Giulio Curone

Relationship between milk cathelicidin abundance and microbiologic culture in clinical mastitis

The availability of reliable tools to enable the sensitive and specific detection of mastitis in dairy cows can assist in developing control strategies and promote the more rational use of antibiotics. We have developed a milk cathelicidin ELISA that shows high sensitivity and specificity for dairy cow mastitis, based on latent class analysis. In this study, we investigated the effect of microbial agents on cathelicidin abundance in the milk of cows with clinical mastitis. We subjected 535 quarter milk samples (435 from quarters showing signs of clinical mastitis and 100 from healthy quarters as a control) to milk cathelicidin ELISA, somatic cell count (SCC), and microbiologic culture. Of the 435 clinical mastitis samples, 431 (99.08%) were positive for cathelicidin, 424 (97.47%) had SCC >200,000 cells/mL, and 376 (86.44%) were culture-positive. Of the 59 culture-negative samples, 58 (98.30%) were positive for cathelicidin and 55 (93.22%) had SCC >200,000 cells/mL. The abundance of cathelicidin and the extent of SCC increase depended on the causative agent: Streptococcus agalactiae and coagulase-negative staphylococci showed the highest and lowest changes, respectively. We also observed differences in behavior between the 2 markers depending on the pathogen: Streptococcus agalactiae induced the highest cathelicidin abundance, and Serratia spp. induced the highest SCC. Nevertheless, the different ability of microorganisms to induce cathelicidin release in milk did not compromise its value as a mastitis marker, given its higher sensitivity compared to SCC or microbiologic culture. All 100 negative control samples (collected from healthy quarters with SCC <100,000 cells/mL and culture-negative) were also negative for cathelicidin, corresponding to 100% specificity in the evaluated sample cohort. This study confirmed the value of the milk cathelicidin ELISA for detecting bovine mastitis, and highlighted the influence of mastitis-causing microorganisms on cathelicidin abundance. This influence did not compromise diagnostic performance; instead, it may have better reflected disease severity and evolution than SCC.

Milk ketone bodies assessment in a local italian cow breed (Modenese) vs. Holstein and characterization of its physiological, reproductive and productive performances.

Several autochthonous cattle breeds characterized by a small territorial diffusion are farmed in Northern Italy. The technical data show that these animals have a good reproductive performance (Communod et al 2010; Communod et al 2011), disease resistance and resilience. The objective of this study was to characterize some productive, reproductive and metabolic parameters (ketone bodies) in the Italian autochthonous cattle breed Modenese, comparing them with those of Holstein and their crossbred (F1=Modenese x Holstein; F2=Modenese x F1) breed in the same farm in order to understand if there is a different metabolic picture that can influence the reproductive performances. Milk samples have been collected at different times of lactation (20th, 40th, 90th day in milk) and analyzed by gas chromatography-mass spectrometry to obtain the ketone bodies concentration. The reproductive (Open Days Period and number of Services Per Pregnancy) and productive (percentage and kg of protein between the 40th and 90th DIM) data have been recovered by the consultation of the farm registers and the APA (Provincial breeder association) data. On days open, number of services per pregnancy, % of proteins in milk, and kg of proteins in milk; a Spearman correlation analysis was applied. In all time points, the Modenese breed showed a significant (p<0,05) lower ketone bodies concentration. The F1, F2 and Modenese showed also better reproductive performances when compared to Holstein, with 80-105 days of days open in average. In conclusion, the better resilience against the negative energy balance and his adverse effects of Modenese cattle could be one of the phenomena underlying their better reproductive efficiency.

Milk microbiome diversity and bacterial group prevalence in a comparison between healthy Holstein Friesian and Rendena cows

Dry and early lactation periods represent the most critical phases for udder health in cattle, especially in highly productive breeds, such as the Holstein Friesian (HF). On the other hand, some autochthonous cattle breeds, such as the Rendena (REN), have a lower prevalence of mastitis and other transition-related diseases. In this study, milk microbiota of 6 HF and 3 REN cows, all raised on the same farm under the same conditions, was compared. A special focus was placed on the transition period to define bacterial groups’ prevalence with a plausible effect on mammary gland health. Four time points (dry-off, 1 d, 7–10 d and 30 d after calving) were considered. Through 16S rRNA sequencing, we characterized the microbiota composition for 117 out of the 144 milk samples initially collected, keeping only the healthy quarters, in order to focus on physiological microbiome changes and avoid shifts due to suspected diseases. Microbial populations were very different in the two breeds along all the time points, with REN milk showing a significantly lower microbial biodiversity. The taxonomic profiles of both cosmopolitan and local breeds were dominated by Firmicutes, mostly represented by the Streptococcus genus, although in very different proportions (HF 27.5%, REN 68.6%). Large differences in HF and REN cows were, also, evident from the metabolic predictive analysis from microbiome data. Finally, only HF milk displayed significant changes in the microbial composition along the transition period, while REN maintained a more stable microbiota. In conclusion, in addition to the influence on the final characteristics of dairy products obtained from milk of the two breeds, differences in the milk microbiome might, also, have an impact on their mammary gland health.

Bioencapsulation of Oocytes and Granulosa Cells

A protocol for the encapsulation in sodium alginate of granulosa cells in primary culture and co-culture of oocyte-cumulus complexes is reported. Sodium alginate forms strong gels when jellified with barium ions, allowing the self-organization of cells into a 3D structure. This method of encapsulation is simple and cheap, allowing the culture of cells in a 3-dimensional fashion.

NMR-based metabolomics to evaluate the milk composition from Friesian and autochthonous cows of Northern Italy at different lactation times

Abstract It is well established that different factors affect milk composition in cows and that milk composition, in turn, affect both technological and nutritional qualities. In this respect the comprehension of the metabolic variability of milk composition in relation to the lactation time as well as to the genetic background may be of paramount importance for the agri-food industries. In the present study we investigated the variations of the metabolic profiles during lactation in milks obtained from Friesian and autochthonous races from Northern Italy by 1H NMR metabolomics. Furthermore, the external factors influencing the milk composition were minimized: the cows were breeded in the same farm, were fed with the same diet and were paired for the lactation interval and lactation stage. Our results showed a difference in milk composition between races and in relation to late lactation. The PLS-DA analysis permitted to distinguish the Friesian and autochthonous cow milks at the investigated different lactation times. Interestingly, the metabolites significantly involved into the discrimination between races appeared to be also technological property parameters, highlighting the importance of maintaining the biodiversity of cow breeds. Therefore, NMR-based metabolomics of milk could represent an informative tool to identify metabolites involved in milk quality both from a nutritional and industrial perspective.

Physiological response to chemical immobilization: a case study of etorphine-azaperone in free-ranging plains zebra (Equus quagga) in Kenya

Predictable immobilization of wild zebras is challenging and there is massive variation in opiate response within different species. Etorphine combined with azaperone is considered the protocol of choice, but no studies have investigated the physiological response to this procedure of immobilization in plains zebras. Eleven free-ranging plains zebras ( Equus quagga ) were immobilized in Kenya using a combination of etorphine 0.019 ± 0.003 mg/kg and azaperone 0.27 ± 0.05 mg/kg administered intramuscularly with a projectile dart. After recumbency, an arterial sample was performed for blood gas analysis and physiological parameters were recorded every five minutes. Descriptive scores were given to the exertion resulting from high-speed chasing and to the quality of induction, immobilization and recovery. Diprenorphine or naltrexone were used for opioid antagonism. In all zebras, the combination induced quick inductions within 3.5 ± 0.8 minutes and provided reliable recumbencies without attempts to stand for the entire duration of the immobilization. The average heart rates, respiratory rates and mean arterial blood pressure recorded were 102 ± 42 beats/minute, 18 ± 4 breaths/minute and 145 ± 28 mmHg respectively. Arterial gas analyses demonstrated mild to severe and partially compensated metabolic acidosis and hypoxia, while electrolytes were within equids range. In particular, higher exertion levels during the chasing were significantly correlated to worse immobilization scores (p=0.008) and hyperthermia occurrence (p=0.0012) and non-significantly to more severe acidosis. Recoveries from anaesthesia were smooth, on average 121 ± 38 seconds after diprenorphine/naltrexone administration. Etorphine-azaperone combination produced physiological alterations in free-ranging plains zebra such as tachycardia, hypertension, metabolic acidosis and hypoxemia. However, these preliminary results indicate that high-speed chase might be responsible for the physiological imbalance and that this drug combination does not suppress the compensatory response. Regardless of the metabolic status, recover from immobilization was uneventful and all zebras went back to normal behavior thereafter.

A comparison study of the inflammatory response in Holstein Friesian versus a local cattle breed (Rendena)

The selective pressure for increased milk production brought about great difficulties in the adaptation of cows to their environment. However, not much is known about the biological mechanisms behind the relationship between genetic selection and higher risk of metabolic and infectious diseases (Oltenacu, P.A., and Broom, D.M., 2010). It is well known that during the calving period, high-yielding dairy cattle are more susceptible to common environmental stressors, affecting disease occurrence and milk production levels (Bach, A., 2011). In this study we compared innate immune response of 6 Holstein Friesian (HF) and 4 Rendena (R) cows reared in the same farm and under the same management conditions. Milk and blood samples were collected at dry-off (T1), 1 day after calving (T2), 7-10 days after calving (T3), and 30 days after calving (T4). Milk samples were subjected to measurement of the inflammation marker cathelicidin and assessment of different innate immune-related mediators; blood samples were used for the analysis of plasma metabolites indicators of systemic inflammation. HF cows showed a more severe systemic inflammatory response at T2 and T3 in comparison with R cows (fig.1). Concerning the milk protein abundance profile, higher levels in R cows were observed in the colostrum (T2). Moreover, at all time points HF showed higher levels of the inflammation marker cathelicidin in milk (fig.2). In addition, the expression of innate immune related genes were different in HF compared with R (fig.3). Our results suggest that HF cows develop a systemic and local mammary inflammatory response that confirms their higher susceptibility to disease compared with R cows. Our findings reveal that fundamental effector activities of innate immunity in the mammary gland could be included in the breeding programs of HF cows and suggest the spread of autochthonous cow farming in order to maintain the biodiversity, reduce the antibiotic consumption and production of high quality dairy products.

Behavioural Profiles of Brown and Sloth Bears in Captivity

Simple Summary Animal personality research is a growing field, since understanding animal personalities has notable implications in ecology and the evolution of animal behaviours. In the current study, we tested different methods described in the literature to obtain robust individual behavioural profiles. Data collected through behavioral observations were categorised into activity budgets, space usage, and social interactions for each individual. In addition, behavioural profile questionnaires were completed by the three zoo keepers who had regular interactions with the bears. The questionnaires included 22 adjectives, which were rated on a scale of 1–12 depending on how well they described each individual bear. The mean ratings of the keepers were used to create the behavioural profiles by adding the adjectives to the appropriate domains, according to the NEO Five Factor Inventory of personality model (NEO-FFI). The data gathered was used to produce behavioural profiles for all animals, in order to clarify the personality characteristics of each subject. Testing and improving existing methodologies to determine animal personality is important for providing optimal welfare and management of captive animals, since it can help to develop more effective management regimes in zoos by remodelling husbandry according to each animal’s personality type. Abstract Three brown bear (Ursus arctos arctos) individuals and two sloth bear (Melursus ursinus inornatus) individuals were observed in captivity to produce behavioural profiles for each individual. Data collected through behavioural observations were used to produce activity budgets, and to identify space usage and certain aspects of social behavior. Behaviour monitoring allowed the researchers to evaluate the welfare of the animals by identifying the occurrence of stereotypic behaviours, which are sometimes associated with stress. Behavioural profiles were created using data obtained through behavioural observations (coding) and keeper questionnaires (rating). The behavioural observations indicated a number of stereotypic behaviours in sloth bears but not in brown bears. The uniformity of zone usage was calculated to investigate if the enclosure size and features were adequate for use, and a social aspect of otherwise solitary animals was also identified. The behavioural profiles generated through coding and rating were compared to determine the reliability between these two methods in Ursids. Profiles were not compared between individuals since this study is not a comparison between different personality types but rather an effort (one of the few ones existing in literature) to select a valid and reproducible methodology capable of assessing personality in bears.