Spatial Distribution of Soil Borne Brucella Species Specific DNA in Punjab, Pakistan
Spatial Distribution of Soil Borne Brucella Species Specific DNA in Punjab, Pakistan
Rais Ahmed1,*, Khushi Muhammad1, Masood Rabbani1 and Muhammad Sarwar Khan2
1Department of Microbiology, University of Veterinary and Animal Sciences, Lahore
2Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore, Pakistan
Brucella species are cause of abortions and other reproductive disorders in animals and human beings. In this project, prevalence and distribution of soil borne Brucella species in nine districts of Punjab including Lahore, Faisalabad, Sheikhupura, Sargodha, D.G. Khan, Chakwal, Sahiwal, Gujranwala and Attock were determined. By using grid based sampling strategy, soil samples (n= 1280) were collected from villages (n=256, 5 samples/village) of the nine districts. Brucella genus specific DNA (bcsp31) was detected using Real Time PCR and prevalence was 2.11%. Out of five species, only two species B. abortus and B. melitensis were detected through conventioneal multiplex PCR. Prevalence of soil borne species specific DNA of B. abortus and B. melitensis was 1.8% and 0.31%, respectively. Out of nine, three districts were positive for Brucella species specific DNA. All districts showed a significant result for detecting Brucella species (χ2 =54.505, df=8, p<0.05, 95% CI). In conclusion, distribution pattern of the soil borne Brucella species in three districts of Punjab puts both human and animal population at a high risk of exposure. Further studies are required to explore molecular diversity of the pathogens together with sero-conversion in animals and humans.
Received 17 February 2017
Revised 26 April 2017
Accepted 08 June 2017
Available online 07 September 2017
RA designed the study, performed experimental work and wrote the manuscript. MR provided funds. KM supervised the study and helped in data analysis. MSK contributed in sample collections.
Brucella, DNA, Prevalence, Real-Time PCR, Soil.
* Corresponding author: firstname.lastname@example.org
0030-9923/2017/0005-1739 $ 9.00/0
Copyright 2017 Zoological Society of Pakistan
Brucellosis is a wide spread zoonosis in world and is caused by various species of Brucella. It is the second major zoonotic disease after rabies (). The organism is categorized as Risk group III pathogen by World Health Organization (WHO) and category B pathogen by Centre of Disease Control and Prevention (CDC). Its species are intracellular facultative pathogens and mostly affect the reticulo-endothelial and reproductive system (). It grows slowly on nutrient media and their growth can be enhanced by the addition of serum or blood. Brucella replicate inside cells and evade the innate and adaptive immunity and cause the disease ().
Lab workers easily acquire Brucella infection. It mainly affects slaughterhouse workers, butchers, and veterinarians. Humans are infected by occupational exposure, oral, respiratory and conjunctival routes but ingestion of unpasteurized dairy milk is the main risk of getting Brucella infection. Signs of disease in humans are undulant fever, musculo–skeletal, cardiovascular, central nervous systems complications, arthritis, orchitis and epididymitis ().
Brucellosis is important disease that also affect economics of the livestock farmers in the developing countries () The component for calculating economic loss were loss due to reduction in meat yield by abortion, still birth, repeat breeding, veterinary expenses and infertility ().
In Pakistan, sheep and goats (small ruminants) are reported for prevalence of the disease by many authors and it was found to be 13.5% to 42.6% (; ), in large ruminants (Cattle and Buffalo) 3.84% to 47.19% (; ) and in human beings was found to be 6.79% to 6.9% ().
Soil provides the complex habitat for the microorganisms but their number is very high in surface soil around the macropores (). The macropores are the channels in the soil which are formed by the activities of earthworms, roots of the plants and other soil biota which is lined with organic matter mostly in top soil (). The bacterial growth and diversity is correlated with organic matter. The microbial diversity and number is very high in top 10 cm of the soil and decreases with the depth (). It is estimated that number of species of bacteria in the soil ranges from 2000 to 18,000 per gram ().
Brucella species are sensitive to heat and can survive for several weeks in water (). Brucella species survive in the moist soil for a long period and can be transferred to relevant host (). Brucella species survive in soil and dust for many weeks (). B. abortus can survive on vegetation, soil and fetal tissues for several weeks depending month, temperature and sunlight (). B. abortus survive up to 135 days (in aborted fetus), more than 60 days (in cool environment) () and up to six months (in shaded fetus) (). Sunlight and temperature has impact on survival of B. abortus in the environment (). B. abortus can survive up to 66 days in wet soil, when humidity increased up to 90%, it can survive for 48 to 73 days and less than 4 days in dry soil (). B. suis survive up to 28 days at 50C or 220C ().
Livestock sector contributes 11.9% share in GDP of Pakistan (). Most of the people earn from livestock. A lot of studies have been done on serological prevalence of brucellosis but no study has been done on the prevalence of soil borne Brucella species in Pakistan.The persistence of Brucella species in soil () is a great risk for animals and humans health.
Therefore present study is designed to investigate the persistence of soil borne Brucella species and to achieve the following objectives: (i) detection of soil borne Brucella genus specific DNA by using Real time PCR and species specific DNA by conventional Multiplex PCR and (ii) mapping of distribution of Brucella species in various villages of Punjab.
Materials and methods
Study area and sample collection
The present study was designed to determine spatial distribution of soil borne Brucella species (B. abortus, B. melitensis, B. suis, B. ovis and B. canis) in Punjab Pakistan. The soil samples were collected from nine districts of Punjab. To select the district for sampling, first of all a map of Punjab was scanned on 600 dpi resolution and geo-referenced with geographic coordinate system. The boundary line of the study area was marked through GPS survey using the ground control points. Other layers including roads, canals, drains and rivers were digitized from Landsat-ETM downloaded from the USGS website (Free source). All of the layers were overlaid and used to make multivariate map. To determine risk areas according to intensity of Brucella species, the geographical mapping of the study area for the soil characteristics, animal and human population with respect to prevalence of pathogen was generated. Furthermore, the maps were generated in the same way district wise of all the districts. The layers including roads, canals, drains and rivers in maps were generated from survey of Pakistan districts maps. Inset map was generated from University of the Punjab, Lahore, Pakistan. All the geographical coordinates of the samples were recorded using GPS receiver (Garmin, Dakota U.S.A.) and mapped using recorded data and sequence. ArcGIS 10.1 (Esri, California) software was used to develop maps to see the spatial distribution of Brucella species in nine districts (Gujranwala, Faisalabad, Sargodha, Sahiwal, D. G. Khan, Chakwal, Sheikhupura, Lahore and Attock).
The soil samples (n=1280) from 10 % villages (n= 256 villages) of nine districts of Punjab including Sargodha, Sahiwal, D.G. Khan, Chakwal, Attock, Sheikhupura, Gujranwala, Faisalabad and Lahore were collected [From each district, 10% of total villages were randomly selected using OpenEpi version 2.3.1 () software using a confidence limit (5%), level of significance 80%] ().
Personal protective equipments (PPEs) were used for soil sampling. Samples were collected by using portable electronic weighing balance and placed in pre-labeled clean polythene bags. From each village five samples were collected. Samples were collected in such a way that four samples (n=4) were taken from each corner of the village where animals and humans were in close proximity while the fifth sample was taken as a control from outside the village, the soil where human and animal populations were not frequently interacting. The soil samples were taken three inches below the ground surface. The soil quantity was taken as 500g, 500g and 250 g and then these were pooled by mixing. One sample about 750g was archived for future reference and second sample (500g) was processed for DNA extraction in University Diagnostic Laboratory (UDL), UVAS Lahore.
A total of 130 soil samples were collected from 26 villages of Lahore. Total 145, 155, 145, 185, 135, 130, 125 and 130 soil samples were collected from 29 villages of Sheikhupura, 31 villages of Faisalabad, 29 villages of Sargodha, 37 villages of Sahiwal, 27 villages of D.G. Khan, 26 villages of Chakwal, 25 villages of Attock and 26 villages of Gujranwala, respectively.
DNA extraction from soil samples
About 0.25 g of soil sample was measured through weighing balance and labelled with proper code. DNA was extracted from the soil samples using PowerMax™ Soil DNA Isolation Kit as per manufacturer’s recommendations. DNA quality and quantity was determined using Nano Drop 1000 spectrophotometer (Nano Drop, USA) and Qubit flourometer (Invitrogen, USA) using DNA BR assay kit (Invitrogen, USA) as per manufacturer’s instructions.
The prevalence of soil borne Brucella genus specific DNA was detected using RT PCR (CFX96™ Real-Time PCR Detection Machine BIO-RAD, U.S.A). A 223 bp fragment of bcsp-31 gene was amplified by RT-PCR. For the amplification, Forward Primer 5’-GCTCGGTTGCCAATATCAATGC-3’, Reverse Primer 5’-GGGTAAAGCGTCGCCAGAAG-3’ and Probe 6-FAM-AAATCTTCCACCTTGCCCTTGCCATCA- BHQ1 was used. Real time-PCR was optimized for detection of soil borne genus Brucella using extracted genome from soil samples. The reaction was optimized for standard concentrations of primers, probe and DNA. The known DNA standards were provided by Friedrich Loeffler Institute (FLI), Germany. The concentration of Known DNA was 100pg/µL. About three different concentrations of Forward primer, Reverse primer and probe like 10, 20 and 30 pmoles per reaction were used, respectively. Along with primers and probe, about four different concentrations (10, 15, 20 and 30 ng/reaction) of DNA were used for optimization. PCR conditions and reaction mixture composition was followed as described by .
Conventional multiplex PCR
The soil DNA samples which were positive for genus Brucella in real-time PCR were again analyzed in conventional Multiplex PCR for detection of Brucella species. The species specific primers were used to detect species like B. abortus by targeting gene IS711 (498 bp) using FP 5’ GACGAACGGAATTTTTCCAATCCC-3’, RP 5’ TGCCGATCACTTAAGGGCCTTCAT-3’, B. melitensis gene IS711 (731bp) using FP 5’ AAATCGCGTCCTTGCTGGTCTGA-3’, RP 5’-TGCCGATCACTTAAGGGCCTTCAT-3’, B. suis by targeting IS711 gene (286 bp) using FP 5’- GCGCGGTTTTCTGAAGGTTCAGG-3’, RP 5’- TGCCGATCACTTAAGGGCCTTCAT-3’, B. ovis by gene IS711 (976 bp) with FP 5’-CGGGTTCTGGCACCATCGTCG-3’, RP 5’ TGCCGATCACTTAAGGGCCTTCAT-3’and B. canis by targeting gene IS711 (276 bp) using FP 5’- CCAGTATAGCTGGGCTGGTG-3’, RP 5’- CGGATATCCTGCGTGTCCAG-3’through conventional Multiplex PCR. The PCR conditions, composition of reaction mixture and agarose gel electrophoresis was performed and followed according to .
The results of real-time PCR and conventional Multiplex PCR analysis were compiled in a single Microsoft Excel spreadsheet. Data was analyzed through chi square test using 95% confidence interval and 5% level of significance. A statistical software SPSS (version 20.0; SPSS Inc., Chicago, IL) was used for statistical analysis. Normality of data was also checked by using Shapiro-wilk test.
Out of total 1280 soil samples collected from 256 villages (10% villages of each district) of the nine districts of Punjab when processed for the detection of Brucella genus specific DNA using genus specific primers and probes through RT-PCR, 27 samples (2.11%) were declared as positive. Threshold cycle (Ct) value was above than threshold for detection of genus Brucella DNA. Out of nine districts, three districts Sargodha, Sheikhupura and Faisalabad were positive for Brucella genus specific DNA. In district Faisalabad, out of 155 soil samples collected from 31 villages when processed through RT-PCR, nine (5.80%) samples were positive (), in district Sargodha, out of 145 soil samples collected from 29 villages, eleven (7.58%) samples were positive () and in district Sheikhupura, out of 145 soil samples collected from 29 villages when processed through real-time PCR, seven (4.82%) samples were positive for Brucella genus specific DNA ().
Soil samples which were positive for Brucella genus specific DNA through RT-PCR, were again run on Conventional Multiplex PCR in order to find out the exact Brucella specie by using species specific primers. Through conventional multiplex PCR, out of five species (B. abortus, B. melitensis, B. suis, B. ovis and B. canis) only two species B. abortus and B. melitensis were detected. Out of 1280 soil samples, 23 (1.8%) soil samples were positive for B. abortus DNA while four (0.31%) soil samples were positive for species specific DNA of B. melitensis. PCR product was run on 2% agarose gel, and bands for B. abortus (498bp) and for B. melitensis (731bp) were seen on gel.
Out of nine districts, three (33.33%) districts Sheikhupura, Faisalabad and Sargodha were positive for soil borne B. abortus DNA and two (22.22%) districts Sheikhupura and Sargodha were positive for B. melitensis DNA. Out of 29 tehsils, 9 (31.03%) were positive for B. abortus DNA and three (10.34%) were positive for B. melitensis DNA. Tehsil wise Brucella genus specific DNA was detected significantly (χ2 =75.977, df=32, p<0.05, 95% CI), B. abortus DNA was also detected significantly (χ2 =76.805, df=32, p<0.05, 95% CI) but soil borne B. melitensis DNA did not show significant result (χ2 =40.128, df=32, p>0.05, 95% CI). Out of 196 union councils, 18 (9.18%) were positive for Brucella genus specific DNA, 14 (7.14%) were positive for B. abortus DNA and four (2.04%) union councils were positive for B. melitensis DNA. In union councils, soil borne Brucella genus specific DNA (χ2 =330.334, df=202, p<0.05, 95% CI) and B. abortus species specific DNA (χ2 =326.052, df=202, p<0.05, 95% CI) were detected significantly but B. melitensis DNA (χ2 =187.839, df=202, p>0.05, 95% CI) was not detected significantly. Out of 256 villages, 19 (7.42%) were positive for B. abortus DNA and four (1.56%) were positive for B. melitensis DNA. village wise detection of B. abortus (χ2 =297.937, df=308, p>0.05, 95% CI) and B. melitensis DNA (χ2 =268.840, df=308, p>0.05, 95% CI) was not significant.
In district Sheikhupura, out of 145 soil samples collected from 29 villages, when processed through Conventional multiplex PCR, five (3.44%) samples were positive for B. abortus DNA while two (1.38%) samples were positive for B. melitensis DNA. In district Sheikhupura three tehsils like Sharqpur, Mureedkey and Ferozwala were positive for B. abortus DNA while two tehsils like Sheikhupura and Ferozwala were positive for B. melitensis DNA. Five (17.24%) villages like Abdalia, Mirza virkan, Muradpur, Kilay and Thatta Alyas were positive for B. abortus DNA and two (6.89%) villages like Chambal and Chak Phannar were found positive for B. melitensis DNA.
In district Faisalabad, out of 155 soil samples collected from 31 villages, when processed through Conventional multiplex PCR, nine (5.80%) samples were positive for B. abortus DNA. None of the samples was found positive for B. melitensis DNA in this district.
Four tehsils in district Faisalabad like Chak jhumra, Samandri, Tandlianwala and Faisalabad were positive for B. abortus DNA. Six (19.35%) villages like Chak 106, Chak 142 GB, Chak 228 GB, Chak 507 GB, Chak 86 JB and Chak 88 JB were positive for B. abortus DNA.
In district Sargodha, out of the 145 soil samples collected from 29 villages when processed through Conventional multiplex PCR, nine (6.20%) samples were positive for B. abortus DNA while two (1.38%) samples were positive for B. melitensis DNA. Three tehsils in district Sargodha like Bhalwal, Kot Momin and Sargodha were found positive for B. abortus DNA and one tehsil Kot Momin was found positive for B. melitensis DNA. Eight (27.58%) villages like Moazamabad, Chak 15/sb, Chak 16/sb, Chak Khana, Rawana, Chanidal, Chak 3/sb and Laluwali were positive for B. abortus DNA while two (6.89%) villages like Dera madansani and Abdal were found positive for B. melitensis DNA.
Out of the 130 soil samples collected from 26 villages of Gujranwala district, 130 soil samples from 26 villages of Lahore district, 185 soil samples from 37 villages of Sahiwal district, 135 soil samples from 27 villages of D.G. Khan District, 130 soil samples from 26 villages of Chakwal district and 125 soil samples were collected from 25 villages of Attock district when processed through conventional multiplex PCR, none of the samples was found positive for species specific DNA of B. abortus and B. melitensis.
Brucella species are mostly diagnosed in the laboratory by serological tests which are easy to perform but these tests lack specificity and Brucella species cross react with other bacteria particularly Yersinia enterocolitica O:9 that results due to O chain antigenic similarity (; ; ; ; ). Therefore, diagnosis through culturing is “gold standard” but due to the zoonotic nature of Brucella species, it poses a great potential hazard to laboratory workers and culturing should be done in well-equipped lab (Biosafety level 3) with highly skilled personnel. In the present study, real- Time PCR is used and Brucella genus specific DNA was detected by targeting bcsp 31 gene. Real-time PCR is used because it is highly specific and sensitive (97%), do not need high biosafety level 3, poses less threat to humans, simultaneous detection of multiple samples and rapid turnaround time.
DNA from bacterial cells can be released into the environment either by active secretion or passively after their death. There is autolysis of dead bacterial cells and release of cytoplasmic contents including DNA (). Other bacteria have access towards DNA of lysed cells (). A significant amount of DNA is released from bacterial cells when they have lethal exposure to antibacterial agents (). Many genera including Acinetobacter, Alcaligenes, Azotobacter, Bacillus, Flavobacterium, Micrococcus, Neisseria and Pseudomonas release DNA during active growth (; ; ; ; ; ; ). Escherichia coli also release extracellular plasmid DNA during co-cultivation with algae (). Deinococcus radiodurans actively excrete its DNA after damage with U.V. light ().
In the present study soil samples were collected from the four corners of the villages where animals were kept in close contact with humans and one sample was collected from the soil which was not inhabited by either animals or humans. Survival of the organisms in the environment poses a great health risk to animals and humans. Soil is an enriched environment with nucleic acid that is consider to be mostly present in upper soil layers (). Regarding the DNA presence in the soil, it was estimated that about 70% DNA present in upper soil is of fungal origin (; ; ). According to , from one gram of the soil, 80 µg DNA can be extracted. DNA in the soil is rapidly hydrolyzed by nucleases (; ; ) and majority of nuclease activity in the soil is of bacterial origin ().
In the present study, the soil of districts Sargodha, Faisalabad and Sheikhupura is positive for genus Brucella species specific DNA. Sargodha City is located in longitude 72o 38′ to 72o 43′ and latitude of 32o 3′ to 32o 7′ and is situated at a distance of about 185 km towards north-west of Lahore. The city is linked with Faisalabad, Jhang, Sheikhupura, Khushab, Gujrat and Sialkot by metaled roads. Total area covered by the city is 5854 KM2 and 3.1 million human population. Two rivers, river Jhelum and river Chenab flows near the city (). Minimum temperature of the city is as low as freezing point in winter and in summer the temperature reaches up to 50°C (122 °F). District Sargodha showed positive soil samples for Brucella species specific DNA as the soil of this district is of sandy loam type and chances of DNA survival and stability are very high in this type of soil as described by .
District Faisalabad is located with latitude 31.3235° N and longitude 73.1822° E and it is situated at elevation 186 meters above sea level and covers an area of 1269 Km2 with human population of 2.5 million, making it the 2nd biggest city in Punjab (). In Faisalabad districts, the positive soil of tehsils Chak Jhumra, Tandlianwala and Samandari is silty, clay and sandy type and this soil supports DNA stability up to months as described by .
District Sheikhupura is located with latitude 31.71660N and longitude 73.98500E. The district covers 5 tehsils, 169 union councils and total land area of 5960 Km2 with human population of 0.28 million (). It is situated at 237m high from sea level and 16th largest city of Pakistan (). The positive soil of tehsils Sharqpur, Mureedkey and Ferozwala of district Sheikhupura is loamy sand type and according to this type of soil supports DNA stability up to months. The contaminated soil by Brucella species (which are excreted through vaginal discharge, aborted fetuses, milk and semen of infected animals) is the source of infection to humans through parenteral, by air droplets, contact and through oral-fecal route (). The organism is highly infectious and cab be aerosolized and very difficult to diagnose due to non-specific symptoms associated with its infection ().
B. abortus survives in aborted fetuses, manure and water for periods of 150 to 240 days (). There is long term survival of DNA in the environment from archaeological and paleontological remains (; ; ). DNA has been amplified from thousands of years old specimens through PCR (; ). DNA resist environmental conditions and survive for a long time and is prevented from degradation by Nucleases. The survival of Brucella species in the environment is a great risk for humans and animals health. Organism can survive up to 122 days at room temperature in cow feces and up to 10 days in aquatic environment at 250C (). The lab workers, veterinary doctors, butchers, veterinary technicians, insemination service employees, zoo technicians, farmers working on multi-herd farms, employees of meat and milk processing enterprises are at great risk of exposure with Brucella species (). Infected people show the symptoms of fever, arthralgia, myalgia, back pain, hepatomegaly, splenomegaly, endocarditis, neurobrucellosis, epididymitis and orchitis (; ; ).
In the present study, overall 2.1 % prevalence of Brucella species was observed while 12.4% was observed in Argentina (), 15.9 % in Brazil (), 8.5 % in Georgia (), 26.3 % in Algeria (), 20.3 % in Cameroon (), 4.98 % in Egypt (), 20.4 % in Ethiopia (), 42.2 % in Libya (), 14.2 % in Niger (), 77.5 % in Nigeria (), 56.4 % in Zambia (), 3.7 % in Iran (), 25.8 % in Jordan (), 12.0 % in Kyrgyzstan (), 4.1 % in Tajikistan (), 35.3 % in Turkey () and 13.6 % in India (; ). Brucella species are not detected in districts Lahore, D.G. Khan, Chakwal, Sahiwal, Gujranwala and Attock. The possible reasons for not detecting DNAs may be (a) there is low animal density (b) less number of households (c) high nucleases activity in the soil (d) exposure to high temperature ().
In conclusion, DNA of B. abortus and B. melitensis is distributed in the soil of districts Faisalabad, Sheikhupura and Sargodha. This pattern of distribution of Brucella species specific DNA in the districts of Punjab puts both human and animal population at a high risk of exposure. Further studies are required to explore molecular diversity (biovars) of the pathogens together with sero-conversion in animals and humans.
Statement of conflict of interest
Authors have declared no conflict of interest.
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