Technical note: Evaluation of a commercial on-farm milk leukocyte differential tester to identify subclinical mastitis cases in dairy cows.

The objective of this study was to validate the precision and accuracy of a milk leukocyte differential tester to identify subclinical mastitis cases in dairy cows. Milk samples from individual quarters (n = 320) of 80 Holstein cows were aseptically collected and analyzed in this study. Each sample was divided into 2 replicate samples after mixing. One replicate was analyzed for somatic cell count (SCC) using the current gold standard of flow cytometry immediately after milking. The second sample was evaluated using the on-farm milk leukocyte differential tester directly after milking, where total leukocyte count (TLC; cells/mL) was obtained. The SCC and TLC were used to calculate somatic cell score (SCS) and TLC score [TLS = log2 (TLC/100,000) + 3]. Two subclinical mastitis thresholds were set: >200,000 (low) and >400,000 (high) cells/mL. First, precision was determined between the 2 methods. Total leukocyte count and calculated TLS from the milk leukocyte differential device were compared with the gold standard using correlation and regression coefficient of determination analyses. Correlation coefficients (r) were 0.97 for TLC and SCC and 0.90 for TLS and SCS. The coefficient of determination for regression (R2) was 0.94 for TLC and SCC and 0.80 for TLS and SCS. Slopes of regression for scores and measures were 0.36 [95% confidence interval (CI): 0.35-0.37] and 0.69 (CI: 0.65-0.73), respectively; both were significantly different from 1. Sensitivity, specificity, and diagnostic accuracy were calculated for correct diagnosis of the 2 SCC thresholds using the gold standard as reference. The sensitivity of the on-farm test was 58% (95% CI: 44 to 71%) and 73% (95% CI: 56 to 86%) for the low and high thresholds, respectively. The specificities for the on-farm test were 100% (95% CI: 99 to 100%) and 100% (95% CI: 98 to 100%) for the low and high thresholds, respectively. Subclinical diagnosis accuracies were 93% (95% CI: 89 to 95%) and 96% (95% CI: 92 to 98%) for the low and high thresholds, respectively. The on-farm milk leukocyte differential tester was precise but not overall accurate for total cell counts; it had high specificity and accuracy for diagnosis compared with a standard diagnostic tool. These results suggest that the tested system is a promising technology to detect subclinical mastitis on-farm.

Comparing dairy farm milk yield and components, somatic cell score, and reproductive performance among United States regions using summer to winter ratios.

Heat stress abatement is a challenge for dairy producers in the United States, especially in the southern states. Thus, managing heat stress is critical to maintain dairy cow performance in the summer. The ability to employ a metric to measure heat stress and evaluate abatement strategies may benefit dairy producers by providing meaningful feedback on the effectiveness of current and future management strategies with the goal of improving heat stress management. Therefore, this study aimed to explore the use of the summer to winter performance ratio metric to quantify and compare farm performance variables among regions of the United States. Monthly performance data recorded by the Dairy Herd Improvement Association from 2007 to 2016, for all US Dairy Herd Improvement Association herds processing records through Dairy Records Management Systems (Raleigh, NC), were obtained. Season dates were based on the astronomical definition of the Northern Hemisphere with summer as June 21 to September 21 and winter as December 21 to March 19. States were grouped into regions based on climate zone classification. Performance records included a total of 16,573 herds [Northeast (n = 7,955), Midwest (n = 6,555), Northern Plains (n = 305), Southeast (n = 1,370), and Southern Plains (n = 388) regions]. Herd test day performance variables energy-corrected milk, somatic cell score, milk fat and protein percentage, conception rate, heat detection rate, and pregnancy rate in summer and winter were used to calculate summer to winter ratios for each region. The MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC) was used to compare test day performance variables. The effects of year, mean days in milk, mean 150-d milk, mean herd size, and number of milkings per day were included as covariates in the models. Dairy cattle performance in all climate regions was negatively affected by summer heat stress, but some regions greater than others. A difference was also observed among regions when comparing summer to winter ratios for each performance parameter. This indicates that summer performance varies by climate region identified by the summer to winter ratio and demonstrates usefulness of the metric to monitor degree of heat stress based on performance.

A short-term in vitro gill culture system to study the effects of toxic (copper) and non-toxic (cortisol) stressors on the rainbow trout, Oncorhynchus mykiss (Walbaum).

A short-term (24 h) method of gill filament culture system was developed to predict the effects of environmental contamination and stress in fish. Gill culture system containing two or three rainbow trout gill filaments in sterile glutamine supplemented Leibovitz 15 (L-15) media was submitted for 24 h to six different treatments: (i) CONT (control, medium only); (ii) CORT (cortisol, 0.28 microM cortisol); (iii) BLOCK (glucocorticoid receptor blocker, 14 microM RU 486); (iv) CORT+BLOCK (cortisol and blocker, 0.28 microM cortisol+14 microM RU 486); (v) CORT+CU (cortisol and copper, 100 microM CuSO4+0.28 microM cortisol); (vi) CU (copper, 100 microM CuSO4). After 24 h, the overall gill structure and cellular components resembled those of salmonids in vivo. Lactate dehydrogenase (LDH) activity in the culture media increased in the CORT+CU and CU groups but was significantly lower in the CORT+CU compared to CU group. Apoptotic cells increased in the CORT and CORT+BLOCK. The numbers of glucocorticoid (GR) receptor-positive cells were lower in the CU group. This short-term culture system seems to be suitable for studying the effects of both external and internal stress effectors (toxicants and hormones respectively), as it contains all cell types found in the gills and the cells give similar biological response as in vivo.

Exposure to water from the lower Rhine induces a stress response in the rainbow trout Oncorhynchus mykiss.

The water quality of the river Rhine has improved in recent years and populations of salmonids are increasing. Nevertheless at present, the water from the lower Rhine still contains a complex mixture of low levels of many pollutants and it is not known whether exposure to such water is stressful to salmonid fish. For 31 days we continuously exposed the trout Oncorhynchus mykiss to water from the lower Rhine in the Netherlands and measured a variety of physiological, biochemical, and histological parameters, including the stress parameters cortisol and glucose. Exposure to Rhine water significantly increased cortisol and glucose after 3 h. At 21 and 31 days, cortisol was lower in exposed fish, indicating inhibition or exhaustion of the hypothalamic-pituitary-interrenal (HPI) axis. Electron microscopical analysis of the skin and gill epithelia revealed stressor-related effects that reflected disruption of the skin epithelium, the interface between the fish and the environment. This had little influence on hydromineral balance, as neither gill Na+/K+-ATPase activity nor plasma Na+ and Cl- were altered, although intestine- and kidney-specific Na+/K+-ATPase activities were affected. Analysis of heavy metal concentrations in the liver, kidney, and intestine indicated no bioaccumulation. Immunostimulation was reflected by increased respiratory burst activity of the head kidney leukocytes. From 7 days onwards, the body weight of the Rhine water fish was significantly lower than that of control fish. Overall, the data show that acute exposure to present day water from the lower Rhine induced a stress response in the fish that, during chronic exposure, was followed by impairment of the HPI axis, reduced growth, and prolonged immunostimulation.

Cortisol influences the host-parasite interaction between the rainbow trout (Oncorhynchus mykiss) and the crustacean ectoparasite Argulus japonicus.

The host-parasite interaction between the rainbow trout Oncorhynchus mykiss and the fish louse Argulus japonicus was investigated by administering low levels of dietary cortisol before infecting the fish with low numbers of the parasite. After 24 h, the dietary cortisol treatment elevated blood cortisol and glucose levels and stimulated the synthesis of secretory granules in the upper layer of skin cells. Infection with 6 lice per fish caused skin infiltration by lymphocytes, also in areas without parasites. The lymphocyte numbers in the blood at 48 h post-parasite infection were reduced. Other changes, typical for exposure to many stressors and mediated by cortisol, were also found in the epidermis of parasitized fish, although neither plasma cortisol nor glucose levels were noticeably affected. Glucocorticoid receptors were localized immunohistochemically and found in the upper epidermal layer of pavement and filament cells, and in the leucocytes migrating in these layers. Cortisol-fed fish had reduced numbers of parasites and the changes in the host skin are likely involved in this reduction. Thus a mild cortisol stress response might be adaptive in rejecting these parasites. Further, the data suggest that this effect of cortisol is mediated by the glucocorticoid receptor in the skin epidermis, as these are located directly at the site of parasite attachment and feeding in the upper skin cells that produce more secretory granules in response to cortisol feeding.

A review of the population biology and host-parasite interactions of the sea louse Lepeophtheirus salmonis (Copepoda: Caligidae).

Lepeophtheirus salmonis is a specific parasite of salmonids that occurs in the Atlantic and Pacific Oceans. When infestations are heavy fish mortality can occur although the factors that are responsible for causing epizootics, especially in wild salmonid populations are still largely unknown. Over the past 20 years this parasite has caused significant economic losses in farmed salmon production and possibly in wild salmonid populations locally. Understanding the connectivity between populations is crucial to an understanding of the epidemiology of infections and for management of infections in aquaculture. Data from genetics, pesticide resistance, larval dispersal models and spatial and temporal patterns of infestation in wild and farmed hosts suggests a spatially highly structured metapopulation the components of which have different levels of connectivity, probabilities of extinction and influence on the development of local infestations. The population structure is defined mainly by the dispersal dynamics of the planktonic stages and the behaviour of the host. Until recently virtually nothing was known about the relationship between the parasite and the host, or how the host may influence lice at local or population level. Typically, impacts on the host have usually been reported in terms of pathological lesions caused by attachment and feeding of the adult stages, as well as localised mild epithelial responses to juvenile attachment. However many studies report pathology associated with severe infestation. Recent new studies on the host-parasite interactions of L. salmonis have shown that this parasite induces stress-related responses systemically in the host skin and gills and that the stress response and immune systems are modulated. In the second part of this review, these new studies are presented, together with results from other host-parasite model systems where data for caligid sea lice are missing. One of the most revealing methods reported recently is the application of a net confinement stressor to examine modulation of the stress response and immune system of the host fish. This approach has shown that although until now, infective stages of L. salmonis were not thought to affect the host, they do induce systematic effects in the host that result in a stress response and modulated immune system. Host-parasite interactions affecting these stress responses and the immune system may be key factors in facilitating epizootics by reducing the host's ability to reject the parasites, as well as reducing disease resistance under some environmental conditions. The host-parasite interaction therefore needs to be incorporated into any model of population structure and dynamics.

Characterization of primary culture of rainbow trout (Oncorhynchus mykiss) skin explants: growth, cell composition, proliferation, and apoptosis.

A trout (Oncorhynchus mykiss) epidermal skin primary explant system was evaluated over 8 d by light and electron microscopy. Three distinct regions of the explant outgrowth were identified on the basis of cell composition. The area immediately adjacent to the founder tissue contained mainly small migrating cells and mucous cells. Of the former. about 20% were mitotic and 6% apoptotic. The middle area was characterized by differentiated pavement cells and mucous cells, with fewer small migrating cells. Proliferation was approximately 30% and apoptosis 5%. Over time, total cell numbers halved as more pavement cells differentiated. The growing front contained many mucous and small migrating cells initially, with few pavement cells. About 50% of the cells were in the proliferative phase, and 5% were apoptotic. Later, there were fewer migrating and mucous cells, with a higher number of pavement cells. About 9% of the cells were apoptotic, and 70% of the cells were proliferating. As in vivo, pavement cells had apical microridges, although they were vacuolated and contained phagocytosed apoptotic bodies. The data and observations are based on the numbers of cell cultures prepared from separate trout giving the sample size n = 7. As this culture system is reproducible and closely approximates the epidermis of trout, it is a powerful tool to study the effects of pollutants, parasites, and endocrine factors on fish skin, eliminating whole-animal factors and reducing the number of experimental animals required.

Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Krøyer) influences the physiological response to an acute stressor.

The influence of infection with the juvenile stages of the sea louse, Lepeophtheirus salmonis (Krøyer) on the response of rainbow trout Oncorhynchus mykiss (Walbaum) to a net confinement protocol was investigated. The experiment consisted of two groups of seawater-adapted rainbow trout, one which was exposed to a total of 4000 nauplii/copepodid stages of L. salmonis 30, 25 and 14 days prior to confinement. Confinement elicited a greater stress response in the lice-exposed fish, than in the controls, as seen by higher plasma cortisol and glucose levels. A reduced spleen somatic index in exposed fish following 6 h confinement coincided with increased erythrocyte and lymphocyte numbers in the blood. Circulating lymphocyte numbers were significantly reduced in both groups 24 h post-confinement, when a lower alternative complement activity was recorded in control fish. Prior to confinement, lice-exposed fish had an elevated serum lysozyme activity and reduced oxygen radical production by blood leukocytes. Following confinement, lysozyme activity was gradually reduced in lice-exposed trout. During confinement, oxygen radical production decreased in control fish and increased in infested fish. Overall, transient exposure to juvenile lice altered the response to a second stressor, which has implications for management procedures of L. salmonis exposed fish.