OCCURRENCE OF BRUCELLA IN MILK AND CREAM

Assiut University web-site: www.aun.edu.eg

OCCURRENCE OF BRUCELLA IN MILK AND CREAM

A.M. EL-KHOLY ¹; A.M.S. MESHREF ¹; N.A. HAMED² and F.E.A. SALEH²

¹ Food Hygiene Department, Faculty of Veterinary Medicine, Beni-Suef University

² Animal Health Research Institute, Egypt

Received: 28 September 2017;       Accepted: 12 October 2017

INTRODUCTION

In Egypt, direct consumption of raw milk is more frequent and more popular than the pasteurized one because it's believed, especially in rural areas, that the raw milk and its byproducts have nutritional advantages over the pasteurized one. Furthermore, milk is produced mainly by individual in small farms that lack proper sanitary measures and may be either consumed fresh, manufactured into dairy products or sell in retail markets that alarming as a major source of food borne brucellosis (El-Sayed et al., 2011) and represent a serious human health problem.

Brucella is excreted in milk, it attains 10⁴/ml at the beginning of the lactation and then it decline to 10/ml but may persist during successive lactation periods (Plommet et al., 1988). The Food and Agriculture Organization (FAO), the World Health Organization (WHO) and the Office International des Epizooties (OIE) considered Brucellosis as one of the widest spread zoonotic diseases of domestic and wild animals throughout the world (Schelling et al., 2003 and Thakur et al., 2002). The World Health Organization (WHO) recently estimated that the median  global  number  of cases of foodborne illness

Corresponding author: Dr. F.E.A. SALEH

E-mail address: fateneid2290@gmail.com

Present address: Animal Health Research Institute, Egypt

due to Brucella infection was 393,239 (Havelaar et al., 2015). The main pathogenic species in both animals and human are Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis (Cloeckaert and Vizcaino, 2004 and Araj, 2010), while Brucella melitensis is currently the predominant species of Brucella present in Egypt (Holt et al., 2011) precisely Brucella melitensis biovar 3 is the most common isolate of Brucella in Egypt (Refai, 2002 and Samaha et al., 2007).

Brucellosis was first reported in Egypt in 1939 and is now considered endemic in most parts of the country (Refai, 2002 and Molina-Flores, 2010), it appear to be of particular risk in rural communities especially in Upper Egypt (Molina-Flores, 2010). Despite its economic and public health importance, the official Egyptian brucellosis control program does not appear to have been fully implemented (Refai, 2002 and Hegazy et al., 2009).

The presence of Brucella organism in milk has conducted by several investigators (Abdel-Hakiem, 1999; Abd-Alla et al., 2000; Meshref, 2000; Abdel-All, 2001; El- Sayed et al., 2011; Abd Al-Azeem et al. (2012); Abosira 2015 and El-Diasty et al., 2016).

Diagnosis of Brucellosis is the corner stone of proper eradication of the disease. Isolation of the causative agent is still the land mark for diagnosis of brucellosis (Alton et al., 1988), however; it is difficult to recover from life infected animals, therefore diagnosis has been based mostly on the results of serological tests (Hamdy, 1997). It is easier for using milk and milk whey for diagnosing brucellosis as injuring animals for collecting blood samples is difficult (Farag, 1998).

There is limited recent data on the prevalence of Brucella organisms in the Upper Egypt. Therefore, the present work was planned to investigate the incidence of Brucella organisms in milk and fresh cream and to throw the light upon the public health significance and preventive and control measures of brucellosis.

MATERIALS AND METHODS

1-Collection of samples:

A total of 200 random samples including Farmers milk (100 raw cow's milk samples were collected from individual farmers in Beni-Seuf and El-Minia cities), Market milk (60 samples were collected from different retail shops and vendors in Beni-Suef and El-Miniacities, Egypt) and Cream (40 samples from the separators in Beni-Seuf and El-Minia cities). All samples were collected in sterilized bottles and transported to the laboratory in an insulated ice box (4-6ºC) and kept in the refrigerator till be examined.

2- Serological examination:

Milk whey samples were prepared according to (Morgan et al., 1978). The assigned tests were carried out on the samples as follow:

2.1. Milk Ring Test (Alton et al., 1988).

2.2. Whey Buffered Acidified Plate Antigen Test (wBAPAT) (Alton et al., 1988).

2.2. Whey Rose Bengal Plate Test (wRBPT) (Alton et al., 1988).

3- Isolation and identification of Brucella organisms:

3.1. Direct culture method:

Briefly, the milk sample was centrifuged at 3000 rpm for 10 minutes to obtain the sediment–creammixture (Alton et al., 1988) which then was cultured on duplicated plates of serum dextrose agar plates containing Brucella selective antibiotics (Oxoid code: SR0083, Hampshire, UK). The plates were incubated in presence of 5-10% CO₂ and aerobically at 37 °C for up to 2 weeks. The plates examined every 2 days for any Brucella growth.

3.2. Indirect cultural method (Brodie and Sinton, 7. 1975):

Two ml of fresh cream were inoculated into bottles (50 ml volume) of serum dextrose broth containing Brucella selective supplements (Oxoid code: SR0083, Hampshire, UK), then incubated at 37◦C in carbon dioxide incubator (5-10% tensions) for 3-5 days. The broth was the sub-cultured onto selective serum dextrose agar plates and the plates as well as control ones were incubated at 37◦C in carbon dioxide incubator (5-10% tensions) for 3-5 days. The plates examined every 2 days for any Brucella growth.

All of the isolates were subjected to standard morphological and biochemical tests, including morphological characters of the colonies, microscopical appearance, CO2 requirement, growth in the presence thionin and basic fuschin dyes, and agglutination with Brucella anti-sera A and M.

4- Molecular examination of Brucella:

4.1. Extraction of DNA for PCR assay:

DNA was extracted from colonies by using QIA amp DNA Mini Kit Catalogue no.51304. It provides silica-membrane-based nucleic acid purification from different types of samples.

4.2 DNA amplification:

Conventional PCR (Bricker, 2002) was carried out for identification of the DNA extracts to confirm the presence of genetic material of genus Brucella. Amplification of target gene (Immunodominant antigen, gene bp26) was carried out in a final volume of 25µl in containing (12.5 μlBiomatik® master mix, 1 μl forward primer, 1 μl reverse primer, 7.5 μl nuclease free water and 3 μl DNA template. The amplification was performed in Labnet® Multigen Gradient thermal cycler, Catalog TC9600-G- 230V. (Labnet international, Inc. Edison, NJ, USA).

Cycling conditions of the different primers during cPCR

Oligonucleotide primers sequences.

4.3 Detection and identification of PCR product.

The PCR products were detected through 1.5 % agarose gel stained with ethidium bromide solution (0.5 μg/ml) and visualized under an ultraviolet trans-illuminator and photographed.

RESULTS

Table 1: Incidence of Brucella organisms in the milk sample based on MRT.

Figure 1: Incidence of Brucella organisms in the farmers and market milk   based on the results of MRT.

Table 2: Incidence of Brucella organisms in the milk samples based on wBAPAT.

Figure 2: Incidence of Brucella organism in the farmers and market milk based on the results of wBAPAT.

Table 3: Incidence of Brucella organisms in the milk samples based on wRBPT.

Figure 3: Incidence of Brucella organisms in the farmers and market milk based on the results of wRBPT.

Table 4: Isolation of Brucella organisms from the milk and fresh cream.

Figure 4: Isolation of Brucella organisms from the milk and fresh cream.

Table 5: Identification of isolated strains.

Conventional PCR for detection of Brucella DNA on genus level

Lane 1:DNA ladder

Lane 2-6, 9: positive sample for Brucella

Lane 7-8: negative

Lane 1:100 bp DNA ladder

Lane 2-6: positive sample for Brucella

Lane 7: negative

Figure5: Gel electrophoresis of PCR products using universal primer set (with 450 bp expected product size).

DISCUSSION

Data presented in Table (1) and Figure (1) showed the incidence of Brucella antibodies in the examined raw milk based on the milk ring test (MRT).Out of 100 Farmers milk samples, 30 (30%) were positive, 7 (7%) were suspicious and 63 (63%) were negative.

This positive result agreed with those recorded by Abosria (2015) and nearly simulated to the finding recorded by El-Gibaly et al. (1995) and Miller et al. (2015) which were 28.2% and 28.7% respectively, while the gained result was higher than those obtained by Mohamed (1989), Zowghi et al. (1990), Hosein and El-Kholy (1993), Kadry (1996), Abdel-Hakiem (1999), El-Sherbini et al. (2002), Shehata (2004), Abd El Hamid (2008), El-Kholy et al. (2008), Shafee et al. (2011) and Ali et al. (2014), whom reported incidences of 22.25%, 25.2%, 4.1%, 0.99%, 8%, 10%, 12.38%, 14.7%, 8.2%, 4.6%, and 6.7% respectively.

The relatively high results obtained by using MRT could be attributed to the fact that MRT is highly sensitive, rapid screening test (Ferguson and Rebortson, 1954; El Gibaly, 1969 and Salem et al., 1987).

On the other hand, it was relatively lower than those reported by Meshref (2000), Abd El-All (2001), Hamdy and Amin (2002), Ibrahim et al. (2002), Hashim et al. (2007), El-Diasty (2009), Abdalla and Hamid (2011), El-Sayed et al. (2011), Ibrahim et al. (2012) and Al-Mariri (2015) whom found incidence of 73.68%, 38.33%, 48.1%, 48%, 80%, 74%, 36.7%, 60.2%, 47.8% and 57%, respectively.

Despite MRT was recommended by (FAO/WHO, 1986) as surveillance test for detection of Brucella in milk due to its economical and practical advantages, the major limitations of the test are mastitis milk, skimmed milk, colostral, the dilution factors as well as milk agglutinations are locally produced in the udder of the infected cases and clustering of fat globules or low level of IgA and IgM tend to yield false negative results (Corbel et al., 1984).

On the other hand the result illustrated in Table (1) and Figure (1) revealed that out of 60 market milk samples, 14 (23.3%) were positive, 5(8.4%) were suspicious and 41 (68.3%) were negative.

Our result was higher than those recorded by Kang'ethe et al. (2004), Bertu et al. (2010) and Ior and Chukwu (2015), whom reported that 3.9%, 12% and 12.5% of their samples, were positive respectively. In the contrary, Abd El Hamid (2008) reported that all market milk samples were negative to MRT.

Concerning whey buffered acidified plate antigen test, the recorded results in Table (2) and Figure (2) showed that the incidence of Brucella antibodies in the examined whey. Out of 100 Farmers milk samples, 44 (44%) were positive and 56 (56%) were negative. Also, out of 60 market milk samples, 32 (53.3%) were positive and 28 (46.7%) were negative.

Many reports dealing with prevalence of Brucella in milk have been accumulated. In those studies, various rates of prevalence were reported as  38.9%, 54.9%, 50%, 4.29%, 6.67%, 2.4%, 20.6%, 14.1%, 56% and 78.1% were obtained by Abdel-Rahman (1991), Hamdy (1997), Meshref (2000), El-Bassiony et al. (2007), Omran (2007); Oraby et al. (2007), Abdel-Hamid (2008) and El-Kholy et al. (2008), El-Diasty (2009) and Abd Al-Azeem et al. (2012)  respectively.

The obtained results in Table (3) and Figure (3) showed that the incidence of Brucella antibodies in the examined whey samples based on the result of whey Rose Bengal plate agglutination test. Out of 100 Farmers milk samples, 42 (42%) were positive and 58 (58%) were negative. On the other hand, in case of market milk, out of 60 examined samples, 29 (48.4%) were positive and 31 (51.6%) were negative.

Lower results were detected by Abdel-Rahman (1991), Hamdy (1997), Abd-Alla et al. (2000), El-Bassiony et al. (2007), Omran (2007), Oraby et al. (2007), Abdel Hamid (2008) and El- Kholy et al. (2008) where as they reported 22.1%, 39.2, 3.53, 4.29%, 6.67%, 2.4%, 15.7% and 11.8% respectively, but  a nearly similar result (44%) was detected by Meshref (2000) and El- Diasty (2009), while higher value (75%) was recorded byAbd Al-Azeem et al. (2012).

The difference in the incidence rate might be due to the variation of the degree of infection, also the appearance of antibody is related to many factors such as size and method of exposure, virulence of organism, stage of pregnancy and previous exposure. The antibody titer usually reach diagnostic level by four weeks after exposure during fourth to sixth month of gestation and at 10 weeks after exposure in non-pregnant or in the first trimester gestation (Nicoletti, 1990). The high results may be correlated with high prevalence in unknown history of animals which might be due to lack of appropriate diagnostic facility at field level and screening of animals for brucellosis prior to purchase.

The result obtained in Table (4) and Figure (4) revealed that 28 (28%) out of 100 Farmers milk samples were found to be contaminated with Brucella organisms. This finding was nearly similar to the result (28.57%) that was reported by Abosria (2015), while higher prevalence (40.6%) was recorded by Abd Al-Azeem et al. (2012) and (33.8%) was recorded by Ibrahim et al. (2012), but lower prevalence (7.5%) was recorded by Abd-Alla and Hamid (2011) and (4%) by Abdel-Kareem et al. (2011).

On the other hand the summarized result in Table (4) and Figure (4) revealed that 36 (60%) out of 60 market milk samples were found to be contaminated with Brucella organisms which was much higher than that reported by Meshref (2000) and Abdel-Hamid (2008) who failed to isolate any Brucella organisms from market milk.

The higher percent in farmers milk may be attributed to the lack of proper sanitary measures under which milk is produced as well as, the high incidence of Brucella infection in the dairy cows. Moreover, higher prevalence in market milk may attributed to the fact that the milk collected from different sources blended before the selling, and explained by the fact that the majority of milk sales are in the hands of farmers who are known to harbor beliefs that milk is inherently hygienic and less likely to get concerned about the hygiene and conservation of milk before sale. (All the collected samples were from bulk milk from various herds and milk sellers each represent). The infected animals serve as sources of infection to healthy animals within the herds as well as other neighboring herds as the animals graze around unrestricted area, making contact between different herds possible, it was understood that most of the milk sold do not undergo any form of heat treatment such as pasteurization or boiling. The recovery of Brucella from milk samples is of great public health significance and presents a particularly serious hazard as previously reported by El-Sayed et al. (2011).

MRT showed lower incidence rate (23.3%) in market milk when compared to that recorded by culture method (60%) and that may attributed to the dilution factor as the milk from different sources may be blended before selling. On the other hand, MRT showed higher incidence rate in farmers milk (30%) when compared to that recorded by culture method (28%) and that may attributed to the intermittent secretion of Brucella in milk (El-Berg, 1981).

As presented in Table (4) and Figure (4) Brucella organisms were detected in 13 (32.5%) out of 40 fresh cream samples. The obtained result was nearly similar to those reported by Meshref (2000) who found that (33.3%) of the cream samples were contaminated. The higher incidence could be attributed to the fact that the cream is usually more heavily infected than whole milk as the Brucella organisms tend to adhere to the surface of fat globules forming a complex which rise to top of milk by means of specific gravity (Champneyz, 1953). In addition, the samples were collected randomly from general separators, which separate the milk of at least 50 small herds/day. Small amounts of milk remains in the separator after each herd are considered as a good mechanical transmitter for pathogenic organisms including Brucella organisms to the following milk. Moreover, poor sanitary measures of the separator whereas washing and sanitation of the separator was done only once/day at the end of day work without using any sterilizer acts as main predisposing factor in mechanical transmission of Brucella organisms from infected to non-infected milk through the separators (Meshref, 2000).

All the obtained isolates from milk and cream were typed as Brucella melitensis biovar 3 as illustrated in Table (5). Isolation of Brucella melitensis biovar 3 from milk was reported by Meshref (2000), Abd El-all et al. (2001), Montasser et al. (2002), Shalaby et al. (2003), Zahran (2004), Abdel Wahab (2005), El-Diasty (2009), Moawad et al. (2013) and Abosria (2015). It is evident that Brucella melitensis biovar 3 is still the prevalent one among cattle in Egypt, also Brucella melitensis is considered the indigenous strain prevalent in sheep and goats in Egypt (El Gibaly et al., 1993 and El- Sheery, 1993). Cattle are readily infected with brucellosis when they are in close contact with infected goats and sheep on communal grazing or at watering (Hellstrom, 1991).

CONCLUSION

Results of the study clearly indicate that the milk and fresh cream in both examined cities play a dangerous role in transmitting infection to man, so efficient boiling or pasteurization of milk before consumption or processing especially in infected areas to safeguard the consumers should be done. Urgent need for effective program for the control of this disease in reservoir animals in Egypt and educational programs to those sharing in milk production and handling as well as processing of dairy products and at risk population should be encouraged. Further studies on brucellosis should be conducted in other areas for setting up priorities for control measures.

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