The outbreak of migratory goat’s brucellosis in the Swat ecosystem of Khyber Pakhtunkhwa

Brucellosis is a major threat to public health especially in developing countries including Pakistan. This study reveals the characterisation of Brucella species affecting humans and goats in the Swat region of Khyber Pakhtunkhwa, Pakistan. Blood samples were collected from shepherds and goats and analysed by Rose Bengal precipitation test (RBPT), standard plate agglutination test (SPAT), polymerase chain reaction (PCR) and Sanger sequencing of 16S rRNA gene. The findings of the study indicated 24% (36/150) and 11.3% (17/150) positivity for Brucella abortus and Brucella melitensis, respectively, in human samples. In samples of goats, 26.66% (40/150) were positive for B. abortus and 16.66% (25/150) samples were positive B. melitensis by SPAT. The species-specific PCR confirmed B. abortus in 24% (36/150) of human samples and 26.66% (17/150) of goat samples by targeting the IS711 locus. The remaining seropositive samples were confirmed as B. melitensis using IS711 M species-specific primer. The sequences of the amplified fragments of the 16S rRNA gene were blasted, and phylogenetic analysis revealed that Brucella species circulating in the Swat district were closely related to B. melitensis and B. abortus reported from India, China, Philippines, and the United States (US) showing the existence of the possible epidemiological linkage among the Brucella species. This study concluded that there was a higher prevalence of B. abortus (26.6%) in humans and goats compared to B. melitensis (16.6%). These results revealed that the Brucella species were circulating in both humans and goats in the study areas. The findings of the study concluded that B. abortus and B. melitensis were circulating in goats and shepherds with a higher prevalence of B. abortus than B. melitensis. Furthermore, the Brucella species identified in Swat were phylogenetically related to the Brucella species reported from India, China, Philippines and the US. Contribution The proposed study covers the scope of the journal. The species of the genus Brucella affect both animals and shepherds. This study investigates the seroprevalence of brucellosis in shepherds and goats in different geographical areas in the Swat district. The phylogenetic analysis of the Brucella spp. identified in Swat showed close relationships to the Brucella species reported in India, China, Philippines and the US, which shows the possible epidemiological linkages between the Brucella spp.


Introduction
Pakistan is a South Asian country with an agrarian economy wherein the livestock industry plays a significant role (Kumar, Mittal & Hossain 2008).The country's livestock population was predicted to be 90.8 million cattle and buffaloes, 109.4msmall ruminants (sheep and goats), 1.1m camels and 6.1m equines in 2019.In 2019-2020, this livestock sub-sector provided 60.6% of the value of the agriculture sector and 11.7% of the national gross domestic product (GDP) (Khan et al. 2021).Pakistan is divided into seven administrative regions: Punjab, Sindh, Balochistan, Khyber Pakhtunkhwa (KPK), Gilgit-Baltistan, Azad Jammu and Kashmir (AJK) and the Islamabad Capital Territory (ICT).The bulk of cattle and buffaloes are found in the country's irrigated parts (Punjab and Sindh), while small ruminants are found mostly in the dry regions.Vaccination and treatment of brucellosis are preferably used for bovines rather than small ruminants (Roth 2011).Khyber Pakhtunkhwa, located in northwest Pakistan, is the third most populous province in terms of both human and livestock populations (Memon et al. 2021).The demography of the area is mostly mountainous, with prominent mountain ranges such as the Hindu Kush and Suleiman mountains.In addition, the population of small ruminants in this area is substantially larger than that of bovines (Khan et al. 2022).
Brucellosis is among the most frequent occupational zoonotic disease in the world, causing tremendous livestock losses and social burdens, particularly in developing countries (Franc et al. 2018).The social burdens of brucellosis include social stigma and discrimination against those infected, as well as economic losses because of decreased productivity of livestock, increased healthcare costs and loss of income.In developing countries, where brucellosis is more prevalent, these social burdens can have a particularly significant impact on the affected individuals' livelihoods and overall well-being (Cleaveland et al. 2017;McElwain & Thumbi 2017).Because of its slow onset and a lack of symptomatic diagnoses in livestock (Khan & Zahoor 2018), it is generally discovered relatively late, and by that time the entire herd is already affected.Aside from immediate public health effects, the presence of brucellosis may obstruct international trade in animals and animal products (Bagheri Nejad et al. 2020).The disease's reservoirs include dairy cattle, sheep, goats and pigs, horses, camels and other wild animals are occasionally infected.Recent findings of Brucella infection in marine animals (Abalos et al. 2009) and birds (Ali et al. 2020) have provided a new perspective.It is the second most important zoonotic disease in humans after rabies in the world (Abubakar, Mansoor & Arshed 2012).Brucellosis has been eradicated in developed countries, but it remains endemic in some developing countries (Robinson & Production 2003).Worldwide, over half a million cases of the disease are reported annually (Avijgan, Rostamnezhad & Jahanbani-Ardakani 2019).Brucellosis is caused by Gramnegative bacteria of the genus Brucella (Mert et al. 2003).Among Brucella species, Brucella abortus (B.abortus) and Brucella melitensis (B.melitensis) are of great importance because of their high prevalence in both humans and animals (Khamesipour & Momeni 2014).Brucella melitensis, in particular, is a reemerging pathogen in the Mediterranean, Arabian Gulf and Middle East regions (Ebid, El Mola & Salib 2020).
Infection in sheep and goats can lead to various health issues such as abortions, weak offspring, reduced milk production, weight loss, infertility and lameness.These problems cause significant economic losses to the global animal industries (Ebid et al. 2020;Franc et al. 2018).Moreover, humans may contract the disease through direct contact with infected animals or consumption of contaminated and unpasteurised dairy products (Mitiku et al. 2021).This can result in acute febrile illness, commonly known as undulant fever.The disease can progress into a chronic form and cause serious complications, affecting the musculoskeletal, cardiovascular and central nervous systems (Galinska & Zagórski 2013) Currently, these conventional techniques are replaced by a molecular technique, polymerase chain reaction (PCR), used for the detection of Brucella and its species because of its high sensitivity and specificity (Marín et al. 2007).Polymerase chain reaction has proven to be the most sensitive diagnostic tool for both B. abortus and B. melitensis in human patients (Kamal et al. 2013).Brucellosis is distributed worldwide and is more common in countries with poor animal and public health hygiene programmes.Although it has been eradicated from many developed countries, it remains an endemic disease in some regions including Pakistan.In addition, the lack of studies on the prevalence of brucellosis in shepherds and migratory goats in the Swat region of Pakistan needs to be updated on the infection rate.The objective of this study was to establish the seroprevalence of brucellosis in shepherds and goats in Swat.Furthermore, the causative agents of brucellosis were characterised based on the Sanger sequencing of the 16S rRNA gene.

Study area
This study was performed on migratory goats and shepherds in different tehsils of the Swat district.It is a cultural tradition for shepherds of this area to migrate with their flocks during the winter months and this practice has been passed down through generations.Previous data about only household domestic small ruminants are available, and there is little knowledge about the seasonal migratory goats in the area.Detailed information regarding brucellosis was collected on a pre-designed questionnaire for both migratory shepherds and goats.Samples were gathered from goats and shepherds who had returned to the area after seasonal migration from seven tehsils, namely Babuzai, Bahrain, Barikot, Charbagh, Kabal, Khwazakhela and Matta, in the Swat district.The demography of the under study area is shown in Figure 1.

Sample collection
Samples were collected from the goat flocks, as well as from the shepherds and their family members who returned to the area after seasonal migration along with their goat flocks.Blood samples (n = 300) were collected from shepherds and goats (shepherds, n = 150 and goats, n = 150) by following standard procedures without any animal harm (Wang et al. 2015).A total of 3 mL to 5 mL of blood samples were collected aseptically from the jugular vein of goats and cephalic vein of shepherds.To maintain the integrity of red blood cells, disposable syringes were utilised to collect blood samples, which were then introduced into both Gel tubes and EDTA tubes without applying any pressure.Proper labeling was employed, and the blood samples were transported to the laboratory of the Centre for Biotechnology and Microbiology at the University of Swat for further analysis.The blood samples were refrigerated overnight until they were processed.

Sample processing
Blood samples were kept in an upright position for 30 min in a cool place and then centrifuged at 3000 revolutions per minute (rpm) for 20 min.http://www.ojvr.orgOpen Access

Rose Bengal Plate Test
The initial screening process involved using the coloured Rose Bengal Plate Test (RBPT) antigens following the protocol outlined by OIE in 2014.To ensure a uniform antigen suspension, both the serum and antigen were equilibrated to room temperature and vigorously agitated.A single drop of 30 µL (0.03 mL) of serum sample and 40 µL (0.04 mL) of Rose Bengal antigen were added to the same slide plate, creating a zone of approximately 3 cm in diameter.Using separate micropipettes, the samples were mixed thoroughly with a mixing stick.The slide plates were then rotated gently in a clockwise and counterclockwise motion for 4 min.Afterwards, the slide plates were examined under a bright light to detect any agglutination, which would appear as small clumps or dots.The results were interpreted based on the specific RBPT kit instructions and established diagnostic criteria for goats, as reported by Ebid et al. (2020).

Standard Plate Agglutination Test
The SPAT was performed following the standardised protocols of OIE in 2014., Briefly, both the antigen and serum were equilibrated to room temperature.An 80 µL sample of each serum was then placed on a glass slide.Next, 30 µL of antigen was added to create a homogeneous mixture.Using a mixing stick, the serum and antigen were mixed thoroughly, forming a circular zone of approximately 3 cm in diameter.
The slide was gently rotated in a circular motion, both clockwise and counterclockwise, for 3 min to 4 min.Positive samples exhibited agglutination, while negative samples displayed no agglutination, as reported earlier by Ebid et al. (2020).

Molecular identification
All samples that tested positive for RBPT and SPAT were further confirmed by PCR.Brucella deoxyribonucleic acid (DNA) was amplified and detected by PCR using the protocol described by Romero et al. (1995).Gel electrophoresis for PCR product was performed by following the standardised methodology of Ahmad and Rahman (2019).Briefly, serum samples were used to obtain genomic DNA using the QIAamp DNA Mini Kit (Qiagen kit, Germany) following manufacturer protocol.The DNA was extracted and purified from the pellet via DNA Purification Kit (GeneJET Genomic; Thermofisher Scientific-K0721).After the DNA purification, a PCR assay was performed for targeting the genus Brucella using the genus-specific primer (BCSP31 gene (BCSP31-PCR).The positive samples were then subjected to PCR (Thermo Fisher Scientific) using species-specific primers (IS711AB locus [IS711-PCR] for B. abortus and IS71M locus for B. melitensis).The thermocycler programme was adjusted for 40 cycles as; DNA denaturation for 1 min at 94 °C -annealing of primer for 30 s at 60 °C -Elongation for 1 min at 72 °C -Final elongation step of 10 min at 72 °C.At the end of the thermocycler programme, the amplified product was run on 2% agarose gel in gel electrophoresis for 60 min at 110 volt.
The positive samples for brucellosis were subjected to 16S rRNA PCR and sequencing.The universal primers of 16S rRNA (Forward 5'-GTG-CCA-GCA-GCC-GCCGTA-ATA-C-3′) and reverse (5'-TGG-TGT-GAC-GGG-CGG-TGT-GTA-CAA-3′) were used.The PCR protocol was optimised for 25 µL final reaction volume, containing 12.5 µL of master mix, DNA template (5 µL), PCR-grade water (5.5 µL), and each primer of 1 µL.The reaction was run for 40 cycles under the optimised condition of PCR (initial denaturation and final denaturation at 95 °C for 5 min and 30 s, respectively, annealing temperature at 52 °C for the 30 s, initial extension and final extension at 52 °C for 30 s and 5 min, respectively).The PCR products were visualised through gel electrophoresis by following the methodology of Ntirandekura et al. (2020) with some modifications.Before sequencing, the PCR products were first purified through a purification kit (GFXTM PCR DNA and Gel band purification) following the manufacturer's protocol.The purified samples were then subjected to Sanger sequencing.The sequencing was performed using Cycle Sequencing Kit (BigDye ® Terminator version 3.1).The final sequencing reaction volume was 10 µL, containing 2 µL of sequencing buffer (5X) and sequencing primers (forward and reverse primers of 16s rRNA gene).
Sequencing was performed twice for each primer with the optimised sequencing condition (96 °C for 1 min, followed by 25 cycles of denaturation at 96 °C for 10 s, 50 °C for 5 s and 60 °C for 4 min).Next, ethanol precipitation was used for cleaning the sequencing products (Ntirandekura et al. 2020).
After precipitation, the tubes containing the sequencing products were centrifuged for 30 min at 13 000 rpm.The pellets were washed with ethanol (70%) after discarding the supernatant and then centrifuged at 13 000 rpm for 30 min.
The samples were then treated with Hi-Di ™ formamide and then loaded on a DNA analyser (ABI 3730).The analysis was performed according to the manufacturer's protocol.

Sequence analysis
The Bioedit software was used to visualise the sequences obtained from Sanger sequencing of BSCP31 isolates from human and goat samples.Chromatogram quality was assessed, low-quality bases were trimmed, and all ambiguities were corrected.Consensus sequences were generated by aligning the forward and reverse sequences of each sample.NCBI BLAST tool was used to identify similar sequences, and sequences with 80% or greater similarity were downloaded from Gene bank.The maximum likelihood method and the Jukes-Cantor model were employed to infer the phylogenetic relationship.Consensus bootstrap with 100 replicates was used to infer the evolutionary history of the eight nucleotide sequences analysed.The final dataset contained 1002 positions for the goat sample and 986 positions for the human sample.Evolutionary analyses were conducted using MEGA X (Girault et al. 2022).

Statistical analysis
The raw data collected from pre-designed questionnaire for both migratory shepherds and goats was manually entered

Ethical considerations
The Ethical Research Committee (ERC) of the University of Swat approved the research project entitled 'The episode of human and migratory goats Brucellosis in the Swat Ecosystem of Khyber Pakhtunkhwa, Pakistan' (No. UoS/ ORIC/2021/13).
No animal was harmed during the sample collection process and the data of shepherds was kept confidential.

Results
The data presented in Table 1  The results presented in Table 3 show the gender-wise seroprevalence of brucellosis in human samples tested using RBPT.Females had a higher prevalence rate (42%, 37/88) compared with males (25.8%, 16/62).Table 3 also displays   There was no significant difference (p ≥ 0.05) found in the gender-wise seroprevalence data for brucellosis in goats.
After performing the initial screening of samples on SPAT, sets of primers were used for the molecular identification and characterisation of Brucella species using PCR assay.The species-specific primer (IS711 AB) targeted the IS711 locus in the genome and produced an amplicon size of 498 base pairs (bp) and thus confirmed the presence of B. abortus in 24% of human samples and 26.66% of goat samples.The remaining seropositive samples (11.3% human and 16.66% goats) were confirmed for the presence of B. melitensis with an amplicon size of 731 bp (Figure 2).The seropositive samples of brucellosis were then subjected to PCR-based amplification of 16S rRNA genes and Sanger sequencing to characterise the Brucella species circulating in the ecosystem of Swat.The amplified fragments of 16S rRNA genes were then sequenced by Sanger sequencing and the sequences obtained were blasted for similarity search.The different parameters of query sequences (E value, percent identity, etc.) revealed homology with the sequences of different species of Brucella (B.melitensis,    B. abortus) deposited in the Gene bank.After cleaning the sequences, phylogenetic analysis was performed to characterise the Brucella species from the Swat region.The phylogenetic tree of the 16S rRNA gene sequences showed that Brucella spp.from the Swat region were grouped into two clades and two branches, all closer to B. melitensis and B. abortus reported from the different areas of the world (Figure 3).

Discussion
The management and diagnosis of brucellosis are challenging because of prolonged culturing time and contagious nature of Brucella species (Shenoy, Jaiswal & Vinod 2016).Therefore, serological and molecular techniques such as RBPT, SPAT and PCR are promising alternatives for the timely diagnosis of fastidious microorganisms such as Brucella species.Serological tests such as RBPT and SPAT were used by previous researchers to find out the seroprevalence of brucellosis in animals as well as in humans (Khan et al. 2022;Niaz et al. 2021).This study also documented the seroprevalence of brucellosis in shepherds and goats based on RBPT and SPAT; the results were further confirmed by species-specific PCR.and confirming brucellosis in both animals and humans, as previously reported (Mol et al. 2020).In this study, we used species-specific primer targeting the IS711 locus in the genome to confirm the presence of B. abortus and B. melitensis in human samples and goat samples.Other researchers have also used the similar set of primers and obtained the similar size of amplified fragment from the genome of Brucella species in PCR assay; thus this study supports the previous finding (Ali et al. 2014).Furthermore, the accuracy and reliability of the species specific primers for detecting the Brucella species in blood sera were also reported by Ahmad and Rahman (2019).
In this study, we are also reporting the PCR-based amplification of 16S rRNA genes and Sanger sequencing for the characterisation of Brucella spp. in goats and shepherds in the Swat region of Khyber Pakhtunkhwa.Positive samples of brucellosis were subjected to Sanger sequencing and the sequences obtained were blasted for similarity search.The E value, percent identity and the query cover of each sequence revealed its similarity with the different species of Brucella (B.melitensis, B. abortus) deposited in the Gene bank, which showed that they shared homology from common ancestry and similar structure (Ntirandekura et al. 2020).The phylogenetic tree of the 16S rRNA gene sequences showed that Brucella spp.from the Swat region were grouped into two clades and two branches, all closer to B. melitensis and B. abortus reported from India, China, Philippines and the US.However, they also showed divergence from other species isolated in India, China and the US.The previous studies reported the identification and characterisation of Brucella in the region by targeting other genes for sequencing (Hoffman et al. 2016;Mathew et al. 2017;Mugizi et al. 2015).
As per the authors knowledge, this is the first research study that reports the identification and characterisation of Brucella species in the Swat region of Khyber Pakhtunkhwa, Pakistan.
Furthermore, the grouping of Brucella spp.into two clades and two branches reported in Swat, representing the existence of genetic heterogeneity among the species in the targeted area.
On the other hand, the phylogenetic tree further revealed that Brucella species circulating in district Swat were closer to B. melitensis and B. abortus reported from India, China, Philippines and the US showing the existence of the possible epidemiological linkage among the Brucella species.

Conclusion
This study concluded that Brucella species were circulating in livestock and shepherds in this region.Brucella abortus was the most prevalent species in both shepherds and animals in the study area.Females of goats as well as humans were more vulnerable to brucellosis.Rose Bengal precipitation test and SPAT can be used for the rapid detection and early diagnosis of brucellosis as their sensitivity and specificity were confirmed by PCR.Furthermore, the Brucella spp.identified in Swat were phylogenetically related to the Brucella spp. reported from India, China, Philippines and the US, thus showed possible epidemiological linkages between Brucella spp.
Brucellosis is a major threat to public health especially in developing countries including Pakistan.This study reveals the characterisation of Brucella species affecting humans and goats in the Swat region of Khyber Pakhtunkhwa, Pakistan.Blood samples were collected from shepherds and goats and analysed by Rose Bengal precipitation test (RBPT), standard plate agglutination test (SPAT), polymerase chain reaction (PCR) and Sanger sequencing of 16S rRNA gene.The findings of the study indicated 24% (36/150) and 11.3% (17/150) positivity for Brucella abortus and Brucella melitensis, respectively, in human samples.In samples of goats, 26.66% (40/150) were positive for B. abortus and 16.66% (25/150) samples were positive B. melitensis by SPAT.The species-specific PCR confirmed B. abortus in 24% (36/150) of human samples and 26.66% (17/150) of goat samples by targeting the IS711 locus.The remaining seropositive samples were confirmed as B. melitensis using IS711 M speciesspecific primer.The sequences of the amplified fragments of the 16S rRNA gene were blasted, and phylogenetic analysis revealed that Brucella species circulating in the Swat district were closely related to B. melitensis and B. abortus reported from India, China, Philippines, and the United States (US) showing the existence of the possible epidemiological linkage among the The outbreak of migratory goat's brucellosis in the Swat ecosystem of Khyber Pakhtunkhwa Read online: Scan this QR code with your smart phone or mobile device to read online.http://www.ojvr.orgOpen Access

FIGURE 1 :
FIGURE 1: Demography of the Swat district.

FIGURE 2 :
FIGURE 2: Polymerase chain reaction amplified fragments of gene BSCP31 and IS711 locus in Brucella genus, Brucella abortus and Brucella melitensis with an amplicon size of 285 bp, 498 bp and 731 bp, respectively.Lane: 1 to 10 positive serum, lane NC: negative control, lane M: DNA ladder.
Microsoft Excel spreadsheet (2016 Version).The data were analysed using the Statistical Package for Social Sciences (SPSS) software version 23 (IBM SPSS version 23).Chi-square test was performed to check the statistical significance between categorical variables and prevalence of brucellosis.The results were considered statistically significant if p ≤ 0.05 at 95% confidence interval.

TABLE 1 :
Area-wise seroprevalence of brucellosis in shepherds.

melitensis No. of positive samples % positive samples No. of positive samples % positive samples
Note: Chi-square test: RBPT p ≥ 0.05 = 3.7, SPAT p ≥ 0.05 = 4.2.There is no significant difference in area-wise seroprevalence of brucellosis as p ≥ 0.05 for both RBPT and SPAT in area-wise seroprevalence of brucellosis in shepherds.RBPT, Rose Bengal precipitation test; SPAT, standard plate agglutination test; No., number.

TABLE 2 :
Age-wise seroprevalence of brucellosis in shepherds.The data inTable 4 also presented the areawise seroprevalence of B. abortus and B. melitensis on SPAT.The highest prevalence rate of B. abortus was observed in Tehsil Bahrain and Tehsil Barikot with 35% (7/20) prevalence each, followed by Tehsil Kabal and Tehsil Khwazakhela with 30% (6/20) prevalence each.The lowest prevalence rate of B. abortus was found in Tehsil Babozai with 13.3% Note: Chi-square test: RBPT p ≥ 0.05 = 2.6, SPAT p ≥ 0.05 = 4.6.There is no significant difference in age-wise seroprevalence of brucellosis as p ≥ 0.05 for both RBPT and SPAT in age-wise seroprevalence of brucellosis in shepherds.RBPT, Rose Bengal precipitation test; SPAT, standard plate agglutination test; No., number.(4/30) prevalence.The prevalence rate of B. melitensis was highest in Tehsil Matta and Bahrain with 25% (5/20) prevalence each, while the lowest prevalence rate of B. melitensis was observed in Tehsil Babozai with 10% (3/30) prevalence.The chi-square test value was greater than p ≤ 0.05, indicating no significant difference in the area-wise seroprevalence of brucellosis in goats.

Table 5
presents data on gender-wise seroprevalence of brucellosis in goat samples using RBPT and SPAT.The prevalence rate was found to be higher in female goats (55/107, 51.4%) than in male goats (10/43, 23.2%).The table also shows the gender-wise seroprevalence of B. abortus and B. melitensis in goats using SPAT.

TABLE 4 :
Area-wise seroprevalence of brucellosis in goats.
Phylogenetic analysis of Brucella spp.from Swat region in relation with other reported species of Brucella based on 16S rRNA gene sequences by maximum likelihood method.
(Corbel 1989;Musallam et al. 2016esults of previous researchers provide evidence that B. abortus is more prevalent in humans than B. melitensis in various regions around the world(Corbel 1989;Musallam et al. 2016).