Evidence of Brucellosis in Hospitalized Patients of Selected Districts of Punjab, Pakistan

Abstract

Brucellosis is a bacterial zoonosis worldwide and a potential health risk in Pakistan due to socio-economic reasons and lack of awareness, veterinary health and modern dairy-processing facilities. The aim of this study was to determine the burden of brucellosis in hospitalized patients of selected districts of Punjab, Pakistan. To this end, a total of 280 serum samples were collected from suspected patients in districts Lahore, Okara and Khushab between 2016 and 2018. Anti-Brucella antibodies were detected in 89 (31.8%) sera by Rose Bengal plate test (RBPT), and Brucella abortus DNA was amplified by polymerase chain reaction (PCR) in 42 (15%) samples. In univariable analysis, the seroprevalence varied significantly (p < 0.05) between districts with Lahore indicating 41/101 (40.6%) Odds ratio (OR) = 53.98 (7.22–403.67; 95% CI), Okara 47/99 (47.5%) OR = 71.4 (9.55–533.65; 95% CI) and Khushab 1/80 (1.3%) as reference. Similarly, exposure to aborted materials and a history of blood transfusion did associate significantly (p < 0.05), posing OR = 2.91 (1.58–5.36; 95% CI) and OR = 4.48 (2.56–7.84; 95% CI) risk. However, consumption of raw milk and history of blood transfusion did associate significantly (p < 0.05) in multivariable analysis indicating OR = 1.91 (1.09–3.34; 95% CI) and OR = 4.92 (2.76–8.75; 95% CI), respectively. The association of history of blood transfusion as a brucellosis risk factor underscores the need to create brucellosis awareness programs both at public and institutional levels in the medical and veterinary fields, as well as facilities and capacity building to delve deeper into the epidemiology of brucellosis in the country. Particular attention should be paid to chronically infected patients. Therefore, laboratory modernization, standardization and validation of the diagnostic tests, and staff trainings are required. Finally, pasteurization of the milk is recommended before consumption.

1. Introduction

Brucellosis is an important bacterial zoonosis of major public health and global economic importance [1]. At least 500,000 cases are reported annually worldwide, but actual cases could be much higher due to a lack of reporting capabilities, particularly in developing countries [2,3]. Brucellosis is caused by Gram-negative intracellular, non-motile, non-spore-forming and coccobacilli bacteria of the genus Brucella. Of them, Brucella abortus (which causes disease in cattle and buffaloes), B. melitensis (sheep and goats), B. suis (pigs) and B. canis (dogs) are potentially zoonotic. The main route of transmission is through accidental exposure (e.g., direct contact with infected animals or indirectly via a contaminated environment) or through the consumption of contaminated unpasteurized milk. Person-to-person transmission through sexual contact, blood transfusion and breastfeeding is rare [4]. It poses a serious health risk at the workplace for veterinarians, slaughterhouse workers, laboratory workers and farmers [5]. In humans, it is usually chronic, but in acute cases, it manifests itself as an undulant fever with nonspecific symptoms, e.g., myalgia, arthralgia and occasional abortions, or in chronic cases such as cardiovascular and central nervous system malaise [6]. In animals, it shows characteristic abortion storm, especially in the last trimester, followed by the retention of the fetal membranes and fever. However, it may be present sub-clinically in animals since carrier and vaccinated animals rarely abort and remain undiagnosed. Brucellosis usually responds well to antimicrobials in both humans and animals, but infected animals remain lifelong carriers, so culling is recommended. There is no licensed vaccine for humans so far.

Brucellosis is mainly diagnosed by serology, e.g., Rose Bengal plate test (RBPT), serum agglutination test (SAT), complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) with addition to the milk ring test (MRT) in lactating animals. Each test has its own applications; however, isolation of the brucellae remains the diagnostic gold standard, but it is not always the preferred choice due to the intracellular-living nature of brucellae, biohazard and time-consuming procedure. Moreover, submitted samples are not always feasible to perform isolation, and this procedure requires advanced biosecurity measures and highly skilled personnel. Molecular testing, e.g., polymerase chain reactions (PCR), are performed to detect/differentiate Brucella-DNA in the samples at the species level or even between vaccine or field strains [7].

Pakistan is a South-Asian country with an agriculture-based economy where the livestock subsector plays an integral role (61.89%) in the agriculture sector and contributes 14.04% of the gross domestic product (GDP) [8]. Punjab province has Pakistan’s largest population in terms of people and livestock and economy, mainly agriculture, which contributed ≥60.58% to the national GDP in the period 2019–20 [9]. There is a strong animal–human relationship in the province as owning livestock is seen as a symbol of financial stability and traditional honor. However, due to the lack of modern veterinary health services, especially in remote rural areas, infectious diseases, mainly of zoonotic origin, continue to pose a serious threat for animal production and public health security [2,10]. Brucellosis is considered endemic in Pakistan and is frequently reported in animals and sporadically in humans [2]. Despite these reports, the public understanding regarding brucellosis still remains low [11]. Keeping in view this background, the authors were interested in investigating and publishing the epidemiology of brucellosis in the hospitalized patients in three selected districts of Punjab, Pakistan.

2. Materials and Methods

2.1. Study Area

Lahore is located between latitude 31.582045 and latitude 74.329376, bordered by districts Sheikhupura to the north and west and Nankana Sahib to the west, Wagah border to the east and Kasur to the south. Ravi river flows to the north of Lahore. With 11,126,285 (2017) residents, it is the most populated, developed and industrialized city in the province. Large herds of cattle and buffaloes prevail on the banks of Ravi and in the rural areas of the district. Moreover, to meet the daily milk demand in the district, large volumes of milk are transported from other districts of Punjab on a daily basis.

Okara is located between latitude 30.81029 and longitude 73.45155, bordered by districts Kasur to the north east, Faisalabad and Nankana Sahib to the north, Sahiwal to the west, Pakpattan to the southwest, and Bahawalnagar to the south. Located between the Ravi and Sutlej rivers, Okara is famous for its fertile land and green fields and is one of Pakistan’s largest milk producing districts with many private and institutional livestock farms. It is also known for its Sahiwal cattle and the Nili Ravi water buffalo breeds. Veterinary facilities, particularly in rural areas, are still largely outdated.

Khushab is located between latitude 32.29667 and longitude 72.3525, bordered by districts Sargodha to the east, Jhelum to the northeast, Chakwal and Talagang to the north, Mianwali to the west, Bhakkar to the southwest and Jhang to the southeast. Located on the bank of the river Jhelum, Khushab has many private and institutional livestock farms. Thal desert also touches this district, where livestock rearing and agriculture remain the main economic sources of the population.

2.2. Sample Collection

From August 2016 to October 2018, a total of 280 sera were collected from clinically ill patients suspected of having brucellosis presented at hospitals in the districts Lahore (n = 101), Okara (n = 99) and Khushab (n = 80) (Figure 1). The suspicion criteria included evidence of persistent fever, fatigue, chills, headaches, sweating, weight loss, joint pain, burning feet and history of abortion (in females). Blood samples measuring 3 mL were each collected into gel-based serum separator tubes, and serum was separated following centrifugation at 3000× g for 5 min. Separated sera were labelled, transported to the Epidemiology and Public Health laboratory, Department of Clinical Sciences, College of Veterinary and Animal Sciences, Jhang, Pakistan and stored at −20 °C until further analysis. The epidemiological data of the participants were collected on a prepared proforma. The study protocol was approved by the bioethics committee of the College of Veterinary and Animal Science, sub-campus University of Veterinary and Animal Science, Lahore, Jhang, Pakistan with number CS.798, dated 9 October 2018.

2.3. Serological Investigation

The samples were subjected to brucellosis screening by Rose Bengal plate test (RBPT) using Rose Bengal antigen (Veterinary Research Institute (VRI), Lahore, Pakistan) as per manufacturer’s instructions [12]. Briefly, 30 µL amounts of both the Rose Bengal antigen and the serum were gently mixed and agitated on white ceramic plate at room temperature for four minutes in the presence of bovine bacterial-positive and -negative control sera obtained from the Friedrich-Loeffler-Institut (FLI), Jena, Germany. An agglutination within four minutes was considered a positive reaction.

2.4. Molecular Screening of the Samples

DNA was extracted from 200 μL of the serum samples using FavorPrep^™ Blood Genomic DNA Extraction Mini Kit (Favorgen, Pingtung, Taiwan) according to the manufacturer’s instructions. The DNA concentration was then determined photometrically using a Nanodrop ND-2000 UV-Vis spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA). Detection of Brucella DNA was performed using a multiplex real-time PCR based detection system using genus specific cell surface salt extractable bcsp31 kDa protein gene primers, i.e., 5′-GCTCGGTTGCCAATATCAATGC-3′ as forward primer and 5-′GGGTAAAGCGTCGCCAGAAG-3′ as reverse primer together with a genus specific probe 5′6-FAM-ACTCCAGAGCGCCCGACTTGATCG-BHQ-3′ and species-specific primers for B. abortus and B. melitensis as previously described (

Table 1. Primer and probes sequence for real-time PCR.

Differentiation (IS711)	Primers and Probes	Sequence
B. abortus	Forward	5′-GCGGCTTTTCTATCACGGTATTC-3′
	Reverse	5′-CATGCGCTATGATCTGGTTACG-3′
		HEX-CGCTCATGCTCGCCAGACTTCAATG-BHQ
B. melitensis	Forward	5′-AACAAGCGGCACCCCTAAAA-3′
	Reverse	5′-CATGCGCTATGATCTGGTTACG-3′
		Cy5-CAGGAGTGTTTCGGCTCAGAATAATCCACA-BHQ

) [13,14]. Brucella abortus (ATCC 23448) and B. melitensis (ATCC 23456) were included in the run as positive controls and nuclease-free water as a non-template control (NTC).

The PCR conditions were as follows: decontamination at 50 °C for 2 min, 1 cycle; initial denaturation at 95 °C for 10 min, 1 cycle; denaturation at 95 °C for 25 s and 57 °C for 1 min to anneal and elongate the primers, both for 50 cycles each. A cycle threshold (Ct) ≤ 39 was considered as positive [14]. The master mix contained 12.5 μL of TaqMan™ Universal Master Mix (Applied Bio-systems, Waltham, MA, USA), 0.75 μL of each (0.3 μM) primer and 0.25 μL of each (0.1 μM) probe. A total of 2 μL of bacterial DNA as template and nuclease-free water were added to give a total reaction volume of 25 μL for each sample.

2.5. Statistical Analysis

In order to find out the connection between potentially associated risk factors and the occurrence of brucellosis, all predictive variables were first tested with univariable analysis at a significance level of p < 0.25 [15]. Afterwards, these predictors—which had produced p < 0.25—were included in the multivariable logistic regression model. Subsequently, all non-significant variables were removed one by one with a stepwise backward selection approach using drop1 function, and the final model contained only significant variables. The quality of the model was assessed using R² Tjur. The final model was fitted with glm function with family = binomial while the odds ratios (ORs) together with 95% confidence intervals (CI) were calculated with exp function. A map was created with ArcGIS (ESRI, Redlands, CA, USA) for spatial representation of the sampling points in the country.

3. Results

Out of 280 investigated serum samples, 89 (31.8%) were positive for Brucella antibodies by RBPT. The seroprevalence of brucellosis in Okara, Lahore and Khushab was 47.5% (47/99), 40.6% (41/101) and 1.3% (1/80), respectively. A total of 42 (15%) (21 seropositive and 21 seronegative) samples amplified B. abortus DNA, while B. melitensis did not amplify in either seropositive or seronegative samples (

Table 2. Seroprevalence of brucellosis and molecular detection of Brucella spp. DNA.

Districts	Serology	Real-Time PCR Positive	Real-Time PCR Negative	Total
Okara	RBPT +ve	13	34	47
	RBPT –ve	14	38	52
	Sub-Total	27	72	99
Lahore	RBPT +ve	7	34	41
	RBPT –ve	7	53	60
	Sub-Total	14	87	101
Khushab	RBPT +ve	1	0	1
	RBPT –ve	0	79	79
	Sub-Total	1	79	80
Total		42	238	280

; Figure 1).

Univariable analysis showed a significantly variable (p < 0.001) seroprevalence across the districts with a higher Odds ratio (OR) of 71.4 (9.55–533.65; 95% CI) and 53.98 (7.22–403.67; 95% CI) in Okara and Lahore, respectively (Table 2). The sex of the participants did not associate significantly (p = 0.639) with the seropositivity although females showed a higher seroprevalence (35.1% vs. 31.3%) (

Table 3. Univariable analysis of risk-associated factors of brucellosis.

Variable	Category	Pos./Tested	Prevalence % (95% CI)	OR	CI	p-Value
District	Khushab	1/80	1.3 (0–6.8)	Ref	-	<0.001 *
	Lahore	41/101	40.6 (30.9–50.8)	53.98	7.22–403.67
	Okara	47/99	47.5 (37.3–57.8)	71.4	9.55–533.65
Sex	Female	13/37	35.1 (20.2–52.5)	1.19	0.58–2.46	0.639
	Male	76/243	31.3 (25.5–37.5)	Ref	-
Age	≤30	48/169	28.4 (21.7–35.8)	Ref	-	0.306
	31–50	35/93	37.6 (27.8–48.3)	1.52	0.89–2.60
	≥51	6/18	33.3 (13.3–59)	1.26	0.45–3.55
Residence	Rural	48/143	33.6 (25.9–41.9)	1.18	0.72–1.96	0.513
	Urban	41/137	29.9 (22.4–38.3)	Ref	-
Recent abortion history (in the female spouse)	No	84/270	31.1 (25.6–37)	Ref	-	0.218
	Yes	5/10	50 (18.7–81.3)	2.21	0.63–7.85
Animals in contact	Single Sp.	9/48	18.8 (8.9–32.6)	Ref	-	0.061
	Multiple Sp.	26/65	40 (28–52.9)	2.89	1.20–6.95
	No contact	54/167	32.3 (25.3–40)	2.07	0.94–4.58
Contact with aborted materials	No	61/226	26.9 (21.3–33.3)	Ref	-	0.001 *
	Yes	28/54	51.9 (37.8–65.7)	2.91	1.58–5.36
Raw milk consumption	No	51/180	28.3 (21.9–35.5)	Ref	-	0.097
	Yes	38/100	38 (28.5–48.3)	1.55	0.93–2.60
History of blood transfusion	No	46/204	22.6 (17–28.9)	Ref	-	<0.001 *
	Yes	43/76	56.6 (67.9–67.9)	4.48	2.56–7.84
Total		89/280	31.8 (26.4–37.6)

* significant association (p < 0.05).

). The age of the participants also varied non-significantly (p = 0.306), whereby the 31–50 years age group showed higher seroprevalence, i.e., 37.6% (35/93), as compared to the ≥51 years and ≤30 years groups, which showed 33.3% (6/18) and 28.4% (48/169), respectively. Residing in an urban or rural area did not represent a significant association (p = 0.513), but rural-population samples showed a higher seroprevalence, i.e., 33.6% (48/143), as compared to the urban samples, i.e., 29.9% (41/137). Out of 37 investigated females in this study, 10 had a recent abortion history, whereas only 5 (50%) were seropositive, which did not show significant association overall (p = 0.218). Contact with animals did not associate significantly (p = 0.061) with the seropositivity of the infection. Contact with aborted material was significantly associated (p = 0.001) with the seropositivity, where high seropositivity 51.9% (28/54) was detected in participants who had contacted aborted materials with higher risk (OR = 2.91: 1.58–5.36; 95% CI) compared to the ones who did not, i.e., 26.9% (61/226). However, this variable did not associate significantly in multivariate analysis.

The consumption of raw milk showed higher seroprevalence in the exposed group 38% (38/100) as compared to the unexposed counterpart, i.e., 28.3% (51/180); however, this did not associate significantly (p = 0.097) in univariable analysis but did associate (p = 0.024) with higher risk (OR = 1.91: 1.09–3.34; 95% CI) with the seropositivity of the infection in multivariable analysis (

Table 4. Multivariable analysis.

Variable	Exposure	Comparison	OR	95% CI	p-Value
Raw milk consumption	Yes	No	1.91	1.09–3.34	0.024
History of blood transfusion	Yes	No	4.92	2.76–8.75	<0.01

Model Fit = Nagelkerke R Square: 0.158; Hosmer and Lemeshow Test: χ² = 3.938, 2df, p = 0.140.

). The history of blood transfusion among participants did associate significantly (p < 0.001) along with higher risks, i.e., OR = 4.48: 2.56–7.84; 95% CI, and OR = 4.92: 2.76–8.75; 95% CI, in univariable and multivariable analysis, respectively (Table 3 and Table 4).

4. Discussion

Brucellosis is a devastating disease in the ruminants of Pakistan, which poses zoonotic threat to the exposed human population [2]. This exposure may include direct contact with infected animals and indirect contact via contaminated environment (e.g., contaminated soil, aborted fetal materials) or fomites (e.g., contaminated milk, syringes, etc.). Serology is the main diagnostic tool where PCR is performed to confirm the etiology. Brucellosis is an established professional health hazard in Pakistan [16].

Based on the location, Lahore and Okara showed the highest seroprevalence rates at 40.6% and 47.5%, respectively, with a significant variability (p < 0.001). Previously, human brucellosis was reported from Lahore (8.3%) and Kasur (3.1%) [16], this study proved that individuals with occupational exposure to farm animals and their products are at a high risk of acquiring brucellosis in an area where farm animal brucellosis is endemic. To our best, no report existed from districts Okara and Khushab. However, brucellosis reports from a variety of animals did exist in these districts, thus indicating a high human risk [17,18,19,20,21,22]. Variability in the location might influence the seroprevalence outcome depending upon the endemicity of disease in the selected areas [23,24].

Contact with livestock was significantly associated with the seropositivity in the univariable analysis. Livestock exposure has previously been associated significantly [25,26,27] and not significantly with the seropositivity of brucellosis [28]. The results may vary based upon the health status of the animals in contact with the participants as carrier animals, or undiagnosed infected animals might be shedding the bacteria in the environment, posing a zoonotic risk. Even healthy animals may shed bacteria in milk. This poses a serious risk to the occupationally exposed population (e.g., working close to livestock) in Pakistan [16]. Contact with aborted material did associate significantly (p = 0.001) with the seropositivity, in our study. This is consistent with previous studies conducted in Pakistan which showed that the exposure to the aborted material should be considered a zoonotic risk [16,28].

Raw milk consumption showed a higher risk of seropositivity in the exposed group. It is already reported as a risk factor for zoonotic transmission in the country [25,26,27,28,29]. The main reason is that the raw milk, especially fresh bovine colostrum, is considered a healthy and nutritive food. Although infected dams do respond well to the antimicrobials, they may still shed bacteria in the milk and body secretions because of the intracellular lifestyle of brucellae. Hence, precautions should be taken in milk production/consumption if the animals are suspected for brucellosis. Moreover, ≥98% of the milk produced in Pakistan is marketed as raw (the milk transport chain is largely not up to the mark), which can pose an additional risk in case of contamination [2].

Blood transfusion was associated significantly with the seropositivity with higher risk (OR = 4.48). Blood transfusion is a well-known practice, although ≤1% of the population donates blood [30]. Before the transfusion, blood is tested for five transfusion-transmitted infections (TTIs), i.e., Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), malaria and syphilis [31]. According to standard guidelines for blood transfusion, brucellosis has not been widely associated with blood-borne transmission as febrile patients with acute bacteremia are rejected as blood donors [4,6,32]. To the best of our knowledge, there exists no reports investigating the transmission of brucellosis by blood transfusion in Pakistan; however, there are reports emanating from neighboring countries [23,24]. Reports even exist, albeit rare, in transplant patients, with cases sometimes being fatal [33,34,35,36]. In this scenario, chronic patients could pose a significant risk.

The results of this study showed that B. abortus DNA was circulating in participants’ sera, with risk factors associated with location, exposure to aborted materials, raw milk consumption and blood transfusions. Although, B. abortus is regarded as the main etiology of brucellosis in animals and humans in Pakistan [27,28,37,38], reports of B. melitensis have increased in the recent past [38,39,40,41]. Based on the animal husbandry practices, socio-economic situation and animal–human relationships in Pakistan, it could be assumed that these brucellae could be of zoonotic origin [2]. Since RBPT is the most common brucellosis screening test in Pakistan, whose antigen consists of inactivated B. abortus (S-99) in Rose Bengal stain at acidic pH and reacts to both anti-B. abortus and anti-B. melitensis antibodies [42], further confirmation is required to differentiate the etiology at species-level, e.g., by species-specific primers using PCR, the later would require standardization and validation. Furthermore, the results may be affected by the DNA extraction methods as well as the timing of sample collection from the animal/participant [2]. PCR and other molecular biology techniques require high-quality DNA, which is only possible from fresh bacterial cultures, which again, in the case of Brucella, is hazardous, skilled and time-consuming. Therefore, special attention needs to be paid to the development of such facilities and capacity building, for which raising awareness of people at both public and institutional levels would be helpful.

5. Conclusions

Brucellosis remains a potential zoonotic hazard in Pakistan. A large part of the population is still unaware of brucellosis. Geographical location, exposure to aborted material, and raw milk consumption were identified as risk factors in this study. Moreover, although rather uncommon, a history of blood transfusion was also identified as a risk factor. In this scenario, it becomes really important to create awareness programs, facilities and capacity buildings to combat this disease and gain acceptance of preventive measures in the country. The isolation of the etiology will aid in understanding the molecular epidemiology of the disease and the relationships between outbreaks. The modernization of laboratories and training of staff are therefore required. Finally, pasteurization of the milk is recommended before consumption.