Investigation of expression of staphylococcal enterotoxin A and TSST-1 toxin genes of S. aureus in milk

S. aureus je ubikvitaran mikroorganizam, široko rasprostranjen u životnoj sredini. Glavni je uzročnik mastitisa kod mlečnih krava, pa tako uzrokuje velike zdavstvene i ekonomske probleme. Može biti prisutan u mleku i proizvodima od mleka koji se termički ne obrađuju, ali i u termički obrađenim proizvodima naknadnom kontaminacijom od strane čoveka. Bitna karakteristika ovog mikroorganizma je sposobnost stvaranja ekstracelularnih enzima i toksina koji predstavljaju glavni uzrok trovanja hranom. Da bi se sintetisala dovoljna količina enterotoksina koja može izazvati intoksikacije kod ljudi, potrebno je da u hrani bude prisutno više od 105 CFU S. aureus/mL.  S. aureus sintetiše više vrsta toksina koji izazivaju različite efekte na ćelije čoveka i drugih sisara. Sinteza toksina zavisi od aktivnosti gena za regulaciju ekspresije stafilokoknih enterotoksina (SEs) i TSST-1. Postoji više faktora koji sprečavaju ili stimulišu sintezu enterotoksina kao što su: temperatura, pH, aw, kiseonik, redoks potencijal i dr. Enterotoksini se sintetišu pri temperaturama od 10°C do 46°C, pri pH od 5 do 9,6, aktivnosti vode od 0,86 do 0,99 i koncentraciji NaCl do 12%. Nalaz enterotoksogenih stafilokoka u hrani ne znači da će doći do trovanja, jer uslovi matriksa hrane i sredine određuju preživljavanje, rast populacije stafilokoka i stvaranje dovoljne količine enterotoksina koja može izazvati oboljenje kod ljudi.  Cilj ovog istraživanja bio je da se ispita uticaj različitih temperatura, vremena čuvanja i matriksa na stepen ekspresije gena za sintezu enterotoksina A i toksina TSST-1, kako bi se došlo do novih saznanja o uticaju paragenetskih faktora na rizik nastanka stafilokokne alimentarne intoksikacije. Materijal za eksperimentalnu inokulaciju predstavljalo je kratkotrajno sterilizovano (UHT) i pasterizovano kravlje mleko sa 3,2% mlečne masti i referentni sojevi S. aureus koji sintetišu stafilokokni enterotoksin A i toksin 1 toksičnog šok sindroma. Eksperimentalni rad odvijao se u dve faze. U prvoj fazi eksperimenta, hrana animalnog porekla (pasterizovano i UHT mleko) koja simulira najčešće izvore toksoinfekcije S. aureus kontaminirana je određenom količinom prekonoćne bujonske suspenzije referentnog soja S. aureus, u kojoj je prethodno utvrđen inicijalni broj. Uzorci su čuvani pri temperaturama od 15°C i 22°C tokom 24, 48 i 72 časa, simulirajući neadekvatne uslove koji nastaju akcidentalnim narušavanjem hladnog lanca. Nakon 24, 48, odnosno 72 časa kontaminirano mleko inokulisano je na Baird-Parker agar (SRPS ISO 6888-1:2009), a nakon inkubacije podloga utvrđen je broj izraslih kolonija S. aureus i upoređen sa inicijalnim brojem. Paralelno sa inokulacijom ove podloge, uzeta je određena količina mleka koja je potom tretirana tečnim azotom da bi se konzervisala iRNK tj. da bi se „zamrznuo“ ekspresioni genetski profil S. aureus. Iz zamrznutih uzoraka ekstrahovana je ukupna RNK, koja je potom metodom reverzne transkripcije - Real Time PCR prevedena u odgovarajuću cDNK svakog od ispitivanih gena. Da bi se ispitala korelacija između ekspresije gena za sintezu stafilokoknog enterotoksina i njegove količine u uzorcima, a na taj način isključila i eventualna posttranslaciona promena u sintezi, stafilokoni enterotoksin A je kvantifikovan pomoću dijalizne koncentracije i ELFA metode. Kao rezultat istraživanja sprovednih u ovoj disertaciji može se uočiti statistički značajna razlika između stepena ekspresije sea gena u pasterizovanom mleku čuvanom pri temperaturi od 22°C tokom 24 sata u odnosu na kalibrator (S. aureus u mleku čuvanom pri 8°C), što se poklapa sa postignutim brojem S. aureus od 105 CFU/mL i imunohromatografski detektovanom količinom enterotoksina A. Stepen ekspresije enterotoksina A u pasterizovanom mleku čuvanom pri temperaturi od 15°C tokom 72 sata statistički se značajno razlikuje u odnosu na kalibrator, što se takođe podudara sa detektovanom količinom enterotoksina A. U UHT mleku čuvanom tokom 24 sata pri temperaturi od 22°C, odnosno tokom 48 sati pri temperaturi od 15°C utvrđena je statistički značajna razlika u stepenu ekspresije u odnosu na kalibrator, što se podudara sa detektovanom količinom enterotoksina A i dinamikom promene broja S. aureus. Na osnovu ovih podataka možemo zaključiti da intezitet ekspresije sea gena odgovara količini fizički „de novo“ sintetisanog enterotoksina A. Stepen ekspresije tst gena pokazao je statistički značajnu razliku u odnosu na kalibrator u pasterizovanom i UHT mleku nakon 24 sata pri temperaturi od 22°C, i 48 sati pri temperaturi od 15°C, dok je broj S. aureus pozitivan na TSST-1 prelazio 105 CFU/mL u pasterizovanom i UHT mleku nakon 24 sata čuvanom pri 15°C, odnosno 22°C. Obzirom na mali broj literaturnih podataka o uticaju sastava hrane, temperature i vremena čuvanja kao i drugih paragenetskih faktora na regulaciju patogenosti S. aureus, dobijeni rezultati mogu predstavljati polaznu osnovu za ispitivanje značaja genetskog profila patogenosti S. aureus u hrani na razvoj infekcije kod potrošača.S. aureus is an ubiquitous microorganism, widely distributed in the environment. It is the main cause of mastitis in dairy cows so it causes major health and economic problems. It can be present in milk and milk products that are not thermally treated, but also in thermally processed products by subsequent contamination by humans. An important feature of this microorganism is the ability to produce extracellular enzymes and toxins that are the major cause of food poisoning. In order to synthesize a sufficient amount of enterotoxin that can induce intoxication in humans, more than 105 CFU of S. aureus / mL should be present in the food. S. aureus synthesizes several types of toxins that cause different effects on the human cells and cells of other mammals. The synthesis of toxin depends on the activity of the gene regulating the expression of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1). There are several factors that prevent or stimulate the synthesis of enterotoxins such as temperature, pH, water activity, oxygen, redox potential, etc. Enterotoxins are synthesized at temperatures from 10°C to 46°C, at pH 5 to 9.6, water activities ranging from 0.86 to 0.99 and a concentration of NaCl to 12%. Finding enterotoxogenic staphylococci in food does not mean that there will come to poisoning, because the conditions of the food and environment matrix determine their survival, the growth of the staphylococci population and the creation of a sufficient amount of enterotoxins that can cause disease in humans. The aim of this study was to investigate the influence of different temperatures, storage times and matrix on the degree of gene expression for the synthesis of enterotoxin A and toxin TSST-1 in order to gain new insights about the effect of paragenetic factors on the risk of staphylococcal alimentary intoxication. The material for experimental inoculation was short-time sterilized (UHT) and pasteurized cow milk with 3.2% milk fat and reference strains of S. aureus which synthesizes staphylococcal enterotoxin A and TSST-1. Experimental work took place in two phases. In the first stage of the experiment, pasteurized and UHT milk, which simulates the most common sources of S. aureus toxoinfection, were contaminated by a certain amount of overnight broth suspension of the reference strain S. aureus, in which the initial number was previously determined. Samples were stored at temperatures of 15°C and 22°C for 24, 48 and 72 hours, simulating inadequate conditions resulting from accidental cold chain disturbance. After 24, 48, and 72 hours, contaminated milk was inoculated to Baird-Parker agar (SRPS ISO 6888-1: 2009), and after the incubation of the substrate, the number of grown S. aureus colonies was compared with the initial number that was determined in beginning of the experiment. At the time this substrate was inoculated, certain amount of milk was taken, which was then treated with liquid nitrogen to conserve iRNA, i.e. in order to "freeze" the expression genetic profile of S. aureus. From frozen samples, the total RNA was extracted, which was then converted by the reverse transcription method - Real Time PCR into the corresponding cDNA of each of the examined genes. In order to investigate the correlation between gene expression for the synthesis of staphylococcal enterotoxin and its amount in the samples, and thereby exclude the possible posttranslational change in synthesis, staphylococcal enterotoxin A was quantified by dialysis and ELFA methods. As a result of the investigations carried out in this thesis, a significant difference between the degree of expression of the sea gene in pasteurized milk stored at 22°C during 24 hours in relation to the calibrator (S. aureus in milk stored at 8°C) can be observed, which matches the achieved S. aureus number of 105 CFU / mL and an immunochromatographically detectable amount of enterotoxin A. The degree of expression of enterotoxin A in pasteurized milk stored at a temperature of 15°C for 72 hours significantly differs from the results of the calibrator, which also match the detected amount of enterotoxin A. In UHT milk stored for 24 hours at a temperature of 22°C, and for 48 hours at a temperature of 15°C, a significant difference in the degree of expression of sea gene relative to the calibrator was found, which matches the detected amount of enterotoxin A and the dynamics of the change in the number of S. aureus. Based on these data, it can be concluded that the intensity of expression of the sea gene corresponds to the amount of physically "de novo" synthetized enterotoxin A. The degree of expression of the tst gene showed a significant difference from the calibrator value in pasteurized and UHT milk after 24 hours at a temperature of 22°C and 48 hours at a temperature of 15°C, while the number of S. aureus positive for TSST-1 exceeded 105 CFU / mL in pasteurized and UHT milk after 24 hours stored at 15°C and 22°C. Given the small number of literature data on the effect of food composition, temperature and storage time, as well as other paragenetic factors on the regulation of S. aureus pathogenicity, the obtained results can be the starting point for examining the significance of the genetic profile of S. aureus pathogenicity in the development of the infection in consumers

Antimicrobial resistance among Salmonella enterica serovar Infantis from broiler carcasses in Serbia

This study aimed to investigate antimicrobial resistance of Salmonella Infantis isolates from poultry carcasses in Serbia. A total of 48 Salmonella isolates were examined for antimicrobial resistance. A panel of 10 antibiotics was selected for testing. Isolates showed resistance to sulfamethoxazole, ceftazidime and cefotaxime (100%). However, the highest number of Salmonella Infantis isolates were sensitive to chloramphenicol. The usage of antibiotics in food producing animals could result in antimicrobial resistance pathogenic bacteria especially Salmonella spp. in poultry, which may be transmitted to humans through the food chain and increase risk of treatment failures

Status of probiotic lactic acid bacteria

Probiotici se definišu kao živi mikroorganizmi koji, kada se unesu, pokazuju blagotvoran efekat na domaćina. Mnogi sojevi iz roda Lactobacillus imaju status probiotika. Nedostatak gena za prenošenje rezistencije na antibiotike jedan je od ključnih uslova kako bi soj dobio GRAS status. Svaki probiotski soj bi trebalo da preživi pasažu kroz gastrointestinalni trakt, kako bi uspeo da ostvari blagotovorne efekte po domaćina. Materijal za ovo ispitivanje predstavljao je 51 soj laktobacila koji su izolovani iz sjeničkog sira. Svi sojevi su ispitivani na rezistenciju na devet antibiotika. Od ukupno 51 soja, šest je pokazalo osetljivost na sve antibiotike, dok je 23 pokazalo prirodnu rezistenciju, koja, kao takva, nije rizična za prenošenje gena. Najčešći profil rezistencije bila je rezistencija na vankomicin i tetraciklin. Od 18 sojeva koji su stekli uslove za dalje ispitivanje, kod devet njih je ispitivana mogućnost preživaljavanja u simuliranim uslovima želuca, odnosno duodenuma. Takođe je ispitivana i antimikrobna aktivnost i sposobnost hemolize. Svih devet sojeva je pokazalo odličnu sposobnost preživljavanja u simuliranim uslovima želuca, dok je svega tri moglo da preživi u prisustvu žučnih soli. Svih devet sojeva je pokazalo antimikrobnu aktivnost protiv Listeria monocytogenes i Staphylococcus aureus, a nijedan nije pokazao hemolizu. Prema rezultatima u ovom ispitivanju, sojevi laktobacila izolovani iz sjeničkog sira pokazali su da imaju potencijal da steknu status probiotika, ali su za to potrebna dalja ispitivanja.Probiotics are live microorganisms that exibit a benneficial effect on the health of the host when ingested. Many strains of lactic acid bacteria from Lactobacillus genus are recognized as probiotics. Absence of transferable genes for antibiotic resistancy is crucial so that probiotic strain can acquire GRAS status. Also every probiotic should survive conditions in digestive tract. Material for this research was 51 strain of Lactobacillus spp. isolated from traditional cheese from Sjenica. All strains were analyzed for resistency to 9 different antibiotics – 6 of them showed susceptibility to all antibiotics, while 23 showed profile of intrinsic antibiotic resistency. Most common resistancy profile was resistancy to vankomycin and tetracycline. For further research, 9 strains were chosen, and they were analyzed for viability in simulated gastric and duodenal conditions, antimicrobial activity and hemolysis. All strains showed excellent survival in simulated gastric conditions, while only 3 of them could survive in presence of bile salts. All 9 strains showed antimicrobial activity toward Listeria monocytogenes and Staphylococcus aureus; none of them showed hemolysis on agar. According to these results, strains of lactobacilli from Sjenica cheese have potential for becoming probiotics, but it requires further research

The common foodborne viruses: A review

Transmission pathways of foodborne viruses include contamination of food by infected food handlers, by contamination of food during the production process and by consumption of products of animal origin harbouring a zoonotic virus. Viral foodborne illnesses, which have become a significant cause of all reported foodborne illnesses in recent years and considered as an emerging risk in veterinary public health. Microbiological genomics studies discovered that Noroviruses and hepatitis A viruses were primarily associated with food-handler transmission and sewage-contaminated foods. In contrast, hepatitis E was associated with consumption of raw or undercooked meat of pig or wild animals. In order to facilitate source attribution and identify risk prevention measures, Routine harmonized surveillance of viral outbreaks, and surveillance of virus occurrence in food commodities, in combination with systematic strain typing, and joint expertise from veterinary, food, and clinical microbiologists would be recommended

Characteristics of traditional production of cheese in monestry rakovica

Као једна од најстаријих намирница, сир, због своје хранљиве вредности, заузима важно место у исхрани људи. Производња сира датира из далеке прошлости и имала је значаја у свим цивилизацијама. Сиреви се традиционално производе у Србији вековима, представљају културно наслеђе и акумулирано искуствено знање, које се преноси са генерације на генерацију. Историјски гледано, у средњем веку главна места где се одвијала производња сирева били су манастири и феудални поседи, тако да многе групе данашњих сирева потичу из тог времена. У Републици Србији, поред доминантне индустријске производње, сиреви се производе у занатским погонима, индивидуалним домаћинствима, али се традиционална производња задржала у малим заједницама као што су манастири. Циљ овог рада био је да се опише производња сира у манастиру Раковица, који се налази на територији Београда, Република Србија. Сиреви су испитани на присуство L. monocytogenes, коагулаза позитивних стафилокока, одређиван је број бактерија млечне киселине, ентеробактерија, као и физичкохемијски параметри (сува материја, маст, маст у сувој материји, вода, вода у безмасној материји, киселост, pH вредност, садржај NaCl, активност воде). Снимање технологије изведено је помоћу анкете састављене од питања, која обједињују основне елементе и технолошке поступке производње сира. Доминантну микрофлору су чиниле бактерије млечне киселине. Средња вредност броја Lactococcus spp. била је 6,34 log cfu/g и Lactobacillus spp. 5,49log cfu/g. У испитаним узорцима није доказано присуство L. monocytogenes и каогулаза позитивних стафилокока, док je средња вредноста броја Enterobacteriaceae била 4,05 cfu/g. Средња вредност за суву материју сира била је 40,19%, за маст у сувој материји сира 39,86%, за воду у безмасној материји 71,21 %, за укупне протеине 14,78%, за киселост 10,80°SH, pH вредност 6,20, за садржај NaCl 0,87% и актвност воде 0,953.As one of the oldest food products,cheese takes an important place in human diet due to its nutritional value. Cheese production dates back to the distant past and had importance in all civilizations. The cheeses are traditionally produced in Serbia for centuries and represent the cultural heritage and accumulated experiential knowledge, which is passed from generation to generation. Historically, in the Middle Ages the monasteries and feudal estates were the main places where the cheese production were carried out, so many groups of nowdays cheeses originate from this time. In the Republic of Serbia, additionaly to the dominant industrial productionat the industrial dairy plants, cheeses are produced in small scale plants and individual households, but traditional production had been kept in small communities such as monasteries. The aim of this study was to describe the production of cheese in the monastery "Rakovica", situated in Belgrade, Republic of Serbia. The cheeses were examined for the presence of L. monocytogenes, coagulase-positive staphylococci, the number of lactic acid bacteria, Enterobacteriaceae was determined and physicochemical parameters (total solids, fat, fat in total solids, moisture, moisture on a freefat basis, acidity, pH value, content of NaCl, water activity) as well. The observation of technology was carried out by the survey consisting of questions, which combines the basic elements and technological operations of cheese production. The lactic acid bacteria represented dominant microbita of cheese. The mean value for the number of Lactococcus spp. was 6.34 log CFU/g and Lactobacillus spp. 5,49 log cfu/g. L. monocytogenes and coagulase-positive staphylococci were not detected in the examined samples, whereas the mean value for the number of Enterobacteriaceae was 4.05 cfu/g. The mean value for total solids of cheese was 40.19%, for fat in total solids of cheese 39.86%, for moisture in free-fat basis 71.21%,for content of total proteins 14,78%, for acidity 10,80 °SH, for pH value of 6.20, for NaCl content 0.87% and water activity 0.953

METABOLIČKA AKTIVNOST TERMOREZISTENTNIH MIKROORGANIZAMA

Termorezistentni mikroorganizmi, zahvaljujući otpornosti na termičke tretmanekoji se primenjuju u industriji mleka, kao i mogućnostima rasta i metaboličkimaktivnostima u uslovima čuvanja gotovih proizvoda, imaju značaja kao mikro -organizmi kvara. Ova osobina deNiniše tehnološki značaj termorezistentnih mikro -organizama.Među populacijom termorezistentnih mikroorganizama, Bacillus spp. pokazujuznačajnu proteolitičku aktivnost, a s obzirom na njihovo svojstvo razlaganja kazeina,potencijal njihove proteolize može se uspešno koristiti u koagulaciji mleka i zrenjasireva. Veliki značaj imaju proteinaze Bacillussubtilis, čiji se proteolitički enzimiuspešno primenjuju u proizvodnji Chedar, Munster i Camembert sira. Suprotnoovome, aktivnost proteina za Bacillus spp. u sterilisanom mleku, u uslovima ne -adekvatnog čuvanja dovodi do slatkog grušanja ili pojave gorkog ukusa mleka, kaorezultat nastanka gorkih peptida, dok preterana aktivnost proteinaza bacila priproizvodnji Chedar sira uslovljava gruš mekše konzistencije, veći sadržaj proteina imasti u surutki, što dovodi do smanjenog randmana sira za 10%. Oslobađanjem nižihmasnih kiselina delovanjem lipolitičkih enzima poreklom od mikroorganizama,razvija se neprijatan ukus i miris, što predstavlja manu poznatu pod imenomhidrolitička užeglost. Od termorezistentnih mikroorganizama, sposobnost hidrolizemasti pokazuju mikroorganizmi roda Bacillus.Cilj ovog rada bio je da se ispita sposobnost termorezistentnih mikro organizamaizolovanih iz mleka da razlažu proteine, masti i skrob, što dovodi do kvara mleka iproizvoda od mleka.Od ukupno 80 izolata termorezistentnih mikroorganizama, proteolitičkuaktivnost je ispoljilo 35 (43,75%) izolata u uslovima inkubacije pri 30oC/72h i 30(37,50%) izolata u uslovima inkubacije pri 20±2oC/72h, dok nijedan izolat nijeispoljio proteolitičku aktivnost u uslovima inkubacije pri 10±1oC/10 dana.Amilolitička aktivnost je utvrđena kod 16 (20,0%) izolata u uslovima inkubacije pri30oC/72h i 15 (18,75%) izolata u uslovima inkubacije pri 20±2oC/72h. Amilolitičkaaktivnost termorezistentnih mikroorganizama izostaje u uslovima inkubacije pri10±1oC/10 dana. Ispitivani izolati termorezistentnih mikroorganizama nisu pokazalilipolitičku aktivnost, bez obzira na primenjene različite uslove inkubacije

Inhibition of Staphylococcus aureus by cinnamaldehyde and its effect on sensory properties of milk

The antibacterial activity of cinnamaldehyde was evaluated against S. aureus experimentally inoculated (10(3) CFU/mL) in UHT-pasteurized milk, which was treated with different concentrations of the cinnamaldehyde (0.1% and 0.05%) and stored at 4 degrees C for 12 days. The MIC of cinnamaldehyde was 160 mu g/ml. During the storage period, S. aureus counts in milk were reduced by 0.35-2.77 log CFU/mL. Significantly greater decreases were observed when cinnamaldehyde was added, regardless of the concentration used, compared with the control. A triangle test showed that panellists could detect the difference between milks with different concentrations of cinnamaldehyde (P<0.01). These results suggest that by adding 0.05% cinnamaldehyde to milk, the safety of the milk can be increased and a pleasant, desirable flavour can be obtained

Prevalencija i molekularna karakterizacija enterotoksin-produkujućih sojeva S. aureus izolovanih iz vimena krava u Srbiji

Staphylococcus aureus is known worldwide as a frequent cause of mastitis in dairy cattle. Due to the production of heath resistant enterotoxins, this pathogen is also a major cause of food poisoning among humans, with symptoms of often severe vomiting and diarrhea. The aim of our study was to determine the prevalence of enterotoxinproducing strains of S. aureus originating from samples of cows with subclinical and clinical mastitis in the Republic of Serbia. Furthermore, we analyzed the type of staphylococcal enterotoxin they produce and phylogenetic relatedness among the S. aureus isolates recovered from milk in this study. Production of staphylococcal enterotoxins A, B, C, D and E was determined by commercial immunoenzyme assay VIDAS® SET2, and presence of corresponding genes encoding enterotoxin synthesis in positive isolates confirmed by Polymerase Chain Reaction. Enterotoxin production was determined in 5 out of 75 (6.67%) isolates of S. aureus and all of them produced staphylococcal enterotoxins C. After analyzing the nucleotide sequence of the gene encoding the synthesis of staphylococcal protein A, S. aureus isolates were assigned into 2 phylogenetic groups, including 7 clusters. All S. aureus isolates with the presence of sec gene formed one cluster even dough they originated from milk samples from different farms.Širom sveta S. aureus poznat je kao čest uzročnik mastitisa krava. Takođe predstavlja i glavni uzrok trovanja hranom nakon konzumiranja hrane kontaminirane njegovim enterotoksinima. Cilj ovog istraživanja bio je da se ispita prevalencija enterotoksinprodukujućih sojeva S. aureus poreklom iz vimena krava sa subkliničkim i kliničkim mastitisom u Srbiji, da se odredi tip enterotoksina koji produkuju i da se odredi filogenetska srodnost ovih izolata. Za određivanje sposobnosti sinteze stafilokoknih enterotoksina A, B, C, D i E korišćen je VIDAS® SET2 immunoenzimski test i utvrđeno je da 5 od 75 (6,67%) izolata S. aureus sintetiše enterotoksine. Lančanom reakcijom polimeraze na prisustvo odgovarajućih gena, utvrđeno je da svih 5 izolata poseduju gen za sintezu enterotoksina C. Analizom nukleotidnih sekvenci gena za sintezu stafilokoknog proteina A, izolati S. aureus grupisani su filogenetski u 2 grupe, odnosno u 7 klastera. Svi izolati S. aureus kod kojih je dokazano prisustvo gena za sintezu enterotoksina spadaju u isti klaster

Molecular identification of Prototheca zopfii genotype 2 mastitis isolates and their influence on the milk somatic cell count

ABSTRACT Algae from the genus Prototheca are the only plant-like microorganisms which can cause inflammation and alterations in the mammary gland. Prototheca mastitis is usually recognized as a chronic and symptomless disease with reduced milk production and a very high somatic cell count. Molecular identification of Prototheca spp. is helpful for the differentiation of pathogenic from non-pathogenic strains which are probably milk contaminants. Genotype-specific PCR assays, based on the 18S rDNA gene sequences, have recently been developed to differentiate three genotypes of Prototheca zopfii, of which Prototheca zopfii genotype 3 was reclassified in a new species: Prototheca blaschkeae. P. zopfii genotype 2 is characterized as the main causative agent of Prototheca mastitis that leads to significant economic losses in primary milk production. The purpose of this study was to give a molecular characterization of Prototheca strains isolated in cases of subclinical and clinical mastitis, as well as to determine the influence of these pathogenic algae on the milk somatic cell count. After microbiological examination, algae from the genus Prototheca were isolated in pure cultures from 1.8% of all tested milk samples, and all 13 (100%) isolates were determined as Prototheca zopfii genotype 2 by a genotype-specific PCR. This study has provided the first molecular identification of Prototheca zopfii genotype 2 in the Republic of Serbia. In the case of subclinical Prototheca mastitis, the somatic cell count was 4,175,244 ± 1,233,685/mL of milk. A distinctly higher somatic cell count (P&lt;0.05) was found in the quarters infected by Prototheca zopfii genotype 2 than in the quarters infected by Staphylococcus aureus, which is the most common mastitis causative agent worldwide. The results from this study support previous observations that P. zopfii genotype 2 is the main causative agent of Prototheca mastitis which leads to a significant increase in the somatic cell count in the milk