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ACUTE BUFFALO MASTITIS CAUSE BY MIXED INFECTION OF
Enterobacter cloacae AND Proteus mirabilis AT BASRAH, IRAQ
Mohanad H. Lafta
Department of Internal and Preventive Medicine, College of Veterinary Medicine University of
Basrah, Iraq.
(Received 9 March 2020 ,Accepted23 May 2020)
Key words: Mastitis, Enterobacter cloacae, Buffalo.
Corresponding Author: kamalsad58@yahoo.com
ABSTRACT
Mastitis caused by Proteus mirabilis and Enterobacter cloacae has been investigated
and diagnosed. Milk samples carried-out from fifteen local buffalo breeds reared in Basrah, Iraq.
Ten clinically healthy local buffalo breeds were considered and controls. Diseased buffalo show
signs of pain, swelling, redness of the udder, watery consistency with the light red color of the
milk, with high systemic reactions including significant increase (p<0.05) in body temperature
,respiratory and heart rate. Furthermore, a significant (p<0.05) reduction of ruminal contractions
also resulted. Hematological changes of diseased buffalo and the controls, reveals Leucocytosis
due to a significant increase of Nurtrophiles. All diseased animals are clinically examined and
the results reveals that Proteus mirabilis and Enterobacter cloacae are the common causes of
mastitis which confirmed by VITEK 2 System. The gram stain from the milk smears show a
clear Gram-negative rods, Moreover, swarm forming and pale colony character was also
indicated of the swab culture of the causative Proteus mirabilis. It has been concluded that acute
buffalo mastitis could be of adverse effect, Nevertheless, the knowledge of the causative agents
of mastitis are very useful for the fast treatment of the disease.
INTRODUCTION
Mastitis is believed to be an important big problem in dairy animals life . The disease
reflect an inflammation of mammary gland mostly caused by bacterial organisms which
contaminated and got entrance thorough teat orifice and multiply (1). Several infectious agents,
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including those caused by microorganism such as (Coli form bacteria, Brevibacterium
erythrogenes, leptospira spp, Micrococcus cerasinus, Serratia marcescens, Micrococcus
hromidrogenes, Micrococcus roseus, Sarcina rubra Lactorubefaciens gruber, and so on .) could
be the causative agents of mastitis (2). Although, some viruses and some species of yeast such
as red yeast (Monascus purpureus) has been cause the disease in the same matter (3). Most of the
above microorganism are present in the animals environment (soil, water, food, bedding, and
even the manure). Those causative organisms can create udder inflammation and a clinical
disease in which an udder abnormality or secretions is detected and notes , Moreover, according
to the severity of clinical manifestations, clinical mastitis can be mild, moderate or severe,
Therefore, animals suffer from the mild type of the disease will have abnormal milk contains
suck as , flakes , clots with abnormality of the inflamed udder like hot and swollen udder,
However, systemic reaction might also detected(1). On the other hand, the acute or severe
clinical inflammation will suddenly started accompanied by acute local inflammatory signs,
abnormalities of milk contains and sever systemic manifestations, Moreover diseased animal
may also show signs of depression, weakness and dehydration (2).
It has been documented that mastitis can have unsatisfactory results and bad
consequences, due to buffalo producing milk which had some abnormal coloration such as
red or pink due to the presence of red blood cells, As, the animal owners could not afford the
economic losses because of the abnormalities of the diseased milk which will always refused by
the consumers , in addition that a premature culling of affected animals (4). It has been
documented that the most important causative agents of mastitis are Enterobacter and Proteus
mirabilis, Since, they are widely distributed in soil, water and faces(5), However, The general
protection could done via good hygienic measure and reducing udder contamination and
trauma(3). The aim of the present work was to investigated the special bacterial causes of
mastitis in inflamed udders of diseased buffalo at Basrah, Iraq.
MATERIALS AND METHODS
The study area and examination of animals:
Fifteen local buffalo breeds was show clinical signs of mastitis .Animals are of
different ages reared in Basrah, Iraq. Ten clinically healthy buffalo breeds was considered as
controls. Complete clinical examinations has been applied for all animals. With special privacy
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developed for udder examination, Since, The animals were properly restrained, and identified,
Moreover, the abnormalities of the diseased udder including, secretions, size, consistency and
udder temperature was examines clinically by inspection and palpation, Furthermore, animal
reaction such as pain during palpation, changes of milk contains was also taken in consideration
as an clinical indications of the mastitis.
Sampling
Blood samples: Blood was take out from jugular vein from each animal mixed with EDTA for
complete blood picture using the automatic cell counter from Beckman, (USA).
Milk samples: Diseased milk was collected under aseptic precautions and the clinical data was
listed from diseased animals, ten(10) ml of milk was aspirated and applied in sterile plastic
vials for further analysis (3).
Staining:Milk smears stained with Gram’s stain. Was used for the primary identification of the
causative bacteria
Culture media: Suspected samples of milk was cultured in Nutrient broth, then incubated for
about 24 h . Furthermore, the sample was transfer to subcultured from nutrient broth medium
into blood agar medium, MB and MacConky agar and incubated for about 24 h. In order to
obtain pure cultures. Moreover, the colony size, shape, consistency and the color was recorded
,On the other hand, a microscopic examination has been applied for the gram stain smears of the
bacterial isolates (6).
Biochemical tests: VITEK 2 systems (Biomerrieux /France) which uses Advanced
Colorimetry™ , was used in this study to confirm the diagnosis of the isolated bacteria.
The statistics: The statistical difference between diseased buffalo and the controls was done and
The significance of variations was analyzed statistically using student t-test (7).
RESULTS
Diseased local buffalo breeds show different clinical manifestations including the local
inflammatory signs which include hotness, redness, swelling and pain of the infected udder
(100%), Partial or complete loss of appetite (80%), Lameness and unable to recumbency
(53.3%), However, fluid exudation, and crepitating was detected on infected quarter area
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(46.6%). Moreover, Infected milk was watery in its consistency with light red color (46%) and
grinding of teeth, table No.1.
Table1: Clinical manifestations of Acute buffalo mastitis.
Clinical manifestations % n=15
Local inflammatory signs which include (hotness, redness,
swelling and pain of the udder )
100
Partial or complete loss of appetite 80
Lameness and unable to recumbency 53.3
Fluid exudation, and crepitating 46.6
Watery milk in its consistency with light red color 46.6
Grinding of teeth 33.3
Significant increase (p<0.05) has been detected in the vital signs including (animal body
temperature , respiratory rates and also the heart rates compared with controls, Furthermore, the
contractions rates of the rumen was significantly lowered (p<0.05) in diseased buffalo than in
controls table No.2.
Table 2: The vital signs and ruminal contractions of diseased buffalo and controls
The parameters Control buffalo n=10 Diseased buffalo n=15
Animal body temperature C° 39.3± 0.28 41.4 ± 0.7*
Respiratory rate/ min 23.2± 1.82 58.6± 7.2*
Heart rate/ min 65.8±1.72 95.7± 11.83*
Ruminal contractions / 5 min 4± 0.54 2± 32 *
(Values are mean ± standard error of mean. * (P<0.05).
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Concerning the blood change parameters, results indicated a significant increase(p<0.05)
in total leukocyte count which occur due to a significant (p<0.05) Nutrophelia, table No.3.
Table 3:Hematological changes of diseased buffalo and control animals
Hematological values Control buffalo n=10 Diseased buffalo n=15
TRCx 106 7.92±1.47 7.94±0.33
Hb mg/dL 13.22 ± 1.77 13.4±0.2
PCV % 32.62 ± 3.63 33.94±1.09
TLC x 103 11.23±1.66 15.74±1.08*
Nutrophiles % 47.22 ± 2.34 53.11± 4.56*
Lymphocytes % 45.56 ± 1.78 41.42± 5.12
Eosinophiles % 3.34 ±0.64 3.54±0.06
Monocytes% 4.46± 0.23 5.61±0.14
Basophiles % 0.56± 0.07 0.32± 5.42
(Values are mean ± standard error of mean. * (P<0.05).
Moreover, Microbiological findings of the cultured media was confirmed by VITEK 2
systems and the results indicated the causative agents which are Enterobacter cloacae and
Proteus mirabilis, figure 1 and 2.
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Fig1: VITEK 2 systems for Proteus mirabilis
Fig2 : VITEK 2 systems for Enterobacter cloacae
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Furthermore, the gram stain of the milk smears showed a clear Gram-negative rods figure
3. However, swarm forming and pale colony character was also indicated of the swab culture of
the causative Proteus mirabilis, figure 4. Moreover, the characteristic feature of cultural media of
was also indicated figure 5.
Fig 3: Milk sample :Gram stain from the milk smear show a clear Gram-negative rods
Fig 4: Swarm forming and pale colony character was indicated of the swab culture of the
causative Proteus mirabilis( blood agar)
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Fig 5:The characteristic feature of cultural media of Enterobacter cloacae was indicated(blood
agar)
DISCUSSION
It has been shown that, the parturition mostly related to udder problems after calving in
the large ruminants like cattle and buffalo which appears to be common due the animal
physiological conditions around the time of parturition, Since, the lower cisternal storage of
secreted milk and milk production might play good roles as a predisposition (8) On the other
hand, the anatomy of the animal udder might add another factors such as , the long teat size and
the thick streak milk canal which was different in both cattle and buffalo , Moreover, the edema
of the udder, the presence of blood in milk and hypogalacia will also occur in cattle and buffalo
with same circumstances (9) .
Mastitis, reveres to the inflammation of tissues of udder (the mammary gland) occur due
to different types of microorganisms, in both cattle and buffalo , such as Streptococcus galactiae
and disagalactia, Staphylococcus aureus, E. coli, Klebsiella pneumoniae, Corynebacteium spp.
, and Mucoplasmas, However, some viruses and some yeast such as Monascus purpureus ( the
red yeast) might also responsible for local udder infection and also systemic invasion resulting
in erythrocyte destructions due to capillary damage leading to discoloration of milk (8,10).
Mastitis will affects the quality of the milk , However, that the production of the diseased
animals with chemical and physical changes of milk contains, However, the extent to
which different alterations will observed and detected (11).
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Diseased buffalo exhibited different clinical signs which also mentioned by (1,2,4), As the local
and systemic clinical signs exhibited by the diseased buffalo confirmed the acute stage of the
disease in infected animals, Moreover, Clinical diagnosis of mastitis were easily confirmed by
the appearance of clinical signs noticed on the inflamed udder (12). Ali and Ahmad (13) has
been mentioned that, The acute type of mastitis might appears suddenly with its local and
systemic reactions like, the chemical, physical, and other changes in the infected milk,
However, the inflamed udder might changed to red, hot and hard palpation. Beside the animals
will feel pain on touching the udder, Moreover, the diagnosis of clinical mastitis could
based on the abnormal appearance of the milk. Milk may be off different colors and
consistency, watery, have blood, serum like , mixed with pus and milk clots, flakes and shreds
containing of cellular and fibrin debris(14).
The abnormal color of milk could be due to the result of changes in vascularity during
acute inflammation and flow of blood from body of animal to the udder. The shape and size of
udder was also changes grossly, Furthermore, Muhammad et al ,1997 (12) was added that, the
presence of blood in milk have different cusses, as hemorrhage occurs when passage of
erythrocytes was detected via the wall of capillaries inside the tissues in which the erythrocytes
exist in the alveolar cells of the mammary tissues occurs after calving. Moreover, When there
are large number of erythrocytes in the milk , then it will give the milk a pinkish or reddish
colors , On the hand udder trauma seems to be an additional important reason for the presence of
blood in the milk (15).
Results was also indicated that the, main cause of the acute buffalo mastitis of the current
study was Enterobacter cloacae and Proteus mirabilis, which confirmed by the VITEK system
2.It have been documented that Proteus spp. are uncommon environmental mastitis causative
agent that have been known to cause an important felid outbreaks (16). It was also shown that
any new infections could occur at any time during lactation period . It was also mentioned that
dairy animals especially at the early stage of lactation will be at high risk for new udder infection
because of depression of immunity and the stress status which related to the postpartum period .
It was indicated that Proteus spp. was always responsible for the great buffalo herds outbreaks,
However, the infection could mostly become chronic (17).On the other hand, the Enterobacter
spp. considered as an important cause for the udder infection which could be isolated easily from
the infected milk. Moreover, it have the similar structure to other coliform type of bacteria with
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an especial odor ( fecal odor ) when the organism grow on the blood agar, The spread of
Enterobacter spp. are indicated firstly via environmental contact, especially when teats are come
in contact with manure or contaminated bedding.(18,19). Infection with Enterobacter could be
created from water supply, animal houses and milk equipments. The organism is thought to be
more resistant to disinfectants , Furthermore The bedding used to animal house could be the
primary source of environmental pathogens, but will contaminated teat dips, intra-mammary
infusions, water used for udder preparation before milking, However, mud holes, skin lesions,
teat trauma, and flies have all been incriminated as sources of infection, .(20).
It has been postulated that, Udder infection due to coliform organisms will cause high economic
losses due to the losses of bad milk quality , Moreover, high mortalities of diseased animals was
also recorded(10).
الھاب الضرع الحاد فی الجاموس المتسبب عن الخمج بجراثیم
Enterobacter cloacae and Proteus mirabilis
فی البصرة، العراق
مھند حسن لفتة
فرع الطب الباطنی والوقائی ،کلیة الطب البیطری ، جامعة البصرة،البصرة ، العراق.
الخلاصھ
شخص التھاب الضرع الحاد فی خمس عشر من الجاموس المحلی فی البصرة، العراق .تم اختیار عشرة من
الجاموس المحلی السوی سریریأ عدت کمجموعة سیطرة .اظھرت الحیوانات المریضة علامات الالم مع تورم واحمرار
الضرع ،وکان الحلیب الملتھب مائی القوام ذا لون احمر خفیف فضلا عن ارتفاع معنوی فی درجات حرارة الجسم ، ترداد
التنفس وضربات القلب وعلى العکس من ذلک فقد انخفضت تقلصات الکرش بشکل معنوی فی الحیوانات المریضة بالمقارنة مع
حیوانات السیطرة . اظھرت نتائج التغیرات الدمویة وجود ارتفاع معنوی فی العدد الکلی لخلایا الدم البیض بسبب الارتفاع
المعنوی للعدلات . فحصت جمیع الحیوانات المریضة سریریأ ومختبریا وبینت النتائج ان المسببات الرئیسة لالتھاب الضرع
اکد تشخیص الشرائح الزجاجیة المصبوغة بصبغة Enterobacter cloacae وProteus mirabilis فی الجاموس ھی
VITEK کرام ظھور المسببات الجرثومیة بشکل عصیات سالبة الکرام والتی تم تأکید تشخیصھا باستخدام برنامج 2
فضلا عن ملاحظة ظاھرة العج والشکل باھت اللون للمستعمرات الجرثومیة . استنتج من ھذه الدراسة ان التھاب .System
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الضرع الحاد فی الجاموس ذا فعل عکسی مؤذی للحیوان المصاب لذا فأن التعرف على المسببات الرئیسة تسرع من عملیة
الاستجابة للعلاج.
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