Seroprevalence of Brucella spp. in Cattle,Molecular Characterization in Milk,and the Analysis of Associated Risk Factors with Seroprevalence in Humans,Egypt

Abstract

The objective of the present study was to estimate the seroprevalence of Brucella spp. in humans and cattle at Sharkia Governorate, Egypt. In addition, identification of Brucella spp. in milk samples by PCR and culture with the evaluation of the risk factors associated with Brucella spp. seroprevalence in humans were carried out. Overall, the seroprevalence of Brucella antibodies in the examined cattle was 23.8%, while in human participants it was 21%. The examination of 205 milk samples using PCR revealed that 6.3% were positive for B. abortus biovar 1 and the results were confirmed by culture methods. Multivariate logistic regression revealed that consumption of unpasteurized dairy products, occupational contact with animals, and knowledge about the disease are risk factors associated with infection in humans. This study documented the endemic status of brucellosis in Egypt. Hygienic measures and increased awareness about the disease are recommended to minimize the spread of infection from animals to humans.

Introduction

Brucellosis is an endemic zoonotic disease in North Africa, especially the Mediterranean region (Pappas et al. 2006). In humans, the disease is commonly referred as “Malta Fever”, which often coincides with livestock infection (Thimm and Wundt 1976). The disease is transmitted to humans by ingestion of unpasteurized dairy products and direct contact with infected animals, aborted fetus, fluids, and membranes (Tsolia et al. 2002). Infection in humans causes debilitating disease, relapsing fever, flu-like symptoms, and involvement of multiple organs (Tsolia et al. 2002). The disease is caused by Brucella abortus, Brucella melitensis, and Brucella suis.

Cattle and buffalo milk production in Egypt is ranked the 11th worldwide according to the Food and Agricultural commodities production statistics in 2012 (FAOSTAT 2012). More than 70% of total livestock population in Egypt is owned by small holders as a source of milk and dairy products for home consumption or for sale in small local markets (El-Rafey 2005). Some commercial dairy products such as white cheese made from unpasteurized milk and the homemade Kareish cheese were reported to be the most frequently consumed types among residences of rural areas in Egypt (El-Ghitany et al. 2014). These types of cheese concentrate large number of Brucella organisms predisposing risk for acquiring the infection through ingestion.

In Egypt, brucellosis was first reported in 1939, now the disease is considered endemic in most parts of the country (Refai 2002). However, the official Egyptian brucellosis control programme is not fully implemented (Hegazy et al. 2009).

Globally, more than 500,000 human cases are reported per year (Pappas et al. 2006). A population-based survey study in Fayoum (a rural Governorate in Egypt) revealed an estimate of 64 and 70 per 100,000 people in 2002 and 2003 were infected, respectively (Jennings et al. 2007). The survey results indicated that contact with animals and consumption of unpasteurized milk products are the factors associated with brucellosis.

Adequate knowledge of brucellosis is of great public health concern to outline control strategies among the general public (Adesokan et al. 2013).

The aim of the current study was to estimate the seroprevalence of Brucella spp. in humans and cattle at Sharkia Governorate, Egypt. Moreover, the study aimed to identify Brucella spp. in milk samples by PCR and culture. Finally, the risk factors associated with Brucella spp. seroprevalence in humans were evaluated.

Materials and Methods

Study area and samples

This study was conducted at Sharkia Governorate, Egypt, and was selected due to convenience in sampling. The study included sampling of serum and milk from cattle from five small-scale dairy farms (each farm consists of less than 50 animals). The farms were included in the study after obtaining formal consent of the farm owner to participate. Serum samples from cattle (n = 143) and milk samples (n = 50) from bulk milk tanks were collected from the visited farms. In addition, 155 retail milk samples were collected from outlets and markets at Zagazig City, Sharkia Governorate. The study also included sampling of 295 serum samples from human participants at Al-Ahrar Fever Hospital, Zagazig City, Sharkia Governorate, Egypt. Human patients were chosen based on their acceptance for participation. The study was carried out during July 2014 and November 2015. Informed verbal/written consent for participation in the study was obtained from all the participants and it was approved by the Committee of Animal Welfare and Research Ethics, Faculty of Veterinary Medicine, Zagazig University, Egypt.

Serum examination

Serum samples collected from the animals and humans were examined using the buffered acidified plate antigen test (BAPAT) (Alton et al. 1988), Rose-Bengal plate test (RBPT) (Morgan et al. 1969), and tube agglutination test (TAT) (Alton et al. 1975). Brucella antigens were obtained from the Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt. Serum samples were considered positive when they tested positive for at least two serological tests.

Milk examination

Fifty milliliters of milk samples were collected for DNA extraction and isolation of Brucella species. DNA was extracted using the QIAamp DNA Mini Kit (catalogue number 51306) according to the manufacturer's guidelines. Brucella species were identified from the extracted DNA by multiplex PCR according to Bricker and Halling (1994). The oligonucleotide primers used in this study were 5′-TGC-CGA-TCA-CTT-AAG-GGC-CTT-CAT-TGC-′3 (IS711-specific primer), 5′-GAC-GAA-CGG-AAT-TTT-TCC-AAT-CCC-′3 (B. abortus- specific primer), and 5′- AAA-TCG-CGT-CCT-TGC-TGG-TCT-GA-′3 (B. melitensis- specific primer). Visible band appropriate size of 495 bp for B. abortus and 730 bp for B. melitensis was considered positive.

For confirmation of the PCR results, Brucella spp. were isolated and identified from milk samples positive by PCR on Brucella Albimi agar according to standard procedures (Alton et al. 1988).

Risk factor analysis

An open- and closed-ended questionnaire was developed to collect information about risk factors associated with seroprevalence in human participants. The questionnaire included sociodemographic factors (sex, age, occupation, residence, and education level), questions about rearing cattle, mixing them with sheep at the same house, slaughtering animals in the house, consumption of unpasteurized dairy products, assessing animals giving birth, disposal of aborted fetus and placentas, and knowledge about the disease. The individual human participants were considered as the unit and the serological status for Brucella spp. (positive vs. negative) as the outcome.

The univariate association of all independent variables with dependent variables was computed by including them individually in a model with brucellosis serological results as an outcome variable. Independent variables that had p-values <0.2 at bivariate analysis and were previously reported to be associated with brucellosis in humans were considered for inclusion in multivariate analysis (Tumwine et al. 2015). A multivariate logistic regression was performed to establish associations between risk factors and brucellosis occurrence in humans.

Bivariate and multivariate logistic regression models were fitted to determine factors associated with Brucella infection using the computer program SPSS, Inc., version 22 (IBM Corp. 2013, Armonk, NY). Odds ratios (OR) and their 95% confidence interval [95% CI] were noted. p-Values less than 0.05 were considered statistically significant in the analysis.

Results

Detection of Brucella spp. in milk samples and seroprevalence of Brucella spp. in the animals

Multiplex PCR applied on 205 milk samples revealed the presence of B. abortus biovar 1 in 13 (6.3%) milk samples, 5 (10%) from farm animals and 8 (7.6%) from milk at retail shops (Table 1). For confirmation of the PCR results, isolation of Brucella spp. from the PCR-positive milk samples was carried out. Brucella spp. were isolated from all PCR-positive samples (n = 13). The biochemical characterization indicated that the isolates belonged to B. abortus biovar 1. Out of 143 examined animals, 34 (23.8%) were seropositive for Brucella spp. (Table 1).

Table 1.

Prevalence of Brucella Antibodies in Animals and Humans

Species	Number tested	Positive
Humans	295	62 (21%)
Cattle	143	34 (23.8%)
Milk	205	13 (6.3%)
Milk from outlets	155	8 (7.6%)
Milk from farms	50	5 (10%)

Seroprevalence and sociodemographic characteristics and risk factors associated with brucellosis in human patients

Table 2 shows that among 295 participants involved in the study, 79.7% (n = 235) were male and the age group ranged from 14 to 60 years, with the majority (92.2%, n = 275) in the 21–60-year age group. Most of the participants (73.6%) were from rural areas at Sharkia Governorate, Egypt, and those with an occupational contact with animals comprised 47.5% of the individuals, while 52.5% had no occupational contact with animals. The majority of the participants had primary education (53.2%), while 32.5% were illiterate (Table 2).

Table 2.

Sociodemographic Characteristics and Risk Factors Associated with Seroprevalence of Anti-Brucella Antibodies Among Human Participants

Characteristics	Number of participants	Percentage of participants	Seropositive (N,%)	Unadjusted OR	95% CI	p-value
Sex
Male	235	79.7	53 (22.6)	1.00	0.28–1.31	0.203
Female	60	20.3	9 (15)	0.61
Age group
14–20	23	7.8	5 (21.7)	1.00	Reference	0.264
21–40	136	46.1	23 (16.9)	0.73	0.25–2.17	0.575
41–60	136	46.1	34 (25)	1.20	0.41–3.48	0.737
Education
Illiterate	96	32.5	29 (30.2)	5.63	1.61–19.69	0.007
Primary	157	53.2	30 (19.1)	3.07	0.89–10.61	0.076
High	42	14.2	3 (7.1)	1.00	Reference	0.01
Occupation
Contact with animals	140	47.5	47 (33.6)	4.72	2.49–8.92	<0.0001
No contact with animals	155	52.5	15 (9.7)	1.00
Residence
Rural	217	73.6	42 (19.4)	1.00	0.378–1.281	0.244
Urban	78	26.4	20 (25.6)	0.696
Rearing cattle
Yes	206	69.8	51 (24.8)	2.33	1.152–4.727	0.02
No	89	27.1	11 (12.4)	1.00
Slaughtering in house
Yes	106	35.9	26 (24.5)	1.381	0.779–2.448	0.269
No	189	64.1	36 (19.04)	1.00
Mixing cattle with sheep
Yes	83	28.1	30 (36.1)	3.184	1.774–5.714	<0.0001
No	212	71.9	32 (15.1)	1.00
Consumption of unpasteurized dairy products
Yes	127	43.1	47 (37)	5.992	3.156–11.377	<0.0001
No	168	56.9	15 (8.9)	1.00
Assist animals giving birth
Yes	127	43.1	35 (27.6)	1.987	1.127–3.501	0.02
No	168	56.9	27 (16.1)	1.00
Improper disposal of aborted fetus and placentas
Yes	106	35.9	24 (22.6)	1.163	0.653–2.072	0.608
No	189	64.1	38 (20.1)	1.00
Knowledge
Yes	172	58.3	22 (12.8)	1.00	1.830–5.900	<0.0001
No	123	41.7	40 (32.5)	3.286

The overall seroprevalence of brucellosis among 295 individuals was 21% (Table 1). The prevalence was higher in male participants (22.6%) than females (15%) and was higher in participants older than 40 years (25%). Participants with no education (illiterate) were 5.63 times more likely to be seropositive compared with participants with high education (OR = 5.63, 95% CI: 1.61–19.69, p < 0.05). The seroprevalence of brucellosis in participants with occupational contact with animals (33.6%) was significantly higher than those with no contact with animals (OR = 4.72, 95% CI: 2.49–8.92, p < 0.0001).

Table 2 shows the bivariate analysis of the potential risk factors associated with the occurrence of brucellosis among human participants. Consumption of unpasteurized milk products has shown to be significantly associated with seroprevalence of brucellosis (OR = 5.992, 95% CI: 3.156–11.377, p < 0.0001). Moreover, rearing cattle and mixing them with sheep are risk factors significantly associated with the seroprevalence of brucellosis (p < 0.05).

Out of 295 participants, 172 (58.3%) heard about brucellosis and the methods of transmission. Those who had no knowledge about the disease showed 3.286 times more likelihood of seroprevalence than participants who heard about the disease (OR = 3.286, 95% CI: 1.830–5.900, p < 0.0001).

Participants who stated that they assist with calving were 43.1% (n = 127), while 106 (35.9%) reported improper disposal of fetal membranes and fetus in water canals.

A final model was fitted to measure the relationship between Brucella sero-positivity and independent variables (Table 3). Occupational contact with animals, consumption of unpasteurized dairy products, and knowledge about the disease increased the odds of being seropositive against Brucella species among human participants.

Table 3.

Multivariable Analysis of Risk Factors for the Seroprevalence of Brucellosis in Humans

Variable	Adjusted OR	p-value	95% CI
Education
Illiterate	1.303	0.704	0.333–5.091
Primary	0.8	0.768	0.181–3.539
High	1.00	0.549	Reference
Occupation
Contact with animals	3.582	<0.0001	1.803–7.116
No contact with animals	1.00
Mixing cattle with sheep
Yes	1.931	0.107	0.868–4.295
No	1.00
Consumption of unpasteurized dairy products
Yes	5.549	<0.0001	2.788–11.047
No	1.00
Rearing cattle
Yes	0.779	0.653	0.263–2.312
No	1.00
Assist animals giving birth
Yes	0.676	0.390	0.277–1.650
No	1.00
Knowledge
Yes	1.00	<0.0001	1.908–7.062
No	3.671

Discussion

The Nile Delta region in Egypt is reported to have the highest human and animal densities in the world (Hegazy et al. 2011). The majority of the households in this area rear small numbers of ruminants in close contact with the household members (Meky et al. 2007).

Brucellosis is mainly a disease of animals, while humans get infected through contact with infected animals or consumption of raw animal products especially dairy products made from unpasteurized milk (Makita et al. 2010, Tumwine et al. 2015).

In the present study, examination of 205 milk samples revealed that 6.3% were positive for B. abortus biovar 1 using PCR and culture methods (Table 1). In Egypt, different studies reported the identification of Brucella spp. in milk samples by PCR and culture. For instance, Hamdy and Amin (2002) identified B. abortus biovar 1 from 55.8% of cattle milk samples, while Abd Al-Azeem et al. (2012) and Wareth et al. (2014) reported that 54% and 17.7% of bovine milk samples were positive for Brucella spp., respectively. The authors attributed the high proportion of positive milk samples to the examination of milk from animals with history of brucellosis. Other studies in Egypt determined the seroprevalence of Brucella antibodies in 14.6% (Holt et al. 2011) and 38.4% (Hegazy et al. 2011) of milk samples from cattle indicating the endemic status of the disease.

In accordance, 5.3% and 3.7% of cattle milk samples from Uganda (Mugizi et al. 2015) and Tanzania (Assenga et al. 2015), respectively, were positive for B. abortus biovar 1 by multiplex PCR examination. The identification of B. abortus in the present study is consistent with other findings in low-income countries of Africa and Asia where B. abortus is more prevalent in cows (McDermott et al. 2013, Assenga et al. 2015). However, in China, 45% of milk samples from cows suspected to be infected with Brucella spp. were positive by PCR (Ning et al. 2012), while in Turkey, 95% of milk samples collected from dairy farms were positive for B. abortus (Arasoğlu et al. 2013). The authors attributed such high percentage to the high contamination of the pasture areas in the study region with miscarriages, fecal waste, and animal secretions.

The isolation of Brucella spp. from milk samples in the current study is of great concern because Brucella organisms in 80% of infected dairy animals colonize the supramammary lymph nodes and mammary glands resulting in continuous excretion of the pathogen in milk throughout the animal's lives (Cordes and Carter 1979). In addition, Brucella organisms can survive in raw milk or water for 4 days, and when the number of the organisms is low, Brucella multiplied by five log units within 3 weeks, therefore, posing risk to human consumers (Falenski et al. 2011).

Seroprevalence in animals provides an estimation on the proportion of animals shedding the organism in milk and during parturition due to the lack of vaccination strategy in the study area (Holt et al. 2011). The seroprevalence of Brucella antibodies in the examined animals was 23.8% (Table 1). In a multicenter study that included animals from different localities in Egypt, 5.4% of cattle were Brucella spp. seropositive (Samaha et al. 2008). Moreover, the prevalence of serological reactors on limited surveys in cows was reported to be between 16.5 and 23.3% (Refai 2002).

The percentage of seropositive cattle was 23.8%, while the percentage of cattle confirmed to have Brucella organisms in milk was 10% (Table 1). This difference can be attributed to several factors; first, the presence of antibodies in the serum samples does not always indicate an active case of brucellosis, as vaccination can yield postvaccinal antibodies or other gram-negative bacteria such as Yersinia enterocolitica may cross react with smooth Brucella spp. (Corbel 1988). Second, the excretion of Brucella organisms in milk is intermittent (O'Leary et al. 2006). Third, some factors might have been affected by the extraction procedure or the amount of the processed sample (Romero and Lopez-Goni 1999). Similar observation was reported by Ning et al. (2012).

The occurrence of brucellosis in humans is correlated to the presence of the disease in animals (Assenga et al. 2016). Humans who participated in the present study showed 21% seroprevalence of anti-Brucella antibodies (Table 1). In Egypt, 1.7% (El Sherbini et al. 2007), 8% (Samaha et al. 2009), 11% (Refai 2002), and 26% (Fouad et al. 1996) of seroprevalence rates in humans were recorded. Moreover, seroprevalence rates of 0.8% in India (Kadri et al. 2000) and 13.3% and 24% in febrile patients in Uganda (Ndyabahinduka and Chu 1984, Mutanda 1998) were previously reported. The relatively higher seroprevalence in the present study could be attributed to the examination of individuals with febrile illness who attended the Fever Hospital at the study area. Such high prevalence strengthens the need for strict hygienic measures to control the disease in animals (Samaha et al. 2009).

Different risk factors have been reported to be associated with brucellosis. Drinking raw milk is a custom in certain countries, for instance, in Yemen, it is a belief that boiling milk “spoils the taste and removes the goodness,” and thus, drinking fresh milk is a significant risk factor (Al-Shamahy et al. 2000). In the present study, although none of the participants declared drinking raw milk, this route could be indirect by the consumption of dairy products made from raw milk (such as Kareish cheese, a traditional type in Egypt, butter, and cream). In the current study, consumption of unpasteurized dairy products was significantly associated with the seroprevalence of brucellosis (p < 0.0001). In Egypt, 62.1% of brucellosis cases were reported to consume Kareish cheese made from raw milk (El-Ghitany et al. 2014). Other studies in Egypt reported the same findings (Abdallah et al. 2007, Holt et al. 2011).

The significant association of Brucella seroprevalence and consumption of unpasteurized dairy products was in concordance with other published studies (Issa and Jamal 1999, Husseini and Ramlawi 2004, Cetinkaya et al. 2005, Kozukeev et al. 2006, Sofian et al. 2008, Ismayilova et al. 2013). In contrast, nonsignificant correlation between consumption of raw dairy products and seroprevalence was reported in Bangladesh (Rahman et al. 2012). In another study in Egypt, the authors reported that eating soft cheese and butter was not significantly related to brucellosis (Meky et al. 2007). They attributed this finding to conducting the study in urban dwelling where cheese and butter are mainly bought from sources that used pasteurized milk for manufacture.

Handling aborted fetus and fetal membranes during assessing abortion or parturition of infected animals is considered an important method for acquiring infection (Corbel 1988). In the present study, individuals assessing animals during parturition were two times more likely to be seropositive for Brucella spp. (95% CI: 1.127–3.501; p = 0.02). In accordance, Holt et al. (2011) and Assenga et al. (2016) reported the same findings. These results could be attributed to the high concentration of Brucella organisms in the placenta of infected animals (Assenga et al. 2016).

Mixing cattle with sheep in the same house has also been found to be significantly associated with brucellosis in the present study (Table 2), thus increasing the possibility of infection spillover among other animals and humans (Assenga et al. 2016). Likewise, keeping cattle and buffalos in a household with sheep and goats had 6.32 (95% CI: 1.44–27.9) times the odds of testing seropositive for Brucella spp. than those kept with neither (Holt et al. 2011).

Rearing cattle was considered a risk factor in acquiring Brucella infection in the present study (p = 0.02). Similar findings were also reported in Iran (Sofian et al. 2008).

The association between the level of education and Brucella seropositivity has been documented earlier in other studies (Al-Shamahy et al. 2000, Assenga et al. 2016). Illiterate participants and those with primary education had no knowledge either about the disease or the methods of acquiring infection. In accordance, Assenga et al. (2016) and Kozukeev et al. (2006) reported similar findings. Seropositivity was not related to educational level as reported in Turkey (Cetinkaya et al. 2005).

Age, gender, and improper disposal of placenta and aborted fetus were factors nonsignificantly associated with seroprevalence of brucellosis in the present study. In contrast, other studies reported significant association of age, gender, and improper disposal of placenta with the prevalence of the disease (Meky et al. 2007, Holt et al. 2011, Jama'ayah et al. 2011).

Awareness about the disease especially among people rearing animals in rural areas is of great public health importance (Assenga et al. 2016). The participants in the present study were knowledgeable about the disease and methods of acquiring infection (58.3%), this is consistent with the endemic situation of the disease (Holt et al. 2011). Knowledge of the mode of brucellosis transmission appeared to be protective against disease transmission (Kozukeev et al. 2006). Poor knowledge about the disease epidemiology among participants from rural areas was reported as a risk factor that will facilitate infection among livestock workers in Nigeria (Adesokan et al. 2013).

Conclusion

The results of the present study revealed a relatively high proportion of animals, milk samples, and human participants at the study area with suspected infection with Brucella species. Consumption of unpasteurized dairy products, occupational contact with animals, and knowledge about the disease are risk factors associated with infection in humans. To minimize the spread of the disease, hygienic measures such as boiling of milk, vaccination of animals, and protective clothing during contact with animals are recommended. In addition, increased awareness of the disease and its methods of transmission and limitation of nonlicensed selling of raw milk should be carried out by authorities.

Footnotes

Author Disclosure Statement

The authors declare that there are no competing interests.

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