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Considering one health concept, human health is thought to be affected by many factors. Heavy metal toxicity is now gaining its place as one of the major factors contributing to detrimental outcomes for human health. The study encompassed to target sites close to the industrial area of Lahore where heavy metal levels are believed to be higher, as industrial waste is drained into the two main drains. Sheep and goats ( n  = 5 from each species) reared in the locality were included in the study, and effects of heavy metal toxicity were evaluated in the selected organs (intestine, kidneys, liver, and muscles) via histopathological examination along with residual concentration of these heavy metals in the aforementioned organs. Heavy metals chromium, copper, zinc, lead, iron, magnesium, manganese, and nickel were detected in sample of selected organs by atomic absorption spectrometry (AAS) along with digestion method. The findings of the study indicated a statistically significant difference of residual concentrations of almost all the selected elements in almost all the tissue samples between the two sites where the values of site 1 (close to the drain) were higher compared with site 2 (away from the drain). Similar trend was depicted in histopathological examination where a higher degree of tissue degeneration, necrosis, and hence organ damage was observed in tissue samples collected from site 1 compared with site 2.

ABSTRACT The objective of the current study was to determine the prevalence of contagious ecthyma along with the association of various risk factors in goats and sheep through qualitative enzyme linked immunosorbent assay (ELISA). This technique is sensitive and cost-effective for detecting disease in large no of animals. For this purpose, serum samples from 350 goats and 91 sheep were collected on the 20th day post-infection for detection of antibodies, and essential information related to potential risk factors was collected through a questionnaire from fourteen districts of Punjab, Pakistan. Serologically positive samples were processed through PCR for confirmation using scab samples by identifying the GIF/IL-2 gene. This study found an overall 13.2% seroprevalence of contagious ecthyma infection, indicating a higher percentage in goats (14.6%) than in sheep (7.7%). In goats, the significant association (P<0.0001) of contagious ecthyma infection with seroprevalence was found in 4 districts; however, contagious ecthyma antibodies titers were found positive in sheep of Muzaffargarh only. The risk factors such as young age, female gender, grazing without stall feeding, presence of lesions on the lips, and injuries on the skin caused by prickly trees and cotton stubbles were found significantly (p<0.05) associated factors in goats; and age and injuries on the skin were important associated risk factors in sheep. The maximum occurrence of contagious ecthyma infection was found from September to December in both goats and sheep; having a significant association in goats (χ2=39.2) (P<0.0001). Likewise, the purchase of new animals and mixing up with the existing animals without quarantine measures cause contagious ecthyma to prevail in the vicinity. Moreover, contagious ecthyma antibodies were detected by ELISA kits up to 50th day post-infection in the current study. This is the first study that reported the seroprevalence of contagious ecthyma in Punjab, Pakistan. Comprehension of risk variables will not only be helpful to develop awareness amongst farmers but will also provide guidelines to government officials about the prevention and control of the disease.

ABSTRACT The present study elucidated sero-prevalence of Mycobacterium avium sub sp. paratuberculosis (MAP) in cattle and buffalo. Diarrhea is a major risk factor for MAP, so 1100 blood samples were collected from diarrheic animals or having history of some diarrheic episodes from four districts of Punjab i.e. Kasur (n=300), Rawalpindi (n=230), Lahore (n=230) and Bahawalpur (n=340). These samples were tested using ELISA and overall seroprevalence found was 39.64%. The major host-associated and non-associated risk factors of MAP were also considered. It was found that positive percentage of MAP was 2.20 times more in buffaloes than in cattle; 21.19 times more in females than in males; 1.04 times more in adult animals having age >6 years; and 1.20 times more in dry animals compared to milking animals. Furthermore, body condition score (BCS), type of housing, and condition of drinking water were also found to have significant (p<0.05) effect on the occurrence of MAP in cattle and buffalo. Moreover, hematological profile of MAP-positive animals showed significantly (p<0.05) lower values of RBCs, hb, and PCV; whereas, levels of MChC showed non-significant (p>0.05) rise in MAP-positive animals as compared to control animals. Thus, findings of the current study depict alarming situation of Johne's disease in Punjab, and help in understanding its associated risk factors and its effects on the health of cattle and buffalos.

Coxiella burnetii, an intracellular zoonotic bacterium, causes query (Q) fever in ruminants. Its role has never been elucidated in small ruminants from Pakistan. The current study is designed to (a) determine the prevalence of coxiellosis in small ruminants, (b) evaluate the association of various potential risk factors and biomarkers in the occurrence of Coxiella burnetii, (c) and determine phylogeny and genetic variability of its various isolates identified during the study. For this purpose, 320 blood samples from sheep (n = 160) and goats (n = 160) were collected from 9 Union Councils of district Kasur, Punjab, and processed for DNA extraction. C. burnetii was confirmed by amplification of IS1111 transposase gene with an amplicon size of 294 bp. The results showed that the overall positive percentage of C. burnetii is 36.87% (sheep: 46.9% and goats: 30%). The phylogenetic tree was also constructed which described the possible origin of this pathogen from environment. Besides, after translation into amino acid, the resultant alignment showed several unique changes at position numbers 18 and 27 in the isolates from goats and at 27 and 66 from those of sheep. These mutations can have major impact on the infectious characteristics of this pathogen. Furthermore, different potential risk factors and clinical biomarkers like age, tick infestation, abortion, mastitis, and infertility were also studied and found that these are significantly (p < 0.05) associated with the occurrence of coxiellosis. It is concluded from the study that C. burnetii is endemic in small ruminants in Punjab, Pakistan. The outcomes of this study are alarming for scientific community as well as for policy makers because coxiellosis is an emerging threat to both humans and animals in this region due to its interspecies transmission ability.

ABSTRACT Coccidiosis caused by Eimeria tenella is a parasitic disease affecting chickens. In Pakistan, there has been no previously published report on phylogenetic analysis of Eimeria tenella. In this retrospective study, tissue samples were collected from a flock of clinically infected chicken followed by haematology, serum biochemistry, and histopathology. Species specific PCR based on polymorphic site of the ITS1 gene was developed and used to identify the organism. haematological examination of the blood demonstrated a decrease in total erythrocytes, packed cell volume, haemoglobin concentration, and red blood cell indices. Differential leukocyte analysis revealed leukocytosis, heterophilia, eosinophilia, monocytosis, and lymphocytosis. Serum biochemistry showed marked elevation in aspartate transaminase, alkaline phosphatase and creatinine and a significant decline in alanine transaminase, total protein, total albumin, globulin, triglycerides and cholesterol values. histopathological examination demonstrated degenerative changes, necrosis haemorrhages, and sloughing off epithelial cells of broad folds of caeca, mild lymphoplasmacytic infiltration in the periportal area of the liver and mild depletion of lymphocyte in the bursa of Fabricius. The seven clades of avian Eimeria species strongly support that E. necatrix and E. tenella were closely associated and placed in the same sister clade with high bootstrap support (98%). Our two isolates RSI and RSII showed a homology index of 99.82% (nucleotide level) and 99.47% (amino acid level) with each other. The maximum similarity percentage indicated that RSI and RSII isolates were closely related to strains reported from India and China. This study is the first report on molecular characterization of E. tenella in Pakistan highlighting the pathological potential and distribution of E. tenella.

ABSTRACT Coxiella burnetii causes query (Q) fever in bovines. Its role has never been elucidated in bovines from Pakistan. The current study was designed to determine the incidence of coxiellosis in bovines, evaluate the association of various risk factors in the occurrence of Coxiella burnetii, determine phylogeny and genetic variability of various isolates identified during the study, and report hematology of the affected bovines. The incidence of coxiellosis was estimated as 32.12% and 12.5% in cows and buffaloes, respectively. The association of selected clinical biomarkers was also ascertained and all of these were found significantly (p<0.05) associated with the incidence of coxiellosis. The results of animal-related risk factors indicated that with increasing age and parity number, poor BCS, or a history of disease, the incidence of coxiellosis became significantly (p<0.05) higher. Various farm-related risk factors were also found significantly (p<0.05) associated with Coxiellosis. Moreover, phylogeny of isolated bacteria showed genetic variability (47%-99%). Lastly, the hematology profile of Coxiella burnetii positive buffaloes showed significant (p<0.05) lymphocytopenia (45.95 ± 13.4), neutrophilia (32.80 ± 11.75), leukocytosis (11.25 ± 3.62), eosinopenia (4.95 ± 2.14), thrombocytopenia (232.50 ± 211.74), and decreased hemoglobin (7.005 ± 0.90) and PCV levels (21.29 ± 2.28). Whereas in affected cows only low hemoglobin level (7.25 ± 1.18), decreased PCV (22.29 ± 3.34), eosinopenia (9.6 ± 5.49), and thrombocytopenia (365.25 ± 227.32) were statistically significant (p<0.05). As far as we know, this study provides the first molecular evidence of coxiellosis in bovines from Pakistan.

ABSTRACT This study was undertaken to investigate the impact of replacement of fishmeal (FM) with soybean meal (SBM) in diets of rainbow trout (Oncorhynchus mykiss). A total of 600 rainbow trout, with an initial mean body weight of 15.92 ± 0.34 g, were distributed randomly into twelve experimental tanks (50 fish per tank). Four isoenergetic and isonitrogenous feed diets were allocated to each treatment, with three replicates/treatment (12 fish/replicate). FM and fish oil were replaced gradually by SBM in the four diets. The inclusion of SBM in diets 1-4 was at the following respective levels of 0% (control), 29%, 35% and 51%. Growth performance of fish fed diet 4 was significantly lower than that of fish fed the other diets (P<0.05). The dietary inclusion of SBM up to level of 35% did not affect the blood biochemical parameters of rainbow trout, and was associated with acceptable growth performance. In conclusion, the SBM can be included in diet of Oncorhynchus mykiss at 35%, while keeping the level of FM and fish oil at 51%, and 1%, respectively.

ABSTRACT Contagious Bovine Pleuropneumonia (CBPP) is an infectious respiratory disease of cattle, characterized by anorexia, fever, dyspnea, polypnea, cough, and nasal discharges and caused by Mycoplasma mycoides subsp. mycoides. This study was carried out in abattoirs of Lahore, Kasur and Jhang districts of Punjab, Pakistan. Different tissue samples were collected from 560 cattle showing respiratory signs at slaughter. Lung samples were used for detection of CBPP through PCR targeting 16S ribosomal RNA. Furthermore, lungs, liver, kidney and lymph node were collected for histopathological examination. Of the 560 samples, 49 (8.25%) were found positive for CBPP. Maximum pathological lesions (50-75% of surface area) were seen in 34.69 percent lung samples, chronic type pleurisy in 97.96 percent, maximum aggregate pathological lesion score in 65.31%, while 48.98% lung samples had 6-10 numbers of sequestra. Besides, infiltration of inflammatory cells, congestion, hemorrhages, necrosis and other degenerative changes were observed in tissue samples of infected cattle. Significantly higher (P<0.05) micro-pathological lesion scores were given to lungs, kidney and lymph node samples from infected cattle as compared to those of non-infected ones. In conclusion, CBPP, being a major factor affecting cattle production and associated economics, warrants the implementation of control measures to mitigate the economic losses associated with the disease.

The aim of this research is to appraise a newly patented method for control of chicken coccidiosis in comparison to a classical approach. The new method implements an invented Disinfectant (WSD) for decontamination of rearing surfaces, and a developed drinking water-herbal coccidiostat (EOBWE) supplement. The experimental design has 8 treatments (TRTs), with four pens/TRT, and 25 broiler chicks/pen. The floors of TRT1 to TRT7 are contaminated with 4.0 × 10 5  sporulated oocysts/m 2 of each of E. acervulina, E. maxima, and E. tenella, while TRT8-floor is left uncontaminated. The floors of TRTs 1 and 2 are disinfected with chlorine, while that of TRTs 3-6 are disinfected by WSD. Floors of TRTs 7 and 8 are deprived of disinfection. Birds in TRTs 1, 6, 7, and 8 are deprived of coccidiostat, while birds in TRTs 2 and 3 are fed salinomycin in their feed; birds of TRT 4 are administered EOBWE in drinking water, while birds of TRT 5 are administered simultameously both, the salinomycin and EOBWE. Birds in TRT 4 (WSD/EOBWE treated) and TRT 5 (WSD/salinomycin + EOBWE) had improved protection (lowest oocyst output and intestinal lesions) and enhanced production (lowest mortality, lowest feed conversion ratio, and highest live body weight) compared to the other five challenged treatments, associated with significant improvements compared to positive controls (TRT 7) (P < 0.05).

Orf virus (ORFV) causes an acute, contagious, skin disease of sheep and goats which is economically important. The objectives of this study were to identify ORFV and to explore its pathological and phylogenetic profiles in 350 goats and 91 sheep of 14 districts of Punjab, Pakistan, from July 2020 to July 2021. Skin scrapings (total no. of samples = 441) of suspected animals were subjected to polymerase chain reactions, phylogenetic analysis, and pathological observations. The partial length of GIF/IL-2 gene (408 bp) was successfully amplified in 58/441 samples. Phylogenetic analysis of GIF/IL2 gene showed that the study isolates belonged to ORFV-cluster I, together with the viruses reported in India and China. Pakistan ORFV isolates were shared 97.6–98.7% nucleotide and 97.6–100% amino acid identities with the reference strain (NC_005336). Moreover, Chinese ORFV-isolates were detected unique multiple amino acid substitutions (F11L, Q21H, D27N, I46V, N49S, N82D, D103N, S129G) with study isolates. Naturally infected animals were anorexic, emaciated, dull, and depressed. The macroscopic lesions included multifocal to coalescing, ulceration followed by proliferative papules, pustules, and crust formation on the epidermis of gums, lips, mouth commissure, muzzles, nose, and udder. Histopathological examination revealed hyperplasia, anastomosing rete ridges formation and degenerative changes, including spongiosis and vacuolation of epidermal cells. Keratinocytes exhibited eosinophilic intracytoplasmic inclusion bodies with pyknotic and karyorrhexis nuclei. This is the first report on molecular characterization of ORFV from Pakistan, with insight into its pathogenesis and comparative analysis of pathological alterations and genetic diversity between ORFV strains reported in different geographical areas.