Proceeding of the Second Scientific Conference
1
CORRELATION BETWEEN CATTLE MASTITIS AND SERUM IRON, ZINC AND COPPER
1Mobarak M. G., 1El- Beahwey M. A. M. 2Maarouf A. A. and 3Abd El-Raof, Y.M.
1Dept.Biochemistry and Deficiency Diseases, Anim. Health Res. Inst. — Benha Provincial Laboratory
2Dept.Bacteriology, Anim. Health Res. Inst. — Benha Provincial Laboratory
3Dept. Anim. Med, Fac. Vet. Med., BenhaUniversity
ABSTRACT
In dairy farms at different localities in Kalubia Governorate, a total of 43 Frisian dairy cows (6-10 years old) were used in this study. Bacterial mastitis was recorded in 36 cows. All affected cattle fed ration contain iron, zinc and copper within that recommended by National Research Council for dairy cattle. The major clinical signs observed on mastitic cows were, general depression, anorexia, fever, ruminal atony, decrease in milk yield marked, changes in the physical characters of milk and signs of inflammation of the affected udder in the form of enlargement, hotness, and pain. Milk samples were taken for bacteriological examination and blood samples were collected for serum preparation and biochemical analysis. According to bacteriological examination,8 cases were diagnosed as coagulase positive Staphylococcus mastitis, 5 cases as E.coli mastitis, and 23 cases as mixed bacterial mastitis. The biochemical data revealed a significant decrease of serum iron and zinc in all mastitic cow compared with cows treated from bacterial mastitis with suitable antibiotic. On the other hand, serum copper showed no significant changes between before and after treatment of bacterial mastitis
INTRODUCTION
Mastitis is one of the most important diseases affecting dairy cattle which have many adverse economic implications represented by decreased in quantity and quality of milk components and shorten the productive life of affected animals (Esron et al, 2005). One of the most important causes of mastitis is the invading microorganism that mostly found in mixed infection .The predominant organisms are Staphylococcusaureus, Streptococci, and E.coli (Waage and Aursjo, 1992).
Lohuis et al, (1990) demonstrated that the numbers of E. colished from inoculated mammary quarters were positively correlated with the decreases of plasma Zn and Fe concentrations during the acute phase reaction. Iron withholding during microbial infection is regarded as host protective and is mediated by a number of host mechanisms(Weinberg, 1984), Binding proteins, such as lactoferrin, or chelators, and deferoxamine, decrease available Fe and Zn, thus decreasing the availability of these divalent cations needed for Gram-negative bacterial growth (Harmon and Newbould, 1980 and Shuster and Harmon 1992). Fe, and possibly Zn, sequestration during acute infections may be a mechanism to eliminate or to reduce pathogenic agents (Bullen, 1981, Klasing, 1984 and Weinberg, 1984). Sequestration of Fe may, also be a mechanism to reduce generation of oxygen radicals, which are potent mediators of tissue damage during inflammation(Hallewell, 1987). Oxygen radicals are released as a result of phagocytic function and from endotoxin-induced inflammatory mediators Free radicals are potentially harmful to host cytosol and membrane systems; thus, defenses to neutralize them exist. Iron is a catalyst for lipid peroxidation and radical formation; therefore, Fe sequestration during acute Gram negative mastitis also plays an antioxidative role (Hallewell, 1987 and Erskine et al, 1989 ).
The aim of this study is to find the correlation between bacterial mastitis and the level of serum iron, zinc and copper. Application of antibiogram for isolated bacterial pathogen to attain effective and specific treatment of the mastitic cases
MATERIALS AND METHODS
Animals: In dairy farms at different localities in Kalubia Governorate, a total number of 43 Frisian dairy cows (6-10 years old), were used in this study. All cows were subjected to clinical examination by visual inspection and palpation of the udder for swelling, redness and pain. Also physical examination of milk was conducted. Mastitis was recorded in 36 cows. According to bacteriological examination 8 cases were diagnosed as coagulase positive Staphylococcus mastitis, 5 cases as E.coli mastitis, and 23 cases as mixed bacterial mastitis.
Ration:
Ration offered to the animals was different in its constituents from farm to farm, but all contained zinc, iron and copper within the requirement of dairy cattle as recommended by National Research Council,(1988) as shown in Table (1). The ration was digested according to Crowell, (1973). The digested feed materials were used for estimation of copper, iron and zinc.
Microbiological Studies:
Under strict aseptic precaution and after discarding the fore milk, two milk samples from each affected quarter were collected in sterile McCartney bottles and transported to the laboratory in ice container for microbiological examination. The mastitic milk samples were incubated for 12 hrs at 37ċo, then centrifuged at 3ooo rpm for 20 minutes .Samples were cultivated into: Nutrient agar, MacConkey agar, blood agar and mannitol salt agar. All plates were incubated aerobically at 37co for 24-72 hrs. The developed colonies were picked up and subculture for purification. The pure colonies were morphologically identified by Gram stain and biochemically according toCarter and Cole, (1990) and Quinn et al, (1994).Further studies on pure isolates of Staphylococciwere done including coagulase activities, different types of plasma (rabbit, sheep, horse, and human) were used for detection of coagulase activities by slide coagulase test and confirmed with tube coagulase test according toKoneman et al, (1997). The isolated strains were subjected to the sensitivity test against 9 different antibiotic disc (oxoid standardized disc), using the agar diffusion method according to Finegold and Martin, (1982). The zone of inhibition was measured and interpretation of results was done according to manufacture recommendation.
Blood Samples:
Blood samples were collected from each animal by jugular vein puncture before treatment and 15days after treatment. The blood samples were collected without anticoagulant for serum preparation and determination ofserum copper, iron and zinc concentration by using the atomic absorption spectrophotometer after Crowell, (1973).
Treatment:
According to sensitivity test and recommended doses for each chemotherapy, treatment of mastitic cows was done by local and systemic treatment. Diclofenate sodium (Declofenil from Arabco) was used as antipyretic and anti-inflammatory at rate of 75 mg/100 kg body weight by intramuscular route once daily. Gentamycin was the most effective drug in both E. coli and mixed mastitis. Gentamast (Premer Pharma–Germany) as 7.5 ml syringe containing 170 mg gentamycin was used twice daily for 7 successive days. Also, this was concurrent with intramuscular injection of gentamycin (El-Nile Co.for Pharmaceuticals and Chemical Industries) as 3 mg/kg B.wt. twice daily. While with coagulase positive staphylococcus mastitis, lincomycin was the most effective drug used in intramammary infusion, as Lincocin Forte (Pharmacia Animal Health Limited): 10 ml syringe contain contain 330 mg lincomycin, 100 mg neomycin and 10 mg prednisolone. This infusion was performed twice daily for 7 successive days. This was concurrent with intramuscular injection of Lincospectin (Pharmacia Animal Health Limited) as 10 mg/kg B.wt. (Prescott and Baggot, 1993). The animal considered treated after amelioration of all clinical signs and milk samples were negative for bacterial pathogens.
Statistical Analysis:
The samples were simultaneously analyzed, using the student (t) test according to Petrie and Weston, (1990).
RESULTS
Clinical Finding:
The major clinical signs observed on mastitic cow included, general depression , anorexia, fever, ruminal atony, marked decrease in milk yield and signs of inflammation observed on the affected udder as enlargement, hotness, and pain. These signs ameliorated and completely disappeared during treatment.
Ration Analysis:
Table (1) Zinc, iron, and copper values in ration offered for mastitic cattle compared with requirements recommended by National Research Council (1988) before and after treatment of mastitis.
parameter / Mean values of the elements in offered ration / Requirements as recommended by National Research Council (1988)zinc / 46ppm ±3.64 / 40ppm
iron / 62ppm ±4.02 / 50ppm
copper / 14ppm ±2.70 / 10ppm
Bacteriological Results:
Table (2) Bacteriological examination of clinical mastitic milk.
Prevalence of single and mixed culture / positive / negative*** / No. of milk samples / No. of examined cowsmixed** / single*
% / No. / % / No. / % / No. / % / No.
69.2 / 45 / 30.8 / 20 / 87.8 / 65 / 12.2 / 9 / 74 ¤ / 43
- *13 coagulase positive Staphylococcus aureus from milk samples of 8 cows.
- *7 E-coli from milk samples of 5 cow.
- **45 mixed cultures from milk samples of 23 cow.
- *** 9 milk samples were negative
- ¤ Milk samples were collected from 43 cow
Table (3) Bacterial species isolated from 65 positive milk samples.
% / No. / Bacterial isolates37.8
27.7
4.2
5.9
17.7
14.3
9.2
6.7
7.6
6.7 / 45
33
5
7
21
17
11
8
9
8 / Staphylococci
Coagulase positive Staph. aureus
Coagulase negative Staph. aureus
Coagulase negative Staph. epidermidis
E.coli
Streptococcus agalactia
Streptococcus dysagalactia
Streptococcus ubreis
Pseudomonas aerogenosa
Actinomyces pyogenes
100% / 119 / Total
Table (4): Efficiency of some antibiotics in treatment of mastitis (sensitivity test).
Bacterial isolates / AntibioticMixed cultures
(23) / Staph. aureus(33)
Coagulase positive / E.coli(21)
% / No. of sensitive / % / No. of sensitive / % / No. of sensitive
56.5
60.9
69.6
73.9
82.6*
78.3
26.1
60.9
47.8 / 13
14
16
17
19
18
6
14
11 / 66.7
63.6
60.6
69.7
81.8
84.9*
48.5
45.5
39.4 / 22
21
20
23
27
28
16
15
13 / 47.6
52.4
76.2
81.0
85.7*
71.4
23.8
57.1
33.3 / 10
11
16
17
16
15
5
12
7 / Amoxycillin
Ampicillin
Chloramphenicol
Enrofloxacin
Gentamycin
Lincomycin
Penicillin
Streptomycin
Tetracycline
Biochemical findings:
Table (5): Mean values of serum iron zinc, and copper in 5 cases of E-coli mastitis before and after treatment.
15 days After treatment / Before treatment / Parameter152.80 ± 7.91
117.40 ± 6.42
94.00 ± 9.93 / 55.60 ± 7.56*
44.60 ± 4.33*
96.60 ± 10.11 / Iron(ug/100ml)
Zinc(ug/100ml)
Copper(ug/100ml)
- *Significant at p ≤ 0.001
Table (6): Mean values of serum iron, zinc, and copper in 8 cases coagulase positive Staphylococcus aureus mastitiscases before and after treatment.
15 days After treatment / Before treatment / Parameter153.37 ± 6.41
123.36 ± 4.2
97.62 ± 9.03 / 120.75 ± 10.25*
104.00 ± 6.38*
96.12 ± 6.45 / Iron(ug/100ml)
Zinc(ug/100ml)
Copper(ug/100ml)
- * Significant at p ≤ 0.001
Table (7): Mean values of serum iron, zinc, and copper in 23 cases of mixed bacterial mastitis before and after treatment
15 days After treatment / Before treatment / Parameter156.86 ± 6.98
119.73 ± 4.49
94.80 ± 6.74 / 94.65 ± 6.95*
56.78 ± 6.92*
96.60 ± 6.89 / Iron(ug/100ml)
Zinc(ug/100ml)
Copper(ug/100ml)
- * Significant at p ≤ 0.001
DISCUSSION
In the present study clinical signs observed on dairy cow suffered from bacterial
mastitis agree with that recorded by Lohuis et al., (1990) and Radostits et al., (1994). They pointed out that systemic clinical signs that are often observed during bacterial mastitis include general depression, anorexia, fever, decrease in feed intake, ruminal atony and marked decrease in milk yield. In addition to hotness, enlargement, and pain were recorded on affected udder.
The bacteriological examination of 74 mastitic milk samples ,Table (2) showed that 65 (87.8%) were positive as 20 (30.8%) had pure single cultures and 45 (69.2%) with mixed ones. Nearly similar results were obtained by Rahman and Boro, (1990), Itman and Abd-Elhalim, (1991).
Table (3) showed that 119 bacterial strain isolated from 65 positive mastitic samples , where Staphylococcus was the most predominant pathogen (37.8%), mainly coagulase positive Staphylococcus(27.7%), followed by E.coli (17.7%), Streptococcus agalactia (14.3%), Streptococcus dysagalctia (9.2%) , Pseudomonas aerogenenosa (7.6%) and finally Actinomyces pyogenes and Streptococcusuberis(6.7%)for each. Nearly similar results were obtained by Rahman and Boro, (1990), Itman and Abd-Elhalim (1991), Barkema, et al, (1998) and Esron, et al, (2005).
Table (4), showed that E.coli was more sensitive to gentamycin (85.7%) followed by enrofloxacin (81.o %), chloramphenicol (76.2%) and lincomycin (71.4%). With respect to coagulase positive Staph. aureus, lincomycin was most sensitive (84.9%) followed by gentamycin (81.8%), enrofloxacine (69.7%), and amoxycillin (66.7%). Meanwhile for mixed bacterial cultures gentamycin was the most effective one (82.6%) followed by lincomycin (78.3%), enrofloxacin (73.9%), and chloramiphenicol (69.6%).Nearly similar results recorded by Rahman and Boro, (1990) Aydin et al, (1995) and Hamouda et al, (2000).
The biochemical analysis for sera from cows with in E.coli mastitis Table (5) showed, significant decrease of serum iron and zinc before treatment) comparing with those estimated after treatment. Nearly similar results obtained by Lohuis et al (1988a), (1988b), (1990) and Erskine and Bartlett, (1993). They attributed that to function of endotoxin release which stimulates the circulating endogenous mediators that cause alteration of these trace minerals (Erskine and Bartlett, 1993).
In coagulase positive Staphylococcus mastitis Table (6) there was significant decrease of serum iron and zinc comparing with those recorded after treatment. Also, we noticed that levels of serum iron and zinc was higher than those with E.coli mastitis. Nearly similar results obtained by Middleton, et al, (2004). They pointed out that inflammatory response following intrammamary staph.aureus infection is localized to the mammary tissue and released inflammatory mediators have little systemic influence.
In mixed bacterial mastitis Table (7) serum iron and zinc were significantly decreased comparing with those after treatment. In mixed infection there were a combination of local inflammatory effect caused by gram positive bacteria and systemic effect of endotoxins of gram negative bacteria that my be alter serum zinc and iron. Nearly similar results were obtained by Erskine and Bartlett, (1993) and Middleton, et al, (2004).
Tables (4, 5 and 6) showed non significant changes in serum copper before and after treatments. Erskine and Bartlett, (1993) documented a decrease in plasma Cu concentrations, to 75%of prechallenge level following experimental infection with E. coli. They attributed this decrease to the fact that the cattle in this experiment were marginally Cu deficient at the beginning of this study. Another study by scaletti, et al, (2002) who recorded that serum copper concentration was greater in copper supplemented group (20ppm) than unsupplemented group (6ppm) during experimental E.coli mastitis. We believe that the level of serum copper during bacterial mastitis depend on supplementation to ration.
From this study we concluded that, the acute infection of the udder by gram positive and gram negative bacterial causea significant decline and sequestration of serum iron and zinc, so these elements must be supplied to the ration in monitoring of those two elements during bacterial mastitis.
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العلاقه بين التهاب الضرع البكتيري في الابقار ومستوي الحديد والزنك والنحاس في مصل الدم
محمد جمال عبدالرازق مبارك1 –معروف عابدين محمد البعلاوي1-أحمد عفيفي عبدالغفار معروف2ويسين محمود عبد الرءوف3
1 قسم الكيمياء الحيوية و امراض النقص الغذائى- معهد بحوث صحة الحيوان - المعمل الفرعى ببنها
2قسم البكتيريولوجيا- معهد بحوث صحة الحيوان - المعمل الفرعى ببنها
3قسم طب الحيوان- كلية الطب البيطرى- جامعة بنها
أجريت هذه الدراسة علي 43 بقره حلابه متوسط اعمارها يتراوح من 6-10 سنوات في مزارع الحلاب في مناطق مختلفه من محافظه القليوبيه. تم تشخيص 36 حالة التهاب ضرع بكتيرى منها ، أهم الأعراض التى سجلت على الحيوان هى خمول وكسل وفقدان الشهية وارتفاع فى درجة حرارة الحيوان وقلة وضعف فى حركة الكرش ونقص فى إنتاج وتغير فى شكل ومواصفات الحليب الطبيعية بالإضافة الى كبر حجم الضرع واحتقانه ووجود الم للحيوان .
كل الحيوانات كانت تتغذى على علائق مركبة تحتوى على تركيزات من الحديد والزنك والنحاس فى الحدود الموصى بها من منظمة بحوث الزراعة العالمية لاحتياجات الأبقار الحلابه.
أخذت عينات لبن ودم لفحصها معملياً . تم عزل البكتيريا المسببة لالتهاب الضرع فوجدت 8 حالات مصابة بالمقورات العنقودية وخمس حالات مصابة بالعصيات القولونية و23 حالة مصابة بخليط من البكتيريا المختلفة . وتم عمل اختبارات حساسيه لاختيار المضاد البكتيري المناسب.
التحاليل البيوكيميائية لمصل الدم أظهرت نقص معنوى ملحوظ فى مستويات الحديد والزنك بمصل الحيوانات المصابة بالتهاب الضرع البكتيرى بالمقارنة ببعد العلاج بالمضادات البكتيرية المناسبة بينما لم يتأثر مستوى النحاس أثناء الإصابة بمقارنته ببعد العلاج .
واستخلصت هذه الدراسه ان يؤخذ في الاعتبار النقص المؤقت لعنصري الحديد والزنك اثناء التهاب الضرع البكتيري.
The Second Scientific Conference, Fac. Vet. Med., BenhaUniversity
Benha - Ras Sedr 25-28 January 2007