Rhipicephalus sanguineus sensu lato (s.l.) is the most widely distributed ixodid tick and is a vector of major canine and human pathogens. High-throughput technologies have revealed that individual ticks carry a high diversity of pathogens, including bacteria, protozoa and viruses. Currently, it is accepted that co-infections (multiple pathogen species within an individual) are very common in ticks and influence pathogen acquisition and transmission as well as host infection risk. However, little is known on the impact of the genetic diversity of pathogens on the incidence of co-infections. Herein, we studied the frequency of co-infections in R. sanguineus (s.l.) and their association with the genetic diversity of Ehrlichia canis. Rhipicephalus sanguineus (s.l.) female ticks (n = 235) were collected from healthy farm dogs in three districts of Pakistan. Microfluidic real-time PCR, a powerful nanotechnology for high-throughput molecular detection of pathogens, was used to test the presence of 25 bacterial and seven parasitic species in individual ticks. The genetic diversity of E. canis was evaluated by characterizing the trp36 gene. A total of 204 ticks were infected with at least one pathogen and 109 co-infected with two (80%) or three (20%) pathogens. Rickettsia massiliae (human pathogen) and E. canis (zoonotic dog pathogen) were the most common pathogens co-infecting (30.4%) ticks. Furthermore, all identified co-infections included R. massiliae and/or E. canis. Multiple correspondence analysis (MCA) revealed that single infections did not show clear regional association whereas some co-infections were restricted to certain geographical regions. The sequence analysis of trp36 in representative samples allowed the identification of three E. canis strains with low genetic diversity, and the strain found in Muzaffargarh district appeared to be more adapted to co-infection with R. massiliae. Rhipicephalus sanguineus (s.l.) harbors multiple co-infections with human and dog pathogens of zoonotic potential. Findings of this study suggest that genetic diversity of E. canis may favor co-infections with different pathogens.

Food adulteration has a direct impact on public health, religious faith, fair-trades, and wildlife. In the present study, a reliable and sensitive assay has been developed for verifying meat adulteration in food chain. The multiplex PCR system was optimised for identification of chicken, cow/buffalo, sheep/goat, horse/donkey, pork, and dog DNAs in a single reaction mixture simultaneously. The primers were designed using 12 S rRNA gene sequences with fragment size in the range of 113 bp to 800 bp, which can be easily visualised on agarose gel electrophoresis making the technique economical. After validation of accuracy, specificity, and sensitivity, commercially available meat products (n = 190) were screened, comprising both raw and cooked meat samples. The results demonstrated a high rate of adulteration (54.5%) in meat products. The technique developed here can be easily used for screening of different meat products for export and import purposes as well as for food inspection and livestock diagnostic laboratories.

Brucellosis is one of the most notorious zoonoses worldwide. The disease is common and endemic in humans and animals of Pakistan, but lack of awareness and lack of research have resulted in an increased incidence in the human population. The present study aimed to determine the seroprevalence and at molecular detection of brucellosis in patients with clinical symptoms in six different hospitals from Lahore, which is the capital city of Punjab province. A total of 218 blood samples were collected from hospitalized patients. The samples were initially screened by the Rose Bengal Plate Test (RBPT), and then quantitative real-time PCR (qRT-PCR) was applied. An overall seroprevalence of 17% (37/218) was found. The highest prevalence was found at the Lady Health center (36.53%), which was followed by the Lady Willingdon Hospital (28.6%). Female patients showed a higher seroprevalence than males and peaked at 34% (n = 32) for women who suffered from abortion. In total, 16.8% of patients younger than 30 years showed seropositive reactions, while the prevalence was 19% in patients between 31 and 50. Thirty-three DNA samples from 24 seropositive and nine seronegative patients tested positive, 32 samples were found positive for B. abortus DNA, and one sample failed to be identified at the species level. Almost all positive cases had direct contact with animals and consumed unpasteurized dairy products. Research on human brucellosis is still scarce in Pakistan. For the diagnosis of brucellosis, serology and molecular tools should be combined if isolation by culture is not possible. Nationwide control activities and increasing awareness for zoonotic brucellosis are needed.

Polycyclic aromatic hydrocarbons (PAHs) are produced as primary environmental pollutants as a result of incomplete combustion of fuels. They harbor mutagenic and carcinogenic properties. Their continuous monitoring is considered a requisite for keeping them into admissible limits. The current study was designed to quantify the levels of PAHs in soil, water and blood samples. One hundred and fifty samples each of soil, water, human and animal blood were collected from highly exposed areas including industrial, highways and incinerators linked areas around Lahore city, Punjab, Pakistan. Amounts of six selected PAHs (Phenanthrene, Biphenyl, Biphenthrene, Naphthalene, Anthracene and P-Ansidine) were quantified by reverse phase-high performance liquid chromatography (RP-HPLC) equipped with UV-VIS photodiodes array detector (PDA) at 247 nm. An isocratic method was optimized and validated for use with soil, water and plasma samples. To obtain the reliable results the HPLC method was validated following the ICH/FDA guidelines. It was found that PAHs exist in large quantity in highly exposed areas particularly the soil samples were extremely contaminated with PAHs. The highest average concentration of Naphthalene (260.85±165.64 μg/kg) was detected in samples from industrial areas. Conversely, the lowest average amount of Biphenanthrene (10.31±3.46 μg/kg) was found in samples from highways. Human and animal plasma samples were also found to carry PAHs in comparatively lower levels than soil and water.

The domestic Nili-Ravi water buffalo (Bubalus bubalis) is the best dairy animal contributing 68% to total milk production in Pakistan. In this study, we identified genome-wide single nucleotide polymorphisms (SNPs) to estimate various population genetic parameters such as diversity, pairwise population differentiation, linkage disequilibrium (LD) distribution and for genome-wide association study for milk yield and body weight traits in the Nili-Ravi dairy bulls that they may pass on to their daughters who are retained for milking purposes. The genotyping by sequencing approach revealed 13,039 reference genome-anchored SNPs with minor allele frequency of 0.05 among 167 buffalos. Population structure analysis revealed that the bulls were grouped into two clusters (K = 2), which indicates the presence of two different lineages in the Pakistani Nili-Ravi water buffalo population, and we showed the extent of admixture of these two lineages in our bull collection. LD analysis revealed 4169 significant SNP associations, with an average LD decay of 90 kb for these buffalo genome. Genome-wide association study involved a multi-locus mixed linear model for milk yield and body weight to identify genome-wide male effects. Our study further illustrates the utility of the genotyping by sequencing approach for identifying genomic regions to uncover additional demographic complexity and to improve the complex dairy traits of the Pakistani Nili-Ravi water buffalo population that would provide the lot of economic benefits to dairy industry.