The empirical use of antibiotics for clinical mastitis is a principal driver of antimicrobial resistance in dairy farming. While on-farm culture systems represent a promising strategy for targeted therapy, robust evidence of their efficacy in heterogeneous commercial settings is still needed. We conducted a randomized controlled trial across 16 commercial dairy farms. Cows with clinical mastitis (CM) were allocated to a Positive Control Treatment (PCT) group (n = 57), receiving immediate empirical intramammary (IMM) antibiotics, or a Culture Based Group (CBG) (n = 46), where treatment was directed by a tri-plate on-farm culture system after 24-hour incubation. The cows were considered experimental unit with mixed-effect models within cluster correlation. Data was statistically analyzed using chi square tests, paired t-tests and Kaplan Meier survival analysis via SPSS (version 20.0). The culture-guided protocol enabled a reduction in antibiotic use, eliminating treatment for the 45.6% of CBG cases (no bacterial growth or Gram-negative infections). The clinical cure rates between the groups (CBG 82.6% vs. PCT 75.4%) were not statistically significant (p = 0.28). Similarly, bacteriological cure rates were comparable between (PCT 71.9% vs. CBG 71.7%, p = 0.987). However, the CBG approach revealed significantly lower treatment failure rate (17.3% vs. 24.5%, p < 0.001) with a shorter median time to clinical cure (3 days vs. 7 days, p < 0.001). At the herd level, the strategy was associated with a significant increase in milk yield (+6.94 L/day, p < 0.001) and reduction in somatic cell count (-56.8%, p < 0.001). The tri-plate on-farm culture system is an effective antimicrobial stewardship tool, facilitating a substantial reduction in antibiotic use while accelerating clinical recovery and improving udder health in commercial dairy operations.

Brucellosis, caused primarily by Brucella melitensis, remains an important yet neglected zoonotic disease in Pakistan, particularly among small ruminant farmers who represent a high-risk occupational group. This cross-sectional study was conducted during 2022–2023 to assess the knowledge, attitudes, and practices (KAP) related to brucellosis among sheep and goat farmers in three agro-ecological zones of Punjab province. A total of 266 households were initially approached, of which 259 complete responses were included in the final analysis. Data on socio-demographic characteristics and KAP variables were collected through structured, face-to-face interviews using a pretested questionnaire. Composite ordinal risk scores were generated for three domains: knowledge of brucellosis, herd management and farm hygiene practices, and personal protective and hygiene practices. A high proportion of participants were classified within the highest-risk category, including 76.1% for knowledge-related risk, 70.7% for herd management and farm hygiene risk, and 52.9% for personal hygiene risk. Descriptive analysis further revealed very low awareness of brucellosis, including its transmission routes and prevention measures. High-risk practices were commonly reported, such as improper disposal of aborted materials and consumption of raw milk. Multivariable ordinal logistic regression analysis demonstrated that lower educational attainment and older age (>50 years) were significantly associated with higher brucellosis risk scores across all three domains. These findings highlight that inadequate knowledge and persistent risky husbandry and hygiene behaviors substantially increase the vulnerability of small ruminant farmers to brucellosis in Punjab. Targeted awareness programs, strengthened veterinary extension services, and promotion of safe farming practices are essential to reduce zoonotic transmission in this high-risk population.

Brucellosis is a major zoonotic disease worldwide. This cross-sectional study aimed to determine the seropositive proportion of brucellosis and identify associated risk factors in small ruminants (sheep and goats) and livestock workers in Punjab, Pakistan. A total of 1783 blood samples were collected from unvaccinated sheep and goats across 219 flocks, along with 122 blood samples from occupationally exposed humans using convenience sampling of villages and flocks. Serum samples were tested using a commercial indirect-ELISA kit. Overall, 2.0 % of animals were seropositive, 9.1 % of flocks had at least one seropositive animal, and 9.8 % of livestock workers were seropositive. Significant animal-level risk factors included flock sizes > 50 (OR: 6.5), extensive production systems (OR: 5.6), introduction of new animals without quarantine (OR: 17.1), and mixed farming with large ruminants (OR: 4.0). In humans, contact with seropositive animals (OR: 8.2), animal abortions (OR: 16.2), and unpasteurized milk consumption (OR: 23.3) were key predictors. Real-time PCR confirmed Brucella abortus in sheep and goats, but no Brucella DNA was detected in humans. These findings underscore the importance of integrated surveillance, farmer education, and biosecurity measures to mitigate zoonotic transmission. Further large-scale studies using advanced molecular tools are recommended to validate these findings and support targeted brucellosis control programs.

Pathogen and stress-related morbidities are the main challenges in tilapia farming.Improving the immune health is an effective approach to protect tilapia against these pathogens.Phenolic content and antioxidant activity of rice husk (RH), sugarcane bagasse (SB), and corn cob (CC) evaluated by Folin-Ciocalteau assay and DPPH assay revealed high total phenolic content and antioxidant activity in RH compared to CC and SB.LCMS analysis of RH, SB, and CC exhibited the presence of hydroxybenzoic acid and hydroxycinnamic acid in agricultural waste samples.Effects of RH, SB, and CC on growth performance, gut microbial flora, innate immunity, and hematology of hybrid red tilapia (Oreochromis aureus X Oreochromis mossambicus) were evaluated in 91 days feeding trial with a test diet supplemented with 10% RH, SB, CC to basal diet (BD) ingredients in optimum conditions of growth.Growth performance data was recorded at an interval of 13 days.At the end of feeding trial, fish gut microflora, skin mucus bactericidal activity, blood serum lysozyme, and bactericidal activity against Shigella sonnei, and hematological parameters were evaluated.Significant difference in growth performance of red tilapia fed with experimental diets was observed.RGR (relative growth rate) was lowest in BD (380.79 ± 46.26) and observed highest in RH (554.01 ± 83.24).Lactobacillus count was increased in all experimental groups.Decrease in Salmonella sp., Shigella sp., and E. coli count was observed in CC.All experimental groups showed enhanced serum bactericidal activity against Shigella sonnei.SB group showed the highest serum bactericidal activity.Skin mucus bactericidal activity was also highest in SB.Least skin mucus bactericidal activity was presented by BD. Results of research work indicated that RH, SB, and CC have bioactive ingredients that positively influenced the growth performance, innate immune response, and hematology of red tilapia.Agricultural by-products are abundant biomass and rich in bioactive and nutraceutical compounds, therefore these are good candidates for maintaining redox balance, health status and growth performance in fish.

Bovine mastitis is a common udder disease in cattle, mainly caused by bacteria and other infectious agents. Traditionally antibiotics are used for their treatment, but the development of antibiotic resistance has increased the importance of using non antibiotic alternative such as probiotic. In current study a previously in vitro characterized isolate Lactobacillus plantarum CM49 infused into two groups of cattle suffering from clinical mastitis (n = 5) and sub-clinical mastitis (n = 5). The bacterial composition and diversity analysis of milk samples before and after probiotic administration was analyzed using 16S rRNA gene base metagenomic analysis and lactobacillus counts were also evaluated using Real time PCR. The results show that there was an increase in abundance of Proteobacteria and decrease in Firmicutes at phylum level in both groups while major mastitogens genera Staphylococcus and Streptococcus abundance was reduced after treatment in sub-clinical mastitis group (SCMG) and clinical mastitis group (CMG) respectively. Lactobacilli counts evaluated through Real time PCR showed an increase in number, furthermore diversity indices showed an increase in diversity after treatment with probiotic. It is concluded from the results that Lactobacillus plantarum CM49 may serve as promising candidate for improving dysbiosis resulting from mastitis and improving microbial diversity.

This study aimed to isolate and characterize Staphylococcus aureus (S. aureus) from samples of mastitis milk taken from dairy cattle in Punjab's Narowal District. 200 milk samples were collected aseptically from different dairy herds in the district, including clinical mastitis (CM) and sub-clinical mastitis (SCM) cows. Blood agar and mannitol salt agar were used for S. aureus isolation and identification. Selective media were then used for additional purification. Observations of morphological and biochemical traits verified the existence of S. aureus. Through questionnaire-based surveys, the prevalence of S. aureus mastitis was identified, and risk variables linked to its incidence were evaluated. The findings showed that S. aureus mastitis was prevalent in 42.5% of cases, with sub-clinical cases having a greater incidence (45.8%) than clinical cases (37.5%). Risk factors such as distance from dung pile to stall(m), source of water, dung removal per day, lactation period (weeks), parity, effect of milk yield (L), traumatic injury of udder, housing type, floor type, teat dipping, and bedding type was identified and their relationship to the occurrence of S. aureus mastitis was studied. Tests for antibiotic sensitivity revealed that S. aureus isolates were responsive to gentamycin, ceftiofur, tetracycline, enrofloxacin, and ciprofloxacin but extremely resistant to penicillin and amoxicillin. Additionally, the Somatic Cell Count (SCC) and California Mastitis Test (CMT) were used at different intervals to assess the effectiveness of the medication. Furthermore, compared to other treatment groups, a larger percentage of cure rates was seen in the groups receiving ceftiofur and enrofloxacin. Overall, this study contributes to the development of more effective management methods for S. aureus mastitis in dairy calves by offering insightful information about the condition's prevalence, risk factors, antibiotic sensitivity, and effectiveness of treatment.

ABSTRACT Climate change, land development, and increased outdoor human activity have increased the prevalence and distribution of tick-borne pathogens, causing public health issues. Asia is a pivotal region of emerging infectious diseases caused by zoonotic disease. Therefore, this study aimed to construct effective surveillance systems and establish preventive strategies against novel tick-borne diseases. Next-generation sequencing (NGS) was performed to detect tick-borne pathogens from animal blood-fed ticks. Ticks (n = 261) were collected from different animals across nine Asian countries between 2022 and 2023. Five genera of adults and nymphal ticks were used, namely, Amblyomma, Haemaphysalis, Hyalomma, Rhipicephalus, and Ixodes. The animals from which the ticks were collected were wild, domestic animals, birds, and reptiles, living in the natural environment. After NGS, clean reads of 18,382,262–30,460,619 DNA viruses, 22,744,384–32,400,471 RNA viruses, 134,506–286,282 16S rRNA of bacteria, and 147,154–352,826 18S rRNA of protozoa were obtained from nine Asian countries. After analysis of the species, 28 RNA, 13 DNA viruses, 16 bacteria, and 4 protozoa were detected. Several tick-borne pathogens, including those in families that cause zoonotic diseases, such as Phenuiviridae, Nairoviridae, Rickettsiaceae, and Borreliaceae, were identified. This study is distinctive in that it involved cooperative studies with nine Asian countries to prevent the spread of tick-borne diseases. The results suggest that pathogens, which were detected in animal blood-fed ticks, can circulate in animals and may be transmitted to humans. In addition, this study can provide a basis for effective surveillance systems to prevent novel pathogens.IMPORTANCESurveillance systems against novel tick-borne diseases are significant for global health. Climate and other environmental changes have contributed to an expanding range of ticks and tick-borne diseases. Areas in Asia constitute key areas of emerging infectious diseases. Through analysis of blood-fed ticks, collected from various animals living in the natural environment, we suggest that tick-borne pathogens may harbor animals and environment and have potential risk of transmission in humans. Understanding the distribution of tick-borne pathogens requires cooperative studying and, thus, can construct standardized surveillance systems.

Bovine mastitis is among the most significant infectious diseases of dairy cattle that cause considerable economic losses of the dairy industry due to cost of treatment, loss of milk production and early culling of cows. Streptococcus uberis is an important environmental pathogen and can cause clinical and subclinical mastitis. Antimicrobial resistance (AMR) is a growing concern for the effectiveness of the treatment for Streptococci. The objectives of the present study are to isolate and characterize Streptococcus spp. from mastitic cow milk, to identify their antimicrobial resistance pattern and to confirm the species using multiplex PCR. 150 milk samples were taken from the cattle at UVAS Ravi Campus Pattoki and its surrounding villages. Samples were screened using the California Mastitis Test (CMT), positive samples were cultured on blood agar for morphological and biochemical identification (Gram staining and biochemical tests). Kirby–Bauer Disk Diffusion method was used to determine antimicrobial susceptibility for the 13 antibiotics as per CLSI guidelines. Multiplex PCR assays to target S. agalactiae (16S rRNA, 317 bp), S. dysgalactiae (16S rRNA, 572 bp) and S. uberis (Cpn60, 400 bp) were performed for molecular confirmation. Of the 150 samples collected 100 (66.9%) were found to be CMT positive, including 12 Streptococcus sp. isolates. A 400 bp amplicon of Cpn60 was found in all isolates, so they were all identified as S. uberis. All the isolates were resistant to penicillin, ampicillin, vancomycin and clindamycin (100%) and 91% sensitive to levofloxacin, azithromycin and ofloxacin. A significant difference in inhibition zones was observed between the different isolates (p ≤ 0.05). Streptococcus uberis was the only mastitis-associated Streptococcal species found and is multidrug resistant highlighting the need of molecular diagnosis, AMR monitoring and rational antibiotic usage in Pakistani dairy industry.

The ostrich meat is considered healthy as it contains low cholesterol and fat levels. Foodborne diseases caused by the bacterium Salmonellaare a significant public health concern. The study was conducted to determine the prevalence, the risk factors involved, and antimicrobial susceptibility patterns of Salmonella isolates recovered from captive ostrich feces in the district of Lahore, Pakistan. There is not a single research on this issue in Pakistan up to date in ostrich. A total of 100 ostrich fecal samples were collected- and specifically cultured on Salmonella Shigella agar. The antimicrobial susceptibility was checked for different antibiotics throughthe Kirby-Bauer disc diffusion method. The 21% fecal samples were found positive for Salmonella. The disease was found to besignificantly(p<0.05) associated with risk factors; water type(p=0.027), bird’s capacity(p=0.042), cage hygiene(p=0.024), age of bird(p=0.041) andother bird species around (p=0.031) while enclosure size and diet were found non-significant. The bacterium was found highly susceptible to Amoxicillin followed by Chloramphenicol, Gentamicin, Cefotaxime, andEnrofloxacin respectively.Further Salmonella species-level diagnostic studies along with susceptibility checks of more therapeutic agents are suggested to future researchers.

Infectious bovine keratoconjunctivitis (IBK) commonly known as pinkeye is a contagious non-fatal bacterial infection of the eye that has tremendous economic impact on the dairy industry. Nevertheless, it is a much-neglected dairy issue in Pakistan. The current study was aimed to find out the prevalence and risk factors associated with pinkeye infection in commercial dairy herds in Pakistan. This study recruited 10,573 cows in 20 commercial dairy herds holding >= 25 cows per herd. The cows were assessed based on eye lesions using a scale from 1 to 4, with 4 being the most severe of all. Culture of subconjunctival swab samples was also undertaken. The overall prevalence of pinkeye was 6.86% with Holstein-Friesian breed being the most affected (7.25%) followed by crossbred (6.89%) cattle. Cow hygiene and farm hygiene scores were indirectly related to the prevalence of pinkeye. Prevalence was also significantly (P<0.05) associated with seasons of the year, being highest in humid summer months while lowest in winter. The prevalence of pinkeye was significantly higher (<0.0001) in cows exposed to sunlight than unexposed ones. Cows on farms performing farm disinfection occasionally had 1.52 times higher odds of being diseased than cows on farms with once-a-week disinfection. Likewise, cows on farms using no fly control program had 2.8 times higher likelihood of pinkeye than those on farms having fly control program. In addition, prevalence of pinkeye was significantly higher (P<0.05) in cows with low body condition scores. It was concluded that Pinkeye is a significant health concern in commercial dairy herds in Pakistan and the risk factors influencing the prevalence of this disease may include cow hygiene, farm management practices, and the environment.

Pathogen and stress-related morbidities are the main challenges in tilapia farming.Improving the immune health is an effective approach to protect tilapia against these pathogens.Phenolic content and antioxidant activity of rice husk (RH), sugarcane bagasse (SB), and corn cob (CC) evaluated by Folin-Ciocalteau assay and DPPH assay revealed high total phenolic content and antioxidant activity in RH compared to CC and SB.LCMS analysis of RH, SB, and CC exhibited the presence of hydroxybenzoic acid and hydroxycinnamic acid in agricultural waste samples.Effects of RH, SB, and CC on growth performance, gut microbial flora, innate immunity, and hematology of hybrid red tilapia (Oreochromis aureus X Oreochromis mossambicus) were evaluated in 91 days feeding trial with a test diet supplemented with 10% RH, SB, CC to basal diet (BD) ingredients in optimum conditions of growth.Growth performance data was recorded at an interval of 13 days.At the end of feeding trial, fish gut microflora, skin mucus bactericidal activity, blood serum lysozyme, and bactericidal activity against Shigella sonnei, and hematological parameters were evaluated.Significant difference in growth performance of red tilapia fed with experimental diets was observed.RGR (relative growth rate) was lowest in BD (380.79 ± 46.26) and observed highest in RH (554.01 ± 83.24).Lactobacillus count was increased in all experimental groups.Decrease in Salmonella sp., Shigella sp., and E. coli count was observed in CC.All experimental groups showed enhanced serum bactericidal activity against Shigella sonnei.SB group showed the highest serum bactericidal activity.Skin mucus bactericidal activity was also highest in SB.Least skin mucus bactericidal activity was presented by BD. Results of research work indicated that RH, SB, and CC have bioactive ingredients that positively influenced the growth performance, innate immune response, and hematology of red tilapia.Agricultural by-products are abundant biomass and rich in bioactive and nutraceutical compounds, therefore these are good candidates for maintaining redox balance, health status and growth performance in fish.

Bovine mastitis results in significant economic losses for the dairy industry globally due to milk production losses and decreased herd efficiency. This research aimed to isolate, select, and characterize indigenous lactobacilli with probiotic properties. A total of 40 lactobacilli were isolated from healthy milk samples of cattle and identified at the species level through 16S rDNA sequencing. All isolates were initially screened for antimicrobial activity, and selected isolates underwent in vitro assessment of probiotic properties. Among the lactobacilli isolates, varying levels of activity (9 to 19 mm) against cattle mastitogens; Stapylococcus aureus (Staph. aureus), Escherichia coli (E. coli) and Streptococcus dysgalactiae (Strep. dysgalactiae) were observed in the well diffusion assay. These isolates demonstrated auto-aggregation (ranging from 14.29 ± 0.96

Theileriosis is a protozoan parasite that is transmitted by ticks and infects a wide range of animals worldwide. This study aims to assess the molecular prevalence and related risk factors of theileriosis in Balochistan, Pakistan. Standard microscopy methods, polymerase chain reaction (PCR), 18S small subunit ribosomal RNA gene sequencing, and phylogenetic analysis were used. For this purpose, a total of 408 blood samples were collected from tick-infested cattle in Zhob, Loralai, and Quetta districts of Balochistan, Pakistan. Microscopy and subsequent PCR analysis confirmed the highest prevalence of Theileria annulata in Loralai district (11.76% and 12.75%), followed by Zhob district (11.27% and 12.25%), and Quetta district (8.34% and 9.56%), respectively. Moreover, the prevalence of T. annulata was higher in young cattle (85.82%), followed by female cattle (58.87%), and exotic crossbred cattle (33.33%) in the study area. However, various variable such as sex, area, and breeds of the cattle were not significantly correlated (P>0.05) with the presence of T. annulata, except for the age of animals (P<0.05). In addition, sequencing and phylogenetic analyses revealed that the isolated T. annulata was closely related to the isolates from Turkiye, Italy, Egypt, Iran, and Pakistan. Hence, these findings will contribute to the development of more effective control strategies for theileriosis in the cattle population of Balochistan, as well as in Pakistan on a broader scale.

Tick infestation poses a serious threat to animal health, leading to significant losses in terms of vector-borne disease transmission, reduced live weight, lower quality hides, decreased milk production, and impaired reproduction in tropical and subtropical regions worldwide. This study aimed to determine the prevalence, seasonal variation, distribution pattern, and associated risk factors of Ixodid family tick species in the cattle and sheep population of three different districts in Balochistan, Pakistan. This study employed a convenient sampling method, collecting 4080 adult ticks from 816 cattle and sheep of various breeds, ages, and sexes. Specific morphological keys were used to identify the ticks up to the genus and species level. Among cattle, the highest prevalence was recorded for R. (B) annulatus (27.01%), followed by R. (B) microplus (24.02%), and H. anatolicum (20.54%). H. dromedarii (5.29%) was the least prevalent species observed in cattle. In the sheep population, H. anatolicum (30.34%) showed the highest prevalence, followed by H. marginatium (22.99%), and R. (B) annulatus (20.88%). H. dromedarii (6.96%) was the least prevalent species observed in sheep. The prevalence of R. (B) decoloratus, H. anatolicum and H. dromedarii was found to be significantly associated (P < 0.05) with the breed, age, and sex of both cattle and sheep. However, the presence of R. (B) annulatus, R. (B) microplus and H. marginatium tick species showed no significant association (P > 0.05) with these factors. In addition, the prevalence of ticks was higher in younger, female, and crossbred Friesian cattle compared to adults, males, and other breeds. Conversely, the prevalence of ticks was higher in adult, female and Hernai breed of sheep in the studied area. In conclusion, R. (B) annulatus and H. anatolicum are the dominant tick species infesting the cattle and sheep population in Balochistan. Consequently, this study provides valuable insights for developing practical and effective control measures against ticks and tick-borne diseases in the sheep and cattle population of Balochistan, Pakistan.