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Jedan od vodećih mikroorganizama koji uzrokuje bakterijsko trovanje hranom su bakterije roda Campylobacter koje prouzroče kampilobakteriozu, zoonozu koja je od 2005. godine najčešće prijavljivana bolest uzrokovana hranom u Europskoj Uniji te kao takva predstavlja javno-zdravstveni problem. Najprisutnijim izvorom ovog patogena smatra se pileće meso s obzirom da su kampilobakteri uobičajeni komenzali u probavnom sustavu peradi. S obzirom da se nalaze u crijevima peradi, osim prilikom procesa klanja i obrade mesa, velika mogućnost kontaminacije javlja se u kasnijim fazama proizvodnje kao što su: čupanje perja, evisceracija, pranje, pohranjivanje i hlađenje te kontaminacija putem ruku radnika, opreme ili same okoline, a pretpostavlja se da upravo koža peradi predstavlja glavni izvor ovog patogena. U ovom je radu određivan broj bakterija Campylobacter spp. u uzorcima kože vratova tovnih pilića, pilećih prsa bez kože, pilećih krilaca i pilećih bataka ‒ zabataka. Za određivanje broja kolonija korištena je standardna metoda za određivanje broja Campylobacter spp. prema ISO normiranoj metodi HRN EN ISO 10272 – 2:2017., One of the leading microorganisms causing bacterial food poisoning are the Campylobacter bacteria that cause campylobacteriosis, a zoonosis that has the most important source of this pathogen, given that campylobacters are common commensals in the digestive system of poultry. They are found in the intestines of poultry, and in addition to the slaughtering and meat processing stages, a high possibility of contamination occurs in the later stages of production, such as plucking feathers, evisceration, washing, storing and cooling, and contamination through the hands of workers, equipment, or the environment itself, and it is believed that the skin of poultry is been the most frequently reported food-borne disease in the European Union since 2005, making it a public health concern. Chicken meat is considered the main source of this pathogen. In this paper, the number of Campylobacter spp. bacteria was determined in samples of the neck skins of broiler chickens, skinless chicken breasts, chicken wings and chicken drumsticks. To determine the number of colonies, the standard method for determining the number of Campylobacter spp. according to the ISO standard method HRN EN ISO 10272 – 2:2017 was used.

Simple Summary: Detection of new diagnostic markers would greatly influence the institution of preventive (control) protocols in bovine mastitis. Using the quantitative proteomics workflows and statistics for milk analysis, we identified that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphy- lococci and streptococci intramammary natural infection and inflammation, and subsequently gained a deeper understanding of the immunopathology of mastitis-related infection and inflam-mation for various etiological agents. Abstract: Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiol-ogy and host immune responses, independent of the mastitis level. After identification of Staphy-lococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantita-tion through the SEQUEST algorithm. Statistical analysis employing R was used to identify dif-ferentially abundant proteins between the groups. Protein classes, functions and function-al-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflam-mation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific- pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

High performance liquid chromatography (HPLC) has proven to be an effective tool for examining the disposition kinetics of florfenicol (FF), a synthetic broad-spectrum antibiotic used to treat infectious diseases in veterinary medicine. Modification and optimisation of the protein precipitation (PPT) sample preparation procedure and HPLC with diode array detector (DAD) instrumental method were carried out to ensure conditions suitable for FF analyses in pig plasma samples. Stable supernatants with good plasma mean recoveries of FF (99.8% ± 0.7%RSD) were achieved using 1% v/v phosphoric acid solution in methanol and 10% w/v sodium chloride in aqueous solution. The PPT-HPLCDAD method’s detection limit of 0.004 μg/mL and quantification limit of 0.013 μg/mL provides high sensitivity for analyses of FF in plasma samples. In addition, the optimisation of method conditions resulted in shorter extraction and analysis time and less solvent consumption, which stresses the sustainability of this method in analytical chemistry. The optimised and validated PPT-HPLC-DAD method was applied in a comparative study of FF in pig plasma after administration of veterinary medicinal products. The study was conducted on fattening pigs following repeated intramuscular administration of two similar solutions for injection at a dose of 20 mg FF/kg bodyweight (test groups 1 and 2). The solutions for injection contained the same FF concentration, i.e., 300 mg/mL, but differed in excipients. The aim was to examine the influence of administrated solutions for injection on the extent of exposure to FF in pig plasma. The dynamics of kinetic profiles of FF in pig plasma from both treatments correspond to the FF kinetic profiles published in similar studies. However, differences were observed in the concentrations of FF, which were constant throughout the study, and statistical differences between the test groups were confirmed (P, Tekućinska se kromatografija visoke djelotvornosti (HPLC, engl. High-Performance Liquid Chromatography) pokazala učinkovitim alatom za ispitivanje kinetike florfenikola (FF), sintetitčkog antibiotika širokog spektra koji se u veterinarskoj medicini koristi za liječenje zaraznih bolesti. Modifikacija i optimizacija postupka pročišćavanja uzoraka krvi tehnikom precipitacije proteina (PPT) i instrumentalne metode sustava HPLC-a uz detekciju UV-detektorom s nizom fotodioda (DAD, engl. Diode Array Detector) provedene su sa svrhom postizanja pouzdane analitičke metode za određivanje FF-a u plazmi svinja. PPT postupkom priprave uzoraka dodavanjem 1% otopine fosforne kiseline u metanolu (V/V) i 10% vodene otopine natrijevog klorida (m/V) dobiveni su stabilni nadtalozi s dobrim srednjim analitičkim povratima FF-a iz plazme (99,8%±0,7%RSD). Postignuta granica detekcije PPT-HPLC-DAD metode od 0,004 μg/mL i granica određivanja od 0,013 μg/mL omogućuju dobru osjetljivost određivanja FF-a u plazmi; optimizacijom uvjeta metode skraćeno je vrijeme ekstrakcije i analize te je smanjena količina potrošnje otapala čime je postignuta održivost metode u analitičkoj kemiji. Primjenom optimirane i validirane PPT-HPLC-DAD metode provedena Tekućinska se kromatografija visoke djelotvornosti (HPLC, engl. High-Performance Liquid Chromatography) pokazala učinkovitim alatom za ispitivanje kinetike florfenikola (FF), sintetitčkog antibiotika širokog spektra koji se u veterinarskoj medicini koristi za liječenje zaraznih bolesti. Modifikacija i optimizacija postupka pročišćavanja uzoraka krvi tehnikom precipitacije proteina(PPT) i instrumentalne metode sustava HPLC-auz detekciju UV-detektorom s nizom fotodioda (DAD, engl. Diode Array Detector) provedene su sa svrhom postizanja pouzdane analitičke metode za određivanje FF-a u plazmi svinja. PPT postupkom priprave uzoraka dodavanjem 1% otopine fosforne kiseline u metanolu (V/V) i 10% vodene otopine natrijevog klorida (m/V) dobiveni su stabilni nadtalozi s dobrim srednjim analitičkim povratima FF-a iz plazme (99,8%±0,7%RSD). Postignuta granica detekcije PPT-HPLC-DAD metode od 0,004 μg/mL i granica određivanja od 0,013 μg/mL omogućuju dobru osjetljivost određivanja FF-a u plazmi; optimizacijom uvjeta metode skraćeno je vrijeme ekstrakcije i analize te je smanjena količina potrošnje otapala čime je postignuta održivost metode u analitičkoj kemiji. Primjenom optimirane i validirane PPT-HPLC-DAD metode provedena FF-a u plazmi svinja.

Milk urea concentration (MU) has been extensively studied and is often used to evaluate energy and protein balance in dairy cattle diets, but not to this extent in dairy goat diets. The aim of this study was to determine the relationship between MU concentration and crude protein content (CP) in the diet of Alpine goats in order to reduce excessive nitrogen excretion from the organism. For this purpose, 72 goats were randomly divided into three equal groups (24 animals) and fed a concentrate mix containing 14%, 16%, and 18% CP, respectively. The chemical composition of milk samples was determined and the ratio of milk fat to protein was calculated. Statistical analysis of the data was performed using the MIXED procedure (SAS V8 software package). Increasing CP from 14% to 16% in the diet increased daily milk yield by 0.4 kg (p, Koncentracija uree u mlijeku bila je predmetom mnogobrojnih istraživanja u svrhu procjene uravnoteženosti udjela energije i proteina u obrocima mliječnih krava, međutim ne u tolikoj mjeri u obrocima mliječnih koza. Cilj ovog istraživanja bio je utvrditi povezanost između koncentracije uree u mlijeku i udjela sirovih proteina (SP) u obroku Alpina koza kako bi se smanjilo prekomjerno izlučivanje dušika iz organizma. Istraživanje je provedeno u stadu od 72 koze koje su slučajnim odabirom podijeljene u tri jednake skupine (24 koza/skupini) od kojih je svaka hranjena krmnom smjesom (KS) s različitim udjelom SP: 14 %, 16 % i 18 %. Prikupljeni uzorci mlijeka poslužili su za utvrđivanje osnovnog kemijskog sastava nakon čega je uslijedilo izračunavanje omjera mliječne masti i proteina. Statistička analiza provedena je korištenjem procedure MIXED računalnog programa SAS V8. Koze hranjene KS sa 16 % SP dnevno su proizvele 0,4 kg mlijeka više (p

BACKGROUND: Bacteria from Staphylococcus and Streptococcus genera are frequent causes of mastitis in dairy cows. We hypothesized that the quantitative changes in the milk proteome differed depending on presence of Staphylococci or Streptococci. METHODS: We analyzed 118 milk samples from cows with subclinical mastitis, clinical mastitis, and positive controls (group with normal somatic cell count and positive bacterial culture), classified into the groups with Staphylococci (SPH, N=51) or Streptococci (STP, N=67). Sample preparation included proteins՚ reduction, alkylation, precipitation using acetone, digestion with trypsin, and labeling with tandem mass tags. The liquid chromatography-tandem mass spectrometry analysis included separation using the linear gradient and scanning in the positive ion mode using the data-dependent analysis Top8 method, on a modular system consisting of Ultimate 3000 RSLCnano system (Dionex®) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific®). The proteome Discoverer® software was employed for proteins՚ identification and relative quantitation following the SEQUEST algorithm. Further, the data were analyzed with the R statistical package and MedCalc® software. RESULTS: Among the 386 identified proteins, 20 had different abundance in SPF and STP groups. Eleven proteins had a higher abundance in the SPF group. They included apolipoprotein E, a regulator of protein kinase C, regulators of the coagulation cascade, complement activity, antigen binding, inflammatory response, etc. Among nine proteins that were more abundant in the STP group, the most important were: apolipoprotein A1, coagulation cascade proteins, and proteins involved in cell proliferation, ion transport, homeostasis of thiol components, antigen processing, and presentation. Furthermore, three proteins showed the highest potential to differentiate between the SPF and STP groups with satisfactory accuracy. Those were protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I, which all had higher abundance in the SPF group. CONCLUSION: The milk proteome pattern differs depending on bacterial culture in milk. Further studies will additionally address pathophysiological changes and clinical utility of the observed differences.

Objective: The occurrence of bovine mastitis (inflammation of the mammary gland) is considered to be one of the biggest challenges on dairy farms, due to reduction of the animal welfare and milk production and, consequently, economic loss for the farm. Therefore, the diagnosis of mastitis at subclinical stage and early treatment is of the utmost importance. However, the lack of clinical symptoms and unambiguous biomarkers for the diagnosis in early disease stage, prevent further advancements. Thus, the objective of this study was to implement the quantitative tandem mass tag (TMT) approach in combination with statistical and bioinformatics data processing for determination of quantitative changes in milk proteomic profiles during development and progression of mastitis on dairy farms. Methods: Milk samples (N = 138) were aseptically collected on dairy farms after physical examination and before the milking. Based on the results obtained for somatic cell count (SCC) and bacterial culture (BC) examination, milk samples were classified into experimental groups: healthy positive control (group with normal SCC and positive BC, N = 20), healthy negative control (group with normal SCC and negative BC, N = 20), subclinical mastitis (moderately increased SCC and positive BC, N = 80), clinical mastitis (high SCC and positive BC, N = 18). Samples were processed using TMT label-based quantitative approach. High resolution LC-MS/MS analysis was carried out using the Ultimate 3000 RSLCnano system (Dionex, Germering, Germany) coupled to Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Protein identification and quantification were performed using Proteome Discoverer software (version 2.3., Thermo Fisher Scientific) with SEQUEST algorithm implemented and database search against Bos taurus FASTA files. Statistical and bioinformatic analysis were done using R and Cytoscape. Results: Altogether, 386 quantifiable proteins were identified. There were 145 proteins with statistically different abundance between the groups. Gene Ontology analysis of proteins with significantly altered abundances showed that milk proteins were involved in biological adhesion, immune system process, biological regulation, interspecies interaction between organism, response to stimulus, signalling and others. Based on the statistical and fold change difference between investigated groups complement component 3, cathelicidin-1, cathelicidin-7, lactoferrin, haptoglobin, and chitinase-3-like protein were identified as potential biomarkers for subclinical mastitis. Conclusion: Shotgun TMT-based high-resolution proteomic profiling allowed identification of potential milk biomarkers for differentiation of mastitis grade in dairy cows. After validation of selected proteins, these findings might provide a valuable contribution to understanding of mastitis pathophysiology and might be applicable to the development of a new cow-side diagnostic tool.

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline., Brzo-rastuće bakterije iz roda Mycobacterium izdvojene se iz dva klinički slična slučaja mastitisa krava u relativno kratkom vremenu. Mikobakterije su izdvojene na krvnom agaru s dodatkom eskulina, obojene po Grammu i Ziehl-Nielsenu te im je provjerena sposobnost rasta pri 25 °C, 37 °C i 42 °C, korištenje željeza, sposobnost rasta na Löwenstein-Jensen agaru bez i s dodatkom 5 % NaCl, tvorba arilsulfataze, sposobnost hidrolize tween 80, redukcije telurita i tvorbe nitrat reduktaze i niacina. Molekularna identifikacija izolata provedena je korištenjem testova Mycobacteria GenoType CM i AS. Odsječci gena hsp65, rpoB, 16S rRNK te tzv. (engl. transcribed spacer sequence (ITS)) jednog izolata su sekvencirani. Osjetljivost izolata provjerili smo pomoću SENSITITRE RAPMYCOL ploča (TREK Diagnostic Systems Ltd.) prema trimetoprim/sulfametoksazolu, linezolidu, ciprofloksacinu, imipenemu, moksifloksacinu, cefepimu, cefoksitinu, amoksicilinu s klavulanskom kiselinom, amikacinu, ceftriaksonu, doksiciklinu, minociklinu, tigeciklinu, tobramicinu i klaritromicinu. Gram-pozitivne acido-rezistentne štapićaste bakterije uočili smo u obojenim razmascima. Oba soja rasla su pri temperaturama 25 °C, 37 °C i 42 °C te na podlozi LJ s i bez dodatka 5 % NaCl. U oba soja utvrdili smo sposobnost korištenja željeza, tvorbe arilsulfataze, hidrolize Tween 80, redukcije telurita i tvorbe reduktaze nitrata. Sojevi nisu tvorili niacin. Oba izolata su testom GenoType Mycobacterium CM identificirana kao Mycobacterium fortuitum II/ Mycobacterium mageritense dok su GenoType Mycobacterium AS testom identificirana kao Mycobacterium smegmatis. Sekvencijskom analizom odsječka gena 16S ribosomske RNK soja DS i usporedbom s javno dostupnim izolatima, utvrdili smo potpunu podudarnost sa sojem Mycobacterium smegmatis soj INHR2. Prema CLSI kriterijima, oba izolata su osjetljiva prema sulfametoksazolu s trimetoprimom, linezolidu, doksiciklinu, amikacinu i tobramicinu, a rezistentna prema klaritromicinu. Razlike između sojeva očitovale su se u osjetljivosti prema cefoksitimu i tigeciklinu.

Bakterija S. aureus najčešći je uzročnik mastitisa krava kod nas i u svijetu. Prevalencija infekcije prouzročene tom bakterijom kreće se od 2 do više od 50%. Meticilin-rezistentni stafilokoki kao uzročnici mastitisa krava prvi su put potvrđeni 1972. godine. Lančana reakcija polimeraze se koristi za identifikaciju i tipizaciju S. aureus, a temelji se na umnažanju spa gena karakterističnog za pripadnike vrste S. aureus. Potvrda otpornosti S. aureus na meticilin (MRSA) dokazuje se umnažanjem gena mecA i mecC dok prisutnost PVL ili LukF PV gena ukazuje na sposobnost bakterije za sintezu čimbenika virulencije, odnosno Panton-Valentine leukocidina. Cilj istraživanja bio je potvrditi vrstu, utvrditi otpornost prema meticilinu te virulentnost bakterije. U istraživanje je uključeno 237 sojeva vrste S. aureus izdvojene iz mlijeka krava sa subkliničkim mastitisom s područja 16 hrvatskih županija u razdoblju 2015-2019 godine. Izdvajanje DNK iz uzoraka provodili smo komercijalno dostupnim kitom QIAcube DNA Mini Kit (QIAGEN, Hilden, Njemačka) prema uputama proizvođača. Korištena je metoda koju preporučuje Referentni laboratorij EU za antimikrobnu rezistenciju (EURL- AR). Od 237 pretražena soja, u 204 (86, 1 %) soja bio je prisutan spa fragment. U pet pretraženih sojeva utvrđen je gen mecA, kao i mecC gen koji je također utvrđen je u 5 pretraženih sojeva. PVL gen nije detektiran ni u jednom pretraženom soju. Unatoč niskoj pojavnosti mec gena (4, 2 %), mlijeko inficiranih krava i bliski kontakt s inficiranim životinjama predstavljaju opasnost za ljudsko zdravlje

Mastitis je među najčešćim bolestima na farmama mliječnih krava za čije se liječenje i prevenciju koristi najveća količina antibiotika. Učestala, neispravna ili nepotrebna uporaba antimikrobnih sredstava predstavlja veliku prijetnju ubrzanom razvoju antimikrobne rezistencije. Prijenos antimikrobno rezistentnih patogena na ljude moguć je putem mlijeka i mliječnih proizvoda te su propisane smjernice za racionalnu primjenu antimikrobnih sredstava u veterinarskoj medicini. U mliječnoj se industriji najviše antibiotika koristi prilikom zasušivanja krava u svrhu prevencije nastanka mastitisa. U isto vrijeme antibiotici se nepotrebno primjenjuje i u životinja koje nisu inficirane. Iz tog razloga sve više zemalja prelazi na model selektivnog zasušivanja krava. Antibioticima se zasušuju samo inficirane četvrti ili vimena krava, odnosno krave s povećanim rizikom za nastanak infekcije. Jedan od većih problema prilikom uvođenja selektivnog zasušivanja krava je odabir kriterija prema kojem će se odabrati krave koje će biti zasušene antibiotikom. Protokoli za odabir krava trebaju biti dovoljno precizni, jednostavni za interpretaciju, jeftini, sigurni, certificirani i dostupni za uporabu u svim dijelovima zemlje. Kriterij za odabir krava može se temeljiti na mikrobiološkoj pretrazi mlijeka, određivanju BSS prilikom zasušivanja, incidenciji kliničkih mastitisa u laktaciji, paritetu i njihovim međusobnim kombinacijama. Bakteriološka je pretraga mlijeka najpouzdaniji pokazatelj, ali se broj somatskih stanica na posljednjoj mužnji ističe kao pouzdan pokazatelj na temelju kojeg se može provesti selektivno zasušenje bez negativnih učinaka na proizvodnju. Granična vrijednost broja somatskih stanica kao učinkovit i održiv model za donošenje odluka iznosi 100.000 st/mL mlijeka za primiparne i 200.000 st/mL mlijeka za multiparne krave. Uporaba antibiotika za zasušenje samo kod inficiranih četvrti krava omogućava dodatno smanjenje primjene antibiotika. Iako je tijekom godina postignut značajan napredak u razumjevaniju epidemiologije, imunologije, dijagnostike i patogeneze intramamarnih infekcija (IMI) u suhostaju u programima suzbijanja mastitisa naglašava se značenje smještaja krava u suhostaju i prijelaznom razdoblju. Važnost kontrole IMI u suhostaju u svakodnevnoj praksi se često podcjenjuje., Mastitis is the most common disease in dairy cows and most antibiotics are used for treatment and prevention. Frequent, inappropriate, or unnecessary use of antimicrobials poses a major risk for accelerated development of antimicrobial resistance. Transmission of antimicrobial-resistant pathogens to humans is possible via milk and dairy products, and guidelines for the rational use of antimicrobials in veterinary medicine are prescribed. In dairy farming, most antibiotics are used to prevent mastitis at drying off. At the same time, antibiotics are used unnecessarily even in uninfected animals. An alternative strategy that promotes lower antimicrobial usage is selective dry cow therapy, where only cows with mastitis receive antibiotic treatment. However, an issue in the implementation of selective dry cow treatment is the criteria applied for selecting the cow for drying off with antibiotics. Cow selection protocols should be sufficiently accurate, easy to perform and interpret, cost-effective, safe, certifiable, and applicable in different areas of a country. The criteria for cow selection can be based on bacteriological culture of milk, somatic cell count at drying off, incidence of clinical mastitis in previous lactation, parity, or any of their combinations. Bacteriological culture of milk is the gold standard, though somatic cell count at the last milking has proven to be a reliable indicator for cow selection to drying off with antibiotics without negative effects on milk production. The thresholds of

Sojevi bakterije S. aureus otporni na meticilin (MRSA) predstavljaju sve veći problem u humanoj i veterinarskoj medicini. Sve je više znanstvenih dokaza da se pojedini sojevi stafilokoka mogu prenositi sa životinja na ljude i obrnuto. Cilj rada bio je utvrditi pripadaju li MRSA sojevi izdvojeni iz mlijeka krava sa subkliničkim mastitisom sekvencijskim tipovima izdvojenima iz ljudi. U radu je istraženo 10 sojeva MRSA izdvojenih iz mlijeka krava s mastitisom. Prisutnost gena mecA i mecC utvrđena je metodom multiplex PCR. Sojevi nositelji mec gena tipizirani su sekvenciranjem na više lokusa (MLST), odnosno umnažanjem 7 house- keeping gena te određivanjem sljedova baza u umnoženim odsječcima gena. Dobiveni sljedovi baza uspoređeni su s izolatima dostupnim u bazama podataka i određen je sekvencijski tip (ST). Analizirani sojevi MRSA pripadali su trima sekvencijskim tipovima. U tip ST97 svrstana su 4 soja, u ST130 tri pretražena soja te u ST398 tri pretražena soja. U literaturi se navodi da je ST130 izdvojen iz slučajeva mastitisa u manjih glodavaca, a ST97 je najčešće utvrđeni uzročnik goveđeg mastitisa. Sekvencijski tip ST398 je prvi i najčešće spominjani alelni profil MRSA izdvojen iz ljudi i povezan sa stokom (livestock associated LA-MRSA). Za potpunije razumijevanje uloge MRSA sojeva u infekcijama ljudi, pored životinja na farmi, u istraživanje bi trebalo uključiti i ljude koji dolaze u kontakt sa životinjama (vlasnike, timaritelje, muzače).

The consequences of infection by Suid herpesvirus type 1 (SuHV-1) that causes Aujeszky’s disease (AD) are well studied, however, the effects of immunomodulators (IMs) of microbial origin (viral and bacterial) when administered solely or in combination with the attenuated SuHV-1 vaccine are less known. The effects of parenteral administration of IMs, inactivated Parapoxvirus ovis (P. ovis) or a combination of inactivated Propionibacterium granulosum (P. granulosum) and detoxified Escherichia coli lipopolysaccharide (LPS) and attenuated SuHV-1, strain Bartha, on the proportion of peripheral blood CD3- CD21+ B cells were analysed in 30 crossbred, 3-month old pigs using flow cytometry (FCM). Specific antibodies for gE and gB of SuHV- 1 were detected using the enzyme-linked immunosorbent assay (ELISA). Data were compared among six experimental groups: (1) pigs that separately received the vaccine, (2) IM of bacterial origin, (3) IM of viral origin, (4) simultaneous administration of the vaccine and bacterial IM, (5) simultaneous administration of the vaccine and viral IM, and (6) the control group of untreated pigs. Comparison of B cell proportions and the detection of specific antibodies in blood samples of vaccinated pigs on Day 11 of the experiment showed a transient decrease in B cell contents, though this could not be assumed to be related since the control group showed a decrease in B cell proportion on the same day. The results showed that the use of IMs alone or in combination with the attenuated SuHV-1 vaccine did not have a significant impact on the proportion of peripheral blood B cells in growing pigs., Posljedice infekcije herpes virusom-1 svinja (SuHV-1) uzročnikom bolesti Aujeszkoga (BA) dobro su istražene, ali, manje je poznat učinak imunomodulatora (IM) mikrobnog podrijetla (virusnog ili bakterijskog) primijenjenih u kombinaciji s atenuiranim cjepivom protiv BA. Učinak parenteralne primjene IM-a, inaktiviranog virusa Parapoxvirus ovis ili kombinacije inaktivirane bakterije Propionibacterium granulosum i lipopolisaharida bakterije Escherichia coli i cjepiva koje sadržava atenuirani virus BA (SuHV-1, soj Bartha) na udio CD3-CD21+ B limfocita periferne krvi analiziran je protočnom citometri¬jom u krvi 30 komercijalnih križanaca svinja u dobi od tri mjeseca bez protutijela za virus BA. Prisustvo je protutijela za glikoproteine B (gB) i E (gE) virusa BA provjereno imunoenzimnim testom (engl. Enzyme Linked Immunosorbant Assay, ELISA). Dobiveni su rezultati uspoređeni između 6 pokusnih skupina tj. između svinja koje su primile: cjepivo (1. skupina), IM bakterijskog podrijetla (2. skupina), IM virusnog podrijetla (3. skupina), svinja koje su istovremeno primile cjepivo i IM bakterijskog podrijetla (4. skupina) ili cjepivo i IM viruisnog podrijetla (5. skupina) te netretirane, kontrolne skupine svinja (6. skupina). Usporedba udjela B limfocita u uzorcima krvi cijepljenih svinja 11. dan pokusa pokazali su prolazno smanjenje sred¬nje vrijednosti udjela B limfocita za koje se nije moglo pretpostaviti da je povezano s pojavom specifičnih protutijela, jer je smanjenje srednje vrijednosti udjela B limfocita bilo i u kontrolnoj skupini istoga dana. Naši su rezultati pokazali da primjena samo IM-a, kao i njihovih kombinacija s atenuiranim cjepivom protiv BA, nisu imali značajan utjecaj na udio B limfocita periferne krvi u tovljene prasadi.

Bovine mastitis is a serious disease with great impact to dairy industry and animal welfare. This study was aimed to characterize milk proteome in naturally occurring subclinical and clinical mastitis. Milk samples from healthy Simmental cows (H, n=10) were compared to those with subclinical (SC, n=12) and clinical mastitis (CL, n=10) using tandem mass tag (TMT) proteomics. Data were analysed using Proteome Discoverer software (version 2.3., Thermo Fisher Scientific). 237 proteins were changed in abundance in both SC and CL showing different change patterns. The greatest fold changes were found in haptoglobin, serum amyloid A, beta-defensin, and serpins B3 and B1 with significant increase in both SC and CL compared to H although without significant difference between SC and CL. Proteins showed a significant increase in SC compared to H and a further significant increase in CL included alpha2-macroglobulin, immunoglobulin heavy chain, pregnancy zone protein and cathelicidin-3. There were also proteins that did not increase in SC but increased in CL including a different variant of alpha2-macroglobulin, calponin cathelicidin-1 and apolipoproteins A1 and A2. Some milk proteins decreased in response to mastitis, i.e. proteins that showed no change in SC but demonstrated a significant decrease in abundance in CL compared to healthy milk with the greatest reduction being shown by thrombospondin along with alphaS2-casein, beta-lactoglobulin and beta-casein among others. Proteins showed significant decreases in SL and further decreases in CL included an ATP-binding transporter, mucin-1 and butyrophilin. Alteration of abundance patterns of milk proteins indicate multiple protein interaction involved in complex reactions of host immune defence, acute phase response, coagulation cascade and amino acid metabolism. Identification of milk proteome is a valuable strategy for revealing potential biomarkers to be used in mastitis diagnostics, disease monitoring and therapeutic possibilities.

Sigurnost i kvaliteta mlijeka te dobrobit životinja koje se uzgajaju radi proizvodnje mlijeka pobuđuju sve veće zanimanje opće i stručne javnosti (EFSA 2009., 2009a., 2012., 2012a., 2015). Uzimajući u obzir takve okolnosti Hrvatska agencija za poljoprivredu i hranu provela je istraživanje koje je imalo za cilj utvrditi stanje dobrobiti krava na farmama u Republici Hrvatskoj, utvrditi sigurnost i kvalitetu mlijeka te njihovu međusobnu povezanost. Za procjenu dobrobiti korištena je metoda talijanskog nacionalnog referentnog centra za procjenu dobrobiti CReNBA (Bertocchi i Fusi, 2014.). Za provođenje istraživanja odabrano je šest farmi, a uzorci za laboratorijske pretrage uzeti su od životinja u drugoj i trećoj laktaciji. Procijenjena je dobrobit na farmama, analizirano je mlijeko na kemijske i mikrobiološke pokazatelje , utvrđen je broj somatskih stanica u mlijeku brzim testovima i laboratorijskom metodom te su određeni biokemijski pokazatelji u krvi krava. Prema ocjenama dobrobiti dvije farme pripadaju gornjem ocjenskom razredu, a ostale sred njem. S obzirom da protokol procjene dobrobiti uvažava ocjenu o životinji, infrastrukturi i menadžmentu, a broj somatskih stanica samo je jedan od pokazatelja dobrobiti, nije utvrđen utjecaj dobrobiti na broj somatskih stanica. Utvrđeno je da je porasla svijest o važnosti provođenja mjera dobrobiti , dok mnoštvo pokazatelja ukazuje da se implementiraju suvremena znanja o hranidbi, kvaliteti vode i zraka u objektima, kao i o postupanju sa životinjama. Međutim, utvrđeno je da još uvijek ima prostora za poboljšanje u kontekstu povećanja površina ležišta po grlu. Evidentno je da postojeća kontrola kvalitete mlijeka mjerenjem broja somatskih stanica mlijeka iz četiriju četvrti zajedno, može maskirati pravo stanje zdravlja vimena. Visoki broj somatskih stanica u jednoj četvrti može biti prikriven(smanjen)ukupnim brojem somatskih stanica (lažno negativni rezultat), a oboljenje neće biti detektirano na vrijeme. Mikroorganizmi uzročnici mastitisa, identificirani su neovisno o broju zahvaćenih četvrti, podjednako u slučajevima kliničkog i supkliničkog mastitisa. Ako se promatra raspodjela povišenog broja somatskih stanica prema četvrtima (>400 000), vidljivo je kako je 34 % krava imalo zahvaćenu jednu četvrt, 22 % dv ije četvrti, 11 % tri četvrti, a 5 % krava imalo je upalu u sve četiri četvrti vimena. Potvrđeno je da su postojeći brzi testovi prikladni za detekciju mastitisa, a u otkrivanju supkliničkog mastitisa valja razviti osjetljivije testove.

Cilj istraživanja bio je odrediti utjecaj količine oborina i temperature zraka na rasplodnu učinkovitost solčavsko-jezerske pasmine u sjeverozapadnom dijelu Hrvatske tijekom sedam uzastopnih godina (2013. – 2019.). Zabilježeno je 2026 ovaca koje su se parile, od kojih je 1876 uspješno koncipiralo i ojanjilo 2321 janjadi. Prosječna veličina legla bila je 1,23. Prosječna plodnost tijekom istraživanja iznosila je 92,31 %. Distribucija janjenja nije bila ravnomjerno raspoređena tijekom godine jer se tijekom zime i proljeća ojanjilo 75 % svih ovaca (zima 52 %; proljeće 23 %; ljeto 8 % i jesen 17 %). Spolna aktivnost najniža je bila od kraja ožujka do lipnja, dok je njezin vrhunac bio od kraja srpnja do listopada. Utvrdili smo pozitivnu ili negativnu korelaciju (P < 0,0001; Kruskall Wallis) između temperature zraka i količine oborina s određenim reproduktivnim svojstvima (broj gravidnih i negravidnih ovaca). Možemo pretpostaviti da temperatura zraka i količina oborina tijekom različitih mjeseci ili godišnjih doba utječu na rasplodnu učinkovitost solčavsko-jezerskih ovaca u sjeverozapadnoj Hrvatskoj., The study aims was to determine the influence of rainfall precipitation and air temperature on the reproductive efficiency of the Jezerska-Solčava breed in NW Croatia for 7 consecutive years (2013-2019). A total of 2026 sheep were mated, of which 1821 successfully conceived and 2321 lambs were born. The average litter size was 1.23. The average fertility during the study was 92.31%. The distribution of lambs was not equally distributed throughout the year because 75% of all sheep were born during winter and spring (winter 52%; spring 23%; summer 8% and autumn 17%). Sexual activity was the lowest from the end of March to June, while the peak of sexual activity was from the end of July to October. There was a positive or negative correlation (p

Cilj ovog rada bio je utvrditi zastupljenost pojedinih oblika vimena u triju mliječnih pasmina ovaca uzgajanih u Hrvatskoj, kao i povezanost oblika vimena s proizvodnjom i kemijskim sastavom ovčjeg mlijeka. U tu svrhu istraživanjem je bilo obuhvaćeno 111 paških ovaca, 93 istarske ovce te 80 istočnofrizijskih ovaca. Tijekom razdoblja mužnje u istraživanim stadima provedena je redovita mjesečna kontrola mliječnosti ovaca primjenom AT metode, dok je prosudbu vimena prema obliku u svih ovaca obavila ista osoba jednom tijekom laktacije, i to prilikom provedbe prve kontrole mliječnosti (nakon odvajanja janjadi). U paških ovaca utvrđena su jasno (izražajno) različita četiri oblika ili tipa vimena (tipovi I, II, III i IV), u istarskih ovaca determinirano je postojanje triju oblika vimena (tipovi II, III i IV), dok su u istočnofrizijskih ovaca utvrđena dva oblika vimena (tipovi II i III). U sve tri istraživane pasmine najzastupljenije su bile ovce s vimenom tipa III. Neovisno o pasmini, u ovaca s nisko postavljenim i (gotovo) vertikalno položenim sisama te s jasno izraženom podužnom brazdom između polovica vimena (tipovi vimena III i IV) utvrđena je veća proizvodnja mlijeka nego u ovaca s visoko postavljenim i horizontalno položenim sisama (tipovi vimena I i II). Međutim, utvrđene razlike u proizvodnji mlijeka između ovaca različitog oblika vimena bile su značajne (P, The aim of this study was to determine the prevalence of individual udder shapes in three dairy sheep breeds, as well as the relationship of udder shape with the production and chemical composition of sheep's milk. For this purpose, the research included 111 Pag sheep, 93 Istrian sheep and 80 East Friesian sheep. During the milking period in all flocks included in the research, milking control was carried out using the AT method, while the udder shape assessment in all studied ewes was carried out by the same person once during lactation when conducting the first milking control (after weaning of lamb(s)). In Pag sheep four distinct forms or types of udder (types I, II, III and IV) were clearly identified, in Istrian sheep the existence of three udder shapes (types II, III and IV) was determined, while in East Friesian sheep were found two udder shapes (type II and III). In all three breeds the most frequent were ewes with the udder type III. in all three breeds, higher production of milk was observed in sheep with low and (almost) vertically positioned teats and with a pronounced longitudinal furrow between the udder halves (udder types III and IV) than in sheep with highly placed and horizontally positioned teats (udder types I and II). However, differences in milk production between sheep of different udder shapes were found to be statistically significant (P

U mliječnoj govedarskoj industriji posljednjih se godina nameće potreba za alternativnim dodacima u prehrani mliječnih krava radi kvalitetnije proizvodnje mlijeka i boljega zdravlja plotkinja. Cilj ovoga istraživanja bio je ustvrditi učinkovitost dodavanja vibroaktiviranog i mikroniziranog zeolita klinoptilolita (Vibrosorb®, Podpićan, Hrvatska) u hrani na kemijski sastav mlijeka i zdravlje vimena mliječnih krava. U istraživanje je uključeno 30 mliječnih krava holštajnske pasmine, dobi od 3 do 5 godina, gravidnih tri mjeseca u početku provedbe istraživanja. Životinje su bile držane na obiteljskom gospodarstvu u okolici Đurđevca u Hrvatskoj. Krave su nasumično podijeljene u dvije skupine: kontrolnu (KON, n = 15) i pokusnu (KPL, n = 15), koja je u obroku za mliječne krave svakodnevno dobivala i 100 g KPL-a. Prvo uzorkovanje mlijeka učinjeno je prije početka dodavanja KPL-a. Ostala četiri uzorkovanja provedena su tijekom četiri mjeseca do suhostaja, tj. sedmog mjeseca gravidnosti. Uzorci mlijeka analizirani su s obzirom na kemijski sastav mlijeka (mliječna mast, bjelančevine, laktoza, bezmasna suha tvar i ureja), broj somatskih stanica (BSS) i mikrobiološkom pretragom. Statističkom obradom ustvrđeno je da su prosječne vrijednosti kemijskog sastava mlijeka bile podjednake u obje skupine tijekom svih mjeseci, sa zanemarivom razlikom u 5. mjesecu u skupini KPL za mliječne bjelančevine. Statistički značajna razlika dobivena je za BSS (P < 0,05). Skupina KPL imala je značajno manji prosjek BSS-a u usporedbi sa skupinom KON (P < 0,05). KPL-om hranjena skupina imala je za 50 % nižu pojavu supkliničkog mastitisa u odnosu na skupinu KON. Dobiveni rezultati upućuju na imunostimulacijski i učinak vibroaktiviranog i mikroniziranog klinoptilolita kao dodatka prehrani na zdravlje vimena mliječnih krava., In recent years, there has been wider usage of alternative substances in the diet for improved performance and udder health of dairy cows. This study aimed to determine the efficacy of the addition of vibroactive and micronized zeolite clinoptilolite (Vibrosorb®, Podpićan, Croatia) to feed regarding the chemical composition of milk, somatic cell count (SCC) and the udder health of dairy cows. The research involved 30 dairy cows of the Holstein breed, aged between 3 to 5 years, and 3 months pregnant at the start of the trial. The animals were kept on a family farm near Đurđevac, Croatia. The cows were randomly assigned into two groups: control (n=15) and test (CPL-fed group) (n=15), which received 100 g of CPL in daily feed. The first milk sampling was done prior to adding CPL to the diet. The other four samples were performed monthly up to the dry period, i.e., the seventh month of pregnancy. Milk samples were analysed for the chemical composition of the milk (milk fat, protein, lactose, non-fatty dry matter, and urea), somatic cell count (SCC) and microbiological examination. Statistical analysis revealed that the average values of the chemical composition of the milk were almost the same in both groups throughout the research, with an insignificant difference in milk protein in the fifth month of research (P>0,05). A statistically significant difference was found in the SCC; the CPL group had a significantly lower average value of SCC as opposed to the control group (P

Fiziološke koncentracije spolnih hormona 17β-estradiola i testosterona varijaju s obzirom na vrstu životinje, dob, spol, pasminu, način držanja, hranidbu, stres i okolišne čimbenike. Navedeni hormoni su nužni za rast i razvoj životinje te njihovu spolnu zrelost i reproduktivnu funkciju. Zbog svog anaboličkog učinka koristili su se u prošlosti u intenzivnoj stočarskoj proizvodnji kao promotori rasta, putem stočne hrane ili u obliku implatanta. No, zbog svojih toksičnih učinaka u životinja i ljudi, uključujući mutagenost, kancerogenost i teratogenost, njihova je uporaba zabranjena u svrhu proizvodnje hrane životinjskog podrijetla. Danas je dopušteno korištenje prirodnih hormona samo u terapeutske svrhe za liječenje poremećaja reprodukcije i gravidnosti. S obzirom na stalni razvoj novih sintetskih tvari koje bi mogle imati anabolički učinak i mogu zlouporabu, nužno je tijekom redovitog provođenja kontrole te poznavanja fizioloških vrijednosti razina ovih hormona. Cilj ovog rada, uzimajući u obzir sve navedeno, bilo je istražiti razine prirodnih hormona 17β-estradiola i testosterona u krvi teladi različitog spola, pasmina, dobi i farmskog podrijetla na području Republike Hrvatske. U tu svrhu s tri različite farme na području Republike Hrvatske nasumično su izabrana 32 teleta različitog spola (5 muških i 27 ženskih), dobi 2 ‒ 5 mjeseci starosti, različitih pasmina i kategorija (13 teladi simentalske pasmine, 2 teladi mesne pasmine, 17 teladi holštajnske pasmine). Prilikom općeg kliničkog pregleda životinje od strane nadležnog veterinara, nisu primijećeni nikakvi znaci bolesti ili stanja koje bi mogle utjecati na razine ovih hormona. Prosječna razina 17β-estradiola iznosila je 28,8±11,0 ng/L. Koncentracija testosterona bila je niža od limita detekcije primjenjene analitičke metode u 24 uzorka plazme teladi, a detektirana je u 8 uzoraka s prosječnom koncentracijom od 126,7±167,5 ng/L, ukazujući na široku varijabilnost u koncentracijama ovog hormona. Statistički značajna razlika u koncentracijama određena je jedino za 17β-estradiol s obzirom na pasminski sastav i farmu podrijetla (P, Physiological levels of the sex hormones 17β-oestradiol and testosterone vary based on animal species, age, sex, breed, rearing technology, nutrition, stress and environmental factors. These hormones are essential for growth and development, and for sexual maturity and reproductive function. Due to their anabolic effects, they have been used as growth promoters in intensive livestock production, applied in feed or as an implant. However, due to their toxic, mutagenic, teratogenic and cancerogenic effects in animals and humans, their use is now banned in food animal production. Today, the use of these natural hormones is permitted exclusively for therapeutic purposes in the treatment of reproductive and pregnancy disorders. Considering the constant development of new synthetic substances that might have anabolic effects and their possible misuse, it is necessary to perform regular controls during the fattening period to determine the physiological values of the levels of these hormones. Accordingly, the aim of this study was to investigate the physiological levels of the natural hormones 17β-oestradiol and testosterone in the blood of calves of different sex, age, breed and farm origin in the Republic of Croatia. For this purpose, 32 calves of both sexes (5 males and 27 females), aged 2-5 months, of different breeds and categories (13 calves of Simmental breed, 2 calves of meat breed and 17 calves of Holstein breed) were randomly selected from three farms for inclusion in this study. During general clinical examination by an authorized veterinarian, no visible clinical changes or conditions were observed that could influence the physiological levels of the investigated hormones. The average level of 17β-oestradiol was 28.83±10.95 ng/L. The concentration of testosterone was lower than the detection limit of the analytical method applied in 24 samples of calf plasma. In eight samples in which this hormone was detected, the average concentration was 126.7±167.5 ng/L, indicating wide variability in the levels of this hormone. A statistically significant difference was determined only for 17β-oestradiol, taking into account the composition of breeds tested (P