THE RELATIONSHIP BETWEEN AFLATOXICOSIS, OXIDATIVE STRESS AND INCIDENCE OF MASTITIS IN DAIRY CATTLE

MONA A. MAMOUD^*, HANAA A. ALLAM^**, A.SH. EL BAKHMY^*** and ENAS M.G. ^****

^* Department of Biology,

^**Department of Mastitis and Neonates Diseases

^*** Department of Field Investigation

^**** Unit of Immunology.

Animal Reproduction Research Institute, Giza, Egypt.

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                                               ABSTRACT

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Key words: Aflatoxicosis, oxidative stress, Mastitis.

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INTRODUCTION

Contamination of animal feed with mycotoxins is a worldwide problem in animal production. The complex diet of ruminants, consisting of forage, concentrates and silage can be a source of diverse mixtures of mycotoxins that contaminate individual feed components concomitantly, there has been an increase in feed intake to meet the greater nutrient demand which often exposes cows to mycotxins contaminated feed (Bennet and Klich, 2003 and Queiroz et al., 2012). The aflatoxins are a group of closely related to mycotoxins that are widely distributed in nature. Aflatoxins constitute toxic metabolites of the fungi as Aspergillus Flavus and Aspergillus Parasiticus (Payne, 1998 and Kourousekos, 2011). The most important of the group is aflatoxin B1(AFB1) which has a range of biological activities and disorders depending on the duration of consumption and quantity of the toxin (Abdel-Wahab et al., 2002 and Eraslan et al., 2004). Such disorders could seriously affect animal production, incidence of metabolic disorders, mastitis and elevated somatic cell count; consequently decrease milk production after aflatoxin is consumed by lactating animals (Singh et al., 1996). It is metabolized to aflatoxin M1 which is excreted into the milk, factors affect carry over into milk include mammary alveolar cell membrane health, low molecular weight of aflatoxins (Masoero et al., 2007).

Consumption of lesser amount of aflatoxin may result in impaired immunity and decreased resistance to infectious diseases and may attack nucleophilic nitrogen, oxygen and sulphur heteroatom in cellular constituents which could be due to the induction of oxidative stress which is classical defined as an imbalance between pro-oxidant and antioxidants resulting in overall increase in cellular levels ofreactive oxygen species(Umberto et al., 2011). As consequence dairy cow could predispose to incidence of mastitis which is directly related to the broad immunosuppressive effect of aflatoxin on cellular and humeral mediated immune response and decrease host resistance which could cause intrammamary infection or make the gland more susceptible to infection (Oswald et al., 2005)                 

The aim of the present study was designed to determine the relationship between aflatoxins as a feed born stress factor, its effect on redox status and their relation to the incidence of mastitis in dairy cattle.

MATERIALS and METHODS

Animal Grouping:

The current study was done in a private farm on 32 lactating cows. All animals in the course of this study were fed total mixed rations (TMR), nutrient concentrations met nutritional requirement for lactation according to NRC (2001), examined periodically for mycotoxins. All cows according to previous considerations were classified into two groups: first group (10 cows) were received TMR mixture, where the percentage of aflatoxins not exceed permissible limits which is 5ppb (FAO, 2004), the second group (22 cows) received ration containing high levels of aflatoxins (16-60 ppb)

Blood Samples:

Two blood samples were collected from each cow via jugular vein puncture the first sample was taken in heparinized vacuum tubes for plasma separation and estimation of oxidant/antioxidant parameters (total antioxidant capacity (TAC), glutathion peroxidase (GSH-Px), catalase (CAT) & superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO) and determination of the cell mediated immune response. The second blood sample was put on plain centrifuge tubes for serum separation and evaluation of serum copper & zinc levels

Oxidant/Antioxidant determination:

Samples were examined using commercial diagnostic kits (Biodiagnostictic) for the following parameters; TAC according to Koracevic & Koracevic (2001), GSH-Px according to Paglia and Valentine (1967),CAT according to Aebi (1984), SOD according to Nishikimi et al. (1972) MDA according to Ohkawa et al. (1979) and NO according to Rajaraman et al. (1980). Copper & zinc levels were estimated by using atomic absorption spectrophotometer (Model, 3300, Parkin Elmer USA)

Cell mediated immune response:

The cellular immune response of first and second groups was assessed by the lymphocyte transformation using MTT lymphocyte blastogenesis micro assay was conducted previously according to Denizot and Lang (1991) with some modifications Maslak and Reynolds. (1995). Isolation from blood by the method described by Rouse et al. (1980). Estimation the percentage of bacteria phagocytised according to Woldehiwet and Rowant (1990) the percentage of killed bacteria was estimated according to the formula described by Woldehiwet and Rowant (1990).

Mycotoxins analysis in feed and milk:

Mycotoxins B1 were analyzed in feed by high performance liquid chromatography (HPLC-CBC-7210H Austerila) with fluorescent detection (FLD). We offer a variety of clean up columns according to Scott (1997). Mycoyoxins M1 were estimated on milk by HPLC technique according to Baccaca et al. (2008).

Milk Sample:

Samples:

A number of 32 milk samples were taken from dairy cows. Each sample were collected in clean, sterile and dry McCartney glasses in duplicate and preserved in ice tank till examination. One sample was examined for bacteriological studies while the other was examined for milk composition and somatic cell count (S.C.C.).

Field Test (California Mastitis Test, CMT):

According to APHA(1992) for detection of subclinical mastitis in cattle, California mastitis test was performed on individual milk samples collected  from each quarter udder of every cattle to detected subclinical mastitis. The C.M.T gives an indirect estimation of S.C.C. and it s based upon a gelling reaction between the nucleic acid of the cells and a detergent reagent depending on the amount of gel formation, samples were assigned to 3 categories negative or positive reaction in 2 grades (++ and+++).   

Bacteriological examination:

All milk samples were collectedfrom infected cows for bacteriological examination were cultured on different specific media (sheep blood media, Manitol salt agar, Edward^,s media MacConky agar media).

Isolation and identification of causative organisms:

Isolation and identification of causative organisms were done according to Toplly and Wilson (1998).

Measurements of milk constituents:

It was done by using infra milk analyzer 150, from Bentley. The following milkconstituents were estimated, in both normal milk and milk from lactating cows suffered from subclinical mastitis: fat (%) and protein (%). 

Statistical analysis:

The redox, milk composition and cell mediated immune parameters were subjected to T test analysis according to Senedecor & Cochran (1982).

RESULTS

The results in Table (1) showed significant reduction at (p<0.05) in antioxidant parameter (TAC, GSH-Px, CAT, SOD,), significant elevation at (P<0.05) in oxidant parameters (MDA, NO), while copper &zinc showed non significant decreased in group received aflatoxin B1 above permissible limit compared to group received aflatoxin B1 within permissible limit. Cell mediated immune response is elucidated in table (2) the data revealed that phagocytosis (%), killing (%) of polymorphnuclear cells and lymphocyte stimulation index in the presence of phyto-haemagglutinine (PHA) display a significant reduction at (P<0.05) due to exposure to aflatoxin B1 above permissible limit.

The present study in table (3) shown that S.aureus was the most prevalent organism (31.25%) followed by E.coli (25%) and str. dysgalactia (15.63%) while coagulase negative staphylococci (CNS) was (15.63%).

Moreover the study identified the changes in CMT scores and SCC levels in cow’s milk in subclinical infections due to some important mastitis pathogens. As shown in table (4) there is a good correlation between CMT and SCC according to the infectious status of mammary quarters of examined cows. In the absence of infection, CMT score was negative and SCC range was 15-159×10³ cells/ml with arithmetic means 85.5×10³. In case of infection with minor pathogens, CMT score was ++ and SCC range was 210-245×10³ with mean 224×10³. While in case of infection with major pathogens CMT score was (+++) and SCC range was 250-800×10³ with mean 623×10³.

Table (5) showed the effect of udder infection on milk fat% and protein% the mean fat (%) in normal milk cows was (5.9±0.38) while in subclinical mastitis cow’s milk the mean was (2.28±0.199) with significant decrease (p>0.05). While the mean protein (%) in normal milk cows was (4.39±0.17) while in subclinical mastitis cow’s milk the mean was (2.65±0.139) with significant decrease (p>0.05).

Table 1: Effect of aflatoxin (B ₁) exposure on oxidant/ antioxidant parameters in of dairy cows.

  Mean significant from healthy at (p<0.05).

Table 2: Effect of aflatoxin (B ₁) exposure on lymphocyte stimulation index (lymph.) phagocytosis % (phag. %) and killing % (killing %) in dairy cows in blood sample.

 Mean significant from healthy at (p<0.05).

Table 3: Prevalence of M.O in cases of subclinical mastitis in cow’s milk

 Mean significant from healthy at (p<0.01).

Table 4: Effect of isolated M.O on CMT and mean S.C.C in examined samples of dairy cows.

Mean significant from healthy at (p<0.01).

Table 5: Effect of aflatoxin (M ₁) exposure on milk: fat (%) and protein (%) of dairy cows

 Mean significant from healthy at (p<0.01).

DISCUSSION

Aflatoxins are highly oxygenated secondary metabolites produced by certain toxigenic strains of Asperagillus flavus and Asperagillus parasiticus growing on a variety of feed stuff (Manjulata, 2011). Data suggests a role for oxidative stress which is  the pathogenesis of aflatoxicosis (Umarani et al., 2008), induced by reactive oxygen species (ROS) which believed to be a primary factor in various cattle diseases including mastitis (Pietro, 2011). In the present study our results of oxidant and antioxidant status revealed that there was a significant decrease of antioxidant net work (TAC, GSH-Px, CAT & SOD) and increase oxidant levels of lipid peroxidation & NO in mastitic animals received contaminated ration with aflatoxin B1 above permissible limit, this results agree with Umberto et al. (2011); Valko et al. (2007); Mclean Dutton (1995). This may be attributed to that aflatoxin B₁ in order to exert its effect, it must be converted to its reactive epoxide by the action of the mixed function mono-oxygenase enzyme system (cytochrome p450-dependent) in the tissues this epoxide is highly reactive and can reacts with several macromolecules including DNA, RNA and protein (Mclean and Dutton, 1995), in addition, Umberto et al. (2011) revealed that the induction of oxidative stress of AFB1on bovine peripheral blood mononuclear cells revealed increase of intracellular the reactive oxygen metabolites (ROM) without any time dependent effect. On the other hand. some mycotoxins may induce the production of free radicals and/or the reduction of antioxidant defense since during toxicity (Meissonnier et al., 2008), leading to the induction of the oxidative stress which has to be regarded as a cause or as a consequence of the action of toxicant on cellular system and variable effects of mycotoxins on different cell systems which has been suggested that this may be due to different sensitivity related to the metabolic characteristic of different cell types (Muller et al., 2004). Whatever, ROM content in animal cells may be increased by several factors including xenobiotics such as mycotoxin with the onset of oxidative stress conditions as a result of increase level of lipid peroxidation (Klaunig and Kamendulis, 2001) (Valko et al., 2007).

AFB₁ has been shown to reduce T-lymphoblastogenesis, impair delayed cutanenus hypersensitivity and graft-versus-host reaction (Williams, 2004). Our results revealed that Cell mediated immune response phagocytosis%, killing % of polymorphnuclear cells and lymphocyte stimulation index reduced this results agree with (Meissonnier et al., 2008; Valko et al., 2007 and Jiang et al., 2005). These results indicated that AFB1 dietary exposure decreased cell-mediated immunity while inducing an inflammatory response these impairments in the immune response increased susceptibility to infectious diseases (Meissonnier et al., 2008). At exposure levels of a strong reduction of the blastogenc potential in bovine lymphocytes hypothesizing a general inhibition of T-lymphocyte functions such as killer, helper, effector or other immune processes which may compromise the immunological surveillance mechanisms (Valko et al., 2007). As the immune system is primary responsible for defense against invading organisms, the sensitivity of the immune system to mycotoxin induced immunosuppression arise from the vulnerability of the continually proliferating and differentiating cells that participate in immune mediated activities and regulate the complex communication network between cellular, humoral components and mycotoxins induced immunosuppression may manifest as depressed T or B lymphocyte activity and impaired macrophage/neutrophil-effctor functions (Oswald et al., 2005), suppressed immune function by mycotoxns may eventually decrease resistance to infectious diseases. Several reports show that mycotoxins such as aflatoxin are able to affect the inflammatory response (Contreas and Sardillo, 2011) Aflatoxin is able to affect the inflammatory response, it act at different levels they can directly affect the viability of phagocytes (macrophages and neutrophils, alternatively they can impair the of the activity of the secretory functions of these cells. AFB₁ihibits phagocytosis, intracellular killing and spontaneous production of oxygen radicals in rat also AFB₁ have suppressive effect on inflammatory cytokines the molecular-cellular basis and general mechanism responsible for the broad immunosuppressive effect of AFB₁ appears to impaired protein synthesis (Oswald et al., 2005)

Regarding to microbial count aflatoxin effect on udder health, resulting in mastitis, somatic cell count and total microbial count change agree with  (Kourousekos, 2011 and Brown et al., 1981), aflatoxin B1 at a high enough dose are often bacterial counts of St.agalciae and S.aureus in milk from infected quarter increased (Valko et al., 2007) who found associated with suppressed immune response of cows, increased the oxidative damage in a cell or tissue, increased SCC as a result of mastitis, moreover These results are in a  harmony with the results recorded by (Dohoo and Meek, 1982 and Sharma, 2003) who found good correlation between CMT score, SCC and infection status of udder halves.

AFM₁ is excreted in milk after aflatoxinB1metabolism in the body. As a consequence of aflatoxin influence on the general condition of the animals together with the excretion into milk. The effect on milk quality, including changes in some milk components, concentrations of the milk fat and protein which reduced significant at (p<0.01) this results agree with (Kourousekos, 2011; Akbar and Majid, 2010; Queroz et al., 2012). Other researcher did not recod any aflatoxin effects on fat values (Kutz, 2009), while the reduction of milk fatty acids in these results were attributed to the inhibition or decrease production of some enzymes (Kourousekos, 2011). Moreover aflatoxin found to cause chemical change in milk attributed to damage of lysosomes by aflatoxinB1and possibly increased capillary permeability. Thus any chemical change in milk caused by aflatoxiin udder may cause obvious physical change in milk (Brown et al., 1981) Opposite result (Kutz, 2009) found that the milk protein content no aflatoxin effects on it. Other make reason for a slight increase of total protein in blood level after aflatoxin consumption as well as for RNA polymerase suspended action, resulting in the inhibition of a lot of metabolic reactions, such as protein synthesis. Another explanation that decrease milk productions are related to decrease dry matter intake from feed contamination to mycotoxin and aflatoxin, however, feed consumption was affect by exposure time of diet with aflatoxin, age of animal and aflatoxin level in diet Dietary aflatoxin could have effect on protozoa population, rumen flora and cause decrease in cellulose utilization, volatile fatty acid, ammonium production and also it leads to increase inactivity of alkaline phosphatase enzyme in the rumen. These factors influence feed consumption, milk yield and composition (Akbar and Majid, 2010). Any chemical change in milk caused by aflatoxin udder resuling in physical change in milk. An increase in SCC during mastitis infection increase the amount may cause obvious of destruction enzymes present in the milk, which increase the rate of deterioration of milk fat and protein, (Jillian, 2006).

The conducted investigations confirmed that, aflatoxin B1 has variable effects on different cell systems resulted in induction of oxidative stress and suppressed immune function which may eventually decrease resistance to infectious disease and exacerbation in existing intrammamary infection and predispose cows for incidence of mastitis, decrease milk production, increase SCC, in turn increase the rate of deterioration of milk fat and protein. 

ACKNOWLEDGMENTS

Extend great thanks and gratitude for Doctor Manal Gallal Chief Researcher in Biology department in Reproduction Research Institute Pyramid on scientific effort that provided for the completion of this study.

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