BackgroundMycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a contagious infectious disease that affects domestic and wild ruminants causing chronic inflammation of the intestine. MAP has proven to be very resistant to both physical and chemical processes, making it difficult to control this pathogen. Based on the recognized antimicrobial properties of copper, the objective of this study was to evaluate the effectiveness of copper ions to reduce MAP numbers and/or MAP viability in a fluid matrix. Besides, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were used as controls of the effectiveness of copper ions. MAP-spiked PBS was subjected to copper ions treatment at 24 V for 5 min and the PBS suspensions were sampled before and after treatment. MAP viability and quantification were determined using three complementary techniques: a phage amplification assay, MGIT culture and qPCR.ResultsModerate numbers (103 CFU ml−1) of the two control bacteria were completely eliminated by treatment with copper ions. For MAP, copper ions treatment reduced both the viability and numbers of this pathogen. Phage assay information quickly showed that copper ions (24 V for 5 min) resulted in a significant reduction in viable MAP. MGIT culture results over time showed statistically significant differences in time-to-detection (TTD) values between PRE and POST treatment. MAP genome equivalent estimates for PBS suspensions indicated that MAP numbers were lower in samples POST-treatment with copper ions than PRE-treatment.ConclusionsThe use of copper ions resulted in a significant reduction of MAP in a liquid matrix, although some MAP survival on some occasions was observed.

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP+), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility.

Clinical manifestations of leptospirosis are diverse and very similar to other febrile diseases, hence early and accurate detection of subclinical infections is a key element in disease control. We evaluated immunomagnetic separation (IMS) capture technology coupled with a standard quantitative PCR (qPCR) system for the detection of pathogenic Leptospira in urine samples from 803 cows from dairy herds with a history of clinical cases of leptospirosis. The urine samples were first processed in a purification step, then subdivided into 2 subsamples, one that continued to DNA extraction and direct qPCR, and one that was pretreated by IMS before continuing to DNA extraction and qPCR. Overall, 133 of 803 (16.6%) samples were IMS-qPCR positive, whereas only 92 of 803 (11.5%) were positive when using direct qPCR. Statistically significant differences were observed between the mean estimated Leptospira load between the IMS-qPCR and the direct qPCR positive urine samples. The IMS-qPCR technology revealed a larger number of positive results and higher bacterial loads than direct qPCR. This difference is most likely the result of the high antigen-binding capacity and capture efficiency of the IMS system. The use of polyclonal antibodies produced by the inoculation of 3 synthetic peptides, which make up the extracellular regions of the LipL32 protein, provided a high detection capacity to the IMS-qPCR technique, resulting in performance superior to direct qPCR.

Lameness in dairy cows is an extremely painful multifactorial condition that affects the welfare of animals and economically impacts the dairy industry worldwide. The aim of this study was to determine the profile of cytokines in the spinal cord dorsal horn of dairy cows with painful chronic inflammatory lameness. Concentrations of 10 cytokines were measured in the spinal cord of seven adult dairy cows with chronic lameness and seven adult dairy cows with no lameness. In all cows lameness was evaluated using a mobility scoring system and registered accordingly. Immediately after euthanasia the spinal cord was removed and 20 cm of lumbar segments (L2-L5) were obtained. After dorsal horn removal and processing, cytokine quantification of tumor necrosis factor-alpha (TNF-α), interleukin-1alpha (IL-1α), interleukin 13 (IL-13), chemokine-10 (CXCL10/IP-10), chemokine-9 (CXCL9/MIG), interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin-21 (IL-21), interleukin-36ra (IL-36ra), and macrophage inflammatory protein-1 beta (MIP-1β) was performed using a multiplex array. Lame cows had higher concentrations of TNF-α, IL-1-α, IL-13, CXCL10, CXCL9, IFN-α, and IFN-γ in their dorsal horn compared to non-lame cows, while IL-21 concentration was decreased. No differences in IL-36ra and MIP-1β concentrations between lame and non-lame cows were observed. Painful chronic inflammation of the hoof in dairy cows leads to a marked increase in cytokine concentration in the dorsal horn of the spinal cord, which could represent a state of neuroinflammation of the Central Nervous System (CNS).

Today, it is theoretically assumed that frozen storage of semen doses in liquid nitrogen guarantees sperm functionality indefinitely. However, there are few studies that objectively evaluate the effects of long-term storage on sperm quality parameters. In this study, we show a freezability analysis of bull semen stored for 1, 10, 25, 40 and 45 years at −196°C. Sperm viability and full sperm motility were analyzed by CASA system, and acrosome integrity was assessed with Coomassie blue staining. Our results showed that sperm viability and total sperm motility were not affected by long-term cryopreservation at −196°C. Specifically, we did not find any significant differences (p > 0.05) associated between different long-time storing analyzed; both parameters showed optimal values of sperm viability and total sperm motility (both over 60%). Additionally, the acrosomal integrity parameter was not affected, showing an optimal range (87±1.6-95±0.5%). We conclude that the sperm quality of bovine semen is not affected by longterm storage at −196°C. However, future field trials will be necessary in order to validate that both fertility and embryo viability are maintained for the times analyzed.

A spermiogramme is an analysis performed to assess the quality of semen. Motility parameters are primarily obtained by subjectively observing samples or by using complex systems, such as computer-assisted sperm analysis (CASA). Here, we describe an easy and low-cost analysis system for obtaining quantifiable kinetic observations using Salmo salar semen model. In this work, we observed and captured video images of both fresh and stored Atlantic salmon semen to describe the possibility of analysis using the ImageJ CASA plug-in application for the kinetic parameters obtained from the videos. The semen is exposed to "powermilt", a commercial activating solution, and the curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP) values are described. When the samples were activated after having been stored, differences were detected in sperm quality, using the low-cost plug-in application. However, this system was not able to detect small variations in the same recorded sample, suggesting limits in sample observation. The results indicate that it is possible to quantify the kinetic parameters of semen samples using a low-cost video system and free software.

Aim The focus of the present study was to evaluate the copper ions treatment on the viability of Mycobacterium avium subsp. paratuberculosis (MAP) and other bacterial communities in cow’s milk. Methods and Results A copper ions treatment was evaluated in naturally contaminated cow’s milk to assay MAP load and/or viability, and relative abundance of other bacterial communities. In addition, physical–chemical analyses of the milk were also performed. All analyses were carried out before and after a copper ions treatment. After copper ions treatment, pH and copper concentration markedly increased in milk; the numbers of viable MAP significantly decreased. The relative abundance of the four target phyla decreased, with the phyla Bacteroidetes and Firmicutes surviving treatment in higher proportions (4 and 2·1% of original populations, respectively). A progressively higher percentage of dead bacterial cells after 5 and 20 min copper ions treatments was found (12 and 35%, respectively). Conclusion With the exception of some MAP‐tolerant strains, we have once again demonstrated that copper ions have a significant inactivating effect on MAP as well as certain other bacterial communities found in naturally contaminated cow’s milk. Significance and Impact of the Study This study showed a significant inactivation of both MAP and other bacteria by copper ions in raw cow’s milk, information that could be useful as a tool for MAP control.

Simple SummaryChronic inflammatory diseases could impact central nervous system homeostasis, being oxidative damage of the dorsal horn, a relevant mechanism mediating central sensitization. Chronic inflammatory lameness in dairy cows is a painful condition that affects animal welfare, affecting dairy production worldwide. This study reveals increased levels of reactive oxygen species, malondialdehyde, and carbonyl groups, indicating lipid and protein damage in the spinal cord of cows with chronic lameness. Moreover, antioxidant system activity was similar between lame and non-lame cows which suggests that antioxidant dysregulation was not the cause of oxidative damage, as has been proposed previously. Based on the fact that nociceptive pathways are strongly conserved between species, there is no reason to neglect that chronic pain in cows promotes Central Nervous System (CNS) alterations, such as oxidative damage. Moreover, lame cows develop central sensitization, as allodynia and hyperalgesia are centrally and not peripherally mediated. Our results support the current assumption that chronic pain is a central nervous system disease and lameness in dairy cows is far beyond an inflammation of the hoof.AbstractLameness in dairy cows is a worldwide prevalent disease with a negative impact on animal welfare and herd economy. Oxidative damage and antioxidant system dysfunction are common features of many CNS diseases, including chronic pain. The aim of this study was to evaluate the levels of reactive oxygen species (ROS) and oxidative damage markers in the spinal cord of dairy cows with chronic inflammatory lameness. Locomotion score was performed in order to select cows with chronic lameness. Dorsal horn spinal cord samples were obtained post mortem from lumbar segments (L2–L5), and ROS, malondialdehyde (MDA), and carbonyl groups were measured along with the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant response (TAR). Lame cows had increased levels of ROS, MDA, and carbonyl groups, while no differences were observed between lame and non-lame cows in SOD, GPx, CAT, and TAR activity. We conclude that painful chronic inflammatory lameness in dairy cows is associated with an increase in ROS, MDA, and carbonyl groups. Nonetheless, an association between ROS generation and dysfunction of the antioxidant system, as previously proposed, could not be established.