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Frequency of brucellosis in high risk human groups in Pakistan detected through polymerase chain reaction and its comparison with conventional slide agglutination test.

Byline: Muhammad Asif Usman Waheed Muhammad Farooq Tayyaba Ali and Qaiser Mahmood Khan

Abstract

The risk of brucellosis in the individuals occupationally exposed to animals was determined in present study by slide agglutination test (SAT) and PCR to find best possible option for diagnosis. Peripheral blood samples (n=95) were collected from the humans in and around Faisalabad district Punjab Pakistan showing clinical signs related to brucellosis of occupationally exposed groups including veterinary professionals (n=33) livestock farmers (n=48) and butchers (n=14). Brucella genus specific primers were designed (targeting BCSP 31 gene) and used in PCR assay. This could amplify 224-bp region. Out of these 95 peripheral blood samples 37(38.94%) and 14(14.7%) were positive by SAT and PCR respectively. PCR is more specific test for detection of Brucella spp. The higher number of positives by SAT as compared to PCR may be due to lack of specificity of SAT. Brucellosis is an occupational hazard in animal handlers and medical practitioners may also focus on this problem while tackling recurrent fever in such population. There is a dire need of more reliable and specific diagnostic facilities like PCR to combat this potentially occupational zoonosis in Pakistan especially where brucellosis is prevalent in animals. Copyright 2014 Friends Science Publishers

Keywords: Brucellosis; Zoonosis; Occupation; Diagnostic PCR; BCSP 31 gene SAT.

Introduction

Brucellosis a zoonotic problem causing abortion and infertility in animals (Corbel 1997; Shabbir et al. 2013) and undulant fever in humans (Corbel and Brinley-Morgan1984). It is caused by the members of genus Brucella which are Gram ve aerobic non-spore forming and facultative intracellular coccobacilli (Moreno and Moriyon 2002). The genus Brucella is classified in to six species viz Brucella (B) abortus (cattle) B. melitensis (sheep and goats) B. suis (pigs) B. canis (dogs) B. ovis (sheep) and B. neotome(wood rat). Brucellosis in bovines is mainly caused byBrucella abortus and less commonly by Brucella melitensis and Brucella suis (Anonymous 2009). Humans are infected by B. abortus B. melitensis B. suis and B. canis (Nicoletti1980; Sikder et al. 2012). Brucellosis is a serious disease that can affect any system or organ of the body. But most of the time the disease mainly affects the reproductive system with reproductive failure in animals. While in humans the disease is generally associated with variable manifestations and typically fluctuating pattern of fever. Mortalities are not common but the disease is extremely debilitating in humans (Poester et al. 2010). Brucellosis is transmitted to humansthrough either direct contact with infected animals their secretions infected carcasses and products of abortion or through utilization of contaminated milk and milk products. So the disease is closely related to certain occupational groups such as dairy farmers shepherds veterinary professionals and butchers (Ayylidiz 2007; Iqbal et al.2013). The Centers for Disease Control in USA has declared brucellosis absolutely an occupational disease for the last three decades. The most affected individuals include abattoir workers meat inspectors veterinarians and animal health technicians (Kunda et al. 2007). Worldwide the accurate incidence of human brucellosis is unknown. Approximately 500000 new cases of human brucellosis are reported annually worldwide but the WHO announced that these figures greatly underrate the true incidence of human brucellosis as the real number of cases is likely to be 10 times the figures officially announced (Pappas et al.2006). In Pakistan the disease is possibly endemic but limited data is available (Abubakar et al. 2012; Gul et al.2013). An effective and safe human vaccine against brucellosis is unavailable up till now despite considerable efforts (Caksen et al. 2002). Therefore the prevention of brucellosis in humans is dependent on the control of this disease in animals by vaccination pasteurization of milk and milk products and hygienic safety measures to avoid occupational contact (Nicoletti 2001).The diagnosis of brucellosis is often difficult to establish on the basis of clinical signs and symptoms due to its unusual presentations and non-specific manifestations. Therefore its diagnosis is based on laboratory tests. Human brucellosis a widespread zoonosis - is clinically characterized by septicemic febrile illness or localizedinfection of bones tissues or organ systems. Accuratedetection of brucellosis depends on culturing of bacteria on growth media but it poses a risk of infection for thelaboratory personnel and is unsuitable for screening of large populations (Lulu et al. 1988). Serological tests can be nonspecific owing to cross-reaction or subsensitive or high immunity reactions depending on subclinical or endemic prevalence of the disease (Weynants et al. 1995; Godfroid et al. 2005). A PCR-based assay has been developed for the diagnosis of genus Brucella from bacterial cultures animal/human tissues/secretions and animal products by targeting gene encoding the 31 kDa Brucella cell surface salt extractable protein (BCSP) (Baily et al. 1992).Pakistan is an agricultural country and nearly 30-35 million of its population earns their livelihood directly or indirectly from the livestock sector. Likewise veterinary professionals livestock farmers butchers and other animal husbandry personnel of Pakistan are particularly at risk of brucellosis. There is scanty information of brucellosis in these occupationally exposed groups. Hence the present study was planned and carried out to evaluate the risk of brucellosis using SAT and PCR in veterinary professionals (veterinarians veterinary assistants artificial insemination technicians etc.) livestock farmers and butchers showing clinical signs and symptoms related to brucellosis. We have selected symptomatic individuals in our study because brucellosis is not considered a common disease in Pakistan and most of our population and even medical practitioners are generally unaware of brucellosis.

Materials and Methods

A total of 95 peripheral blood samples were taken from the individuals occupationally exposed to the animals from different areas of Faisalabad district of Pakistan. The selection of samples was based on appearance of clinical signs in past or presently continued. The clinical signs included were; undulating fever orchitis joint and muscle pains reproductive disorders etc. While during sample collection we also kept in to account like duration of clinical signs or symptoms. The number of female and male was 38 and 57 respectively. The age of the subjects ranged from 18-56 years with a mean of 40.5 years (Table 1). They comprised of veterinary professionals (33) farmers (48) and butchers (14). All these individuals had varied clinical signs and symptoms associated to brucellosis. Peripheral blood (5 mL) was drawn from each individual

Table 1: Information about the collected blood samples

Characteristics###Values

No. of patients studied###95

Female : Male###38 : 57 (1:1.5)

Mean age in years (range)###40.5 (18-56)

Clinical and Laboratory Diagnosis

Mean duration of chronic cases in years (range)###4.5 (1-10)

No. of patients with fever###40/95 (42.1%)

No. of patients with joint and muscle pain###46/95 (48.4%)

No. of patients with problems relating to###52/95 (54.7%)

aseptically. Serum was separated out of the blood andscreened by SAT for the detection of antibodies against Brucella while total genomic DNA was isolated from blood and used in PCR.

Slide Agglutination Test

For SAT we used the method described by Alton et al. (1975). The principle of this test is an immunologic reaction (agglutination) between the antibodies produced to viable bacteria (agglutinins) and their counterpart febrile antigens. The Brucella antigen used in this study was purchased from Immunostics Inc. USA. Briefly in SAT a drop of serum and antigen was mixed on a clean transparent glass slide to observe the agglutination.

DNA extraction from blood samples

Genomic DNA purification kit (MBI Fermentas Graiciunau8 Vilmius 2028 Lithuania) was used to extract total DNA from all the blood samples as per the directions of manufacturer. Purified DNA pellet was dissolved in 100 l of double distilled deionized water and stored at -20oC till further processing.

DNA amplification by PCR

A set of primers A1 (5'TGGCTCGGTTGCCAATATC3') and A2 (5'CGCGCTTGCCTTTCAGGTC3') was designed from Brucella cell surface salt-extractable protein (BCSP31 gene) of Brucella spp. (Baily et al. 1992) with modifications. These primers amplified 224bp DNA fragment within the BCSP31 gene that encodes 31-kDA membrane protein conserved in Brucella genus (Mayfield et al. 1988). In positive control DNA isolated from B. abortus culture was used whereas in negative control PCR amplification was carried out without adding any DNA to monitor performance of the reaction and confirmation of absence of cross contamination. About 50 L volume amplification reaction mixture containing 10 mM Tris-HCl (pH 8.4) 50 mM KCl 1 mM MgCl2 0.5 M each primer200 M each deoxyribonucleotide triphosphate 0.5 U ofTaq DNA polymerase and 2 g of total DNA isolated from blood or 150 ng of DNA from the positive control was used in an Eppendorf (Mastercycler gradient Eppendorf AG) thermal cycler. The PCR profile was set to initial denaturation at 93C for 5 min followed by 35 cycles each of template denaturation at 94C for 1 min primer annealing at 60C for 30 sec and primer extension at 72C for 1 min. Final extension was carried out at 72C for 10 min. Amplified DNA products were examined on 1.5% (w/v) agarose gel in 0.5x TAE (Tris base glacial acetic acid0.5 M EDTA (pH 8.0) buffer containing ethidium bromide(10 mg/ml). Amplified PCR products were run under the60V for 30-60 min. Fragment sizes were estimated by comparing with 100bp ladder (Fermentas). The 6x DNA loading dye (0.4% bromophenol blue 25% ficoll 400 and0.4% xylene cyanol FF) by MBI Fermentas Graiciunau 8 Vilmius 2028 Lithuania was used as tracking dye. Finally the gel was observed under UV light and photographed.

Results

Application of SAT (for screening) and PCR (for confirmation) assays on blood samples (n=95) collected from Brucella symptomatic patients revealed 37 (38.94%) individuals positive by SAT. Positive reaction was indicated by the presence of agglutination (Fig. 1) where as negative reaction showed no such agglutination. Among those positives 7/33 (21.2%) 23/48 (44.2%) and 7/14 (50%) were veterinary professionals livestock farmers and butchers respectively (Table 2). The PCR assay detected 14 (14.73%) individuals positive for presence of Brucella spp. Among these PCR positive cases 2/33 (6.06%) 9/48 (17.30%) and3/14 (21.4%) were veterinary professionals livestock farmers and butchers respectively (Table 2). Each sample was checked thrice by PCR in order to avoid false negative and false positive results (Fig. 2). The comparison of the tests was given in Table 3 and Table 4. Both SAT and PCR results showed higher incidence of brucellosis in the order of butchers followed by livestock farmers and then veterinary professionals.The specificity of both SAT and PCR assay was checked on a control group of 20 apparently healthy non- occupationally exposed individuals. It was found that out of these 20 individuals 3 were positive by SAT (85% specificity) while none of them was positive by PCR (100% specificity). Difference between the results of SAT and PCR on a control group (Table 3) was also found to be significant. Thus it may be concluded that the PCR assay is more specific and reliable than SAT. For enhancing the reliability of diagnosis of brucellosis both tests may be performed simultaneously. Out of these 3 healthy non- occupationally exposed seropositive individuals two had a history of febrile illness in the past.

Discussion

Brucellosis is a major public health issue worldwide especially in developing agricultural countries like Pakistan where 60-70% of the population reside in rural areas whoare in close contact with animals. Brucellosis remains in the background due to lack of awareness. The disease is possibly endemic in Pakistan but unfortunately data regarding human brucellosis is not available (Pappas et al.2006). Brucella species are secreted by the infected animals in their milk urine and vaginal discharge therefore occupational contact and consumption of unpasteurized contaminated dairy food items remained the major sources of infection.The gold standard for the detection of brucellosis is isolation but it is time consuming and requires BSL-3 laboratory requirements along with skilled personnel due to potential exposure to the causative agent. In such conditions PCR-based diagnosis is a best option due to benefits of improved specificity sensitivity and lower costs (Yu and Nielsen 2010). The use of primers targeting BCSP31 gene is frequently used for the PCR-based diagnosis of brucellosis in human with enhanced sensitivity (Baddour

Table 2: Summary of the results of SAT and PCR on field samples and the percentage of positive samples

Occupationally Exposed###No. of###Positive by###Positive by

Group###Samples###SAT No.###PCR No.

###Screened###(%)###(%)

Veterinary Professionals###33###7(21.2%)###2(6.06%)

Livestock farmers###48###23(44.2%)###9(17.30%)

Butchers###14###7(50%)###3(21.4%)

Total###95###37(38.94%)###14(14.7%)

Table 3: Evaluation of the results of SAT and PCR

Occupationally###Samples SAT +ve SAT +ve SAT ve SAT - ve

Exposed Group###screened PCR +ve PCR -ve PCR +ve PCR - ve

Veterinary###33###2###5###0###26

Professionals

Livestock###48###9###16###0###23

Farmers

Butchers###14###3###4###0###7

Total###95###14###25###0###56

Table 4: Comparison of the results of SAT and PCR

###No. of samples###Positive by SAT###Positive

###screened###by PCR

Test Population###95###37###14

Control Group###20###3###0

Control Group###85%###100%

Specificity

showing significant difference (pless than 0.001) in two testsand Alkhalifa 2008). We used the same primer set withminor changes in nucleotide sequences and increased cycle number for better amplifications of DNA (224bp) as shown in Fig. 2.In present study we applied SAT and PCR assays both to all 95 blood samples for the diagnosis of brucellosis in different subjects occupationally exposed to animals. The assays detected 37(38.94%) and 14(14.7%) of exposed workers positive by SAT and PCR assay respectively. The fundamental reasons of higher number of individuals positive by SAT as compared to PCR may be due to low specificity of SAT especially in the occupationally exposed individuals or in the areas where brucellosis is endemic and is the potential cause of high occurrence of Brucella antibodies in the healthy population (Al-Attas et al. 2000). Moreover most of serological tests used for diagnosis of brucellosis including SAT produce cross reactions with other Gram ve bacteria e.g. Francisella tularensis (Francis and Evans 1926) Yersinia enterocolitica (Feeley 1969) Vibrio cholerae (Ahvonen et al. 1969) Salmonella and Escherichia coli (Stuart and Corbel 1982).The present study both SAT and PCR results indicated higher incidence of brucellosis in butchers among the occupationally exposed workers. Cadmus et al. (2006) Araj and Azzam (1996) reported higher seroprevalence of brucellosis in butchers when compared with other exposed workers and they reported this to 63.3% and 54% respectively. This high rate of prevalence in butchers in Pakistan is multi-factorial and includes mainly; poor stateof meat inspection services and the unhealthy practicesduring slaughtering (e.g. smoking and eating withcontaminated hands putting of contaminated knives intomouths during skinning and cutting of carcass etc).

Generally the butchers do not adopt any protectivemeasures while slaughtering leaving them exposed toinfected materials such as blood urine vaginal dischargesetc. of the slaughtered infected animals. The chances of attracting the brucellosis are even higher when these discharges are in contact with broken skin especially of hands and feet. Furthermore splashing of infected blood or other fluids while slaughtering in to the eyes exposethem to high risk of acquiring brucellosis and otherinfections. Moreover in Pakistan especially in rural areas mostly diseased and non-productive animals wereslaughtered. Most of these animals have problems relatedto reproductive system (e.g. repeat breeding abortionmetritis etc.) and Brucella is one of the major pathogenaffecting the reproductive system.

The subjects from livestock farmers community are persistently exposed to brucellosis due to milking and

rearing of the infected animals. Poor hygienic practicesadopted by the farmers further encourage the transmission of Brucella spp. Furthermore hand washing is not a common practice among farmers following contact withinfected material. As a result of lack of awareness about the disease those who develop symptoms like relapsing feverjoint and muscle pain fatigue and weakness always relate them with other diseases like typhoid malaria tuberculosis rheumatoid arthritis etc.The veterinary professionals are aware of zoonotic potential of brucellosis. They may contract the diseaseduring insemination of the infected animals removal of retained fetal membranes exposure to the aborted fetuses and newborn calves and accidental splashing of B. abortus stain 19 vaccine in to the eyes.Brucellosis is a curable disease. In 1986 the WorldHealth Organization (WHO) issued therapeutic guidelines for brucellosis which consist of two regimens both consisting of doxycycline for six weeks in combination with either rifampin for six weeks or streptomycin for 2-3 weeks (Pappas et al. 2005). It was suggested to medicalpractitioners to prescribe the above antibiotic combinationsto the patients in whom brucellosis has been diagnosed by PCR. We have received favorable response from the patients which completed full course of antibiotic therapy.After the completion of therapy these patients were again checked by SAT and PCR. None of them was found to be positive by PCR while all these patients remained positive by SAT. The main reason behind this is that the PCR detects only the nucleic acid of the infectious agent which is eliminated from body after completion of successful therapy. While SAT detects only the antibodies produced in the body in response to exposure of an antigen. The antibody titers in serum of individuals often remain positive for a prolonged period after therapy even in cases of complete recovery from brucellosis (Pellicer et al. 1988). Therefore we can support this idea that SAT is inferior to PCR in terms of specificity and sensitivity and is not recommended if other procedures (like PCR) are available (Robinson 2003).In conclusion brucellosis is an occupational hazard in veterinary professionals butchers dairy farmers shepherds and other animal husbandry personnel. Butchers are at higher risk compared with other groups because of adopting poor personnel hygienic measures and frequently higher exposure to the blood body secretions and tissues of the infected slaughtered animals. Medical practitioners miss many cases of brucellosis due to its atypical and nonspecific clinical signs and symptoms and also it is not regarded as a common disease in Pakistan. Medical practitioners should consider the possibility of brucellosis in cases of relapsing fever arthritis muscle pain and orchitis etc. Our results also suggest that commonly used serological tests like SAT lack specificity. Therefore use of SAT as a sole tool for diagnosis of human brucellosis is not reliable. There is a need of more reliable and specific diagnostic tests like PCR and national surveillance programme especially for the occupationally exposed workers to combat this re-emerging zoonosis in an agricultural country like Pakistan where brucellosis has been reported in animals (Nasir et al. 2004).

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Author:Muhammad Asif; Usman Waheed; Muhammad Farooq; Tayyaba Ali; Qaiser Mahmood Khan
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9PAKI
Date:Oct 31, 2014
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