In-depth immunophenotyping reveals significant alteration of lymphocytes in buffalo with brucellosis.

Water buffalo (Bubalus bubalis) has a prominent position in the livestock industry worldwide but still suffers from limited knowledge on the mechanisms regulating the immune against infections, including brucellosis (BRC), one of the most significant neglected zoonotic diseases of livestock. Seventy-three buffalo were recruited for the study. Thirty-five were naturally infected with Brucella spp. The aims of the study were to (i) verify the cross-reactivity of 16 monoclonal antibodies (mAbs) developed against human, bovine, and ovine antigens; (ii) evaluate lymphocyte subset alterations in BRC positive buffalo; (iii) evaluate the use of the canonical discriminant analysis (CDA), with flow cytometric data, to discriminate BRC positive from negative animals. A new set of eight mAbs (anti CD3e, CD16, CD18, CD45R0, CD79a; CD172a) were shown to cross-react with water buffalo orthologous molecules. BRC positive animals presented a significant (p < 0.0001) decrease in the percentage of PBMC (29.5 vs. 40.3), total, T and B lymphocytes (23.0 vs. 35.5, 19.2 vs. 28.9, 2.6 vs. 5.7, respectively). In contrast, they showed an increase in percentage of granulocytes (65.2 vs. 55.1; p < 0.0001) and B lymphocytes CD21neg (22.9 vs. 16.1; p = 0.0067), a higher T/B lymphocyte ratio (10.3 vs. 6.4; p = 0.0011) and CD3+ /CD21+ (14.7 vs. 8.3; p = 0.0005) ratio. The CDA, applied to 33 different flow cytometric traits, allowed the discrimination of all BRC positive from negative buffalo. Although this is a preliminary study, our results show that flow cytometry can be used in a wide range of applications in livestock diseases, including in support of uncertain BRC diagnoses.

Relationship among Milk Conductivity, Production Traits, and Somatic Cell Score in the Italian Mediterranean Buffalo.

The measurement of milk electrical conductivity (EC) is a relatively simple and inexpensive technique that has been evaluated as a routine method for the diagnosis of mastitis in dairy farms. The aim of this study was to obtain further knowledge on relationships between EC, production traits and somatic cell count (SCC) in Italian Mediterranean Buffalo. The original dataset included 5411 records collected from 808 buffalo cows. Two mixed models were used to evaluate both the effect of EC on MY, PP and FP and EC at test-day, and the effect of EC on somatic cell score (SCS) by using five different parameters (EC_param), namely: EC collected at the official milk recording test day (EC_day0), EC collected 3 days before official milk recording (EC_day3), and three statistics calculated from EC collected 1, 3 and 5 days before each test-day, respectively. All effects included in the model were significant for all traits, with the only exception of the effect of EC nested within parity for FP. The relationship between EC and SCS was always positive, but of different magnitude according to the parity. The regression of EC on SCS at test-day using different EC parameters was always significant except when the regression parameter was the slope obtained from a linear regression of EC collected over the 5-day period. Moreover, in order to evaluate how well the different models fit the data, three parameters were used: the Average Information Criteria (AIC), the marginal R2 and the conditional R2. According to AIC and to both the Marginal and Conditional R2, the best results were obtained when the regression parameter was the mean EC estimated over the 5-day period.

Changes in the lipidome of water buffalo milk during intramammary infection by non-aureus Staphylococci.

This study aimed to determine the lipidome of water buffalo milk with intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted lipidomic approach. Non-aureus Staphylococci are the most frequently isolated pathogens from dairy water buffalo milk during mastitis. A total of 17 milk samples from quarters affected by NAS-IMI were collected, and the lipidome was determined by liquid chromatography-quadrupole time-of-flight mass spectrometry. The results were compared with the lipidome determined on samples collected from 16 healthy quarters. The study identified 1934 different lipids, which were classified into 15 classes. The abundance of 72 lipids changed in NAS-IMI milk compared to healthy quarters. Significant changes occurred primarily in the class of free fatty acids. The results of this study provided first-time insight into the lipidome of dairy water buffalo milk. Moreover, the present findings provide evidence that NAS-IMI induces changes in water buffalo milk's lipidome.

Peptidomic changes in the milk of water buffaloes (Bubalus bubalis) with intramammary infection by non-aureus staphylococci.

Mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In NAS-positive milk, the differential peptides belonged mainly to caseins, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, these belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant.

Determination of 20 Endocrine-Disrupting Compounds in the Buffalo Milk Production Chain and Commercial Bovine Milk by UHPLC-MS/MS and HPLC-FLD.

Bisphenol A (BPA) and some of its analogues are well known as endocrine-disrupting chemicals (EDCs), i.e., compounds that can affect the physiological hormonal pathways in both animals and humans, causing adverse health effects. The intake of these substances through diet represents a public concern, compounded by the scarce data in the literature about contamination levels in food. In the framework of a research project, funded by the Italian Ministry of Health, we determined the contamination levels of BPA and 19 EDCs in the production chain of buffalo milk, analysing feed, drinking water, buffalo milk, and blood sera. Overall, 201 feed, 9 feed additive, 62 drinking water, 46 milk, and 190 blood serum samples were collected from 10 buffalo farms in the Campania region, Southern Italy, between 2019 and 2020, and analysed. Moreover, 15 retail bovine milk samples packaged both in Tetra Pak and in PET were analysed to further evaluate consumers' exposure to EDCs. The results of our work showed no contamination by EDCs in drinking water samples, whereas in 43% of all of the other samples from the farms at least one bisphenol was detected. The most abundant bisphenol detected was BPA (32% of the samples from the farms and 80% of the retail milk samples), thus proving that this compound is still widely used for plastic production.

Italian Tracing System for Water Buffalo Milk and Processed Milk Products.

This document describes the development of a tracing system for the buffalo supply chain, namely an online computer system in which farmers, dairies, and brokers must maintain records of the production of milk through to the production of derivatives. The system is jointly used throughout the Italian national territory by the Istituto Zooprofilattico Sperimentale del Mezzogiorno (IZSM) and the Sistema Informativo Agricolo Nazionale Italiano (SIAN), after being made mandatory and regulated with the publication of the Ministerial Decree of 9 September 2014. Farmers are obligated to communicate their daily production of bulk milk, the number of animals milked, the number of the delivery note of the sale, and the name of the purchaser; within the first week of the month, they must communicate the milk production of each animal milked. Dairies are required to communicate the milk and the processed product (mozzarella, yogurt, etc.) purchased on a daily basis. The intermediaries are required to communicate the daily milk purchased, both fresh and frozen, the semi-finished product, and the sale of the same. The tracing system linked to the project authorized by the Ministry of Health, called "Development, validation and verification of the applicability of an IT system to be used for the management of traceability in the buffalo industry", provides operators with the monitoring of production and sales in real time through alerts and access logs. Currently, there are 1531 registered farmers, 601 non-PDO dairies, 102 PDO dairies, 68 non-PDO intermediaries, and 17 PDO intermediaries in Italy. The system provides support for the recovery of the buffalo sector; from the analysis of the data extrapolated from the tracing system of the buffalo supply chain for the years 2016 to 2019, this paper highlights that the application of the Ministerial Decree No. 9406 of 9 September 2014 and the tracing of the supply chain have increased the price of buffalo milk at barns from EUR 1.37/kg to EUR 1.55/kg from 2016 to 2019.