Members of Enterobacteriaceae are known to produce extended-spectrum beta-lactamases (ESBL) which hydrolyze the beta-lactam group of antibiotics. The existence of ESBL-producing Salmonella enterica (S. enterica) and Escherichia coli (E. coli) harbored by urban avifauna was investigated in this study. Dropping samples (n= 180) were collected from six different bird species in the district Jhang, Punjab province, Pakistan. Isolation and identification of ESBL isolates were made by using cefotaxime-(4 mg/L) supplemented MacConkey agar and double disc synergy test (DDST). Polymerase chain reaction (PCR) was performed for the detection of four different ESBL genes including bla CTX-M , bla TEM , bla SHV and bla OXA . A total of 42.69% isolates were confirmed as ESBL via DDST including 30.64% S. enterica and 49.54% E. coli. The incidence of ESBL S. enterica and ESBL E. coli was found highest in egret (Ardea alba) and pigeon (Columba livia) as 64.28% and 78.95%, respectively. The bla CTX-M gene was detected in 57.89% and 64.81% of isolates of S. enterica and E. coli, respectively. Among other genes in S. enterica and E. coli, bla TEM (21.05%, 20.4%); bla SHV (15.78%, 9.26%), and bla OXA (5.26%, 5.56%) were detected, respectively. All of the tested isolates were found resistant to at least one of the thirteen antimicrobial agents except meropenem. To the best of our knowledge, this is the first study reporting the incidence and genetic diversity of ESBL bacteria associated with urban avifauna in Pakistan. The urban avifauna can serve as a potential subject of bio-surveillance to monitor the emergence of antimicrobial-resistant bacteria.

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.

Toxoplasmosis is a zoonotic infection caused by a pathogenic protozoan, Toxoplasma gondii, responsible for huge economic and health losses in developing countries. The current study was conducted to assess the seroprevalence of Toxoplasmosis and associated risk factors in sheep and goats in District Jhang, Punjab, Pakistan. Blood samples (n=400) were collected from both genders of goats (n=219) and sheep (n=181) from four Tehsils of District Jhang alongwith a comprehensive questionnaire to evaluate the risk factors associated with the disease endemicity and spread. For assessing the seroprevalence, the samples were  examined using Latex agglutination test. Additional data regarding hygienic conditions, water source, gender, breed, age of animal was also collected on a predesigned questionnaire. The overall seroprevalence of Toxoplasma gondii was found  34.25% (137/400) in District Jhang. Higher seroprevalence was recorded in goats {36.52% (80/219)} as compared to sheep {31.49% (57/181)}, however, it was non-significant (p>0.05). Gender-wise seroprevalence was found 32.59% (44/135) and 35.09% (93/265) in male and female animals, respectively (p>0.05). Further, the association of Toxoplasmosis between different age groups was significantly higher in older animals having age >24 months 42.75% (62/145) than younger animals with age <12 months 26.60% (29/109) and 11-24 months 31.50% (46/146) (p<0.05). The seroprevalence was also higher 40.81% (80/196) in animals drinking water from outdoor water source than in animals drinking from indoor water source 27.94% (57/204) (p<0.05). Moreover, seroprevalence was significantly higher 43.11% (97/225) in animals  kept in vicinity of cats than in absence of cats 22.85% (40/175) (p<0.05). However, reproductive status, breeds, flock size had non-significant impact on the prevalence of T. gondii.  Thus, it is concluded that the presence of cats near animals, larger flock size, older age of animals, and poor hygienic conditions are main risk factors of Toxoplasmosis in sheep and goats and these could be a potential threat of infection for livestock industry and public health.

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