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.

Toxoplasmosis is a globally distributed disease of warm-blooded animals. It is caused by the opportunistic parasite Toxoplasma gondii (T. gondii). One-third of the global human population is believed to be infected with T. gondii. Cats serve as final host of T. gondii and are the main source of contamination of soil and water. This study aimed to detect genotypes of T. gondii in cats. Fecal samples (n = 400) were collected from districts of South Punjab (Khanewal and Sahiwal), and were processed by polymerase chain reaction (PCR) followed by sequencing and phylogenetic analysis. The obtained oligonucleotide sequences (T. gondii) were submitted to the GenBank database, and the evolutionary tree was constructed using MEGA-X software. Seven fecal samples (3.5%) from cats were positive. Five out of thirteen fecal samples (38.46%) found to be positive for T. gondii with microscopy were confirmed by PCR. After phylogenetic analysis with 3 clonal types and atypical strains, isolates of T. gondii in current study were more closely linked to a typical strain (AF249696). Besides genotyping from cats, seroprevalence from humans and ruminants is still considered to be the best and easiest way to identify the Toxoplasma. Blood samples were collected from sheep and goats (n = 2000 each), and human blood samples (n = 400) were collected from the same vicinity. Seroprevalence was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit. In Khanewal, the blood samples of 292 goats (29.2%) and 265 sheep (26.5%), and 6 fecal samples from cats (3%) were positive. Out of 200 human blood samples, 52 were positive, with a seroprevalence of 26%. In the Sahiwal district, the blood samples from 49 humans, 235 sheep and 348 goats were positive, with seroprevalence of 24.5%, 23.5% and 34.8%, respectively. The present study revealed the current circulating genotype of T. gondii from cats in the districts Khanewal and Sahiwal and the seroprevalence of the organism in small ruminants and humans living in the same vicinity. Further genotype analyses of the organism from ruminants and humans are needed.

Canine babesiosis is a serious threat to dogs’ health worldwide, caused by the intra-erythrocytic Babesia species. The present study was carried out in pet dogs presented at three clinics of Lahore and one of Narowal in Punjab, Pakistan. Two hundred blood samples (50 from each clinic) were collected and screened by microscopy for Babesia spp. Out of 200 samples, 84 (42%) were found to be positive for babesiosis. The highest number of positive cases (50%) was recorded in dogs at Narowal clinic. Non-significant variation (p > 0.05) was observed in the prevalence of babesiosis in dogs in relation to sex and age. Positive samples were further confirmed by a polymerase chain reaction using 18S-rRNA genus-specific and species-specific primers. Amplicons were further analyzed by nucleotide sequencing for genetic diversity. Babesia canis and gibsoni were confirmed by genome sequencing in all diseased dogs. These isolates closely resembled each other, but differed from previous reported strains. In conclusion, pet dogs suffering from babesiosis were infected with B. canis and gibsoni, while in other countries, other Babesia species are also prevalent.

Background and Aim: Ticks are blood-feeding ectoparasites that transmit pathogens to animals and humans. One of the most important hard ticks in animals is Rhipicephalus microplus, which transmits Babesia and Anaplasma spp. Although many potential tick vaccine candidates have been identified, no effective vaccine that can provide sterile immunity against R. microplus tick infestations has been developed. This study aimed to design a construct using different computational tools to identify and predict immunogenic epitopes within protein sequences and to prepare a messenger RNA (mRNA) vaccine against R. microplus based on lipid nanoparticles (LNPs). Materials and Methods: The R. microplus proteins (Bm86, Subolesin, and ATAQ) were selected and their consensus sequence was obtained from the National Center for Biotechnology Information in FASTA format. The Immune Epitope Database and Analysis Resource (IEBD) server was used for the prediction of helper T-cell epitopes, the NetCTL 1.2 server was used to predict cytotoxic T-cell epitopes, and the ABCpred server was used for B-cell epitope prediction. Antigenicity testing, allergenicity assessment, and toxicity screening were immuno-informatic techniques used to identify potent epitopes within protein sequences. The multi-epitope construct was prepared and cloned into the pVAX1 plasmid. Plasmids were transformed in compatible competent cells, and restriction analysis was performed. After restriction analysis of the transformed plasmid, in vitro transcription was performed to prepare mRNA. The mRNA was purified, quantified, and converted into complementary DNA, and gene-specific primers were used to confirm the in vitro transcription of mRNA. A mixture of four lipids containing 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), Distearoylphosphatidylcholine (DSPC, cholesterol, and 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DMG PEG-2000 was used to prepare LNPs. LNPs were characterized using a scanning electron microscope, Zeta potential, and Zeta Sizer tests. Results: More than 1000 epitopes were predicted, from which only nine helper T-lymphocytes, 18 cytotoxic T-lymphocytes, and nine B-cell epitopes of all three proteins were selected with high antigenic scores of 0.958 for Bm86, 0.752 for Subolesin, and 0.964 for ATAQ, respectively. An adjuvant was used to enhance immune responses, all of which were linked to one another using GPGPG, AAY, and KK linkers, respectively. The physiochemical properties predicted that the instability index of the construct would be <40%, indicating that the construct is stable. Plasmids were transformed in compatible competent cells, and white-transformed colonies were observed. Restriction analysis was performed, DNA was transcribed into mRNA, and LNPs were prepared and characterized. Conclusion: More than 1000 epitopes were predicted using immune informatic tools, and only high-scoring epitopes were selected. A multi-epitope construct was designed using bio-informatic tools, and its physicochemical properties were predicted. The design construct was inserted into the pVAX1 plasmid, and in vitro transcription was performed to prepare the mRNA. LNPs of mRNA were prepared and characterized to be used as vaccines. It was found that LNPs were stable and nanometer-sized. Keywords: immuno-informatic tools, lipid nanoparticles, multiepitope construct, Rhipicephalus microplus.

Coccidiosis is a protozoan disease that is characterized by diffuse diarrhea, dehydration, emaciation accompanied by moderate morbidity and mild mortality in animals and birds. The current study targeted the molecular characterization of Eimeria isolates in captive deer from different localities in Lahore. The host species was the Cervidae family, such as Hog deer (Axis porcinus) and Punjab urial (Ovis aries vignei). The Eimeria crandallis was isolated from zoo animals. The DNA was extracted from oocysts and amplified by using reported oligonucleotide primers that exhibited the 809 bp product. These were analyzed by using the small subunit 18S rRNA gene-based evolutionary relationship with 36 other Eimeria species reported in caprine, cervinae, bovines, avians, and rodents. Light microscopic examination exhibited 3.29% (7/213) Eimeria-positive fecal samples with morphological features, including sub-spherical forms, the presence of micropyle with polar cap, and oocysts diameters (μm) ranging from 24.32 ± 1.61 to 18.94 ± 1.51. The phylogenetic tree constitutes four distinct clusters with relatively higher values. The evolutionary network showed that sequences were clustered in the monophyletic group of Eimeria species reported in caprine and cervinae. The nucleotide and amino acid sequence similarity matrix analysis exhibited 99.5–99.9% identity of the study isolates with Eimeria crandallis (AF336339). This study provides relevant baseline data to develop strategic control measures for coccidiosis in zoo animals. However, further investigations are required to place the hog deer and Punjab urial-derived E. crandallis into the caprine-originated cluster.