Abstract: Brucella abortus, one of the major pathogen causing abortions in cattle worldwide and a zoonotic agent, need to be detected earlier in order to prevent its spread among animals. The present study aimed at to know the prevalence of B. abortus in cattle population of three states (Uttar Pradesh, Uttarakhand and Tamil Nadu) of India by serological (Rose Bengal Plate Test (RBPT) and Serum Tube Agglutination Test (STAT)) and molecular (polymerase chain reaction) detection in sera samples and whole blood (n = 370), respectively. Out of a total of 370 sera samples, 61 (16.49%) were positive by RBPT and 59 (15.94%) by STAT. Screening of the whole blood samples by genus specific bcsp31 gene based PCR as well as species specific IS711 gene based PCR revealed that 56 (15.13%) samples were positive for brucellosis. None of the serologically negative sample showed positivity by PCR; however few positive samples were tested negative by PCR. Sensitivity and specificity of PCR compared with RBPT was 100 and 92.4% while with STAT these were 100 and 95.16%, respectively. Results are promising that whole blood can be used for studying the molecular epidemiology of B. abortus in cattle and particularly detecting the active phase of infection and PCR can be well adopted as a valuable test for mass screening of animals for this purpose. The present study adds to the prevalence data available regarding to B. abortus infection in cattle population and highlights the usefulness and advantages of molecular tool of PCR over serological tests.
INTRODUCTION
Brucellosis, caused by a member of gram negative bacterium, is a well documented disease in animals and also a potential zoonotic agent (Gupta et al., 2014). Brucella abortus is the major abortion (usually in third trimester) causing agent in cattle and has been reported to affect cattle industry worldwide (Seleem et al., 2010). Brucellosis is endemic in most parts of India (Kumar et al., 2009; Islam et al., 2013) and it was found to be highest in Assam as per the earlier reports by sero-epidemiology and prevalence in Uttar Pradesh was 6.37% in cattle (Islam et al., 2013).
Diagnosis of a disease is of prime significance in order to identify, prevent and control a disease. Rapid and confirmatory diagnosis is more important in case of diseases which are highly infectious in nature, have high economical significance and possess zoonotic threats which cause havoc to the mankind (Atluri et al., 2011). A diagnostic method combined with appropriate sample for diagnosis at any stage of infection is the requirement to keep brucellosis in check. Conventional cultural isolation and identification of the agent is the gold standard test for B. abortus but is time consuming, laborious and also needs skills as well as biosafety measures to prevent zoonosis (Taleski, 2010). Various serological tests like Rose Bengal Plate Test (RBPT), Standard Tube Agglutination Tests (STAT), enzyme linked immuno sorbent assay (ELISA) and others are commonly employed for mass screening of animals for brucellosis at farm level (Yu and Nielsen, 2010; Priyadarshini et al., 2013). The major drawback of these assays are they are not always specific, can cross react with other gram negative bacteria (Yersinia enterocolitica O:9, Francisella spp.) and antibodies are not produced at the acute stage of infection (Moussa et al., 2011). In this view, DNA detection methods like Polymerase chain reaction (PCR), versions of PCR (nested PCR, real time PCR, multiplex PCR) and loop mediated isothermal amplification test (LAMP) are now being employed for raid and confirmatory detection of brucellosis in cattle (Yu and Nielsen, 2010; Soleimani et al., 2013; Dhama et al., 2014; Gupta et al., 2014). B. abortus remains intracellularly and this poses a problem for selection of a suitable sample (Wattam et al., 2009). Only during acute phase of infection it circulates in blood, mostly inside the white blood cells and hides itself in mammary organs, genital organs and lymph nodes (Morgan and MacKinnon, 1979). Hence sample should be selected according to the phase of Brucella’s life pattern which is not practically possible to find out.
The present study aimed at to know the prevalence of B. abortus in cattle population of three states (Uttar Pradesh, Uttarakhand and Tamil Nadu) of India by serological [RBPT and STAT] and molecular (polymerase chain reaction) detection in sera samples and whole blood. Besides comparing the sensitivity and specificity of serological versus molecular tool of PCR, the suitability of the whole blood as a sample for detection of B. abortus in cattle using PCR was also evaluated.
MATERIALS AND METHODS
Bacterial strains: Two standard strains of B. abortus namely B. abortus S99 and B. abortus S19, obtained from Division of Veterinary Public Health and Division of Biological Standardization, Indian Veterinary Research Institute (IVRI), Izatnagar, India, were used in the present study. Both the cultures were tested for its family/genus characters and tested to be pure.
DNA template preparation: Bacterial DNA of both the strains was extracted using modified Cetyl trimethylammonium bromide (CTAB) method (Rodriguez et al., 1997). Briefly, 2 mL of Brucella culture previously inoculated in Brucella selective broth (Hi Media, India) was taken in a microfuge tube and centrifuged at 12,000 rpm to settle the pellet. Using 200 μL of 1% TE (Tris EDTA), 50 μL of 10% SDS and 5 μL of proteinase K, the pellet was resuspended and rapid pipetting was carried out to mix the contents and incubated at 37°C for 1 h. The incubated mixture was added with 5M NaCl (500 μL) and CTAB (100 μL) and kept in water bath at 63°C for 10 min, followed by addition of equal volume of phenol:chloroform:isoamylalcohol (25:24:1) and centrifugation at 10,000 rpm for 10 min. Aqueous phase was carefully transferred to another microfuge tube to which 1/10 volume of 7.5 M ammonium acetate and 2 volume of chilled absolute ethanol followed by centrifugation at 10,000 rpm; final washing was given with 70% ethanol. Finally, 50 μL nuclease free water (NFW) was added to resuspend the DNA and the DNA was stored in -20°C till further use.
Sample collection: The serum samples and whole blood samples (n = 370 each) were collected separately from cattle population of different parts of three states [Uttar Pradesh (220), Uttarakhand (100) and Tamil Nadu (50)] of India, from various sources viz., cattle farms, polyclinics and slaughter houses. The serum samples were collected in vacutainers without anticoagulant; while whole blood samples were collected in heparin coated vacutainers. Most of the samples were collected from areas where there was a history of abortion or suspicion of Brucella occurrence. All the serum and whole blood samples were subjected for serological and molecular detection of B. abortus.
Serological tests: Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Tests (STAT) for detection of B. abortus antibodies in all the serum samples (n = 370) was carried out according to the standard protocol of Alton et al. (1975). RBPT and STAT antigens were procured for Division of Biological Standardization, IVRI, Izatnagar.
Molecular Test
PCR detection of B. abortus: DNA from the whole blood was extracted
according to protocol of Sambrook and Russell (2001) with
slight modifications. Briefly, 300 μL of whole blood along with 700 μL
of lysis buffer (1 M Tris-Cl, 5 M NaCl, 0.5 M EDTA, 10% SDS) and 5 μL proteinase
K was taken in a 2 mL microfuge tube. The contents were mixed thoroughly and
incubated at 56°C in water bath overnight. Tris saturated phenol was added
in equal volume to the lysed mixture and centrifuged at 12,000 rpm for 15 min.
Phenol: Chloroform: Isoamylalcohol (25:24:1) was added to the supernatant and
centrifuged at 12,000 rpm for 10 min. To the aqueous phase, isopropyl alcohol
(double the volume) was added to precipitate the DNA. The DNA pellet obtained
after centrifugation at 10,000 rpm was washed twice with 70% chilled ethanol;
air dried and added with 50 μL NFW to resuspend the DNA. The DNA thus obtained
was stored in -20°C till further use.
Primers targeting for the bcsp31 gene were used for Brucella genus specific PCR (Baily et al., 1992) and primers targeting IS711 gene were employed for B. abortus species specific PCR (Doust et al., 2007). The primer sequences used and the expected product sizes are presented in Table 1. The bcsp31 gene based genus specific PCR was optimized with initial denaturation at 95°C for 10 min followed by 35 cycles of denaturation (94°C for 1 min), annealing (55°C for 1 min) and extension (72°C for 1 min). Final extension was carried out at 72°C for 7 min. Similarly, IS711 gene based species specific PCR was optimized with the same conditions used for bcsp31 PCR, except for the annealing condition which was kept at 58°C for 1 min.
| Table 1: | Details of PCR primers |
All the 370 whole blood samples were screened for the presence of Brucella genus first with the bcsp31 primers based PCR and the samples which were found positive were screened further by IS711 primers based PCR for detecting the B. abortus at species level.
RESULTS
Serological testing of the 370 serum samples revealed that 61 (16.49%) samples were positive by RBPT and 59 (15.94%) samples were positive by STAT. Screening of the whole blood samples by genus specific bcsp31 gene based PCR as well as species specific IS711 gene based PCR revealed that 56 (15.13%) samples were positive for the Brucella genus as well as the B. abortus species, showing expected 223 bp (Fig. 1) and 498 bp sized amlpicons (Fig. 2), respectively. None of the serologically negative sample showed positivity by PCR; however few positive samples were tested negative by PCR.
| Fig. 1: | bcsp31 gene based PCR for detection of Brucella spp. at genus level. Lane 1-5 DNA samples from whole blood, Lane M-100 bp ladder |
| Fig. 2: | IS711 gene based PCR for detection of B. abortus at species level. Lane 1-5 DNA samples from whole blood, Lane M-100 bp ladder |
| Table 2: | Serological and molecular detection of Brucella abortus in clinical samples by RBPT, STAT and PCR |
The clinical sensitivity and specificity of PCR compared with RBPT was 100 and 92.4% while with STAT these figures were 100 and 95.16%, respectively. The results of the serological and molecular detection and the prevalence rate of B. abortus in different areas under study are presented in Table 2. Among the three states, Uttar Pradesh showed a higher prevalence rate of 15.9% followed by Uttarkhand and Tamil Nadu both having 14% prevalence rate as detected using PCR.
DISCUSSION
Brucellosis is a zoonotic disease which needs to be diagnosed at the earliest and PCR is an exceptional tool which can diagnose a disease within 3 h accurately (Habtamu et al., 2013). Various diagnostic assays are used for the detection of B. abortus from cattle which includes basic bacteriological, serological and molecular assays (Da Silva Mol et al., 2012). Basic bacteriological techniques though remain as the standard tests they are laborious and involve risk to the laboratory person as the organism is zoonotic (Sam et al., 2012). This study was aimed at the diagnosis of B. abortus in three different states of India using serological test like RBPT, STAT and molecular assay of PCR and also the whole blood was evaluated as a sample of choice for mass screening of cattle for B. abortus. Serological tests recorded more positivity in the present study compared to PCR but these are not specific always because there are chances of cross reaction with other bacterial species (Moussa et al., 2011). The status of animal with respect to Brucella infection was not known during the time of sample collection and hence organism might have lodged in organs like lymph node, mammary glands or uterus and so DNA was not recovered from whole blood sample (O’Leary et al., 2006; Moussa et al., 2011). During acute phase of infection when the circulating organisms are higher, good quantity of DNA can be extracted and used for PCR (Gemechu et al., 2011). The prevalence of B. abortus from whole blood of cattle using PCR was 15.13% showing that B. abortus can be detected from whole blood sample. Similar results that DNA can be extracted from whole blood and used as a sample for screening animals for brucellosis were reported by Guarino et al. (2000) in buffaloe, Keid et al. (2010) in dogs and Khamesipour et al. (2013) in cattle and sheep. Lot of studies demonstrated that Brucella can be detected by PCR from whole blood of human samples but there are some contradictory findings in case of cattle (O’Leary et al., 2006; Bhanu Rekha et al., 2013). Al Nakkas et al. (2002) and Leal-Klevezas et al. (2000) used buffy coat instead of whole blood for DNA extraction as the macrophages take up the Brucella organisms. But buffy coat separation needs additional steps (Mitka et al., 2007) and hence the use of whole blood as such was tried in this study and it showed good results. Conventional DNA extraction protocol was carried out during the study which might be another reason for lower quantity and quality of DNA. Use of commercially available kits can improve the quantity and quality of extracted DNA (Queipo-Ortuno et al., 2008; Keid et al., 2010).
The prevalence of brucellsois in India in this study was lesser as compared to earlier study by Trangadia et al. (2010) but higher in Uttar Pradesh compared to Kumar et al. (2009). Various factors like time of sample collection, infection status of the animal, condition of the farm, number of samples collected can all influence on the detection results. In order to know the exact scenario, an extensive epidemiological survey should be conducted state wise which can help in planning the control and eradication measures for this important pathogen. The specificity and sensitivity of the PCR assay was compared with RBPT and STAT which was promising that PCR can be employed as a test for rapid screening of herds.
CONCLUSION
In conclusion, PCR can be used as a rapid and confirmatory diagnostic tool for mass screening of herds with brucellosis. The whole blood samples can be used as a valuable field sample of choice which can be collected easily unlike aborted foetal stomach contents needing aborted foetus or lymph node samples. Also, whole blood samples have utility in knowing the active/acute phase of infection B. abortus using PCR. This study adds to the available prevalence data regarding B. abortus infection in cattle. Extensive molecular epidemiological studies are suggested for knowing the magnitude of B. abortus infection in the cattle population of the country which would help to devise and adopt appropriate disease prevention and control measures against this economical important animal pathogen possessing public health concerns.