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Detection of Rota and Corona Viruses in Raw Milk and Milk Products
Hamdi, Abd-ELsamei Mohameda, Adham, M. Abdoub, Ekbal , M. Adelc, Sayed
Ahmed, H. Salemd, Mervat, I. M. EL-Hassaninee
a Professor of Milk Hygiene and Head of Food Control Department, Faculty of Veterinary
Medicine, Banha University; b Professor of Milk Hygiene, Faculty of Veterinary Medicine,
Banha University; c Professor of Milk Hygiene, Faculty of Veterinary Medicine, Banha
University; d Chief Researcher of Virology Department, Animal Health Research Institute
Dokki, Giza; e Food Control Department, (Milk Hygiene) Faculty of Veterinary Medicine,
The spread of viral disease in Egypt in last years had a great effect on public healthy and on
economic loss in animals and it's milk products
So, it is very important to study some viruses as rota and corona viruses . This study included
of 100 raw milk samples , 60 cheese samples and 50 yoghurt samples were collected randomly
from markets at El-Gharbia Governorate from December 2011 till June 2012 for detection of rota
virus and corona virus antigens by ELISA.
We could concluded that the raw milk and its products may play an important role in infecting
human with rota virus and corona virus and play an importance role in spreading of these virus.
Processing of Kareish cheese, soft white cheese and Balady yoghurt could not eliminate rota virus
and corona virus when infected milk is used due to insufficient heat treatment and bad hygienic
We can also concluded that canned milk products free from rota virus and corona virus this
indicate sufficient heat treatment and application of HCCP.
Milk is one of the complete food for human from birth to senility, as it contains all nutrients
required for growth and maintenance of the body health. On the other hand milk and its products are
very suitable media for growth of all types of microorganisms. They subjected to contamination
either directly or indirectly from different sources during the chain of production that make them, at
time unfit for human consumption or even a dangerous source of human infection with several
types of microorganisms, including members of bacteria, viruses, moulds and yeasts (Jay, 2000).
Food of animal origin could be contaminated by exogenous and endogenous means. Virus
involved in the exogenous contamination are specific pathogenic to man,( zoonotic viruses )such as
enteroviruses –Rotavirus and Coronavirus. Rota and corona viruses either singly or in combination
are associated with diarrhoea in neonatal calves (Faheem et al., 2008).
The economic losses due to neonatal calf diarrhoea have been estimated to 1.7 billion $
world wide (Saif and Heckert, 2000). Such losses are not only due to mortalities, but also reflect
the veterinary costs and medication as well as subsequent chronic ill thrift and poor growth that
represent a significant economic loss to dairy industry. (Crouch et al., 2001a).
Rota viruses are one of nine genera belonging to the family Reoviridae, order
Caudovirales(James et al.,1998). The intact virion is 70 nm in diameter and the genome consists of
11 segments of double stranded RNA (Van Regenmortel et al., 2000). Rota viruses are
transmitted by the oral-fecal rout. Clinical sing of rota virus infection range from mild to sever
diarrhoea resulting in depression, dehydration and occasionally death (Jank, 2006).
Bovine corona virus (BCV) is single stranded, positive sense, enveloped RNA virus. It is
spherical about 120 to 150 nm in diameter (Lin et al., 2000). BCV spread efficiently among cattle
by both fecal, oral and respiratory routes. It causes yellow watery diarrhoea, mild depression,
reluctance to stand and dehydration, while in cattle, it manifested by acute onset of winter
dysentery, dark, liquid hemorrhagic diarrhoea, decreased milk production, with extension to
respiratory tract affections (Hasokusuz et al., 2005).
The two viruses rota and corona viruses were frequently identified in the faeces of
diarrhoeic and apparently healthy calves and cattle from different localities in Egypt (Byomi et al.,
1996 and Hussein et al., 2001). The rota and corona viruses had a great effect on general health
and reproduction in dairy herd and act as parameters measurable on herd level and to explore the
association between antibody status and some herd characteristics (Anna Ohlson et al.,2010).
Milk contaminated by rota and corona viruses by fecal contamination due to bad hygienic
measure in milking place. Infected milk and milk products act as vehicles for transmission of rota
and corona viruses. The rota and corona viruses could survive in the contaminated milk after high
temperature short time (HTST, 71.7C for15s) pasteurization and induce public health hazard in
dairy products such as yoghurt and cheese (Vanden Berg, 1986, Panon et al., 1988 and Hazel
Transmission of rota and corona viruses occurs as a consequence of the ingestion of
contaminated unprocessed milk or dairy products. The disease affected children among 6 years and
the onset of the illness usually begins 3 days after exposure to the virus. Rotavirus usually starts
with fever, upset stomach, and 1-3 days of vomiting, followed by 5-8 days of watery diarrhea which
has an extremely foul odor. Children can lose body fluids and electrolytes very rapidly with this
disease. This is especially dangerous for children under 2 years of age (Robert Hamilton,2002 and
Glass et al., 2002).
So the present study based on virological studying on raw milk and some dairy products
which marketing in Gharbia Governorate, (Egypt).
The present investigation was planned to study the following items:
1- Detection of rota and corona viruses antigen in raw milk.
2- Detection of rota and corona viruses antigen in cheese and yoghurt. .
MATERIALS AND METHODS:
1- Milk samples:
One hundred individual randum milk samples were collected from markets of three districts
in El-Gharbia Governorate(Cottour, El-Mehalla El-Kobra and Tanta) under hygienic condition in
sterile tubes (Venoject). The samples immediately placed in ice box and sent to the laboratory. The
milk samples (100 samples) were stored at (-20oC) in the form of whole milk for detection of Rota
and Corona viruses antigens.
2- Cheese samples:
60 samples of different types of cheese ( 30 kariesh cheese -20 from unknown source and 10
from cand white cheese) were collected from the markets of the same examined area in EL-Gharbai
Governorate (Cottour, El-Mehalla El-Kobra and Tanta) . The samples were placed in ice box and
were sent to the laboratory with a minimum of delay. The cheese samples were ground in clean
sterile mortar and diluted 1/4 times w/v with phosphate buffer solution (PBS). The suspension of
each sample was centrifuged at 1000 rpm for 10 minutes, the supernatant fluid of (60) samples were
aspirated for detection of Rota and Corona viruses antigen .
3- Yoghurt samples:
50 samples of different types of Yoghurt (40 samples of balady yoghurt and 10 samples of
canned yoghurt) were collected randomly from the markets of the same examined area in EL-
Gharbai Governorate (Cottour, El-Mehalla El-Kobra and Tanta) The samples were placed in ice
box and were sent to the laboratory with a minimum of delay. The yoghurt samples were ground in
clean sterile mortar and diluted 1/4 times w/v with phosphate buffer solution (PBS). The suspension
of each sample was centrifuged at 1000 rpm for 10 minutes, the supernatant fluid was aspirated for
detection of Rota and Corona viruses antigen
Enzyme-linked immunosorbert Assay (ELISA) for Rota and Corona viruse (Vollar et al. 1976):
Composition of the kit:
1. Microplates: Two 96- well microtitration plates. Rows A, C, E, G are sensitized by anti-
Rotavirus specific antibodies or anti-Coronavirus specific antibobies, while rows,B, D, F, H are
sensitized by non specific antibodies.
2. Washing solution : One 100-ml bottle of 20x concentrated washing solution. The solution
crystallizes spontaneously when cold. If only part of the solution is to be used, bring the bottle
to room temperature until disappearance of all crystals. Mix the solution well and remove the
necessary volume. Dilute the buffer 1:20 with distilled or dematerialized water. Store the diluted
solution at 4oc.
3. Dilution buffer: One 50-ml bottle of 5x concentrated buffer for diluting samples and conjugate.
Dilute this concentrated dilution buffer 1:5 with distilled or dematerialized water. Store the
diluted solution at 4oc. If a deposit forms at the bottom of the container filter the solution on
Whatman filter paper.
4. Conjugate: One vial of anti-rotavirus- peroxidase conjugate(horseradish peroxidase- labeled
anti-rotavirus monoclonal antibody) or anti- coronavirus-peroxidase conjugate.This solution is
ready to use.
5. Positive reference : 2 bottles containing the Rotavirus reference antigen or coronavirus reference
antigen. Reconstitute this antigen with 0.5 ml of distilled or dematerialized water. The
reconstituted serum may be kept at -2oc. Divide the reconstituted antigen into several portions
before freezing in order to avoid repeated freezing and thawing. If these precautions are taken
the reagent may be kept for several months.
6. Chromogen solution: One 2-ml drop- dispenser bottle of the chromogen tetramethylbenzidin.
Store at 4oc.This solution is ready to use.
7. Substrat solution: One 30-ml bottle of the hydrogen peroxide substrate solution. Store this
reagent at 4oc.
8. Stopping solution : One 15-ml bottle of the 1 M phosphoric acid stop solution.
ELISA kit procedure for detection of Rota and Corona viruses antigen (BRV – BCV):
Principle of the test:
The test uses 96-well microtitration plates sensitized by specific antibodies for rota virus or
corona virus. These antibodies allow a specific capture of the corresponding pathogens, which are
present in the samples. Rows A, C, E, and G have been sensitized with these antibodies and rows B,
D, F, and H are containing non-specific antibodies. These control rows allow the differentiation
between specific immunological reaction and non-specific binding so as to eliminate false positives.
Samples are diluted in dilution buffer and incubated on microplate for 60 minutes at room
After this first incubation step, the plate is washed and incubated for 60 minutes with the
conjugate. After this second incubation, the plate is washed again and the enzyme substrate
(hydrogen peroxide) and the chromogen (TMB)are added. This chromogen has the advantages of
being more sensitive than other chromogens and not carcinogenic.
If viruses are present in the tested samples, the conjugate remains bound to the
corresponding microwells. And the enzyme catalyses the transformation of the colorless
chromogen into a pigmented blue compound. Enzymatic reaction can be stopped by acidification.
Table (1): Detection of rota and corona viral antigen in milk samples from randomly markets
by ELISA test at El-Gharbia Governorate in Egypt.
Districts Number of
Detection of rota virus
antigen in Milk
Detection of corona virus
Antigen in Milk
+ve -ve +ve -ve
Couttor 25 4 21 2 23
El-Mehalla El Kobra 50 9 41 3 47
Tanta 25 2 23 - 25
Total 100 15 85 5 95
Table (2): Comparison between rota and corona virus antigen in the same examined milk
samples by ELISA test.
Districts Number of
infection Rota Carona
Couttor 25 2 4 2
El-Mehalla El Kobra 50 2 9 3
Tanta 25 - 2 -
Total 100 4 15 5
Table (3): Detection of rota and corona viral antigen in different types of cheese from
randomly market at El-Gharbia Governorate by ELISA test.
Types of Cheese Number of
Detection of Rota
Virus Antigen in
Detection of Corona
Virus Antigen in
+ve -ve +ve -ve
Kariesh cheese 30 5 25 2 28
White cheese from
20 2 18 1 19
Canned white cheese 10 - 10 - 10
Total 60 7 53 3 57
Table (4): Detection of rota and corona viral antigen in different types of Yoghurt at El-
Gharbia Governorate by ELISA test.
Types of Cheese Number of
Detection of Rota
Viral Antigen in
Detection of Corona
Viral Antigen in
+ve -ve +ve -ve
Balady yoghurt 40 3 37 - 40
Canned yoghart 10 - 10 - 10
Total 50 3 47 - 50
The results given in Table (1) showed that the antigen of rota virus and corona virus in raw
milk samples collected from randomly markets in El-Gharbia Governorate could be detected in
15samples and 5 samples out of 100 samples respectively.
The results pointed out that the milk contaminated by fecal contamination (exogenous mean)
because the rota virus and corona virus not shedding in milk from infected and carrier animals but
the two viruses was shedding in faces of diarrheic neonatal calves or from apparent healthy calves
and cattle these results were in agreement with those reported by Lu et al. (1991), Abou El
Hassan et al. (1995), Barboi and Turcu (1995), Abd El Rehim (1997), Sahna et al. (2005) and
Abrams and Hilmers (2008). The positive results obtained my be due to seasonal factors during
collection of samples, and bad hygienic measures.
the data presented in Table (2) showed the comparison between rota virus antigen and
corona virus antigen in the same examined raw milk samples (100) and revealed that the level of
infected milk samples by rota virus was higher in percentage than corona virus this results were
supported by Panon et al., (1988) and Erdogan et al., (2003) they concluded that rota virus is more
stable than corona virus and also these results revealed that the milk samples may be contaminated
either single or mixed by rota and corona virus this results were in agreement with Barrett, (1986) ,
Abd El Rehim (1997), Hasoksuz-M, et al.,(2005) and Anna Ohlson et al., (2010)..
Results in Table (3) pointed out that, the incidence of rota virus and corona virus in
different types of cheese collected randomly from markets at El Gharbia Governorate. the rotavirus
were detected in kariesh cheese 4 samples out of 30 samples and in white cheese from unknown
source were detected in 2 samples out of 20 samples but the incidence of rota virus in white canned
cheese were zero. But corona virus were detected in kariesh cheese 2 out of 30 samples, and in
white cheese from unknown source 1 out of 20 samples but not detected in white canned cheese
These data showed that presence of two viruses in kareish cheese and in white cheese from
unknown source is attributed to preparation of these types of cheese from infected unheated milk or
may infected during manufacturing and selling of cheese which may induce public health hazard in
children under 6 years when ingested the infected milk and milk products . These were in
agreement with Glass et al., (2002) and Robert Hamilton, (2002).
The obtained results confirmed the importance of rota virus and corona virus in public
healthy and the diarrhea by these virus responsible for more than 2 million hospitalizations and
500,000 deaths annually Linhares and Bresee (2002), Parashar et al. (2003) Umesh et al.
(2006), WHO( 2008), Parashar et al. (2009), Merck and Dohme (2011) and Weiss and Leibowitz
Results recorded in Table (4) revealed that the rota and corona virus antigen could be
detected in balady yoghurt 3 out of 40 samples and zero out of 40 respectively. But rota and corona
virus antigen could not detected in canned yoghurt. It is evident from the obtained results that milk
products (different types of cheese and different types of yoghurts) play an important role in
spreading of diseases among animals and human and had public healthy important. Our conclusion
that rota virus and corona virus persistence and survive through the manufacture of cheese and
yoghurts when these products prepared from raw milk and also concluded that high thermal
processing of milk during the manufacturing of canned cheese and yoghurts and also application of
HACCP system is very important to obtain products free from any disease these results were
supported by Cliver (1973), Vanden Berg (1986) and Panon et al. (1988).
The points for control of rota and coron viruses reported by Tucker et al. (1999), Zarzosa
and Margueriti (1999), Marquardt and Freiberg (2000), Glass et al. (2002), Doaud et al.
(2003), Robert (2007) and Abrams and Hilmers (2008) they concluded that to control rota virus
and corona virus, early ditection was essential for effective control and required rapid and sensitive
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