.

Nonpooled bovine plasma was derived from 2 clinically healthy Holstein blood donor cows. The plasma was evaluated for sterility and considered free of transmissible blood‐borne pathogens. An aliquot (5 mL) of colostrum or plasma to be administered was collected before administration for IgG concentration determination. Plasma was administered through the jugular vein using an aseptically placed intravenous catheter1 at a dosage of 34 mL/kg. Infusion of plasma was performed at a rate of 10 mL/kg/h over the first 20 min. Monitoring for transfusion reactions included monitoring heart rate, respiratory rate, mucous membranes color, and abnormal behavior. In the absence of an immediate transfusion reaction, the remainder of the plasma was transfused over 20–30 min. In the event of a plasma transfusion reaction, transfusion was discontinued for 10 min and resumed at 5 mL/kg/h. Calves enrolled in the CL group received 3 L of pooled, pasteurized colostrum, from 1 batch collected from the farm of study, through an oroesophageal tube, once. Colostrum was pasteurized at 60°C for min using a batch pasteurizer. All calves received colostrum or bovine plasma within 2 h after birth. Thereafter, all calves were fed 2 L of nonmedicated milk replacer2 twice daily, 0.5 kg of nonmedicated commercial concentrate feed twice daily and water was available ad libitum. Calves were monitored 3 times daily. Daily calf monitoring procedures by trained personnel included assessment of rectal temperature, appetite for milk replacer, and concentrate feed and evidence of diarrhea or coughing. Decisions to medically treat calves were made by a UC Davis campus licensed veterinarian and not by the investigators. Calves that died during the study period were submitted for necropsy at the California Animal Health and Food Safety Laboratory in Davis, CA.

Blood and fecal samples from each calf were collected by jugular venipuncture and digitally, respectively, at 0 h (before procedures), 6 h, 12 h, 24 h, 48 h, 5 d, and 7 d of age. Serum was harvested from the blood samples after centrifugation at 2,880 × g for 5 min at 4°C. Serum total protein concentration was determined by a hand‐held refractometer.3 Serum samples were then stored at −20°C until IgG determination. Fecal samples were immediately frozen at −20°C until IgG determination. Serum and fecal IgG determinations were performed by single radial immunodiffusion (SRID). All calves were enrolled within a 2‐wk period and the study was performed from June 2014 to July 2014.

Bovine respiratory syncytial virus (BRSV) is an important respiratory pathogen in cattle, detrimentally affecting the economy and animal welfare. The virus is distributed worldwide and is a major pathogen of the bovine respiratory disease complex [1, 2]. Viral respiratory infections are also of concern with regards to antibiotic resistance, as they predispose cattle to secondary bacterial infections that are commonly treated with antibiotics. Bovine respiratory disease is traditionally handled with management measures, vaccination and metaphylactic antibiotic treatment. Another possible strategy is to prevent inter-herd transmission of the main pathogens by increasing biosecurity measures at herd level. Because live animal transport is considered one of the main modes of BRSV transmission between herds [5, 6], proper mitigation must ensure that live animal transport be performed without compromising biosecurity. This requires knowledge on transmission risk associated with animal contact at different stages of infection. Knowledge of BRSV shedding related to clinical features would also be useful in order to assess the transmission risk of an infected herd without the use of viral diagnostic assays. For both of these areas, several knowledge gaps exists. Although way of infection may affect both viral shedding and clinical signs compared to naturally exposed animals, challenge studies are superior in the sense that aetiology and time of exposure is known and clinical features and virus excretion can be followed closely. Challenge studies, many of them aiming to evaluate the efficacy of vaccines [7–11] seldom last longer than one to two weeks. Grissett et al. and Gershwin concluded that shedding of BRSV begins on day three or four post-infection (p.i.) and usually lasts until day nine or ten. Grissett et al. summarized that the median time to appearance, peak and resolution of clinical signs was 3, 6 and 12 days, respectively, based on information from 22 inoculation studies [7–11, 14–22]. As studies outlasting the acute phase of infection are lacking, it is not known how long an animal can transmit infectious viruses to other animals. Appearance of clinical signs is usually the only information available in the field, and finding a clinical parameter that indicates shedding of infectious BRSV would be valuable. The existence of chronic or persistent infections in individuals is likewise still unclear [23–26].

During the acute phase of a BRSV infection, immunological protection develops, but it is assumed to be short-lived. This might enable early reinfection and new shedding of the infective virus, which complicates the risk assessment. A few BRSV studies have been performed to shed light on this. In a study by Kimman et al. they reported a strong local IgA response in the respiratory tract, but no virus shedding, when calves were re-exposed 3–4 months after primary BRSV infection. Stott et al. indicated, referring to their own unpublished results, that reinfection in calves and heifers may occur as early as three weeks post-infection. However, early reinfection with BRSV is not well-documented, and more precise knowledge of the occurrence is needed.

The existing literature on BRSV shedding and transmission is based on various laboratory methods, such as detection of viral RNA and culturing of the virus. Although resource-demanding, virus transmission studies are preferably performed using live animals in sentinel trials.

The aim of the present study was, therefore, to study basic features of BRSV infection in calves infected by exposure to BRSV-shedding calves. This was performed by:Investigating the shedding of viral RNA and infective virions:related to clinical outcome during the experimental period, lasting for two monthsin calves rechallenged by inoculation seven weeks p.i.Investigating whether the calves and their environment are not infectious to naïve in-contact calves four to nine weeks post-infection despite rechallenge with BRSV and mild stress induction.

Results of our study indicate that supplementation of 20 g of immunoglobulins twice daily in milk did not reduce the time to resolution of diarrhea, treatment events, or mortality rate in dairy calves with diarrhea. The hypothesized benefit of conferring local gastrointestinal immunity by addition of immunoglobulins in milk was not evident. We suggest further studies to determine the effective dose of immunoglobulins that might confer local gut immunity in dairy calves with diarrhea.

Cells were plated on glass coverslips in 6-well tissue culture plates and incubated at 37°C until they were 90% confluent. The cells were inoculated with VZV-infected cells at a ratio of 1 infected cell to 8 uninfected cells. The infected cells were treated with 10 nM bafilomycin at 24 or 48 hpi. At 72 hpi, the cells were fixed with 2% paraformaldehyde with 0.02% Triton X-100 at room temperature for 1 h. The fixed cells were washed 5 times for 5 min each time in PBS and blocked for 30 min in PBS with 5% nonfat dry milk. The coverslips were incubated in primary antibodies diluted 1:2,000 in PBS-1% milk for 1 h at room temperature and washed 5 times for 5 min each time in PBS. The coverslips were then incubated in goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 546, washed 5 times for 5 min each time, and mounted on glass slides. Images were collected on a Zeiss 710 laser scanning confocal microscope (6). Z-stacks of 2D images were converted into 3D images with Imaris software (Oxford Instruments).

Lactoferrin is a highly glycosylated protein that was first isolated from bovine milk in 1939 by Sorensen and Sorensen, and later identified in human milk in 1960 by Johanson. It has been identified in secretions from exocrine glands as well as in specific granules of neutrophils. Lactoferrin is present in large amounts in milk, and in mammalian exocrine secretions such as saliva, tears, mucus, white blood cells, seminal fluid, and bronchial secretions. Lactoferrin content in milk varies depending on the mammalian species and the stage of lactation. Lactoferrin is the second most abundant whey protein in human milk, with a concentration of 2–4 mg/mL (6–8 mg/mL in colostrum). Its concentration in bovine milk ranges between 0.02 and 0.2 mg/mL, and between 0.2 and 2 mg/mL in pig, mouse, and horse milk, whereas rat, rabbit, and dog milks contain less than 0.05 mg lactoferrin/mL. Oral administration of lactoferrin has been proposed to exert various beneficial health effects in humans and animals, including anti-cancer, anti-inflammatory, and anti-infective activities. Lactoferrin is also used for the prevention of lipid oxidation and the improvement of microflora.

Milk lactoferrin possesses varying numbers of potential glycosylation sites, depending on the species of origin. Although the contribution of conjugated glycans to the functions of lactoferrin is not fully understood, some connections between glycans and physicochemical and biological roles of lactoferrin have been reported. Lactoferrin has been produced from various microorganisms, transgenic animals (cows, goats) and recombinant plants, and through large-scale isolation methods from bovine cheese whey. Cation-exchange chromatography is the most common method used to isolate lactoferrin from dairy products. Lactoferrin exhibits anti-oxidant and anti-microbial properties, and has many applications in the food industry. For example, bovine lactoferrin (bLF) is used to supplement food products such as cakes, pastries, yogurts, and drinks, and non-food products such as cosmetics. To develop lactoferrin-enhanced foods, and infant formulas in particular, a natural source of lactoferrin from dairy milk is used. An appropriate design of infant formula requires the addition of lactoferrin that mimics the properties of human milk lactoferrin, and improves the immune responses of newborns. bLF is also approved by the Food and Drug Administration in the USA as an ingredient in anti-microbial sprays for use on uncooked beef carcasses to eliminate pathogens and extend shelf life. Lactoferrin is used to inhibit lipid oxidation due its iron-binding capacity, as iron-promoted lipid oxidation is responsible for rancidity, and decreases the shelf life of commercial products, including infant formula and skincare cosmetics.

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03281a

All IG and EG calves showed signs of respiratory disease, varying from mild to severe (Table 2). In IG, the first clinical sign was observed three days after inoculation (D1), when one calf had sparse serous nasal discharge. Four days later, both inoculated animals showed mild respiratory signs with mild depression, normal to seromucous nasal discharge and a sporadic cough. Nine days after inoculation (D7), the total clinical score was at its highest for the IG calves, and the median of the total clinical score value was 4.0. On D1, the first clinical sign appeared in three of the six EG calves as they showed mild signs of respiratory disease, including a sporadic cough, mild depression and/or sparse serous to mildly opaque nasal discharge. Calves E5 and E6 started to show severe signs of respiratory disease on D10 and D15, respectively, which qualified for medical treatment with penicillin and anti-inflammatory drugs. The peak outbreak was on D11 with all EG animals showing sign of disease and the highest total clinical score with a median value of 2.5 (min 1, max 13). During the trial, five out of six of the EG calves had a temperature equal to or higher than 40.0 °C. The highest median rectal temperature of the calves in EG occurred on D12 and D15, with the median temperature being 39.6 °C both days (D12: min 38.6 °C, max 39.7 °C, D15: min 38.9 °C max 40.6 °C). The SG calves did not develop respiratory disease.

The association of the BRSV RT-ddPCR results versus scores of clinical parameters and total clinical score of the EG calves is shown in Fig. 2.

Baby hamster kidney fibroblast cells (BHK-21:ATCC) were grown to 80% confluence in high-glucose Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 1X Penicillin-Streptomycin. The BHK-21 cells were infected with 1 mL of Modified vaccinia Ankara (MVA) virus (kindly provided by Dr. Jingxin Cao, National Microbiology Laboratory) and incubated for 48 hours at 37 °C with 5% CO2. The infected cells underwent 3 freeze-thaw cycles in the presence of the growth media, alternating between −80 °C and room temperature. MVA was collected in the supernatant after removing cells and debris by centrifugation at 3000 × g for 3 min.

Authors declare no conflict of interest.

Advances in microfluidics for nanotechnology-based sensing methods have been met with serious challenges in the creation of diagnostic devices that allow for the simultaneous detection of several types of biotargets on a single platform for environmental monitoring. The need to rapidly detect and characterise micro-organisms in environmental samples is imperative in many different industries, among which food and agriculture, healthcare, environmental monitoring, and biodefense are key players [1–3]. The inability to cultivate the majority of naturally occurring micro-organisms despite the demonstrated need necessitates a fast, sensitive and reliable platform, such as a microfluidics-based lab-on-chip (LOC) system. In the field of environmental monitoring, serious attention is needed in the evaluation of microbial cells in water, soil and the environment. A list of some biological threat agents compiled by the Centers for Disease Control and Prevention (CDC) in the United States includes such notable agents as Bacillus anthracis (anthrax), Francisella tularensis (tularaemia), Yersinia pestis (plague), Variola major (smallpox), botulinum toxin (botulism), Coxiella burnetii (Q fever), Brucella spp. (brucellosis), Vibrio cholera (cholera), ricin, Shigella and Salmonella spp. These biological agents are transmitted via food, water, insect vectors, as aerosols or by direct contact (for extensive details, see [5–7]). The study of microorganism evolution and populations under conditions, such as during bio-waste composting, also requires highly sensitive devices. Microfabrication technology has led to the miniaturisation of biosensors in response to increased demand for their use in environmental and medical diagnostic applications for environmental monitoring.

Global Industry Analysts, Inc., have indicated that biosensors provide low-cost, compact, and low-power devices for environmental monitoring and point-of-care (POC) medical applications. Point-of-care testing (POCT), which is commonly described as bedside, near-patient, ancillary, and decentralised laboratory testing used for clinical diagnostics, is considered one of the main driving forces for the future in vitro diagnostic market. The demand for dissolved-oxygen (DO) biosensors will continue to grow with increasingly poor water quality and the desire to preserve natural resources to maintain the health of people and the environment. The October 2001 anthrax attacks in the United States, outbreaks of severe acute respiratory syndrome (SARS), bovine spongiform encephalopathy (BSE, commonly known as mad-cow disease), Iraq’s acknowledgement following the Gulf War that it possessed loaded biological weapons, and many other threats and biological “incidents” worldwide have increased global demand for the tools to rapidly identify causative agents and infected individuals before the agents spread beyond control. This need for detection necessitates the development of biodefense devices using a microfluidics approach to monitor and control food sources, water sources, and suspect powders, and to test for decontamination after the treatment of equipment, personnel, and key environments. The advent of microfluidic chips has enabled the application of biosensors in warfare threat detection and security. Microfabrication and newer manufacturing techniques will continue to increase the number of applications for current biosensors in environmental monitoring and health care. The use of inexpensive, transistor-based biosensors has recently transformed the medical research field.