Camila Costa Baccili

A robust segmentation method for counting bovine milk somatic cells in microscope slide images

A two-step methodology for background removal of bovine milk images is proposed.A well-tuned k-means algorithm removes all sort of debris from background.A new thresholding removes the debris-free and statistically varying background.The background-removed cells under Watershed transform are robust-segmented.High accuracy somatic cell counting algorithm for bovine milk quality control. Mastitis is an infectious disease associated with the increased number of somatic cells in cows milk, and it is one of the most relevant cause of economic losses in dairy farming industries. In this paper, we propose a method capable of determine, with 99.7% accuracy, the number of these cells in microscope slide images. This level of accuracy is achieved by changing the image original RGB format to Lab color space and applying k-means clustering algorithm to remove debris and other background features. A new gray level thresholding is proposed, and the remaining bound cells are separated in the final segmentation step applying Watershed transform. Many microscope slide images with debris, contrast, and hue variation were used to validate the experimental results. Comparison between the proposed method and manual counting indicates that this new approach is a robust and promising solution to be incorporated in a future automated somatic cell counting system using optical microscopy.

Effect of maternal cells transferred with colostrum on the health of neonate calves

Abstract The objective of this research was to evaluate the influence of cells from colostrum on the health of neonate calves. Animals were distributed in 2 groups: COL+ (n =9) which received fresh colostrum from their own damns; and COL− (n =10) which received frozen colostrums from donors. Heifers were assessed before colostrum intake – D0; D2; D7; D14; D21 and D28. Heifers were monitored by clinical examination, hematological profile and serum iron. COL− had a higher diarrhea intensity score (typically 3) on D7. Moreover, a single case each of bronchopneumonia and navel inflammation were observed in COL− calves. COL− had fewer red blood cells (RBC) (6.5±0.8×106/μL) and less hemoglobin (Hgb) (8.3±1.4g/dL) than COL+ (RBC=7.2±0.8×106/μL; Hgb=9.6±1.3g/dL) at D14 (P ≤0.05). COL− had more anemia on D21 (P =0.03) and on D28 (P =0.02). Iron was lower in COL− (5.6±2.7μM/L) than COL+ (10.7±6.2μM/L) (P =0.03) on D7. Lymphocytes was lower in COL− than COL+ on D7 (3.8±1.0×103/μL COL+ and 5.4±2.2×103/μL COL−, P =0.02). COL− calves had more anemia and lower serum iron concomitant with diarrhea on D7. The number of leukocytes was relatively consistent in the COL+ calves, while COL− calves showed an increasing number of of lymphocytes starting on D7.

Innate immune response in neonate Holstein heifer calves fed fresh or frozen colostrum

Abstract The aim of this research was to evaluate the influence of maternal cells from colostrum on the development and function of the innate immune response in Holstein calves. Calves were divided into 2 groups: COL+ (n =10) received fresh colostrum; and COL− (n =10) which received frozen colostrum containing no viable cells. The calves were assessed before colostrum intake (D0), 48h of age (D2), and weekly from D7 up to D28. Blood samples were collected for analysis of the distribution of leukocytes, cellular phenotype and in vitro granulocyte function. COL+ calves tended to have a high number of neutrophils on D7 (p =0.073). COL− calves took up significantly more Escherichia coli (measured as MFI) on D7 (p =0.034). Endogenous production of radicals (as percentage of cells) tended to be higher in COL− calves on D14 (p =0.061). The intensity of endogenous reactive oxygen species (ROS) produced by granulocytes tended to be higher in COL+ calves on D21 (p =0.094). Overall, ROS production (percent of cells, and MFI) induced by Staphylococcus aureus and Escherichia coli were higher in COL+ calves than COL− calves. It was our observation that COL+ calves developed an innate immune response more quickly and efficiently after natural exposure to pathogens after birth. In contrast, COL− calves mounted an innate response more slowly that yielded a persistent inflammatory response after natural exposure to these bacteria agents. This research provides evidence of an advantage to the calf of receiving fresh colostrum on the development and function of the innate immune system.

Contagem de células somáticas como indicador de infecção mamária em vacas periparturientes

The somatic cell count (SCC) is a screening test for the evaluation of intramammary infection; however, changes in mammary secretion during colostrogenesis can promote a physiological increase in the SCC, potentially reducing its reliability in the diagnosis of mastitis. Therefore, the aim of this study was to evaluate bovine colostrum SCC as an indicative parameter of breast infection in Holstein periparturient cows. A total of 80 samples were harvested from the first milking colostrum of 20 cows and were subjected to manual SCC and bacteriological examination. Bacterial growth was present in 36.62% of the crops; coagulase-negative Staphylococcus (CNS) was the predominant microorganism (76.92%). The median SCC in infected cows (1.8 × 106 cells/mL) was significantly higher than in uninfected cows (0.9 × 106 cells/mL) (p = 0.0451). The sensitivity (100– 15%), specificity (100–2.2%), and false positive (100–2.2%) of the SCC decreased gradually when thresholds of 0.2–10.0 × 106 cells/mL were adopted. In contrast, the proportion of false negatives (0–84.6%) revealed an opposite trend. The threshold of greatest concordance between SCC and bacteriological examination was 10.0 × 106 cells/mL; however, the sensitivity rates (15.4%), specificity (2.2%), and false positive (2.2%) were very low. Based on these results, we conclude that SCCs increased prior to the infectious processes of the mammary gland, particularly in the CNS group. However, physiological changes caused by colostrogenesis resulted in poor concordance between the SCC and bacteriological examination of the colostrum.

Interface between Maternal Antibodies and Natural Challenge for Bovine Viral Diarrhea Virus (BVDV) in Holstein Heifers

Background: Newborn calves are agammaglobulinemic, immunosuppressed and immunologically immature at birth. The passive immune transfer is fundamental to protect calves against pathogens. The decay of maternal antibodies precedes the immune maturation at puberty enhancing the susceptibility of calves to infections caused by BVDV. Then, the objective of this research was to evaluate the interface between passive and active immunity for Bovine Viral Diarrhea Virus (BVDV) in Holstein dairy heifers in the first 13 months of age to detect susceptibility periods and establish prophylactic measures on prevention of Bovine Viral Diarrhea. Materials, Methods & Results: Sera were analyzed from 585 heifers by serum neutralization (SN) and enzyme linked immunosorbent assay (ELISA) for the p80 protein of BVDV. Heifers were categorized according to their age by the month of life. Heifers were seropositive (100%) from 1st to 13th. Median of neutralizing antibodies (Ab) titers obtained from 1st up to 13th month were 316.2; 125.9; 63.1; 50.1; 50.1; 39.8; 63.1; 63.1; 39.8; 79.4, 100.0; 74.4; and 79.4, respectively. The neutralizing Ab titers obtained in 1st month were different of the values observed from 2nd until 13th (P < 0.001), furthermore the Ab titers from 2nd month was statistical different of 4th (P = 0.01) and 6th (P = 0.05). The frequencies (%) of positive heifers for p80 from 1st up to 13th were 24.7; 18.2; 10.4; 11.8; 73.3; 73.8; 72.4; 58.1; 45.9; 48.4; 46.2; 43.8 and 61.5, respectively. The correlation observed for neutralizing Ab titers and age was negative and weak (ρ= -0.299; P < 0.001). On the other hand, the correlation between positive heifers for p80 and age was positive and moderate (ρ= 0.319; P < 0.001). Discussion: The newborn calves had higher titers of neutralizing antibodies than other age groups and some calves were seropositive for the p80 protein. This profile points to the transfer of maternal antibodies produced by vaccination and/or natural exposure to BVDV. The exposure of the cows to the inactivated and live virus stimulates the production of neutralizing antibodies to the structural proteins of the virus, particularly the glycoprotein E2, detected by the serum neutralization test. The titers of serum neutralizing antibodies and the frequencies of seropositive for p80 protein decreased gradually from the first to the 4-6th month of life due to the metabolization of maternal immunoglobulins acquired by ingestion of colostrum. Frequencies of seropositive animals for protein p80 increased from the fifth month of life, which is the same moment that was observed declined of neutralizing maternal antibody titers. The phase of higher frequency of p80 positive animals coincides with greater rates of Babesiosis and Anaplasmosis. This history could justify the importance of the BVDV immunodepression as a risk factor for concomitant diseases. In general, the neutralizing antibody titers increased after the peaks of positive reactions to the p80 protein, but this moment coincides with the primo-vaccination in calves. Therefore, it is not possible to state the origin of these antibodies. Correlations between ages and serologic tests are consistent with previous data reporting the decrease in antibody titers and increase of seropositive animals for p80 protein, from the first month of life to puberty. In conclusion, maternal neutralizing Ab titers had gradual decreased whereas the frequency of positive heifers for p80 had increased values. The inversion observed between the maternal antibody titers and the increase in antibody for p80 indicates the moment of greatest risk for natural infections caused by BVDV.