Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
761
INVESTIGATIN OF M. agalactiae AND STUDY ITS EFFECTS ON
SOME HEMATOLOGICAL AND BIOCHEMICAL PARAMETERS IN
SHEEP AND GOATS INFECTED WITH MASTITIS
Bashar Sadiq Noomi *, Khalid Ahmed Hadi**, Hiba Younis Khalaf ***,
Ahmed Abdulaali Azeez****, Nihad Abdalussain Jaafar*****
, Hameed Ali Naji Al-refaai*******
*,***,***** Department of Microbiology , faculty of Veterinary Medicine,University of
Tikrit , Iraq
** Department of Physiology , Pharmacology and Biochemistry , faculty of Veterinary
Medicine,University of Tikrit , Iraq
**** Department of Physiology , Pharmacology and Biochemistry , faculty of Veterinary
Medicine,University of Kirkuk , Iraq
*****Department of Microbiology , faculty of Veterinary Medicine,University of Hama ,
Syria
Corresponding Author: [email protected]
ABSTRACT
This study was performed to investigate M. agalactiae as a cause of mastitis in sheep and
goat and study some hematological and biochemical parameters, for this aims (102) milk
samples and (102) blood samples from mastitic sheep and goat were collected . our results
showed that detection of M. agalactiae in ratio of (10.87 %) and (16.6 % ) by culture and
PCR technique. Regarding biochemical parameters we noted that zinc decrease significantly
(P ≤ 0.05) in infected group, while copper, Total protein, Albumin and Globulin levels not
affected. As far as hematological parameters we noted no significant differences between
Infected and Non Infected group in the level of WBC and Hb while significant decrease in
the level of RBC and PCV in infected group in compare with control group . we conclude
from this study M. agalactiae consider as one of mastitis cause in Salahaldeen province and
it affects on hematological and biochemical parameters in studied group.
INTRODUCTION
Mastitis defines as inflammation of parenchymal tissue of mammary glands by
microorganism leads to chemical, physical and microbial changes, its consider an
important economic and public healthy disease (1,2). Mastitis may be clinical or subclinical
and according to its causes either contagious mastitis like Staphylococcus aureus,
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
762
Streptococcus agalactiae and mycoplasma spp. or Environmental mastitis like coliform
bacteria (3). Mycoplasma is smallest free living prokaryotics , loss of cell wall so that its
highly polymorphic shape and it has fried egg appearance colony, many species of them
causing mastitis in sheep like M. capricolum subsp capricolum and M. mycoides subsp.
mycoides but main of them M. agalactiae (4). Mycoplasma agalactiae cause many
economic enzootic diseases and main complication are septicemia, mastitis, arthritis,
pleurisy, pneumonia, and kerato conjunctivitis (5).
regarding tissues infection with Mycoplasma agalactiae are mostly associated with
exudations and cellular infiltration which alter blood cellular counts, Serum biochemical
changes are of paramount importance in accessing the level of cellular and systemic
responses of tissues and organs to damage (6) . Nelson and others (7) reported serum zinc
levels of (4.86-6.08 μmol/l ) in a herd which suffered from cases of anorexia, wool eating,
alopecia, hyperkeratosis and parakeratosis, Çama إ and others (8) have reported a level of
(3.80-7.30 μmol/l) for similar symptoms. In ruminants, normal serum zinc levels are
between (11 and 18 μmol/l) and animals with levels below (10.5 μmol/l) are considered
deficient (9). Normal serum copper levels for sheep as reported by various researcher (8) are
(9.26-15.86 μmol/l ).
MATERIALS AND METHODS
This study performed in Salahaldeen province
Milk sample: 64 milk samples were collected from mastitic ewes (43 from clinical cases
and 21 sub clinical cases which positive to CMT). And 38 milk samples were collected from
mastitic goats (26 from clinical cases and 12 sub clinical cases which positive to CMT).
After disinfected of teats and removing first drops, 10 ml of milk were collected in septic
tube.
blood samples: 102 blood samples were collected from same mastitic ewes and goats that
milk collected. Each sample divided in to two parts, first for hematological and second for
biochemical tests.
Culture methods : 0.1ml of homogenized milk sample were culturing in Freyś agar
medium (Himedia- India) with additives 150ml horse serum (collected from jugular vein
than centrifuged and filtrated by using 0.22μM filter), 100ml of yeast extract (10). 5ml of
Cystin hydrochloroide (Himedia- India), 5ml of NAD (Himedia- India(0.1g:5ml), 20mlof
Thalium acetate (1g:100ml), 5 ml of Penicillin solution (1000000I.U.Lml) (Segmi),
dextrose (50g:100ml) and 1% phenol red (1g:100ml). final pH 8.9 (10).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
763
Genetic methods:
DNA extraction: 500μl of samples were centrifuged at 13,000 rpm for 20 mints then 100μl
of lyses buffer was added and inculpated at 56°C 4h. 200μl of phenol added and centrifuged
at 13,000 rpm for 20 min. supernatant were taken and mixed with Phenol/ Chloroform (1:1).
centrifuged supernatant mixed with pure Chloroform then recentrifuged at 13,000 rpm for
5 min. supernatant mixed with 1/10 volume of acetate sodium then kept at -20°C refrigerator
with 2 fold volume of cool and pure ethanol (20 min), then the tube was centrifuged at
13,000 rpm for 15 min. 200μl of ethanol (70%) was added and the tube centrifuged at
13,000 rpm for 5 min, DNA was dried and redissolve in distal water (11) .
- The Compounds used in preparation of PCR reaction mixture as in table (1).
Table (1):. Compounds used in preparation of reaction Mixture
Compounds used in preparation of reaction mixture Volume
(microliters)
Reference
Taq PCR Master Mix KIT: Which contain Taq DNA
Polymerase (2.5 Unit), PCR Buffer with 3mM MgCL2,
200μMdNTP
25 (Qiagen, Germany).
Forward primer
FS1 FS1:5/-AAAGGTGCTTGAGAAATGCC-3/
2 from 100pM
Solution
Santos et al., 2018
(12)
Reverse Primer
FS2: 5/-GTTGCAGAAGAAAGTCCAATCA-3/
2 from 100pM
Solution
DNA Template 2 (Qiagen, Germany)
DNA free water 19 (Qiagen, Germany)
Total 50
Table (2): Thermo cycler programs
Stage Temperature (c) Time No.of
cycles
First Denaturation 95 60second 1
Denaturation step 95 60second
Primer-annealing step 50 60second 35
DNA extension step 72 60second
Final DNA extension 72 5 mint 1
End Temperature 4
Serum copper and zinc measurements were carried out with a Shimadzu Atomic absorption
Spectrometry model AA-680 in accordance with the technique described in the references
(13,14). In order to prevent contamination from glassware, plastic materials were used
during the measurements of trace elements. Total protein and Albumen were measured by
using special kit supplied by (RANDOX) company, while globulin value was obtained by
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
764
this equation (Total protein – Albumen = globulin ) (15). Blood parameters were measured
by using Vet. Hematological coulter apparatus .
Statistical analysis
The results were analyzed using the SPSS program for values representing the rate
and the standard error; the data were analyzing using ANOVA Analysis of variance One
Way. The differences between the groups were determined using the Duncan multiple range
test, at a probability level (P ≤ 0.05).
RESULTS
According to colony morphology, M. mycoides isolated from 11 milk samples with ratio
10.78% (11:102). As in table (3). In figure (1) shows morphology of mycoplasma colony.
Table (3): isolation ratio of M. mycoides from milk samples
Animal Case No. of
sample
No. of positive
sampled
Ratio
Sheep Clinical 43 5 11.6%
Sub clinical 21 1 4.7%
Goats Clinical 26 4 15.3%
Sub clinical 12 1 8.3%
Total 102 11 10.78%
Figure (1): shows colony morphology of M. agalactiae
By using of PCR technique, M. mycoides detected in rate of 16.6% (17:102) from mastitic
sheep and goat milk. As in table (4). Figure (2) show positive result of PCR test.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
765
Table (4) M. mycoides detection ratio from mastitic sheep and goat milk
Animal Case No. of
sample
No. of positive
sampled
Ratio
Sheep Clinical 43 8 18.6%
Sub clinical 21 2 9.5%
Goats Clinical 26 6 23.0%
Sub clinical 12 1 8.3%
102 17 16.6%
Figure(2): Electrophoresis on 2 % agarose gel and ethidium bromide staining, showing the
results of PCR procedures. M: DNA marker, , wells 1-6 positive samples band in size 375
bp. (M. agalactiae), CN: control negative
From table (5) noted no significant differences between Infected and Non Infected group in
the level of copper, Total protein, Albumin and Globulin, while significant decrease in the
level of zinc in infected group in compare with control group
Table (5) : Biochemical parameters of studied cases
Groups
Parameters
Infected group Non Infected group
Mean ± S.D Mean ± S.D
Copper (μmol/l) 13.22 ±1.43 a 12.54 ±2.12 a
Zinc (μmol/l) 8.92 ±1.76 a 10.86 ±1.54 b
Total protein (g/dl) 72.2 ±9.60 a 75.0 ±10.22 a
Albumin (g/dl) 33.0 ±3.50 a 32.6 ±4.10 a
Globulin (g/dl) 39.2 ±9.28 a 42.4 ±12.04 a
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
766
From table (6) noted no significant differences between Infected and Non Infected group in
the level of WBC and Hb while significant decrease in the level of RBC and PCV in
infected group in compare with control group
Table (6) : Blood parameters of studied cases
Groups
Parameters
Infected group Non Infected group
Mean ± S.D Mean ± S.D
WBC(x103/μl) 10.90 ±1.64 a 9.59 ±1.56 a
RBC(x106/μl) 13.29 ±2.15 b 15.27 ±2.60 a
Hb(g/dl) 10.70 ±1.77 a 12.24 ±2.41 a
PCV (%) 28.67±4.13 b 33.25 ±7.11 a
DISCUSSION
In current study Mycoplasma agalactiae detected in rate of 16.6% , that ratio is less than
ratio recorded by (16).which is 57%, and less than result recorded by (17), which 44% in
Iran , and more than ratio recorded by (18) which 8.26% in Syria. In Jordin mycoplasma
isolate in milk with rate of 13%(19) that’s due to deference in geographic area and using
techniques. In our study Mycoplasma agalactiae we detected high rate in goat milk in
compare with sheep milk , that’s agreed with (20). In compare between results of culture and
PCR techniques we found that PCR techniques more efficiency than culture methods, this
results agreed with result of (16,21). In the other hand there was a considerable decrease in
the level of zinc and our findings agreed with (22) who reported that decreasing in the level
of zinc in case with mastitis infection due to disturbance in the immune system . and we find
in this study there is no significant differences in the level of copper between studied groups.
In the other hand we noted no differences observed in the concentration of total protein,
albumin and globulin in the serum (23). There is no significant differences between the
infected group in compare with control group in the values of total WBC. We find In the
current study, there were significant decreases in the values of packed cell volume and RBC
count in the infected group with mastitis in compare with control group. These results were
similar to the findings of (24) . who observed a decrease in total erythrocyte count and
attributed his results may be due to that RBC important determinants for accessing responses
in systemic tissue injury depending on the stage and type of inflammation present (25) .
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
767
الکشف عن المایکوبلازما القاطعة للحلیب وتاثیرھا على بعض المعاییر الفسلجیة والکیموحیویة فی
الاغنام والماعز المصابة بالتھاب الضرع
بشار صادق نومی*، خالد احمد ھادی** ، ھبة یونس خلف*** ، احمد عبد العالی عزیز****
نھاد عبدالحسین جعفر ***** ، حمید علی ناجی الرفاعی ******
*****،***،* فرع الاحیاء المجھریة ، کلیة الطب البیطری ، جامعة تکریت ، تکریت ، العراق .
** فرع الفسلجة والادویة والکیمیاء الحیاتیة ، کلیة الطب البیطری ، جامعة تکریت ، تکریت ، العراق .
**** فرع الفسلجة والادویة والکیمیاء الحیاتیة ، کلیة الطب البیطری ، جامعة کرکوک ، کرکوک ، العراق .
***** فرع الاحیاء المجھریة ، کلیة الطب البیطری ، جامعة حماة ، سوریا .
الخلاصة
کاحد مسببات لالتھاب الضرع فی M. agalactiae أجریت ھذه الدراسة للتحری عن المایکوبلازما القاطعة للحلیب
الأغنام والماعز فی محافظة صلاح الدین. اذ تم جمع ( ١٠٢ ) عینة من الحلیب و ( ١٠٢ ) عینة دم من الأغنام والماعز.
کان بنسبة ( ١٠.٨٧ ٪) بواسطة الزرع M. agalactiae أظھرت النتائج أن الکشف عن المایکوبلازما القاطعة للحلیب
الجرثومی و ( ١٦.٦ ٪) بواسطة تقنیة البولمرات المتسلسلة . وقیاس بعض المعاییر الدمیة والکیموحیویة، اذ لوحظ ان
فی عنصر الزنک فی المجموعة المصابة فی حین أن مستویات النحاس والبروتین (P ≤ ھنالک انخفاضا معنویا ( 0.05
الکلی والألبومین والجلوبیولین لم تسجل ای تغییر معنوی. وفیما یتعلق بالمعاییر الدمیة لوحظ عدم وجود فروقا معنویة فی
مستوى خلایا الدم البیض وخضاب الدم بینما لوحظ انخفاضا معنویا فی مستوى کریات الدم الحمر وحجم الخلایا
المرصوصة فی المجموعة المصابة بالمقارنة مع المجموعة السلیمة. نستنتج من ھذه الدراسة ان المایکوبلازما القاطعة
للحلیب تعد احد اسباب التھاب الضرع فی محافظة صلاح الدین ویؤثر على المعاییر الدمیة والکیموحیویة فی الحیوانات
المصابة .
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