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

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.

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

Efficiency of two protocols of resynchronization of estrus and ovulation in high-producing dairy cows at peak lactation

The reproductive efficiency of the cows was monitored after two resynchronization protocols: Ovsynch (OVS) and Double Ovsynch (DOS). The research initially included 70 HF cows who entered the first synchronization protocol - Presynch. Cows that did not conceive after the first synchronization were divided into two groups and introduced to two resynchronization protocols. In the first group of cows (n=35), the DOS protocol began with the application of GnRH on day 22 after the Presynch TAI (Timed Artificial Insemination), and seven days later pregnancy check-up was done and PGF2α was applied only to non-pregnant cows (n=23), which remained in the study. In the second group of cows, the OVS protocol started on day 32 after Presynch TAI only in non-pregnant animals (n=20). Progesterone (P4) concentration was determined at the time of application of GnRH1, PGF2α and GnRH2 in both groups of cows, and then 30 days after Resynch TAI, ultrasound pregnancy diagnosis was done. A higher percentage of pregnant cows were recorded in the OVS group compared to the DOS group (45% and 35%, respectively). The concentration of P4 in the serum of cows in the DOS group during the first measurement (GnRH1) was significantly higher than the value in cows that did not conceive (p<0.05), while in the third measurement (GnRH2) the average concentration of P4 in conceiving cows was significantly lower (p<0.001) compared to cows that did not coincive. The open days period was significantly longer in pregnant cows that were resynchronized using the DOS protocol compared to cows from the OVS protocol. In conclusion, the OVS protocol of estrus resynchronization in dairy cows proved to be more successful than the DOS protocol. However, considering the advantages the OVS, it is needed to determine which day of the sexual cycle is the best to start resynchronization

The influence of selenium and zinc addition in food on concentration of these elements in blood and milk, on somatic cells number and histological characteristics of cows udders

The experiment included 30 cows of Holstein-Friesian breed, out of which 15 were receiving selenium and zinc in optimal doses before calving, while the others had never been supplemented with these micronutrients. There was analysed the concentration of selenium and zinc in blood and milk serum as well as the average number of somatic cells in corresponding lactation. After the cows exclusion from production, histological characteristics of cows udders were examined. The results of the investigation have shown that addition of selenium and zinc before calving has a positive effect on the values of these microelements in the blood and milk during the period of early lactation, that is, the concentration of these elements was significantly higher in the blood and milk of the cows that obtained selenium and zinc supplements. Also, in these cows there was significantly lower number of somatic cells during the following lacation period. In the parenchyma of the udder there was found less pronounced infiltration of leukocytes, notably thicker keratin layer of ductus papillaris and less expressed repairing processes that indicate a chronic inflammation of the udder in the samples after exclusion of the cows from production. There was a significant positive correlation between selenium in blood and milk, while there was not observed such a correlation for zinc. On the other hand, there was a significant negative correlation between the concentration of selenium in the blood and milk with the average number of somatic cells and the degree of infiltration of leukocytes, while its influence on the keratin layer of ductus papillarus was not shown. Zinc from blood and udder had a negative correlation with the number of somatic cells, had a positive correlation with the thickness of ductus papillaris keratin layer and had no influence on the level of leukocyte infiltration of udder parenchyma. Zinc demonstrates a positive influence on the formation of ductus papillaris keratin layer and protects the udder from pathogens penetration, while selenium stimulates the immunological response of the udder. Their positive impact can be defined as additive, because athough they have effect on two morphologically separate udder parts, adding both of them significantly decreases the number of somatic cells in milk. [Projekat Ministarstva nauke Republike Srbije, br. TR31062

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

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

Thermal comfort of cows and temperature humidity index in period of 2005-2016 in Vojvodina region (Serbia)

The aim of this study is to determine value of temperature-humidity index (THI) in period of 2005-2016. in Vojvodina and their correlations with milk production and temperature of body surface of animals. THI index is calculated according to the following formula THI=(1,8×Temperature)-(1-Relative Humidity) × (Temperature- 14,3)+32. Temperature and humidity data were collected directly from Hydrometeorological Institute of Serbia. Average THI values were calculated for every month for every year from 2005 to 2016. Measuring spots were cities Vršac, Zrenjanin, Kikinda, Palić, Sombor, Rimski Šančevi and Sremska Mitrovica. Temperature and humidity are noted at 14h, so they represented maximal average values of THI index. Trend of THI values was represented for each day in every month in years from 2005. to 2016. Average production of drinking milk for year of 2016 was calculated. This year was chosen because is the last year of research and represents cumulative effect of heat stress in cows. Data were collected from Statistic Institute of R. Serbia. Thermo-vision camera was used for determining skin temperature of udder and skin in eye region. Results were showed that temperature ranged from 40.3 in January to 77.5 in July. Average maximal THI value in period of 2005-2016 showed that heat stress was presented in May, June, July and August. Drinking milk production was the greatest in January. After that it started dropping. The lowest production of drinking milk was noted in August with an increase afterwards till December. Body surface temperature was ranged from 34.1 to 38.5ºC. The highest body temperature was measured at August and the lowest in winter time. Significant negative correlation between ten-year average THI value and milk production was noted. Positive correlation was noted between THI and body temperature. From 2005 to 2016 positive linear trend of THI values that were measured in hottest period of day was noted. In every month statistically significant linear trend of rising THI value was founded, except in January, October and November. Extremely hot summers in Serbia appear in every five years when cows are affected the most. Showed data indicated global warming and climate changes which affect biological adaptation of cows

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

Relationship between milk production and metabolic adaptation in dairy cows during heat stress

The aim of this study is to determine correlation between change in milk production and metabolic adaptation of cows during heat-stress. Experiment included 30 Holstein-Frisian cows. Cows exposed to heat-stress, which is expressed in high THI index, have lower milk production, higher concentration of insulin and lover concentrations of glucose and NEFA. Indexes of insulin resistance-RQUICKI, insulin: glucose and insulin: NEFA relations are higher in cows under heat stress. Grouping cows in accordance to reduced milk yield have showed that trend of reduction of glucose and NEFA levels, followed by increased insulin level, higher RQUICKI, greater insulin: glucose and insulin: NEFA ratios, started and increased during the reduction in milk production. Among this, significant correlation was founded between these parameters and milk yield. Significant correlation was also noted between those parameters. Change in metabolic value is of great importance for prediction of cows that would have decreased milk yield (above 18%). With almost 90% certainty cows with great reduction in milk yield can be detected. These cows have had following relative changes in values of metabolic parameters during heat-stress compared to thermo-neutral period: insulin increased for ≥12.5%, reduction in NEFA values for ≤14.1%, reduction in glucose for 21.5%, RQUICKI index increased for ≥9.6%, insulin: NEFA ratio increased for ≥20.1% and insulin: glucose for ≥20.3%. Dynamical changes in metabolites and insulin resistance values have great influence on milk yield in cows under heat stress. Decreasing in glucoses followed by increased insulin level and increased insulin sensitivity indicate that glucose is transferred from udder to other tissues which can cause decreased milk production