Research of equine influenza with different epizootic and clinical findings of infection

Influenca kopitara je ozbiljno, akutno, visoko kontagiozno, respiratorno oboljenje kopitara sa karakterističnom kliničkom slikom u tipu respiratornog simptoma. Ovo oboljenje izazivaju dva suptipa influenca A virusa (EIV – ekvini influenca virus) – H7N7 (ranije poznat kao tip 1) i H3N8 (ranije poznat kao tip 2) iz familije Orthomyxoviridae. Cilj istraživanja je bio da se uporede različite dijagnostičke metode za dijagnostiku influenca A virusa kod kopitara sa različitim vakcinalnim statusom i kliničkim nalazom. U eksperimentu je učestvovalo 61 grlo. Sprovedena je epizootiološka anketa, grla su klinički pregledana, analizirana je kompletna krvna slika, urađen HI, Direktigen i RT-PCR test. Rezultati su pokazali prisustvo oba suptipa virusa. Registrovane su seropozitivne jedinke kod kojih je dokazano prisustvo antigena virusa influence, ali bez dokazanog prisustva genoma virusa influence.Equine influenza is a serious, acute, highly contagious, respiratory disease, equine with characteristic clinical picture in the type of respiratory syndrome. This disease is caused by two subtypes of influenza A virus (EIV - equine influenza virus) - H7N7 (formerly known as type 1) and H3N8 (formerly known as type 2) from the family Orthomyxoviridae. The aim of the research was to compare different diagnostic methods for the diagnosis of influenza A virus in equine vaccine with different status and clinical findings. The experiment involved 61 throat. Conducted epidemiological surveys, clinical throat examined, analyzed the complete blood count, done HI, Directigen and RT-PCR test. The results showed the presence of both subtypes of the virus. Seropositive individuals are registrated in which the presence of an influenza virus antigen is confirmed, but without evidence of the presence of the influenza virus genome

THE EFFECT OF BLOOD AND MILK SERUM ZINC CONCENTRATION ON MILK SOMATIC CELL COUNT IN DAIRY COWS

The objective of this study was to evaluate the effect of blood and milk zinc concentration on somatic cell count and occurrence of subclinical mastitis cases. The study was performed on thirty Holstein cows approximate same body weight, ages 3 to 5 years, with equally milk production. Blood samples were taken after the morning milking from the caudal vein and milk from all four quarters was taken before morning milking. All samples of blood and milk were taken to determined zinc, using inductively coupled plasma mass spectrometry. 37.67% (11/30) cows have blood serum zinc concentration below 7µmol/l, and 63.33% or 19/30 cows have blood serum zinc concentration higher then 13µmol/l. Also 30% (9/30) cows have somatic cell count lower then 400.000/ml which indicate absence of subclinical mastitis, but 70% (21/30) cows have somatic cell count higher then 400.000/ml which indicate subclinical mastitis. Results indicate that cows with level of zinc in blood serum higher then 13 µmol/l have lower somatic cell count. Cows with lower zinc blood serum concentration then 7 µmol/l have high somatic cell count and high incidence of subclinical mastitis. According to results in this research there is no significant effect of milk serum zinc concentration on somatic cell count in dairy cows

Analysis of Different Diagnostis Methods of Influenza in Horses

Background: Equine Influenza is a serious, acute respiratory illness with characteristical clinical signs. The disease is caused by family of Orthomyxoviridae, genera Influenza virus A by two subtypes H7N7 and H3N8. Currently, there is believe that H7N7 has been replaced as a predominant subtype with the H3N8. Horse infection with influenza virus can be detected by serological tests on paired sera using HI test. Commercial rapid tests could be used for the detection of influenza virus. Recently it is widely use a PCR method as fast and more specific methods.Materials, Methods & Results: Fifty horses and one pony, age between one and 22 years have been included in experiment. Horses were of different race, sex, and age and vaccination status. Ten out of total 51 (10/51) have been regularly vaccinated against EI. Prior to initiation of these study epidemiological survey has been performed. The clinical examination has been followed by blood sampling for blood cell and serum extraction. The serums were evaluated by HI method. Nasal swabs are taken from both nostrils twice, one was frozen for virus detection by RT-qPCR while another was used for detection of EI virus by Directi-gen™ FLU A rapid test. Analysis of titers of antibody reveled that 7 horses (14%) had specific antibodies (IgG) against subtype H7N7, while 9 horses (18%) had specific antibodies against H3N8. In the same time 4 horses had specific antibodies against both subtypes. Serological data confirmed that from 48 horses (96%) had the titer of antibodies greater than 16 against H7N7, while 40 horses (80%) had the specific antibodies (IgG) against H3N8. We found quite unexpected presence of specific antibodies (IgG) for H7N7 in horses that have not been previously vaccinated with H7N7 subtype. These horses never been utilized for sport activity and there was no legal requirements for their vaccination. Could horses with specific antibodies for H7N7 be transfected from vaccinated horses we could not confirme with scientific evidence either we could not have evidence that wild and domestic birds played a significant role especially knowing that horses are dead end of further we could confirm it. Our findings unequivocally confirmed that EI virus subtype H7N7 antibodies (IgG) were present in horses that have not been vaccinated and this is serological evidence that virus H7N7 is circulating  in these geographical areas.Discussion: Fast and reliable diagnosis and isolation of suspicious horses represent the first line of defense against pandemic influenza. Recognition of clinical signs (fever, depression, sharp cough and nasal discharge) along with epizootical survey provides the basis for the early detection of infection. In some cases, cough and rapid spread of symptoms of cough in a group of horses that were either unvaccinated against influenza virus or have been in contact with influenza virus infected horses can clearly point out to the EI virus. The definitive confirmation of EI virus could be done by virus isolation on tissue culture or embrionated eggs followed by detection of virus nucleic acid by RT-PCR. Our data suggest that a substantial number of horses (90%) that have not been vaccinated or vaccinated irregularly had specific antibodies against both subtypes of EI, what suggest that those horses have been exposed to viruses sometimes during their lifetime. Additionally, despite the fact that 20% of horses had some signs of respiratory disease that resemble EI we were not able to confirm EI nucleic acid by RT-PCR while Directi-gene™ assay confirmed virus presence just in two horses. Failure to detect virus nucleic acid could be due to fact that nasal swab samples have been taken at the end of the clinical symptoms, other authors have simular PCR negative patient which displayed a significant rising titre to influenza type A

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.

Hematological Parameters in Cows in Early Lactation Treated with Ketoprofen and their Relationship with Lipid Mobilization and Ketogenesis

Background: Dairy cows are exposed to numerous hematological and biochemical changes, what is bringing cows into the state of increased metabolic activity and physiological adaptations. These adaptive processes have resulted in increased lipid mobilization and ketogenesis with increased concentration of non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB). As a non steroidal anti-inflammatory drug, ketoprofen produces anti-inflammatory effects. The main objective of the present study was to found relationship between hematological parameters and ketoprofen administration and high lipid mobilization and ketogenesis in cows treated with ketoprofen immediately after calving.Materials, Methods & Results: Ketoprofen was used (3 mg/kg body weight) intramuscularly for three consecutive days post-partum on 15 cows of Holstein-Friesian breed. Cows of the control group (n = 15) were not treated with ketoprofen. Blood samples were collected at the day of calving, in the first and in the second week after parturition from the coccygeal vein of the both groups. Hematological parameters (erythrocytes, hemoglobin, neutrophils, lymphocytes and mean platelet volume) were determined on the automatic hematological counter. Metabolic parameters (NEFA, BHB) were determined by standard colorimetric kits using a semi-automatic biochemistry analyzer. Student’s t-test was used to determine the influence of the application of ketoprofen to the hematological parameters. The data analysis was performed using SPSS, version 19.0, software package for Microsoft Windows (IBM, Armonk, NY, USA). The results of comparison with (P < 0.05) were considered to represent statistically significant differences. Difference in correlation test between NEFA and BHB and other blood parameters in experimental and control group of cows were determined by the Fischer r-to-z transformation. The research results show that there was a increased red blood cell count and a higher concentration of hemoglobin (P < 0.05), fewer neutrophils (P < 0.01), lower number of lymphocytes (P < 0.01) and fewer mean platelet volume in cows that received ketoprofen after calving than those of the control. Determined hematological parameters and values of NEFA and BHB are in correlation. The correlation coefficients were significantly lower in the experimental group of cows. The intensity of the connection between hematological parameters and value of NEFA and BHB decreases.Discussion: It is well known that dairy cows with excessive adiposity manifest a low-grade inflammation and that elevated NEFA concentrations present positive risk factors for many proinflammatory diseases. Analyzing the results in this examination, it has been noticed that the increase of NEFA and BHB after calving were significantly lower in group of cows treated with ketoprofen. Also, determined hematological parameters and values of NEFA and BHB are in correlation. The intensity of these connection decreases in ketoprofen treated cows. In current study, in cows treated with ketoprofen there is decrease in concentrations of inflammatory mediators such as neutrophils and lymphocytes. Red blood cell and hemoglobin concentration decreased in the peripartal period as a result of inflammation. In cows treated with ketoprofen these concentrations increased. In many inflammatory conditions mean platelets volume is increased while there is a decrease in ketoprofen treated cows. Presented results indicate strong relationship between the concentration of NEFA, as well as BHB and hematological parameters indicating that the dependence of hematological parameters of intensity of lipid mobilization and ketogenesis was significantly lower in cows treated with ketoprofen immediately after calving

Hematological Parameters in Cows in Early Lactation Treated with Ketoprofen and their Relationship with Lipid Mobilization and Ketogenesis

Background: Dairy cows are exposed to numerous hematological and biochemical changes, what is bringing cows into the state of increased metabolic activity and physiological adaptations. These adaptive processes have resulted in increased lipid mobilization and ketogenesis with increased concentration of non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB). As a non steroidal anti-inflammatory drug, ketoprofen produces anti-inflammatory effects. The main objective of the present study was to found relationship between hematological parameters and ketoprofen administration and high lipid mobilization and ketogenesis in cows treated with ketoprofen immediately after calving.Materials, Methods & Results: Ketoprofen was used (3 mg/kg body weight) intramuscularly for three consecutive days post-partum on 15 cows of Holstein-Friesian breed. Cows of the control group (n = 15) were not treated with ketoprofen. Blood samples were collected at the day of calving, in the first and in the second week after parturition from the coccygeal vein of the both groups. Hematological parameters (erythrocytes, hemoglobin, neutrophils, lymphocytes and mean platelet volume) were determined on the automatic hematological counter. Metabolic parameters (NEFA, BHB) were determined by standard colorimetric kits using a semi-automatic biochemistry analyzer. Student’s t-test was used to determine the influence of the application of ketoprofen to the hematological parameters. The data analysis was performed using SPSS, version 19.0, software package for Microsoft Windows (IBM, Armonk, NY, USA). The results of comparison with (P < 0.05) were considered to represent statistically significant differences. Difference in correlation test between NEFA and BHB and other blood parameters in experimental and control group of cows were determined by the Fischer r-to-z transformation. The research results show that there was a increased red blood cell count and a higher concentration of hemoglobin (P < 0.05), fewer neutrophils (P < 0.01), lower number of lymphocytes (P < 0.01) and fewer mean platelet volume in cows that received ketoprofen after calving than those of the control. Determined hematological parameters and values of NEFA and BHB are in correlation. The correlation coefficients were significantly lower in the experimental group of cows. The intensity of the connection between hematological parameters and value of NEFA and BHB decreases.Discussion: It is well known that dairy cows with excessive adiposity manifest a low-grade inflammation and that elevated NEFA concentrations present positive risk factors for many proinflammatory diseases. Analyzing the results in this examination, it has been noticed that the increase of NEFA and BHB after calving were significantly lower in group of cows treated with ketoprofen. Also, determined hematological parameters and values of NEFA and BHB are in correlation. The intensity of these connection decreases in ketoprofen treated cows. In current study, in cows treated with ketoprofen there is decrease in concentrations of inflammatory mediators such as neutrophils and lymphocytes. Red blood cell and hemoglobin concentration decreased in the peripartal period as a result of inflammation. In cows treated with ketoprofen these concentrations increased. In many inflammatory conditions mean platelets volume is increased while there is a decrease in ketoprofen treated cows. Presented results indicate strong relationship between the concentration of NEFA, as well as BHB and hematological parameters indicating that the dependence of hematological parameters of intensity of lipid mobilization and ketogenesis was significantly lower in cows treated with ketoprofen immediately after calving

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

Contamination of Cow Milk by Heavy Metals in Serbia

Background: Dairy cows are exposed to numerous environmental hazards, such as heavy metals. Milk and dairy products could be harmful to humans when maximum tolerable amounts of heavy metals are exceeded. Analysis of heavy metals in milk is important because milk is a source of essential nutrients and is an indicator of environmental contamination. Some heavy metals are essential to maintain proper metabolic activity in living organisms, but at exceeded levels they could be toxic to living organisms. The main objective of the present study was to determine the residue levels of ten heavy metals (As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn) in cow milk in Serbia.Materials, Methods & Results: The experiment was conducted at three farms at Vojvodina district, Serbia. Cows from all three farms were on pasture during spring and summer time. Randomly 50 cows from each farm were taken for this experiment. The cows have similar body condition score they were in their third or fourth lactation and gave approximately the same amount of milk in the previous lactation. Total 150 milk samples were collected from cows during the morning milking, in the period between April to May in 2016. All 150 milk samples were transported to laboratory as soon as possible and analyzed for the heavy metals. Heavy metals were analyzed by Inductance coupled plasma - optical emission spectrometry. Data are presented as mean values ± sd. Statistical analysis was done by one way repeated measures analysis of variance (ANOVA). The level of significance was set at P < 0.05. The highest average concentration in cow milk was determined for Iron (283.9 mg/kg), then for Zinc (60.21 mg/kg) and Copper (4.404 mg/kg), while the lowest concentration was for Cobalt and Lead (less than 0.005 mg/kg).Discussion: Bioaccumulation is one of the biggest problems with heavy metals. Heavy metals residues in milk can be very dangerous for the calves and humans as well. The existence of trace elements and heavy metals in dairy products and milk has been recorded in various countries since it is considered as one of the most dangerous aspects of food contamination. The presence of heavy metals in cow's milk may be attributed to contamination due to exposure of lactating cow to environmental pollution or consumption of contaminated feeding stuffs and water. Heavy metals after intake in cow organism distributed to all organs and glands such as udder. By that, secretory udder tissue gets intoxicated with heavy metals and after that they can be found in milk. After analyses the results in this experiment the highest average concentration of Fe in cow milk it might be because cows were fed with hay from pasture rich in iron between April to May. Some researchers found the amounts of Cu in the milk of individual varied from 0.2 to 0.8 mg/kg. Contrary to this finding, in current study the average value of Cu in cow milk was 4.404 mg/kg. It is found that the highest concentration of Zn in cow milk is 10.75 mg/kg, lower compare to result in current study (60.21 mg/kg). Arsenic (As) concentration was 0.058 mg/kg, cadmium (Cd) was 0.01 mg/kg, cobalt (Co) was 0.002 mg/kg, chrome (Cr) was 0.018 mg/kg, manganese (Mn) was 0.493 mg/kg, nickel (Ni) was 0.119 mg/kg and lead (Pb) was 0.08 mg/kg what is just above recommended values by International Dairy Federation. The results showed that most of the milk samples from the different farm contained all the studied metals with concentration higher than those recommended by International Dairy Federation and Codex for cow milk

Ispitivanje veze između koncentracije selena i cinka u krvi i broja somatskih ćelija u mleku kod krava u ranoj i srednjoj laktaciji

Selenium and zinc are important components of the antioxidant system of the udder. Selenium has a direct impact on the status of immune cells by increasing the efficiency of the immune tissues. Zinc is important in the reparation of the terminal part of the duct papilarisa that devastated daily after milking and also protects against mechanical penetration of harmful agents in the environment. The cows were examined Se and Zn concentrations in early lactation and middle lactation. In parallel with these trials were measured somatic cells. After sorting the data examined the correlation between the values of Se and Zn and somatic cells. Somatic cell count and concentration are significantly negatively correlated. In addition there is a clear negative correlation between the concentration in the first month of lactation and somatic cell count in the sixth month of lactation. The correlation between the somatic cells and the concentration of the zinc is found in early lactation, and zinc significantly correlated with the number of somatic cells in the middle of the lactation period.Selen i cink predstavljaju značajne komponente antioksidantnog sistema vimena. Selen direktno utiče na status imunoloških ćelija povećavajući imunološku efikasnost u tkivima. Cink je značajan u reparaciji završnog dela duktusa papilarisa koji se devastira svakodnevno posle muže i tako štiti od mehaničkog prodora štetnih agenasa iz spoljašnje sredine. Kod krava je ispitana koncentracija Se i Zn u ranoj laktaciji i sredini laktacije. Paralelno sa ovim ispitivanjima vršeno je merenje broja somatskih ćelija. Posle sortiranja podataka ispitana je korelacija između vrednosti Se i Zn i broja somatskih ćelija. Broj somatskih ćelija i koncentracija Se značajno negativno koreliraju. Pored navedenog postoji jasna negativna korelacija između vrednosti koncentracije Se u prvom mesecu laktacije i broja somatskih ćelija u šestom mesecu laktacije. Korelacija između vrednosti somatskih ćelija i koncentracije cinka nije pronađena u ranoj laktaciji, dok cink značajno korelira sa brojem somatskih ćelija u sredini laktacije

Effects of Ketoprofen Administration on Relation between Acute Phase Proteins and Metabolic Parameters in Cows during Early Lactation

Background: Acute phase proteins (APPs) are clinically useful parameters for measuring the occurrence and severity of inflammatory responses in cattle. As large group of proteins, APPs are mainly secreted by hepatocytes whose concentration increase or decrease as response to tissue injury, inflammation or infection. The transition from late pregnancy to early lactation is associated with a compromised immune status coupled with increased acute phase response. Non-steroidal anti-inflammatory drugs (NSAIDs) are used in treatment of inflammatory conditions. The main objective of the present study was to analyze the effects of a preventive administration of the NSAID (ketoprofen) in post-calving dairy cows on concentration of APPs (haptoglobin and fibrinogen) and their relationship with indicators of liver function.Materials, Methods &amp; Results: The treatment group (n = 15) was given an intramuscular injection of ketoprofen in the concentration of 3 mg/kg during three consecutive days after calving. The control group (n = 15) was not treated with ketoprofen. Blood samples were taken from coccygeal vein, on the first day of treatment and in the first and second week postpartum and they were analyzed for metabolic parameters (albumin and aspartat aminotransferaze) and APPs such as haptoglobin and fibrinogen. Compared with control, ketoprofen administration decrease the levels of haptoglobin and fibrinogen and AST activity. Increase in albumin concentration was recorded in experimental group of cows compared with control. Negative correlations (P &lt; 0.01) were found between the haptoglobin concentration and albumin concentration and aspartate aminotransferaze activity. Discussion: As inflammatory indicator in dairy cows, it is recorded that serum concentrations of haptoglobin is increased during mastitis. Beside haptoglobin, fibrinogen represents one of the APPs whose serum concentration increases during response in the acute phase. During the first and second week after partus, in cows with acute puerperal metritis, an increase in fibrinogen concentration was observed in relation to clinically healthy animals. Based on our results, it can be concluded that an intramuscular injection of ketoprofen (3 mg × kg. bw.-1) administered in cows in the first days following parturition reduces the concentration of some APPs (haptoglobin and fibrinogen). In regard to Hp concentration, the treatment success agreed with previous results using i.m. acetyl-lysine salicylate during the first 5 days of lactation. Reduction in APPs concentration in our research results could be attributed to the anti-inflammatory effect of ketoprofen. In addition, use of ketoprofen reduces the intensity of relationship between inflammatory markers and indicators of liver funcition. Impaired functional capacity of the liver may be associated with reduced albumin concentration in the peripartal period, and during inflammatory or infectious diseases. Increase in albumin concentration in experimental group of cows compared to the control in our research could be attributed to the anti-inflammatory effect of ketoprofen. Hence it can be concluded that the use of ketoprofen immediately after calving reduces the intensity of relationship between inflammatory marker liver function and that NSAID treatment could be used in in improving animal well-being and controlling of the occurence and intesity of postpartum diseases