A CONTRIBUTION TOWARDS MILK ENZYMES, SOMATIC CELL COUNT AND BACTERIAL PATHOGENS ASSOCIATED WITH SUBCLINICAL MASTITIS COWS MILK

A CONTRIBUTION TOWARDS MILK ENZYMES, SOMATIC CELL COUNT AND BACTERIAL PATHOGENS ASSOCIATED WITH SUBCLINICAL MASTITIS COWS MILK

ZEINHOM, M.M.A^*; ABED, A.H.^** and HASHEM, K.S^***.

^* Food Hygiene and Control Department, Faculty of Veterinary Medicine, Beni-Suef University, Egypt.

^**Bacteriology, Mycology and Immunology Department, Faculty of Veterinary Medicine, Beni-Suef University, Egypt.

^*** Biochemistry Department, Faculty of Veterinary Medicine, Beni-Suef University, Egypt.

INTRODUCTION

Mastitis is the most costly disease of the dairy industry in Egypt (Seleim et al., 2002) and throughout the world affects both quality and quantity of milk (Ashfaq and Muhammad, 2008). Annual losses in the dairy industry due to mastitis are over 1.7 billion dollars a year in losses in the US alone (Sahoo et al., 2012). Economical losses are due to loss in milk production, discarding abnormal milk and milk withheld from cows treated with antibiotics, degrading of milk quality and price due to high bacterial or somatic cell count (SCC), costs of drugs, veterinary services and increased labor costs, increased risk of subsequent mastitis, herd replacement, and problems related to antibiotics residues in milk and its products (Abdel-Rady and Sayed, 2009).

Different types either environmental or infectious mastitis and also some types of mastitis pathogens are more virulent than others. The severity of the inflammation can be classified into subclinical, clinical and chronic forms; however detection of subclinical mastitis (SCM) is difficult to be detected due to the absence of any remarkable indications (Mohammadian, 2011). Karyak et al. (2011) summarized that the higher percentages of mastitis losses were due to subclinical mastitis where milk production decreases by 10-26% (Dhakal, 2007). Moreover, cows with subclinical mastitis should be considered as a risk for spread of mastitis pathogens within and between herds and are as such of national concern (Persson et al., 2011).

Various method of detection of SCM have been found including California Mastitis Test (CMT) which is considered the best cut off to correctly identify SCM mastitis (Zaki et al., 2008). Also, estimation of somatic cell counts (SCC); is an indication of inflammation, measurement of biomarkers or enzymes associated with the onset of the disease and identification of the causative microorganisms (Viguier et al., 2009).

Identifying and eliminating intramammary infection (IMI) may have significant economic benefits as preventing clinical mastitis and decreasing the amount of discarded milk (Dingwell et al., 2003). Additionally, knowledge of the clustering of IMI, both within quarters of a cow or within a herd, may be of considerable interest and may lead to further understanding of the dynamics of the disease (Lam    et al., 1996). Otherwise, from public health view, the assessment of SCM etiological pathogens aids to classify the healthy sound milk samples from those of public health hazard as the limits recommended by European countries standards (IDF, 1996) and (Egyptian Standards, 2001).

Pathogenic organisms in milk can be derived from the cow itself, the human hand or the environment (Bradely, 2002). About 150 species of microorganisms were found as the etiological agent of mastitis (Ebrahimi and Nikookhah, 2005). The major mastitis causing organisms are S. aureus, Streptococci, E. coli, Corynebacterium species and Klebsiella species (Shrestha and Bindari, 2012).

Antimicrobials are the most important tool in mastitis control programs. Therefore, identification of mastitis pathogens and their antimicrobial susceptibilities is important when selecting appropriate antimicrobial therapy (Ebrahimi and Taheri, 2009) to ensure optimal results of antimicrobial’s use and minimize the risk for selection and spread of antimicrobial resistance (Moroni et al., 2006).

For many years there has been an interest in using different enzymes in milk as biomarkers for mastitis. Many studies have revealed that enzyme activities in the udder epithelium change markedly due to mastitic inflammation (Andrei et al., 2011). More practical attention has been given to detection of enzyme activity in milk, and many enzymes have been proposed and listed as reliable markers for early diagnosis of subclinical mastitis (Babaei et al., 2007; Guha et al., 2012). Among these enzymes, lactate dehydrogenase (LDH) has been suggested as a biomarker for udder health disturbances as well as a statistical model for mastitis detection (Chagunda     et al., 2006). The connection between milk alkaline phosphatase (ALP) and mastitis has been recognized for decades (Bogin and Ziv, 1973; Babaei et al., 2007). However, in practice only a few studies on this enzyme have been published although it has recently been suggested as a biomarker for human mastitis (Rasmussen et al., 2008). Larsen et al., 2010, concluded that milk enzymes ALP and LDH were markedly increased in case of experimentally induced mastitis and they considered both are the early indicators of acute mastitis.

Antioxidant activity of milk is due to the presence of antioxidant enzymes such as catalase and glutathione peroxidase (GPx) and other enzymes which catalyze the reduction of different peroxides aided by glutathione or other reducing substrates. The activity of milk antioxidant enzymes increases when SCC increases (Andrei et al., 2011). Measurement of the activity of such enzymes in milk has been used to monitor udder health in dairy cows (Fox and Kelly, 2006; Andrei et al., 2011).

To our knowledge, the correlation between bacterial infection, SCC and milk enzymes activity has not yet been fully considered in cows. Therefore, the aim of the present study was to evaluate possible relationships between milk SCC, isolated pathogens and enzymes activity; LDH, ALP, catalase, and GPx in dairy cows with subclinical mastitis.

MATERIALS and METHODS

Animals: A total of 50 clinically healthy dairy cows of different breeds (Holstein Friesian and native breeds); free of clinical mastitis and any other udder lesions, were examined in a special dairy farm in Beni-Suef governorate, Egypt.

Milk sampling: Milk samples were collected from 200 quarters before morning milking. Cows had no evidence of clinical mastitis at the time of sampling. Teat orifices were cleaned firstly then swabbed with cotton soaked in 70% ethyl alcohol, discarding the first streams of foremilk and then 20 ml of milk was collected aseptically from each teat into sterile tubes. Milk samples were kept cold during transportation, at 4°C and reached to the laboratory to be examined within 2 hours after collection. Milk samples were not collected from cows, which were treated with antibiotics by any route, till 96 hours after last treatment.

California mastitis test (CMT): CMT was applied directly in the farm to milk samples from each quarter using the method of Schalm et al. (1971). It is based on the principle that the addition of a detergent to a milk sample with a high cell count will lyse the cells, release nucleic acids and other constituents and lead to the formation of a ‘gel-like’ matrix consistency. According to the visible reactions the results were classified in four scores; 0 "negative or trace", 1 "weak positive", 2 "distinct positive" and 3 "strong positive". A cow was considered mastitic if one or more quarters were CMT positive with or without isolation of microorganisms.

Somatic cell counts: The SCC was applied to all milk samples. Milk samples showed positive CMT were used to detect any possible SCC variation as well as bacteriological examination to correlate and investigate the possible associated causal agent while the samples showed negative CMT were used as control. The samples were warmed in waterbath 40ºC for 5min, then mixed autonomically before autonomic reading of SCC by Bentley Soma count 15º for dispersion of fat globules (Zecconi et al., 2002). The SCC measures the number of WBCs including neutrophils, macrophage, lymphocytes, eosinophils and various epithelial cell types of mammary gland in milk that were present in large number in case of subclinical mastitis.

Bacteriological examination of milk samples: Milk samples (10 μl) were cultured from each positive CMT milk sample onto 10% sheep blood agar, nutrient agar and MacConkey agar plates according to Abdel-Rady and Sayed (2009). Plates were incubated aerobically at 37°C for 24-48 h. The plates were examined for gross colony morphology, pigmentation and haemolytic characteristics at 24-48h. Presumptive identification of bacterial isolates was according to their colonial characteristics, Gram's reaction and morphology. Identification was confirmed by additional laboratory tests according to Quinn et al. (1994); Abera et al. (2010) and Persson et al. (2011).

Enzymatic assays: It was applied to all milk samples. Firstly, milk samples were defatted by centrifugation at 3000×g for 10min at 4°C. Defatted milk samples were used for enzymatic estimation. LDH, ALP, catalase and GPx enzymes activities were estimated using commercial colorimetric assay kits (Biodiagnostic Company, Egypt). The standardized protocol provided with the kit was followed for estimation. The final results were reported in units per ml of milk (U/ml milk).

Antimicrobial susceptibility testing: Antimicrobial susceptibility test was performed on the isolates using Mueller Hinton agar by disc diffusion method according to NCCLS Standards (2002). The isolates were tested for 10 antibiotic discs. The following antimicrobial discs (Oxoid, Basing Stoke, UK) with their corresponding concentration were used: Ampicillin (AMP, 10μg), Spiramycin (SP, 100μg), amoxacillin (AML, 10μg), Clindamycin (DA, 2μg), Doxycycline (DO, 30μg), Florophenicol (F, 30μg), Spectinomycin (SH, 100μg), Enrofloxacin (ENR, 5μg), Ciprofloxacin (CIP, 5μg) and Flumequine (UB, 30μg). The inhibition zone was reported as the diameter of the zone surrounding the individual disk in which bacterial growth was absent. Based on this, the isolates were defined as resistant, intermediate and susceptible according to the guide lines of the NCCLS Standards (2002).

Statistical analysis: Statistical analysis was performed using Sigmastat version 3.5 (Systat Software Inc., London, UK). For all treatments, data are the means ± the standard deviation of the results. Data were analysed using analysis of variance (ANOVA). A significant difference was defined as a P value of ≤ 0.05. In case of a statistical difference, treatment means were compared with the Holm–Sidak or Tukey test for multiple pairwise comparisons.

RESULTS

Results of CMT: Out of the 50 clinically healthy dairy cows examined by the CMT, 14 (28%) were subclinically mastitic. Results of positive CMT realized on quarters showed that out of 200 quarters, 31 (15.5%) were positive with variable scores; 3 (21.5%) showed degree 3, 16 (8%) showed degree 2, 12 (6%) showed degree 1 and the rest; 169 (84.5%) showed degree 0 (Table 1).

Table 1: Relationship between the positive CMT and SCC, enzymes activities and the infection status of the quarters.

Fig. 1: Relation between CMT score and SCC, enzymes activities and the infection status.

Results of somatic cell counts: Out of the 31 positive CMT quarters, 27 (87.1%) were high in SCC with an average of 9.24±2.0×10⁵cells/ml. The other 173 quarters (4 positive CMT and 169 negative CMT) were normal SCC with an average of 1.2± 0.21×10⁵cells/ml (Tables 1 and 2).

Table 2: Changes in SCC in both normal and sub clinical mastitis cow’s milk according to CMT.

Somatic cell counts and bacteriological analysis: Of the 27 quarters with high SCC, 17 (63%) gave positive bacterial isolation while 10 (37%) were negative. On the other hand, out of the 173 quarters with normal SCC, 4 (2.3%) showed positive bacterial isolation while 169 (94.4%) were negative (Table 3).

Table 3: Classification of quarters according to SCC and bacteriological findings in SCM cases.

Results of bacteriological examination of milk samples. Out of the 31 SCM cases, 21 (67.7%) showed positive bacterial isolation of which 16 (51.6%) had single bacterial infections and 5 (16.1%) had mixed bacterial infections (Tables 1&4). Of the 16 single bacterial infections, 5 (16.1%) were Str. dysgalactiae, 4 (12.9%) E. coli, 2 (6.5%) Str. agalactiae, 2 (6.5%) were S. aureus, 2 (6.5%) were Bacillus subtilis and 1 (3.2%) were Corynebacterium bovis. The 5 mixed infections were 2 (6.5%) Str. agalactiae+ E. coli and one (3.2%) for each ofS. aureus+ E. coli, S. aureus+ Str. dysgalactiae and S. aureus+ B. subtilis (Table 4).

Table 4: Incidence of bacterial pathogens in SCM quarters milk samples in subclinical mastitis.

Results of enzymatic assays: All the 31 mastitis milk showed samply high levels of milk enzymes compared with the 169 normal milk where the average levels of LDH, ALP, catalase and GPx in SCM cases were 236.9±40.9, 268.5±53.3, 631.6±87.9, and 344.2±43 U/ml, respectively, compared with 66.6±13.7, 32.8±8, 125.2±10.2, and 125.2±20.7 U/ml in normal cases, respectively (Tables 1 and 5).

Table 5: Changes in milk enzymes in both normal and subclinical mastitis cow’s milk samples.

Correlation between bacterial infections, SCC and enzymatic activities in subclinical mastitis milk: Among the bacterial infections, the mixed S. aureus+ Str. dysgalactiae infection was the highest in SCC with an average of 12.44×10⁵cells/ml. Moreover, there were 4 bacterial infections associated with normal SCC including 2 E. coli isolates (1.5±0.04×10⁵cells/ml) and 2 B. subtilis isolates (1.39±0.04×10⁵ cells/ml), but having high levels of milk enzymes. On the other hand, there were 10 cases with negative bacterial isolation but with high SCC with an average of 7.18±0.62×10⁵cells/ml. Concerning milk enzymes, the mixed S. aureus+ Str. dysgalactiae infection also gave the highest levels of LDH, ALP and GPx with levels of 307.6, 408 and 454 U/ml, respectively, while catalase enzyme showed the highest level with the mixed S. aureus+ E. coli infection; 781 U/ml (Table 6).

Table 6: Correlation between bacterial infection, SCC and enzymatic activities in subclinical mastitis milk.

       No.= Number of bacterial isolates,  ^* = Latent infections (Bacterial isolates associated with normal SCC).

^** = Non specific mastitis (High SCC with no bacterial isolation).

Results of antimicrobial susceptibility testing: Using 10 antimicrobials; all Streptococcus species (Str. agalactiae and Str. dysgalactiae) as well as E. coli were sensitive to spectinomycin, enrofloxacin and ciprofloxacin while Streptococci were more resistant to flumequine; 60%, and clindamycin; 50, meanwhile 71.4% of E. coli isolates were resistant to ampicillin. All S. aureus isolates were sensitive to clindamycin, florophenicol and enrofloxacin but 80% of them were resistant to ampicillin. Moreover, all B. subtilis isolates were sensitive to spiramycin, doxycycline and florophenicol while they were more resistant to ampicillin and amoxacillin; 66.7% for each. C. bovis was resistant to clindamycin only (Table 7).

Table 7: Sensitivity of bacterial isolates to different antimicrobial drugs.

    R= Resistant,  I= Intermediate, S= Sensetive

DISCUSSION

Mastitis is one of the most critical diseases in dairy production as it is an economically devastating disease causing immense economic losses in the dairy industry. Subclinical mastitis is the most serious type as the infected animal shows no obvious symptoms and secrets apparently normal milk for a long time, during which causative organisms spread infection in herd, so it is an important feature of the epidemiology of many forms of bovine mastitis (Abdel-Rady and Sayed, 2009). Early detection of mastitis is of utmost importance in order to take steps toward isolation and/or treatment of the cow (Larsen et al., 2010). Clinical mastitis is easy to detect but also cows with SCM should be identified. The diagnosis of mastitis according to the IDF recommendations is based on SCC and microbiological status of the quarter (Guha et al., 2010).

CMT is still the superior screening diagnostic aid for subclinical mastitis, while bacteriological examination is still the most suitable, accurate and reliable method to confirm the causative organisms (El-Balkemy et al., 1997). Tests for indicators of inflammation are therefore necessary as screening tests to identify quarters with IMI and to select cows for subsequent bacteriological sampling. Over the past 50 years, many studies have been conducted in attempts to validate the CMT as a predictor of IMI. So, the CMT is a screening test for subclinical mastitis that can be used easily at cow-side (Leslie et al., 2002). It is widespread used in dairy fields and recommended as rapid and characteristic indicator for the infection of mammary gland (Al-Anbari et al., 2006). CMT can estimate SCC indirectly based on estimation of the DNA content of the milk; the reagent is simply a detergent (Sandholm et al., 1995) and the results were scored from 0 to 3 where 0 represents SCC less than 500,000cells/ml.

From the aforementioned results presented in Table 1, the recorded overall quarter incidence of SCM by the CMT was 15.5% (31 quarters out of totally 200). Nearly the same incidence was obtained by Abdel-Rady and Sayed (2009) in Egypt. Higher incidences were obtained by Mahmoud (1988) in Egypt, Ismail and Hatem (1998) in Saudi Arabia, Abera et al. (2010) in Ethiopia and Heleili et al. (2012) in Algeria.

Furthermore, among positive CMT milk samples, elevated enzymatic activity was shown in 100% of them while 87.1% only of them showed elevation in SCC. Meanwhile, 67.7% only were accompanied with bacterial isolation (Table 1 and Fig. 1). These results indicated that enzyme activity were more proportional with CMT, followed by SCC and finally bacterial isolation.

Milk SCC has been used extensively as indicator of IMI. SCC has been included as a component of the definition of mastitis, and the original limit for SCC of a healthy quarter is 500,000cells/ml (IDF, 1971). The definition was a guide for diagnosis, even though 50% of truly infected quarters could at any time have a cell count less than the break-point of 500,000cells/ml (IDF, 1971). The somatic cells present in the milk of a healthy cow belong mainly to the macrophages (66-88%); in addition there are neutrophils (1-11%); which increases up to 90% and more in case of IMI, as well as epithelial and mononuclear cells (Sandholm et al., 1995).

The obtained results from tables 1 and 2 revealed that 87.1% of the positive CMT samples were high in SCC with an average of 9.24±2.0×10⁵cells/ml which were significantly different (P<0.001) comparing to the normal ones. These results were higher than that of Heleili et al. (2012) who found that 34% only out of positive CMT milk samples were high in SCC. The rest of samples; 12.5%, were normal SCC although they were positive CMT and bacterial isolation and that occurs due to presence of latent infections (IDF, 1971). Consequently, SCC is always compared with bacteriology, and these tests can never completely agree (Pyorala, 2003). It is evident that a healthy quarter with no bacterial growth contains less than the original limit, and if the cell count exceeds it, the quarter is very likely to be infected. It is not relevant if the bacteria are minor or major pathogens, even though the so-called minor pathogens cause less elevation in the SCC (Hillerton, 1999).

The terms latent infection; positive bacterial isolation with normal SCC, and non specific (non infectious) mastitis; negative bacterial isolation with high SCC, have been in use (IDF, 1971). The comparison of SCC and bacteriological analysis results, illustrated in Table 3, showed that 63% of quarters with high SCC suffered from infectious mastitis while 37% suffered from non-infectious mastitis. On the other hand, latent infections were observed in 2.3% of the quarters with normal SCC. These results were nearly similar to those of Gianneechini et al. (2002) but differed from that of Heleili et al. (2012) who found that infectious mastitis cases were 94.4% while non infectious mastitis represented 5.6% only and latent infections were 83.8%.

Additionally, Dingwell et al. (2003) had assured that bacteriological culture is the gold standard method for identifying IMI but till nowadays the bacteriological sampling is not feasible as a routine test to identify subclinical mastitis. The indirect tests of mastitis seem to be more suitable for selecting cows with IMI (Pyorala, 2003). Also, Sargeant et al. (2001) added that logistic and financial considerations involved with sampling all fresh cows for bacteriological culture have precluded the widespread adoption of this strategy in the dairy industry.

Concerning the results of bacteriological examination shown in table 4, 67.7% of SCM milk samples showed positive bacterial isolation. These results were higher than that of Kapronezai et al. (2005); 26.8 %, Argaw and Tolosa (2008); 56.7%, Abdel-Rady and Sayed (2009); 40% and Rahman et al. (2010); 51.3%. On the contrary, our results were lower than that of Heleili et al. (2012); 87.3%. There were 10 positive CMT without isolation of the causative agent may attributed to that single milk sample has been criticized for not being sufficiently sensitive and for requiring more than one bacteriological sampling to determine whether the quarter is infected (Pyorala and Pyorala, 1997). Also, Heleili et al. (2012) reported that some of the microorganisms like listeria,mycoplasma and fungi require specific culture media. They also added that freezing of milk samples destroys some germs or reduces their number and makes the isolation of bacteria difficult. Persson et al. (2011) elucidated the reason for the number of colony forming units in milk is below the detection limit of the assay as well as the spontaneous bacteriological recovery. Additionally, the presence of antibiotic residues may explain falsely negative bacteriological results (Longo et al., 1994) because the withdrawal time is not respected in our herds.

Of the positive bacterial isolation, 51.6% had single bacterial infections and 16.1% had mixed bacterial infections. These results were higher than that of Rahman et al. (2010) who found that 17.1% of samples had mono-bacterial infection and 10.8% had mixed bacterial infections. Among the bacterial infections, Str. dysgalactiae was the most prevalent; 16.1%, followed by E. coli (12.9%), then Str. agalactiae, S. aureus, Bacillus subtilis and mixed Str. agalactiae+ E. coli infections (6.5% for each), and Corynebacterium bovis as well as mixed infections of S. aureus+ E. coli, S. aureus+ Str. dysgalactiae and S. aureus+ B. subtilis (3.2% for each). These bacterial infections were recorded also by Kapronezai et al. (2005); Elango et al. (2010); Rahman et al. (2010) and Shrestha and Bindari (2012) but they all differed in that S. aureus was the most prevalent. Our results also are in agreement with Andrei et al. (2011) who reported that most mammary gland infections are caused by only a few types of bacteria, including streptococci, staphylococci, coliforms and Corynebacterium bovis

Earlier investigations have revealed that enzyme activities in the udder epithelium change markedly due to mastitic inflammation (Bogin and Ziv, 1973; Kitchen, 1981). Many of the indigenous enzymes increase in milk during inflammation. The enzymes dealing with the synthesis of milk decrease and the enzymes related to inflammation increase. This increase is due to that the enzymes originating from phagocytes increase exponentially and they include NAGase, catalase and beta-glucuronidase (Pyorala, 2003). Also, the activity of the enzymes originating from the blood increase and leak from the blood into the milk due to an increased permeability (Pyorala, 2003). Moreover, Kitchen (1981); in his review article, listed many enzymes as reliable markers of bovine mastitis such as ALP, LDH, different lipases, esterases, etc. Recently, it had shown that LDH possesses comparable qualities for mastitis detection (Chagunda et al., 2006).

Regarding the results of enzymatic analysis in milk samples; reported in Tables 1and 5, it was observed that enzyme activity of LDH, ALP, catalase and GPx were significantly increased (P< 0.001) in mastitis (positive CMT) milk compared to normal milk. Our results of higher activity of LDH and ALP in SCM milk were supported with that of Babaei et al. (2007) and Batavani et al. (2007). Also, LDH and ALP were found to be elevated in mastitis buffalo milk infected with both gram positive and negative bacteria (Guha et al., 2012).

The early investigations indicated that elevated LDH activity in mastitic milk would not originate from blood plasma alone, but were more likely from parenchyma cells as well as disintegrated leukocytes (Bogin and Ziv, 1973; Kitchen, 1981). Kato et al. (1989) confirmed the contribution of LDH activity in mastitic milk from leukocytes and further specified the major increase in activity to originate from granulocytes and lymphocytes. Furthermore, Heyneman and Burvenich (1992) concluded that the leukocyte ALP enzyme from healthy and mastitic cows displayed very similar characteristics, suggesting that the increase in activity during mastitis is most probably related to the enhanced expression of the normal leukocyte ALP enzyme under direct or indirect influence of inflammatory mediators. Recently, Batavani et al. (2003) attributed that to the blood enzymes leaking from the blood into the milk due to an increased permeability. Katsoulos et al. (2010) and Mohammadian (2011) reported that the origin of elevated LDH and ALP activity was from leukocyte and mammary epithelial and interstitial cells damaged during inflammation, particularly from disintegrated leukocytes.

Catalase activity present in milk varies according to diet, lactation stage and especially for mastitis. It was demonstrated that catalase activity can be used as a marker of mastitis, in these cases the activity of catalase is much higher compared with normal milk (Fox and Kelly, 2006). Our results confirmed those of Hamed et al. (2008) who found a positive correlation between the catalase activity and the milk SCC. They signalled that catalase plays a central role in milk redox control. Especially during mastitis, catalase activity increases markedly, making it a useful indicator of mastitis. Also, they demonstrated that the catalase activity increased more with neutrophils than with macrophages and lymphocytes, indicating its role in free-radical decomposition in the case of bacterial infection resulting in an important increase of milk somatic cells. Elevated catalase activity also originated from the mammary gland and or bacterial cells (Fox and Kelly, 2006).

Glutathione-peroxidase is widespread in the cytoplasm of animal cells. The function of this enzyme is to protect cells against the damaging effects of peroxides, as part of an antioxidant enzymatic system (Andrei et al., 2011). Milk GPx varies according to species and diet (Fox and Kelly, 2006). Two different classes of GPx; selenium-dependent and selenium-independent, are known and both utilize glutathione for reducing hydroperoxides, but selenium-dependent enzymes are also capable of reducing hydrogen peroxide (Andrei et al., 2011). Our results of increased GPx activity in SCM milk compared to normal milk were supported with that of Andrei et al. (2011) who issued three hypotheses to explain this variation: (1) increased activity in milk is due to increasing enzyme concentration/activity in blood; (2) in milk, the enzyme is associated with caseins, an intensive hydrolysis process of casein can release the enzyme, and thus increase its activity; (3) peroxidases can be produced in milk by pathogens.

The correlations between bacterial infections, SCC and enzymatic activities in SCM milk were illustrated in Table 6. Concerning SCC, the mixed bacterial infection showed higher level than single infections and the mixed S. aureus+ Str. dysgalactiae infection were the highest in SCC with an average of 12.44×10⁵cells/ml while the 4 latent infections; 2 E. coli and 2 B. subtilis, showed normal SCC. On the other hand, the 10 cases of non specific mastitis showed high SCC with an average of 7.18±0.62×10⁵cells/ml. Concerning the milk enzymes, all bacterial infections; even latent infections, as well as non specific mastitis showed high milk enzymatic activity. Generally, the mixed infections were associated with the highest activity and the mixed S. aureus+ Str. dysgalactiae infection gave the highest levels of LDH, ALP and GPx with a level of 307.6, 408 and 454 U/ml, respectively, while catalase enzyme showed the highest level with the mixed S. aureus+ E. coli infection (781 U/ml). The high levels of mixed infections may be due to the more powerful effect of two or more pathogens than single pathogen especially if they were highly virulent, therefore this point needs more investigations.

Results of antimicrobial susceptibility testing illustrated in Table 7 showed that the susceptibility of Streptococci and E. coli to the tested antibiotics has revealed a complete (100%) susceptibility to spectinomycin, enrofloxacin and ciprofloxacin. Streptococci were more resistant to flumequine; 60%, and clindamycin; 50, while 71.4% of E. coli isolates were resistant to ampicillin. Concerning S. aureus strains, we noted a complete susceptibility against clindamycin, florophenicol and enrofloxacin but 80% of them were resistant to ampicillin. B. subtilis isolates were completely susceptible to spiramycin, doxycycline and florophenicol while they were more resistant to ampicillin and amoxacillin; 66.7% for each. Corynebacterium bovis was sensitive to most antimicrobials while it was resistant to clindamycin only.

CONCLUSION

SCM influences milk enzyme activity. From the current study it is evident that there is not a single indicator to detect SCM.  Although, LDH, ALP, Catalase and GPx were found to be indicators of SCM, all are being better and more reliable in the present study as they showed the highest agreement amongst all with IDF criteria of SCM diagnosis. It is recommended that all of mentioned enzymes are considered as markers for screening large herds for SCM. Standardizing easy biochemical methods or qualitative tests for estimating these indicators is recommended to develop kit for diagnosing SCM in the field. An attempt to develop a pathophysiological explanation of the ascertained association is also recommended for further study.

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