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 bla_CTX-M (51.55%), bla_TEM (20.62%), bla_OXA (6.18%) and bla_SHV (2.06%). In addition, gene combinations bla_CTX-M + bla_TEM, bla_TEM + bla_OXA and bla_CTX-M + bla_SHV 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 global demand for protein is on the rise owing to the exponential increase in the world population and to meet the global protein requirements, it is imperative to seek alternative sources of proteins in farm animal feeding. Recognizing the importance of proteins and the fact that a major portion of human protein requirements is derived from livestock in the form of meat, milk, and eggs, the available protein-feeding stuff in the form of soybean meal cannot be regarded as sufficient for feeding to livestock. Oil seeds such as rapeseed meal and canola meal have a crude protein content of 30%-40% and are widely used and hence a potential alternative protein source to soybean meal. Going forward, Grain legumes such as peas, faba beans, and lupins, another alternative source of proteins have the potential to replace traditional protein feeds completely or partially such as bone and fish meals. Duckweed with a protein content of 20%-45% is another plant-based potential protein source that can be employed in livestock feeding particularly pigs owing to its huge potential as a growth promoter as evidenced by studies in pigs and piglets. Because of their nutritional qualities and possible environmental advantages, insects represent another class of alternative protein sources that have enormous potential to function as sustainable protein sources. Several insect species have been assessed for use as animal feeds; the most promising ones include the yellow mealworm (Tenebrio molitor, TM), the common house fly (MD), and the black soldier fly (Hermetia illucens, HI). Byproducts from aquaculture and fisheries are abundant in macro- and micronutrients, and their utilisation can provide fishmeal and fish oil, which can then be further adapted for use as a source of protein in animal nutrition. Employing microalgae as an alternative source of protein in animal feeding is somehow a new concept. Many nutritional and toxicological studies have demonstrated the potential of algae biomass as a valued feed supplement or substitute for conventional protein sources such as soybean meal. While these alternative protein sources in livestock feeding may serve as useful tools, parameters such as feed safety and acceptability should be monitored based on feed safety regulations.

The emergence of SARS-CoV-2 variants has posed a challenge to disease control efforts worldwide. This study explored the genomic diversity and phylogenetic relationship of SARS-CoV-2 variants reported in Pakistan. Our objective was to understand the transmission dynamics of different lineages within the country. We retrieved and analyzed spike protein sequences from Pakistan and compared them with reference sequences reported worldwide. Our analysis revealed the clustering of Pakistan-origin isolates in nine different clades representing different regions worldwide, suggesting the transmission of multiple lineages within the country. We found 96 PANGO lineages of SARS-CoV-2 in Pakistan, and 64 of these corresponded to 4 WHO-designated variants: Alpha, Beta, Delta, and Omicron. The most dominant variants in Pakistan were Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2, AY.108), and Omicron (BA.2.75, BA.5.2), and the N-terminal domain and receptor binding regions were the most hypervariable regions of the spike gene. Compared to the reference strain, characteristic substitutions were found in dominant variants. Our findings emphasize the importance of continuously monitoring and assessing nucleotide and residue substitutions over time to understand virus evolutionary trends better and devise effective disease control interventions.

Impact of Mycotoxin Binders on Humoral Immunity, Lymphoid Organs and Growth Performance of Broilers