1 COMPARISON OF CULTIVATION TESTS TO DETECTION MASTITIS IN DAIRY COWS Farkašová Zuzana 1 , František Zigo* 1 , Arvaiová Juliana 1 , Šimon Halás 1 , Mária Vargová 2 , Silvia Ondrašovičová 3 , Jana Záhumenská 4 , Rawia M Ibrahim 5 , Ibrahim F. Rehan 6,7 Address(es): DVM. František Zigo, Ph.D. 1 University of Veterinary Medicine and Pharmacy, Department of Animal Nutrition and Husbandry, Komenského 73, 041 81 Košice, Slovakia. 2 University of Veterinary Medicine and Pharmacy Department of Hygiene, Technology and Health Food Safety, Komenského 73, 041 81, Košice, Slovakia. 3 University of Veterinary Medicine and Pharmacy, Department of Biology and Physiology, Komenského 73, 041 81 Košice, Slovakia. 4 University of Veterinary Medicine and Pharmacy, Department of the Public Veterinary Medicine and Animal Welfare, Komenského 73, 041 81 Košice, Slovakia. 5 Division of Clinical Laboratory Diagnosis, Animal Medicine Department, Faculty of Veterinary Medicine, South Valley University, Qena, 83523, Egypt. 6 Menoufia University, Faculty of Veterinary Medicine, Department of Husbandry and Development of Animal Wealth, Shebin Alkom, Menoufia, 32511, Egypt. 7 Meijo University, Faculty of Pharmacy, Department of Pathobiochemistry, Yagotoyama 150, Tempaku-Ku, Nagoya-Shi, Aichi, 468-8503, Japan. *Corresponding author: frantisek.zigo@uvlf.sk ABSTRACT Keywords: Cows, Detection, Mastitis, Cultivation, MicroMast test, Udder pathogens INTRODUCTION Milk from ruminants is among the most widely used animal products globally due to its specific composition and nutritional value. Approximately 150 million farmers around the world are involved in its production, catering to over 6 billion people. Obtained milk is a traditional raw material to produce a wide range of dairy products, which are unique in their composition. However, EU regulations emphasize that such products must originate from healthy animals (Audarya et al., 2022; FAO, 2022). Currently, mastitis, an inflammation of the mammary gland, is one of the most significant and challenging diseases in dairy cattle. Its occurrence has a substantial impact on the economy of production farms, despite improvements in milk production hygiene and zootechnical control. Mastitis affects health and utility parameters such as decreased milk production, increased somatic cell count (SCC; directly affecting milk price), reduced qualitative components (lactose and casein, essential for cheese processing), and also increases undesirable elements (lipase and plasmin, affecting milk longevity). Additionally, indirect losses caused by mastitis include increased culling of diseased cows, loss of premiums, premature drying off, animal welfare aspects, and other related health issues. For clinical forms of mastitis, treatment costs can reach up to 200 EUR per case (Cobirka et al., 2020). To date, more than 135 different causative agents of intramammary infection (IMI) in ruminants are recorded, including bacteria, viruses, yeasts, and algae. Up to 95% of IMIs are most caused by bacteria (Tančin, & Uhrinčať, 2014). Due to the polyetiological nature of mastitis, this disease cannot be completely eradicated from livestock, especially due to environmental pathogens such as staphylococci, streptococci, mycoplasmas, or Escherichia coli (E. coli), which attack the mammary gland from the surroundings. The disease can have clinical manifestations with apparent changes in milk appearance (results of inflammatory response) and signs of mammary gland inflammation, but it can also be subclinical, meaning that the infection is present (and can spread to other animals) but without visible clinical symptoms (Foltys, & Kirchnerova, 2003). Diagnostic tests for the detection of mastitis are divided into indirect, by which we determine SCC in milk in cases of subclinical mastitis, and direct so-called culture methods serving to identify the causative agents of inflammation of udder in all forms of mastitis. According to the place of use, we divide them into farm and laboratory methods. Laboratory diagnostics consists of bacteriological, cytological, biochemical examination and laboratory tests to determine the number of cellular elements in milk. Milk samples from individual dairy cows (quarter, half, mixed) or average samples obtained from dairy cow groups (cisterns, pools) are examined (Škarda, & Škardová, 2000). It is carried out on blood agar or on special agars. Various identification tests based on DNA analysis can be used to characterize pathogens at different phylogenetic levels. These methods can detect either DNA or RNA. Currently, mastitis can be detected using on-farm tests, with the most common being the California Mastitis Test (CMT), but it doesn't allow pathogen identification. The presence of udder pathogens is most frequently detected in the laboratory through cultivation on various selective agars, which is time- consuming. Lately, the goal is to introduce tests that will be as effective as laboratory cultivation and can be performed by a trained person directly on the farm, saving farmers time associated with sample transportation and evaluation (Prášek, 2023). The aim of the study was to compare two tests for the cultivation of udder pathogens from samples of raw cow's milk using classical laboratory cultivation on Columbia agar with 5% addition of sheep's blood and the MicroMast plates. MATERIAL AND METHODS Dairy Cow Production, Housing, and Milking For the practical purposes of the study, a farm of 270 Slovak Spotted Cattle was monitored. The farm was in eastern Slovakia. The farming environment consisted of a modern building with high air ventilation and automated waste removal. The cows were housed in a stable divided into 2 sections using a feeding table, with Mastitis is an inflammation of the mammary gland which has a particular importance in ruminants. The purpose of this study was to evaluate and compare two methods for cultivation of udder pathogens: classical laboratory cultivation on Columbia agar with 5% sheep blood versus MicroMast rapid plates. The results allowed assessing the incidence of mastitis and the prevalence of the pathogens. During the study have been investigated 227 cows in a dairy farm localized in the east of Slovakia. Subsequently, 141 quarter milk samples from the positive cows with California mastitis test score 1-4 have been undergone the laboratory culture on Columbia agar and MicroMast test in accordance with their respective steps. The values obtained from these tests showing sensitivity of positive samples using the MicroMast test at the level of 64.5%, and sensitivity of cultures on Columbia blood agar at the level of 61.7%. After biochemical identification of cultured isolates, the Columbia blood agar and MicroMast test identified both as the main pathogen present Staphylococcus aureus (S. aureus) with results of 13.4% and 14.9%, respectively. On the base of results, both tests are comparable and therefore a test that speeds up testing is more useful in practice. It will be a relevant importance in the following decades to succeed in the development of tests with directly detection of udder pathogens. ARTICLE INFO Received 21. 8. 2023 Revised 8. 12. 2023 Accepted 15. 1. 2024 Published 1. 2. 2024 Regular article https://doi.org/10.55251/jmbfs.10507