Funmilola C. Thomas

Early post parturient changes in milk acute phase proteins.

The periparturient period is one of the most critical periods in the productive life of a dairy cow, and is the period when dairy cows are most susceptible to developing new intramammary infections (IMI) leading to mastitis. Acute phase proteins (APP) such as haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) have been detected in milk during mastitis but their presence in colostrum and milk in the immediate postpartum period has had limited investigation. The hypothesis was tested that APP are a constituent of colostrum and milk during this period. Enzyme linked immunosorbent assays (ELISAs) were used to determine each APPs concentration in colostrum and milk collected daily from the first to tenth day following calving in 22 Holstein-Friesian dairy cows. Haptoglobin was assessed in individual quarters and composite milk samples while M-SAA3 and CRP concentration were determined in composite milk samples. Change in Hp in relation to the high abundance proteins during the transition from colostrum to milk were evaluated by 1 and 2 dimension electrophoresis and western blot. In 80% of the cows all APPs were detected in colostrum on the first day following parturition at moderately high levels but gradually decreased to minimal values in the milk by the 6th day after calving. The remaining cows (20%) showed different patterns in the daily milk APP concentrations and when an elevated level is detected could reflect the presence of IMI. Demonstration that APP are present in colostrum and milk following parturition but fall to low levels within 4 days means that elevated APP after this time could be biomarkers of post parturient mastitis allowing early intervention to reduce disease on dairy farms.

Omic Approaches to a Better Understanding of Mastitis in Dairy Cows

Mastitis, which is caused by infection of the mammary gland is the most important disease problem facing dairy farmers. While the disease has been studied for decades in order to determine better diagnosis and treatment, it is only recently that the full panoply of advanced biotechnological methodologies has been applied that are needed to bring a systems biology approach to investigations. Molecular investigations using analyte-specific immunoassays, such as for the acute phase proteins haptoglobin and mammary-associated serum amyloid A3 in milk have introduced possibilities for monitoring the host inflammatory response. The omics revolution in biology, with genomics being harnessed especially for identification of the causative pathogens of mastitis, has enhanced dissection of the mammary microbiome. The application of proteomics, peptidomics and metabolomics to the diagnosis and pathophysiology of mastitis, in contrast, is in its infancy though the potential of these advanced tools of biological research is clear as they are applied in a systems biology analysis of this major health problem of dairy cows.

Blood serum acute phase proteins and iron dynamics during acute phase response of Salmonella enterica serotype Dublin experimentally infected buffalo calves

The study aimed to evaluate clinical signs, blood serum acute phase proteins (APP) and iron dynamics during the acute phase response (APR) of Salmonella Dublin experimentally infected Murrah buffalo calves. Six buffalo calves constituted the control group (CNT) and six were orally inoculate with 108 CFU of S. Dublin (INF). Clinical evaluation was performed, rectal swabs to detect S. Dublin strains were collected and venous blood was sampled before and throughout seven days after inoculation. The APP fractions β-haptoglobin, α-haptoglobin, ceruloplasmin and transferrin were analyzed by 1-D and 2-D electrophoresis. Proteins were identified using LC/ESI-MS/MS and NCBI database. Plasma fibrinogen, serum iron and serum haptoglobin concentrations were measured. The inoculation of 108 CFU of S. Dublin was effective in inducing clinical signs of Salmonellosis, such as hyperthermia and diarrhea. 1-DE showed that β and α-haptoglobin increased 204% (p = 0.008) and 184% (p = 0.022) 48 h after inoculation (HAI), respectively, with highest concentrations 120 HAI (498% increased, p = 0.012; 431% increased, p = 0.011) and 168 HAI (492% increased, p = 0.019; 523% increased, p = 0.028). 2-DE showed that the expression of two spots, identified as β-haptoglobin, were increased 693% (p = 0.0006) and 580% (p = 0.0003) 168 HAI, respectively, while one spot, identified as α-haptoglobin, increased 714% (p = 0.040). Haptoglobin concentrations increased 1339% (p < 0.0001) 168 HAI. 1-DE showed that ceruloplasmin increased 42% (p = 0.034) 48 HAI, with highest concentration 120 HAI (133% increased, p = 0.022). 2-DE showed that the expression of two spots, identified as ceruloplasmin, were increased 218% (p = 0.0153) and 85% (p = 0.0143) 168 HAI, respectively. Fibrinogen increased 78% (p = 0.012) 96 HAI, with highest concentration 120 HAI (increased 114%, p = 0.002). Iron decreased 33% 24 HAI (p = 0.015) and 37% 72 HAI (p = 0.029), and began to be restored 96 HAI. 1-DE showed that transferrin decreased 23% 120 HAI (p = 0.047), and that values were restored 168 HAI. 2-DE showed that expression patterns of transferrin comparing 0 h and 168 HAI were similar, evidencing that values were restored 168 HAI. In conclusion, the inoculation of 108 CFU was effective in inducing hyperthermia and diahrrea. β and α-haptoglobin, ceruloplasmin and fibrinogen worked as positive APP during the APR to S. Dublin infection and are potential biomarker candidates. Concentrations of iron and transferrin decreased during the infection, highlighting the fact that mechanisms for restricting iron availability are part of the APR triggered against S. Dublin infection in buffalo calves.

A pilot study of acute phase proteins as indicators of bovine mastitis caused by different pathogens

This study analysed three acute phase proteins in milk from natural cases of bovine mastitis and compared their profiles across different pathogens causing the infection. Their ability to differentiate subclinical and clinical mastitis from normal (uninfected) milk samples was also examined. Samples from various dairy farms across Scotland submitted to the Veterinary Diagnostic Services unit of the University of Glasgow were used for this study. They were subjected to microbiological examination for mastitis pathogens, evaluation of somatic cell counts and analyses by ELISAs for haptoglobin, C-reactive protein and mammary associated serum amyloid A3. Each acute phase protein (APP) was compared across pathogens and form of mastitis. Significant differences (P = 0.000) were observed for each APP between causative pathogen and form of mastitis. There were significant correlations between the pathogen and the form of mastitis and the 3 APP showed similar profile for the different pathogen type and forms of mastitis. It can be concluded that the aetiological pathogen of mastitis to a large extent influences the clinical form of the disease, this, ultimately being reflected in the degree and course of secretions of the acute phase proteins; Hp, M-SAA3 and CRP into milk during mastitis. Variations of which, show correspondent patterns with related pathogen/form-of-mastitis.

Reference 1D and 2D electrophoresis maps for potential disease related proteins in milk whey from lactating buffaloes and blood serum from buffalo calves (Water buffalo, Bubalus bubalis)

The aim of this study was to identify potential disease related proteins in milk whey of lactating buffaloes and blood serum of buffalo calves, in order to define a reference electrophoresis map for 1-DE and 2-DE. Additionally, changes in some protein patterns from buffalo calves during salmonellosis and lactating buffaloes during mastitis are presented. Milk samples were collected and distributed into groups: Milk samples from healthy buffaloes (SCC < 100.000 cells/ml, negative microbiology and CMT) (G1, n = 5) and buffaloes with subclinical mastitis (SCC > 500.000 cells/ml, positive microbiology and CMT) (G2, n = 5). Blood samples from buffalo calves (n = 6) were collected, and three calves were experimentally infected with Salmonella Dublin and samples analyzed before (M0) and 72 h after inoculation (M1). 1-DE was accomplished by loading 10 μg of TP into SDS-PAGE, stained with Coomassie blue. 2-DE was accomplished by loading 200 μg of TP into 11 cm, pH 3-10 non-linear IPG strips, followed by SDS-PAGE, stained with Coomassie blue. Protein bands/spots were excised, subjected to tryptic in-gel digestion and analyzed by LC/ESI-MS/MS. Protein identity was assigned using NCBI databases. After bands/spots from 1-DE and 2-DE were analyzed, a protein map with 35 and 40 different identified proteins in blood serum and milk whey, respectively, was generated. Significant changes in patterns of haptoglobin were observed in buffalo calves with salmonellosis and in patterns of IgLC, β-lactoglobulin and α-lactalbumin of lactating buffaloes during mastitis. The establishment of a protein map for 1-DE and 2-DE, identifying potential disease related proteins, can help to address alterations during diseases in buffaloes.

Immunohistochemical localization of Haptoglobin in the Bovine Mammary Gland

Haptoglobin (Hp), an acute phase protein has been recognized as an important indicator of intramammary infections that can be assessed in milk. In this study, we utilized immunohistochemical techniques on naturally infected and healthy bovine mammary glands in order to determine the source of Hp found in milk during bovine mastitis. Mammary glands sections were harvested from udder of two cows, one showing signs of clinical mastitis and the other healthy. Hematoxylin and eosin as well as immunohistochemistry (IHC) for anti-Hp staining were carried on these sections. It was revealed that the mammary epithelial cells around the alveolar ducts of the gland as well as with infiltrating neutrophils had dense staining for Hp in mastitic sections while in the healthy section there was minimal cytoplasmic positivity for Haptoglobin on the ductal epithelial cells. These observations support the conclusion that Hp arises locally from mammary epithelial cells and from circulation from migrating neutrophils during episodes of mastitis, and can therefore serve as a sensitive indicator of bovine mastitis.

Acute phase proteins in dogs naturally infected with the Giant Kidney Worm (Dioctophyme renale).

BACKGROUND Dioctophyme renale is a nematode parasite of dogs, usually found in the right kidney, causing severe damage to the renal parenchyma. OBJECTIVES The objective was to evaluate the acute phase response in dogs naturally infected with this Giant Kidney Worm and the possible effects of nephrectomy on circulating concentrations of select acute phase proteins (APP) such as serum amyloid A (SAA), C-reactive protein (CRP), and haptoglobin (HP). METHODS Nephrectomy was performed in infected dogs and the worms were collected for identification. Blood samples were taken 24 hours before surgery, and 4, 8, and 12 hours postoperatively on the following 10 consecutive days, and 28 days after surgery. Acute phase protein concentrations were determined at all time points. Cortisol concentrations were determined 24 hours before surgery and at recovery (28 days after surgery). One-way ANOVA and Friedman test were used for multiple comparisons; the Wilcoxon-signed rank test was used to compare variables, and Spearmans rho rank test was used to assess the correlation between the number of parasites recovered from the dogs and the APP concentration. RESULTS Forty-five parasites were recovered from the 12 dogs evaluated in this study. Dogs showed significantly increased HP concentrations (P < .05) but lower CRP and SAA concentrations before surgery, and cortisol concentrations were significantly higher at admission when compared to recovery. No significant correlations were found between the number of parasites and APP concentrations. CONCLUSION There is a particular acute phase response profile in dogs with kidney worm infection. Nephrectomy induced a short-term inflammatory process.