Search

With over 200 zoonotic diseases and the emergence of antimicrobial-resistant (AMR) microbes, a substantial threat looms over public health. If we are unable to cope with AMR, it suggests a potential of 10 million global fatalities by 2050. The evolving landscape of AMR transcends geographic boundaries and species, necessitating collaborative and multidisciplinary efforts. The interplay between humans, animals, and the environment highlights the profound significance of One Health in addressing these interconnected challenges. The imperative of multidisciplinary efforts to safeguard the health and well-being of our shared world, comprised of humans, animals, and the environment, can pave the way toward success. The role of surveillance, education, and public awareness in combating AMR and fostering responsible antimicrobial use is inevitable in addressing this global concern. Key Words: Antimicrobial resistance, Zoonoses, One health

Tick infestation poses an important challenge to livestock in Pakistan. Farm management practices and environmental variables can influence tick infestation prevalence in animals. To this end, a cross-sectional survey of 96 farms in four different geographical districts (24 farms from each district) was conducted in Punjab, Pakistan, between October 2021 and January 2022. An epidemiological questionnaire was designed focusing on farm management practices and their impact on tick infestations at these farms. Data were collected via in-person interviews. Regional and farm-specific variables’ associations were evaluated using Pearson’s chi-square test and Fischer’s exact test, respectively. A multivariable logistic regression model was used to identify significant risk factors. This study identified that using soft bedding materials, e.g., wheat straw, leaf litter or plain soil posed a significant risk of tick infestation. Additionally, the absence of quarantine measures, open sheds and inadequate drainage were found as contributing factors in univariable analysis. Higher tick prevalence in the hotter seasons highlighted the influence of Punjab’s extreme weather on tick infestation. Despite regular veterinary visits and the use of acaricidal drugs, the prevalence of tick infestation at these farms suggested potential drug resistance in the ticks. The study recommended establishing quarantine practices, improving farms’ drainage systems and bedding and using a combination of chemical and traditional remedies to tackle drug resistance in ticks. Education and awareness programs on tick-borne diseases and control measures are advocated to reduce the tick infestation burden on animals. Further research on longitudinal studies to better understand tick population dynamics and develop effective acaricides is encouraged. This called for collaborative control efforts among farmers, veterinarians and research institutions.

Peste des petits ruminants (PPR) is an extremely transmissible viral disease caused by the PPR virus that impacts domestic small ruminants, namely sheep and goats. This study aimed to employ a methodical approach to evaluate the regional occurrence of PPR in small ruminants in Pakistan and the contributing factors that influence its prevalence. A thorough search was performed in various databases to identify published research articles between January 2004 and August 2023 on PPR in small ruminants in Pakistan. Articles were chosen based on specific inclusion and exclusion criteria. A total of 25 articles were selected from 1275 studies gathered from different databases. The overall pooled prevalence in Pakistan was calculated to be 51% (95% CI: 42–60), with heterogeneity I2 = 100%, τ2 = 0.0495, and p = 0. The data were summarized based on the division into five regions: Punjab, Baluchistan, KPK, Sindh, and GB and AJK. Among these, the pooled prevalence of PPR in Sindh was 61% (95% CI: 46–75), I2 = 100%, τ2 = 0.0485, and p = 0, while in KPK, it was 44% (95% CI: 26–63), I2 = 99%, τ2 = 0.0506, and p < 0.01. However, the prevalence of PPR in Baluchistan and Punjab was almost the same. Raising awareness, proper surveillance, and application of appropriate quarantine measures interprovincially and across borders must be maintained to contain the disease.

Abortion is one of the leading causes of economic losses in the livestock industry worldwide. Chlamydia abortus, Coxiella burnetii, and Brucella spp. are the leading cause of late fetal loss in small ruminants. This study determined the seroprevalence of these agents in small ruminants in district Jhang. A total of 385 serum samples were taken from the sheep and goats from different flocks with a history of abortion and subjected to i-ELISA. Further, samples were analysed for liver enzymes and total serum protein using a semi-automated chemistry analyzer. The result of indirect commercial ELISA showed 13.0, 4.2 and 11.2% prevalence for Coxiella burnetii, Chlamydia abortus, and Brucella spp., respectively. Values of different serum parameters (ALT, AST, and total protein) of seropositive animals were also determined. There was a significant rise in AST and ALT values of infected animals (p ≤ 0.05). Total protein decreased for all three infections, but a significant drop was noted in Brucella positive sheep serum samples. Various risk factors were studied. Binary logistic regression proved a significant role of ticks for coxiellosis and brucellosis. Age, parity, and species did not impact the prevalence of diseases studied.

Anaplasma (A.) ovis is the most important cause of anaplasmosis in small ruminants. The current study was planned to estimate the molecular prevalence, risk factors, and phylogenetic analysis of A. ovis infection in sheep and goats from different agro-climatic regions of Central and Southern Punjab, Pakistan. A total of 400 jugular blood samples were collected from asymptomatic goats (n = 200) and sheep (n = 200) from the Jhang and Dera Ghazi Khan districts from January 2021 to February, 2023. Two hundred blood samples were collected from each district. Ten union councils (UC) were randomly chosen from each district, and 20 samples were collected from each UC based on the multistage cluster sampling technique. The samples were analyzed with PCR targeting the major surface protein (msp4) gene of A. ovis. The overall molecular prevalence of anaplasmosis was 57.5%. The disease occurrence was higher in Dera Ghazi Khan (61.5%) than in the Jhang district (53.5%). Infection positivity was greater in goats (65.5%) than in sheep (49.5%). Multivariate logistic regression analysis indicated that host species [sheep; Odds Ratio (OR) = 3.212; p = 0.000, Confidence Interval (CI) = 1.968–5.242], age (adult; OR = 2.606; p = 0.003, CI = 1.398–4.858), and acaricide use (never; OR = 13.671; p = 0.000, CI = 6.414–26.283) were significantly higher risk for A. ovis in small ruminants (p< 0.05; OR > 1). The sequencing and phylogenetic analysis of four representative isolates in the current study (Genbank numbers; Goats: OQ302202, OQ302203; Sheep: OQ319592, OQ319593) revealed novel strains of A. ovis with 97–100% similarity from different countries. The msp4-based goat isolates showed greater genetic diversity, while sheep genotypes showed homology with isolates from Italy, Spain, Hungary, Cyprus, Spain, Iran, and China. The current surveillance study will help in devising prevention and control strategies regarding anaplasmosis in small ruminants. However, there is a need for further study on the clinicopathological and vector competence aspects of these genotypes.

Extended-spectrum β-lactamases (ESBL) give rise to resistance against penicillin and cephalosporin antibiotics in multiple bacterial species. The present study was conducted to map genetic determinants and related attributes of ESBL-producing bacteria in three wild aquatic bird species and chickens at the “Trimmu Barrage” in district Jhang, Punjab province, Pakistan. To study the prevalence of ESBL-producing bacteria, a total of 280 representative samples were collected from wild bird species; cattle egrets (Bubulcus ibis), little egrets (Egretta garzetta) and common teals (Anas crecca) as well as from indigenous chickens (Gallus gallus domesticus) originating from a local wet market. The isolates were confirmed as ESBL producers using a double disc synergy test (DDST) and bacterial species were identified using API-20E and 20NE strips. A polymerase chain reaction (PCR) was used to detect ESBL genetic determinants and for genus identification via 16S rRNA gene amplification. A phenotypic antimicrobial susceptibility test was performed for ESBL-producing isolates against 12 clinically relevant antibiotics using the Kirby–Bauer disk diffusion susceptibility test. A phylogenetic tree was constructed for the sequence data obtained in this study and comparative sequence data obtained from GenBank. The overall prevalence of ESBL-producing bacteria was 34.64% (97/280). The highest percentage (44.28%; 31/70) of ESBL-producing bacteria was recovered from chickens (Gallus gallus domesticus), followed by little egrets (Egretta garzetta) (41.43%; 29/70), common teal (Anas crecca) (28.57%; 20/70) and cattle egrets (Bubulcus ibis) (24.28%; 17/70). Five different ESBL-producing bacteria were identified biochemically and confirmed via 16S rRNA gene sequencing, which included Escherichia coli (72; 74.23%), Enterobacter cloacae (11; 11.34%), Klebsiella pneumoniae (8; 8.25%), Salmonella enterica (4; 4.12%) and Pseudomonas aeruginosa (2; 2.06%). Based on PCR, the frequency of obtained ESBL genes in 97 isolates was blaCTX-M (51.55%), blaTEM (20.62%), blaOXA (6.18%) and blaSHV (2.06%). In addition, gene combinations blaCTX-M + blaTEM, blaTEM + blaOXA and blaCTX-M + blaSHV were also detected in 16.49%, 2.06% and 1.03% of isolates, respectively. The ESBL gene variation was significant (p = 0.02) in different bacterial species while non-significant in relation to different bird species (p = 0.85). Phylogenetic analysis of amino acid sequence data confirmed the existence of CTX-M-15 and TEM betalactamases. The average susceptibility of the antibiotics panel used was lowest for both Klebsiella pneumoniae (62.5% ± 24.42) and Salmonella enterica (62.5% ± 31.08) as compared to Enterobacter cloacae (65.90% ± 21.62), Pseudomonas aeruginosa (70.83% ± 33.42) and Escherichia coli (73.83% ± 26.19). This study provides insight into the role of aquatic wild birds as reservoirs of ESBL-producing bacteria at Trimmu Barrage, Punjab, Pakistan. Hence, active bio-surveillance and environment preservation actions are necessitated to curb antimicrobial resistance.

The increasing incidence of extended-spectrum β-lactamase (ESBL)-producing Escherichia (E.) coli in backyard chicken farming in Pakistan is of serious concern. This study aimed to assess the prevalence, antimicrobial resistance patterns and risk factors associated with ESBL avian pathogenic E. coli (APEC) isolated from backyard chickens in the Jhang district, Punjab, Pakistan. In total, 320 cloacal swabs were collected from four breeds of backyard chicken (Aseel, Golden, Misri and Necked Neck). ESBL E. coli were phenotypically identified using double disc synergy test (DDST) and corresponding genes were confirmed by multiplex polymerase chain reaction (mPCR). Out of the 320 samples, 164 (51.3%) were confirmed as E. coli, while 74 (45.1%) were characterized as ESBL E. coli. The frequency of isolation of ESBL E. coli was highest in Aseel chickens (35.1%). Of the 164 confirmed E. coli, 95.1%, 78.6%, 76.8%, 71.3%, 70.1%, 68.9%, 60.4% and 57.3% were resistant against tylosin, doxycycline, cefotaxime, enrofloxacin, colistin, trimethoprim/sulfamethoxazole, chloramphenicol and gentamicin, respectively. The ESBL gene types detected and their corresponding proportions were blaCTX-M (54.1 %, 40/74), blaTEM, (12.2%, 9/74) and co-existence (blaCTX-M and blaTEM) were shown in 33.8% (25/74). The blaCTX-M gene sequence showed homology to blaCTX-M-15 from clinical isolates. The mean multiple antibiotic resistance index (MARI) was found to be higher among ESBL E. coli (0.25) when compared to non-ESBL E. coli (0.17). Both free-range husbandry management system (p = 0.02, OR: 30.00, 95% CI = 1.47–611.79) and high antimicrobial usage in the last 6 months (p = 0.01, OR: 25.17, 95% CI = 1.81–348.71) were found significantly associated with isolation of ESBL-producing E. coli in the tested samples using binary logistic regression analysis. This study confirmed the potential of backyard chickens as a reservoir for ESBL E. coli in the Jhang district, Punjab, Pakistan.

Brucellosis is a neglected zoonotic disease of ruminants. It causes severe health problems in humans and significant economic loss. Only a limited number of studies have been conducted in Pakistan to determine the prevalence of human brucellosis and related risk factors. The objectives of the current cross-sectional study were to determine the prevalence of anti-Brucella antibodies in sera collected from symptomatic patients at three hospitals of Abbottabad using a commercial slide agglutination test (SAT) and to determine risk factors for brucellosis for these patients. Five hundred blood samples were collected. A questionnaire was filled in for each patient to obtain information on age, gender, living area, brucellosis associated symptoms, associated risk factors, pregnancy and abortion history. A total of 13.6% (n = 68) patients were found to be SAT positive and in 83.3% (n = 57) of these samples Brucella DNA was detected by genus specific RT-PCR for BCSP-31 gene. Statistical analysis was performed to determine odd ratios, risk ratios, 95% confidence intervals, and p-values. The prevalence of brucellosis by SAT was reported to be higher in women (14.6%, n = 44) than in men (12.1%, n = 24). The age group 25–50 years was found to be at higher risk for brucellosis (14.5%, n = 50) “animal contact” was reported as the main risk factor followed by “consumption of raw animal products.” Out of 131 pregnant women and 21 patients had abortion, the seropositivity of Brucellosis was 9.9% and 23.8%, respectively. The present study reports a striking prevalence of brucellosis among patients including pregnant women at three hospitals of Abbottabad. These findings may foster strategies for controlling human brucellosis at household level, raising of awareness about brucellosis in hospital and family doctors, and finally in setting up an eradication program in the dairy industry.

Brucellosis is a bacterial zoonotic disease that affects many animal species and can be transmitted to humans via direct contact or via contaminated food. Although brucellosis is a serious health hazard, its public health concern has been neglected in many countries. In some developing countries, such as Pakistan, where brucellosis is endemic, this disease continues to be of importance. A literature search for the past 11 years (2011–2021) provided a comprehensive insight into brucellosis in Pakistan. In this review, particular emphasis was placed on occurrence, diagnostic tests used, and prevention, treatment, and control in the context of the “One Health” approach.

Bovine brucellosis is a global zoonosis of public health importance. It is an endemic disease in many developing countries including Pakistan. This study aimed to estimate the seroprevalence and molecular detection of bovine brucellosis and to assess the association of potential risk factors with test results. A total of 176 milk and 402 serum samples were collected from cattle and buffaloes in three districts of upper Punjab, Pakistan. Milk samples were investigated using milk ring test (MRT), while sera were tested by Rose–Bengal plate agglutination test (RBPT) and indirect enzyme-linked immunosorbent assay (i-ELISA). Real-time PCR was used for detection of Brucella DNA in investigated samples. Anti-Brucella antibodies were detected in 37 (21.02%) bovine milk samples using MRT and in 66 (16.4%) and 71 (17.7%) bovine sera using RBPT and i-ELISA, respectively. Real-time PCR detected Brucella DNA in 31 (7.71%) from a total of 402 bovine sera and identified as Brucella abortus. Seroprevalence and molecular identification of bovine brucellosis varied in some regions in Pakistan. With the use of machine learning, the association of test results with risk factors including age, animal species/type, herd size, history of abortion, pregnancy status, lactation status, and geographical location was analyzed. Machine learning confirmed a real observation that lactation status was found to be the highest significant factor, while abortion, age, and pregnancy came second in terms of significance. To the authors' best knowledge, this is the first time to use machine learning to assess brucellosis in Pakistan; this is a model that can be applied for other developing countries in the future. The development of control strategies for bovine brucellosis through the implementation of uninterrupted surveillance and interactive extension programs in Pakistan is highly recommended.

Brucellosis is a highly contagious zoonosis that occurs worldwide. Whole-genome sequencing (WGS) has become a widely accepted molecular typing method for outbreak tracing and genomic epidemiology of brucellosis. Twenty-nine Brucella spp. (eight B. abortus biovar 1 and 21 B. melitensis biovar 3) were isolated from lymph nodes, milk, and fetal abomasal contents of infected cattle, buffaloes, sheep, and goats originating from nine districts in Egypt. The isolates were identified by microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differentiation and genotyping were confirmed using multiplex PCR. Illumina MiSeq® was used to sequence the 29 Brucella isolates. Using MLST typing, ST11 and ST1 were identified among B. melitensis and B. abortus, respectively. Brucella abortus and B. melitensis isolates were divided into two main clusters (clusters 1 and 2) containing two and nine distinct genotypes by core-genome SNP analysis, respectively. The genotypes were irregularly distributed over time and space in the study area. Both Egyptian B. abortus and B. melitensis isolates proved to be genomically unique upon comparison with publicly available sequencing from strains of neighboring Mediterranean, African, and Asian countries. The antimicrobial resistance mechanism caused by mutations in rpoB, gyrA, and gyrB genes associated with rifampicin and ciprofloxacin resistance were identified. To the best of our knowledge, this is the first study investigating the epidemiology of Brucella isolates from livestock belonging to different localities in Egypt based on whole genome analysis.

This study examined the relationship between perceived stress, academic performance participants and pattern of energy drink consumption in the context of higher education universities and medical students of Khyber Pakhtunkhwa province of Pakistan. Primary data was collected through self-designed Questionnaires including the Perceived Stress Scale and question about energy drink consumption, academic performance and demographics. Convenient sampling was used to select the universities and medical colleges of the province. To obtain the participants of study, random sampling technique was used. Consistent with the study hypotheses, the relationship existed between perceived stress and selected energy drink consumption items. Participants’ perceived stress and academic performance was negatively correlated. The relationship between participants’ perceived academic performance and energy drinks consumption was also found. It was found that medical students used more energy drinks as compare to non-medical students. There is a significant difference between male and female energy drink users. The participants who study in private sector institutions consume more drinks as compared to public sector students per month. More stress was found in female participants as compare to male. Comparatively medical students’ stress level is high then non-medical students. Similarly stress level increases as one move from junior to senior level at his/her college or university.

Brucellosis is one of the most important worldwide zoonoses of many countries including Egypt. Camel brucellosis has not gained much attention in Egypt yet. This study is focused on the three governorates with the highest camel populations and the largest camel markets in the country to determine the disease seroprevalence and identify the Brucella species in local camel holdings. In total, 381 serum samples were collected from male and female camels from Giza, Aswan, and Al-Bahr Al-Ahmar (the Red Sea) governorates. Samples were serologically examined using the Rose–Bengal plate test (RBPT), indirect ELISA (i-ELISA), competitive ELISA (c-ELISA) and complement fixation test (CFT). Brucella antibodies were detected in 59 (15.5%), 87 (22.8%), 77 (20.2%) and 118 (31.0%) of sera by RBPT, i-ELISA, c-ELISA and CFT, respectively. Using real-time PCR, Brucella DNA was amplified in 32 (8.4%) seropositive samples including Brucella abortus (25/32), Brucella suis (5/32) and Brucella melitensis (2/32), defining a complex epidemiological status. To the best of our knowledge, this is the first study reporting Brucella suis DNA in camel serum. The risk-associated factors including age, sex, breed and geographical distribution were statistically analyzed, showing non-significant association with seroprevalence. The results of this study will raise awareness for camel brucellosis and help develop effective control strategies.

Brucellosis is a highly contagious zoonosis worldwide with economic and public health impacts. The aim of the present study was to identify Brucella (B.) spp. isolated from animal populations located in different districts of Egypt and to determine their antimicrobial resistance. In total, 34-suspected Brucella isolates were recovered from lymph nodes, milk, and fetal abomasal contents of infected cattle, buffaloes, sheep, and goats from nine districts in Egypt. The isolates were identified by microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differentiation and genotyping were confirmed using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR. Antimicrobial susceptibility testing against clinically used antimicrobial agents (chloramphenicol, ciprofloxacin, erythromycin, gentamicin, imipenem, rifampicin, streptomycin, and tetracycline) was performed using E-Test. The antimicrobial resistance-associated genes and mutations in Brucella isolates were confirmed using molecular tools. In total, 29 Brucella isolates (eight B. abortus biovar 1 and 21 B. melitensis biovar 3) were identified and typed. The resistance of B. melitensis to ciprofloxacin, erythromycin, imipenem, rifampicin, and streptomycin were 76.2%, 19.0%, 76.2%, 66.7%, and 4.8%, respectively. Whereas, 25.0%, 87.5%, 25.0%, and 37.5% of B. abortus were resistant to ciprofloxacin, erythromycin, imipenem, and rifampicin, respectively. Mutations in the rpoB gene associated with rifampicin resistance were identified in all phenotypically resistant isolates. Mutations in gyrA and gyrB genes associated with ciprofloxacin resistance were identified in four phenotypically resistant isolates of B. melitensis. This is the first study highlighting the antimicrobial resistance in Brucella isolated from different animal species in Egypt. Mutations detected in genes associated with antimicrobial resistance unravel the molecular mechanisms of resistance in Brucella isolates from Egypt. The mutations in the rpoB gene in phenotypically resistant B. abortus isolates in this study were reported for the first time in Egypt.

Brucellosis is considered as endemic disease of animals and humans since thousands of years in Egypt. However, brucellosis in pigs has never been reported in Egypt. Thus, serological and molecular assays were applied to detect anti-Brucella antibodies and DNA in serum samples collected from pigs. In total 331 blood samples collected from male and female pigs at slaughterhouses of Cairo and Giza governorates were investigated using Brucella c- and i-ELISA and Brucella real-time PCR. Anti-Brucella antibodies were detected in 16 (4.83%) and 36 (10.8%) sera by i-ELISA and c-ELISA, respectively. Brucella DNA was detected in 10 (3.02%) seropositive samples and identified as Brucella melitensis (7/10) and Brucella suis (3/10). A higher prevelance was found in boars. This is the first study investigating pig brucellosis in Egypt. The results of this study will raise awareness for brucellosis in these farm animals and will help to develop effective control strategies.

Brucellosis is an infectious disease caused by bacteria of the genus Brucella (B.), affecting both animals and humans, causing severe economic loses and severe illness, respectively. The objective of the present study was to determine the seroprevalence and the risk factors associated with caprine, ovine, and bovine brucellosis in selected districts of Punjab, Pakistan. A total of 1083 blood samples were randomly collected from animals (goats = 440, sheep = 203, cows = 206, and buffaloes = 234). Questionnaires were used to collect data on risk factors associated with brucellosis on the sampling day. All samples were initially screened for anti-Brucella antibodies using the rose bengal plate test (RBPT). The seropositive serum samples were confirmed by a quantitative real-time polymerase chain reaction (PCR) assay for the detection of the Brucella genus- and Brucella species-specific DNA (B. abortus and B. melitensis). Univariant and binary logistic regression were used to identify important risk factors of brucellosis. Anti-Brucella antibodies and DNA were detected in 35 (3.23%) serum samples. Thirty-four (97.1%) DNA samples were confirmed as B. melitensis by qRT-PCR. Abortion history and natural mating were found to be potential risk factors. Brucella melitensis was identified as the causative agent of caprine, ovine, and bovine brucellosis in the selected districts of Punjab, Pakistan. Diseased animals may act as a source of infection for other animals. The elimination of positive seroreactors, development of control strategies for brucellosis, and education programs regarding the control of zoonotic disease are highly needed in developing countries like Pakistan.