Klinički i ultrazvučni pregled vimena krava nakon primenelaktoferina u periodu involucije

Kontrola zdravlja vimena krava je bitan element u procesu proizvodnje zdravstveno bezbednog mleka, te se na farmama visokomlečnih krava, kroz program kontrole mastitisa, redovno sprovode mere otkrivanja i prevencije bolesti vimena. Klinički pregled vimena predstavlja osnovni metod koji pruţa korisne informacije o zdravstvenom statusu vimena krava, ali nailazi na poteškoće u otkrivanju patoloških promena unutar parenhima i papile vimena. U cilju otkrivanja promena u parenhimu vimena moţe se primeniti ultrazvučni pregled koji omogućava vizualizaciju strukturnih promena vimena nastalih kao posledica upalnih procesa i tako olakšava dijagnostiku oboljenja. Tokom poslednjih godina, javio se problem povećanja rezistencije bakterija na antimikrobne lekove, što oteţava lečenje bolesti, ali i ugroţava zdravlje ţivotinja i ljudi. Najčešći uzroci toga su nepravilna upotreba i eventualna zloupotreba antimikrobnih lekova. Mnoga istraţivanja vršena su u in vitro i in vivo uslovima na primeni laktoferina samog ili u kombinaciji sa antibioticima u terapiji i prevenciji mastitisa krava. Laktoferin, gvoţĎe vezujući antimikrobni glikoprotein koji se nalazi u mleku i drugim sekretima, predstavlja bitan deo sistema odbrane mlečne ţlezde. Cilj istraţivanja u okviru ove disertacije je procena dijagnostičke mogućnosti ultrazvučnog pregleda u detekciji subkliničkog mastitisa i poremećene sekrecije vimena, kao i razmatranje opravdanosti primene laktoferina u prevenciji i lečenju mastitisa. Kliničkim pregledom izvršena je procena opšteg zdravstvenog stanja krava, kao i ispitivanje mlečne ţlezde adspekcijom i palpacijom. Za otkrivanje poremećene sekrecije vimena i subkliničkih mastitisa korišćeni su brzi testovi, Kalifornija mastitis test i Draminski test, kao i ultrazvučni pregled mlečne ţlezde krava. OdreĎivanje broja somatskih ćelija u uzorcima mleka uraĎeno je metodom protočne citometrije. Za identifikaciju uzročnika mastitisa korišćene su klasične mikrobiološke metode. Krave sa pozitivnim bakteriološkim nalazom podeljene su u dve ogledne grupe. Krave ogledne grupe I su tretirane intramamarnom aplikacijom antibiotika, dok je kravama ogledne grupe II aplikovana kombinacija antibiotika i laktoferina. OdreĎivanje koncentracije imunoglobulina G u mlečnom serumu krava vršeno je metodom radioimunodifuzije, a odreĎivanje koncentracije laktoferina u mleku krava ELISA testom. U istraţivanjima u okviru ove disertacije, najčešće izolovani major mastitis patogeni bile su bakterije Staphylococcus aureus i Streptococcus agalactiae, a najčešće izolovani minor mastitis patogeni Corynebacterium spp. i koagulaza negativne stafilokoke. Ultrasonografija mlečne ţlezde krava pokazala se kao efikasna metoda u dijagnostici poremećaja sekrecije vimena. Veće vrednosti koncentracije imunoglobulina G u mlečnom serumu krava uočene su tokom perioda predzasušenja i zasušenja, u odnosu na period rane laktacije. Najveći uticaj na porast koncentracije laktoferina u mleku krava su imali bakterijski uzročnici mastitisa. Efikasnost antibiotske terapije primenjene tokom perioda zasušenja kod krava ogledne grupe I iznosila je 52,7%, dok je efikasnost primenjene terapije sa goveĎim laktoferinom i antibiotikom kod krava ogledne grupe II iznosila 60%. Aplikacija laktoferina tokom perioda zasušenja doprinela je efikasnosti terapije intramamarnih infekcija, ali nije imala uticaj na koncentraciju laktoferina u mleku tokom perioda rane laktacije. Dalja istraţivanja su neophodna kako bi se u potpunosti potvrdila efikasnost intramamarne terapije sa goveĎim laktoferinom i antibiotikom u cilju eliminacije uzročnika mastitisa i da se precizno odredi terapijska koncentracija laktoferina.Control of udder health is an essential element in the process of safe milk production, thus, through the mastitis control program, dairy farms regularly conduct measures of detection and prevention of udder diseases. Clinical examination of the udder is a basic method that provides useful information about the health status of cow udders, but this method is limited in its ability to detect pathological changes in the udder parenchyma and teat. In order to detect changes in the parenchyma of the udder, an ultrasound can be applied which allows visualization of udder structural changes incurred as a result of inflammatory processes, thus facilitating diagnosis of diseases. Over the last few years, the problem of increasing resistance to antimicrobial drugs has appeared, making it difficult to treat disease and also threaten the health of animals and humans. The incorrect and widespread use of antimicrobial drugs are the most common cause of this. Many studies were conducted in vitro and in vivo conditions on the use of lactoferrin alone or in combination with antibiotics in the treatment and prevention of mastitis. Lactoferrin, an iron-binding antimicrobial glycoprotein which is found in milk and other secretions, represents an important part of the mammary gland defense system. The aim of the research within this thesis was to evaluate the diagnostic possibilities of udder ultrasonography in the detection of subclinical mastitis and udder secretion disorders, as well as considering the feasibility of lactoferrin application in the prevention and treatment of mastitis. The general condition of the animals was evaluated by clinical examination, as well as udder examination by adspection and palpation. The California mastitis test, Draminski test and ultrasound examination of the cow's mammary glands were used for detection of udder secretion disorders and subclinical mastitis. Somatic cell count in milk samples was determined by flow cytometry method. Classical microbiological methods were used for the isolation and identification of mastitis causative agents. Cows with positive bacteriological findings were divided into two experimental groups. Cows in experimental group I were treated with intramammary applications of antibiotics, while the cows in experimental group II were treated with a combination of antibiotics and lactoferrin. Immunoglobulin G concentration in bovine milk serum was determined by the immunodiffusion method, while lactoferrin concentration in bovine milk was determined using the ELISA test. In this study, the most common isolated major mastitis pathogens were Staphylococcus aureus and Streptococcus agalactiae, while Corynebacterium spp. and coagulase-negatice staphilococci were the most commonly detected minor mastitis pathogens. Ultrasonography of the bovine mammary gland proved to be an effective method in the diagnosis of udder secretion disorders. Higher immunoglobulin G concentrations were observed in milk serum from cows during pre-dry and dry period, relative to early lactation period. The biggest influence on the increase in the concentration of lactoferrin in the bovine milk had mastitis pathogens. The efficacy of antibiotic therapy during the dry period in cows of experimental group I was 52.7%, while the efficacy of the applied therapy with lactoferrina and antibiotics in cows of experimental group II was 60%. Application of lactoferrin during the dry period contributed to the effectiveness of the treatment of intramammary infections, but had no influence on lactoferrin concentration in the milk during the early lactation period. Further studies are necessary to in order to fully confirm the efficacy of intramammary therapy with bovine lactoferrin and antibiotic to eliminate the mastitis pathogens and to determine the therapeutic concentration of lactoferrin

Transmission of Coxiella burnetii to Calves from Infected Cows

Background:Coxiella burnetii is the causative agent of a very important disease with zoonotic potential. Infected cows represent risk for spreading of infection to humans and to other animals on farm and also to their offspring. There is possibility for calves from infected cows to be infected nearly after parturition or during intrauterine life. Studies have shown that Coxiella burnetii initially infects the placenta and subsequent spread to the fetus may occur either by haematogenous or by the amniotic-oral route providing congenital infection. The main objective of the present study is to determine the presence of Coxiella burnetii genome in milk serum of infected cows and blood serum of calves.  Materials, Methods & Results: A total of 200 blood serums from dairy cows were tested for presence of antibodies to Coxiella burnetii and nine of those were found positive. These animals compiled experimental group. From animals in experimental group milk samples during lactation, pregnancy and the postpartum period were collected. Samples were used for performing PCR test for determination of Coxiella burnetii presence in milk serum. On calving of each cow blood samples were taken from calves during first 24 hours after calving, from jugular vein. These blood samples were also used for PCR test to determine the presence of Coxiella burnetii. Milk serum analysis showed presence of Coxiella burnetii genome in serum, indicating on intermittent excretion. During lactation, the excretion of bacteria was greatest in the second stage when 80% of milk serum samples were positive for Coxiella burnetii. In the colostrums stage, there was a high percentage of Coxiella burnetii excretion through milk (50% of positive milk serum samples). The lowest percentage of excretion through milk was in the first stage of lactation. Analyzing blood serum samples from calves, taken on first day at calving using PCR method, all serums were positive for presence of Coxiella burnetii genome. Discussion:In animals, Coxiella burnetii is found in the reproductive system, both uterus and mammary glands, and may cause abortion or infertility. The high prevalence of Coxiella burnetii infection in dairy cattle with reproductive problems showed that these infected cattle play an important role in maintaining the infection and in disseminating of pathogenic agent to environment. The lowest percentage of excretion of Coxiella burnetii was in the first stage of lactation, amounting to 16.6%. With the transition to the next stage of lactation, a striking increase in the percentage of excretion was noticed. In the second stage it was 80%. In the third stage there was decrease to 40.6%. In colostrums stage percentage of excretion through milk was 50%. Similar results claims that excretion of Coxiella burnetii through milk starts after eight to twelve weeks of lactation in most cows. This period coincides with second stage of lactation as we divide it. Blood serums taken from calves were proven positive on Coxiella burnetii which indicates on intrauterine infection as described in the literature. Intrauterine infection takes place after placenta infection when bacteria penetrate the placenta and contaminate the amniotic fluid and gets aspirated or swallowed by fetus. Besides this,haematogenous spread can also occur.

Lactoferrin and Immunoglobulin G Concentration in Bovine Milk from Cows with Subclinical Mastitis during the Late Lactation Period

Background: Lactoferrin and immunoglobulin G in milk have an important role in udder resistance to infection in the involution period. Both proteins express antimicrobial activity- lactoferrin by the binding and sequestration of iron ion; and immunoglobulin G by complement activation, bacterial opsonization and agglutination. Many factors affect lactoferrin and immunoglobulin G concentrations in bovine milk, such as the stage of lactation, milk production, and intramammary infections. The aim of this study was to determine concentrations of lactoferrin and immunoglobulin G in milk from healthy cows and subclinical mastitic cows during the late lactation period, and to evaluate the relationship between them.Materials, Methods & Results: A total of 150 quarter milk samples from 41 cows (Holstein-Friesian breed) in late lactation period were reviewed in this study. Milk samples were collected during morning milking, using aseptic techniques in sterile test tubes. From each sample, 0.1 mL of milk was plated on Columbia blood agar base with 5% defibrinated ovine blood, MacConkey agar and Sabouraud dextrose agar and incubated for 24 h - 48 h (bacteria) and 5 days (yeasts, mould) at 37oC. Milk samples for detection lactoferrin and immunoglobulin G concentration were skimmed at 1,400 g for 45 min and stored at -20°C until analysis. Lactoferrin concentration in bovine milk was determined using the Bovine Lactoferrin ELISA Quantitation Set. Milk samples were diluted at a ratio of 1:10,000. Plates were read at 450 nm absorbence values. Immunoglobulin G concentration was determined by the immunodiffusion method using radial immunodiffusion (RID) plates. Milk samples were diluted in a ratio of 1:30. Reading of results was done after incubation for 48 h by measuring the diameter of the precipitation ring. The highest mean lactoferrin concentration was observed in udder quarters infected with contagious pathogens (Streptococcus agalactiae and Staphylococcus aureus), while the highest mean immunoglobulin G concentration was detected in milk samples where minor mastitis pathogens (coagulase-negative staphylococci and Corynebacterium spp.) were isolated. Milk samples where Staphylococcus aureus was isolated had the lowest immunoglobulin G concentration, and the lowest lactoferrin concentration was observed in samples infected with enviromental pathogens (Streptococcus dysgalactiae).Discussion: This study showed that lactoferrin and immunoglobulin G concentrations are higher in milk samples from subclinical mastitic cows than in milk from normal lactating cows. Lactoferrin concentrations in milk samples from udder quarters infected with major mastitis pathogens were significantly higher than in milk infected with minor mastitis pathogens. The lowest concentration of immunoglobulin G was detected in milk samples where Staphylococcus aureus was isolated, while the highest immunoglobulin G concentration was observed in milk samples from quarters infected with minor mastitis pathogens. Lactoferrin and immunoglobulin G concentrations were significantly and positively correlated in all milk samples. This means that cows with high lactoferrin concentrations have high immunoglobulin G concentrations. In quarter milk samples infected with Staphylococcus aureus, lactoferrin and immunoglobulin G concentrations were negatively correlated. The cause of these findings could be the suppression of local immune response of mammary gland

Biosecurity and mastitis in intensive dairy production

Newly purchased animals that enter a herd with high milk production can be infected with pathogens of the mammary gland and are a potential risk of infection to the cows on the farm. This risk cannot be avoided entirely, but it can be minimized by taking biosecurity measures that should be written as a policy developed for biosecurity oversight of veterinary service: when older cows are purchased, they should be bought with complete lactations and SCC records, and bacterial examination of milk from the udder quarters must be negative for pathogens of the udder; newly purchased cows should come from herds in which the geometric mean somatic cell count is less than 200,000. The herd must have individual cow SCC recorded at least bimonthly for the previous 6 months; the herd must not have had any history of Strep. agalactiae infection in the last 2 years, the herd should be BVDV-free or vaccinated, and the herd owner must be honest and willing to provide all this information. Our country has accepted the standards for milk quality and hygienic properties that comply with EU standards. The proposed biosafety measures presented in this paper enable the determination of the health status of the herd and the biosecurity level of mastitis in commercial farming in intensive dairy production. [Projekat Ministarstva nauke Republike Srbije, br. TR-31034

Koncentracija laktoferina u mlijeku krava tijekom involucije mliječne žlijezde s različitim bakteriološkim nalazima.

Lactoferrin is an iron-binding glycoprotein of the transferrin family, present in high concentrations in secretions from the mammary glands during the involution period, and has antimicrobial ability. To determine lactoferrin concentrations in bovine milk with different bacteriological findings, 151 quarter milk samples were collected on a dairy farm of the Holstein-Friesian breed in Vojvodina, Republic of Serbia. Classical microbiological methods were used for bacteria isolation, and ELISA analysis was used for lactoferrin concentration quantification. The most common isolated bacteria in bovine milk samples were Corynebacterium spp. (32.45%) and coagulase-negative staphylococci (4.64%) with lactoferrin concentrations of 6.0497 ± 1.6774 mg/mL and 5.2961 ± 1.3633 mg/mL, respectively. The lowest mean value of lactoferrin concentration was observed in uninfected quarters and quarters infected with environmental pathogens, while the highest concentration of lactoferrin was in udder quarters infected with Streptococcus agalactiae. One in four milk samples where Staphylococcus aureus was isolated had much lower lactoferrin concentrations (1.1736 mg/mL) than the other three samples (6.2089 ± 0.5016 mg/mL), which requires further research.Laktoferin, glikoprotein koji veže željezo, pripadnik porodice bjelančevina transferin, prisutan je u visokoj koncentraciji u sekretu mliječne žlijezde tijekom involucije i posjeduje antimikrobnu sposobnost. Radi utvrđivanja koncentracije laktoferina u mlijeku krava s različitim bakteriološkim nalazima, prikupljen je 151 pojedinačni uzorak mlijeka na farmi holštajn-frizijske pasmine na području Autonomne pokrajine Vojvodine Republike Srbije. Za identifikaciju bakterija korištene su klasične mikrobiološke metode i ELISA za određivanje koncentracije laktoferina. Najčešće izdvojene bakterije u uzorcima mlijeka krava bile su Corynebacterium spp. (32,45%) s koncentracijom laktoferina 6,0497 ± 1,6774 mg/mL i koagulaza negativni stafilokoki (4,64%) s koncentracijom laktoferina 5,2961 ± 1,3633 mg/mL. Najniža srednja vrijednost koncentracije laktoferina zabilježena je u negativnim četvrtima vimena i četvrtima inficiranim bakterijama iz okoliša, dok je najviša koncentracija laktoferina bila u četvrtima vimena inficiranima vrstom Streptococcus agalactiae. Jedan od četiriju uzoraka mlijeka gdje je bio izdvojen Staphylococcus aureus imao je znatno nižu vrijednost laktoferina (1,1736 mg/mL) od ostala tri uzorka (6,2089 ± 0,5016 mg/mL), što zahtijeva daljnja istraživanja

OSNOVNI PRINCIPI TERAPIJE MASTITISA KRAVA

Mastitisi predstavljaju, u savremenom govedarstvu, jedan od najvažnijihzdravstvenih problema kod muznih krava. Pored zdravstvenog aspekta i očiglednog uticaja nadobrobit životinja mastitisi se ubrajaju u najveće finansijske troškove na farmi krava zbog troškovasame terapije i odbačenog mleka zbog karence kao i trajno smanjene proizvodnje mleka ili potpunogprekida laktacije kod teških formi mastisa. Ovome treba dodati i trošak nastao zbog mogućihuginuća ili preranog izlučenja krava iz proizvodnje. Postupak terapije mastitisa i ishod lečenja zaviseod forme zapaljenja vimena, stepena alteracije tkiva i na kraju od vremena početka terapije i primeneadekvatnog preparata ili adekvatnog postupka. Ishod terapije može biti potpuna restitucijaparenhima, oporavak funkcije ili propadanje žlezdanog parenhima i popunjavanje vezivnim tkivom.U današnje vreme, u uslovima intenzivne proizvodnje mleka terapija mastitisa je samo deo programakontrole mastitisa koji u prvi plan stavljaju prevenciju mastitisa. U slučaju pak pojave kliničke formemastitisa neophodno je pristupiti terapiji u laktaciji

TRICHOPHYTOSIS IN BEEF CATTLE

Trichophytosis is one of the most common skin diseases in cattle.Trichophytosis is a transmissible infectious skin disease caused most often byTrichophyton verrucosum with spores which can remain alive for years in a dryenvironment. This is a zoonotic disease, as it can equally infect people and animals.The infection spreads rapidly, and this fungal disease leads to certain economiclosses. Direct contact with infected animals is the most common method of spreadingthe infection. During this research, beef cattle aged 4 months were transported fromsouthern Serbia to the part of Vojvodina - Mačva. One month after the transportation,the characteristic gray-white round changes occured on the skin of beef cattle.Affected animals from five individual farms did not receive any treatment, whileanimals from other five farms were treated with Decanol® solution. As a conclusion,application of therapy or vaccination along with a high quality diet is the best wayto to suppress triphophytosis

BASIC PRINCIPLES OF MASTITIS THERAPY

Mastitis is one of the most important health problems in dairy cows. In addition to thehealth aspect and the apparent impact on animal welfare, mastitis is the largest financial cost in dairyfarms due to treatment costs and rejected milk because of the withdrawal period, as well aspermanently reduced milk production or complete lactation interruption in heavy form of mastitis.The procedure and the outcome of the treatment depend on the form of udder inflammation, degreeof tissue alteration, timing of treatment initiation and the application of adequate preparations or theappropriate procedure. The outcome of therapy can be full restitution of parenchyma and its functionor deterioration of the glandular parenchyma and filling with connective tissue. Nowadays, inconditions of intensive milk production, mastitis therapy is only part of a mastitis control programthat puts mastitis prevention at the forefront. In case of clinical mastitis, it is necessary to applytherapy during lactation

Prevalencija gena za rezistenciju na meticilin i Panton-Valentine leukocidin u izolatima bakterije Staphylococcus aureus podrijetlom od goveda i ljudi.

Using the California Mastitis Test (CMT), on 46 highly productive dairy farms in Serbia, cows with milk secretion disorder were identified. Milk samples were taken from cows with positive CMT and from cows with clinical mastitis. Standard microbiological analysis of milk samples and API Staph confirmed the presence of 75 isolates of Staphylococcus aureus. Those 75 isolates, as well as 11 isolates of Staphylococcus aureus originating from humans were analyzed for the presence of genes encoding Panton-Valentine leukocidin (PVL) and PBP2A protein, responsible for methicillin resistance. The presence of gene encoding PVL was determined by PCR in 5 out of 75 (6.67%) and in 7 out of 11 (63.63%) bovine and human isolates of Staphylococcus aureus, respectively. The presence of the mecA gene was determined by PCR in 1 of 75 (1.33%) and in 2 of 11 (18.18%) bovine and human isolates of Staphylococcus aureus, respectively. The presence of the mecC gene was not determined in analyzed isolates. Further research is needed to investigate the genetic relationship between bovine and human Staphylococcus aureus isolates, to determine the exact impact of bovine Staphylococcus aureus strains from the cow udders on animal and public health.Uporabom kalifornijskog testa za upalu vimena na 46 visoko produktivnih mliječnih farmi u Srbiji identificirane su krave s poremećajem sekrecije mlijeka. Uzeti su uzorci mlijeka krava pozitivnih kalifornijskim testom i krava s kliničkim mastitisom. Standardnom mikrobiološkom pretragom uzoraka mlijeka i identifikacijom kompletom API Staph potvrđena je prisutnost 75 izolata bakterije Staphylococcus aureus. Tih 75 izolata, kao i 11 izolata te bakterije podrijetlom iz ljudi bili su analizirani lančanom reakcijom polimerazom na prisutnost gena koji kodiraju za Panton-Valentine leukocidin (PVL) i PBP2A protein odgovoran za meticilinsku rezistenciju. Prisutnost gena koji kodira za PVL dokazana je u pet od 75 (6,67%) izolata podrijetlom iz goveda i u sedam od 11 (63,63%) izolata podrijetlom iz ljudi. Prisutnost mecA gena dokazana je u jednog od 75 (1,33%) izolata podrijetlom iz goveda i u dva od 11 (18,18%) podrijetlom iz ljudi. Prisutnost mecC gena nije utvrđena u analiziranim izolatima. Potrebna su daljnja istraživanja kako bi se istražio odnos između izolata Staphylococcus aureus-a podrijetlom iz ljudi i iz goveda i odredio točan utjecaj sojeva vrste Staphylococcus aureus iz vimena krava na zdravlje životinja i zdravlje ljudi

High infection rate of zoonotic Eucoleus aerophilus infection in foxes from Serbia

The respiratory capillariid nematode Eucoleus aerophilus (Creplin, 1839) infects wild and domestic carnivores and, occasionally, humans. Thus far, a dozen of human infections have been published in the literature but it cannot be ruled out that lung capillariosis is underdiagnosed in human medicine. Also, the apparent spreading of E. aerophilus in different geographic areas spurs new studies on the epidemiology of this nematode. After the recognition of the first human case of E. aerophilus infection in Serbia, there is a significant merit in enhancing knowledge on the distribution of the nematode. In the present work the infection rate of pulmonary capillariosis was investigated in 70 red foxes (Vulpes vulpes) from the northern part of Serbia by autopsy. The estimated infection rate with Eucoleus aerophilus was 84%. In contrast, by copromicroscopic examination only 38% of foxes were positive. In addition, 10 foxes were investigated for the closely related species in nasal cavity, Eucoleus boehmi, and nine were positive. Our study demonstrates one of the highest infection rates of pulmonary capillariosis in foxes over the world