While both human and animal trypanosomiasis continue to present as major human and animal public health constraints globally, detailed analyses of trypanosome wildlife reservoir hosts remain sparse. African animal trypanosomiasis (AAT) affects both livestock and wildlife carrying a significant risk of spillover and cross-transmission of species and strains between populations. Increased human activity together with pressure on land resources is increasing wildlife–livestock–human infections. Increasing proximity between human settlements and grazing lands to wildlife reserves and game parks only serves to exacerbate zoonotic risk. Communities living and maintaining livestock on the fringes of wildlife-rich ecosystems require to have in place methods of vector control for prevention of AAT transmission and for the treatment of their livestock. Major Trypanosoma spp. include Trypanosoma brucei rhodesiense, Trypanosoma brucei gambiense, and Trypanosoma cruzi, pathogenic for humans, and Trypanosoma vivax, Trypanosoma congolense, Trypanosoma evansi, Trypanosoma brucei brucei, Trypanosoma dionisii, Trypanosoma thomasbancrofti, Trypanosma elephantis, Trypanosoma vegrandis, Trypanosoma copemani, Trypanosoma irwini, Trypanosoma copemani, Trypanosoma gilletti, Trypanosoma theileri, Trypanosoma godfreyi, Trypansoma simiae, and Trypanosoma (Megatrypanum) pestanai. Wildlife hosts for the trypansomatidae include subfamilies of Bovinae, Suidae, Pantherinae, Equidae, Alcephinae, Cercopithecinae, Crocodilinae, Pteropodidae, Peramelidae, Sigmodontidae, and Meliphagidae. Wildlife species are generally considered tolerant to trypanosome infection following centuries of coexistence of vectors and wildlife hosts. Tolerance is influenced by age, sex, species, and physiological condition and parasite challenge. Cyclic transmission through Glossina species occurs for T. congolense, T. simiae, T. vivax, T. brucei, and T. b. rhodesiense, T. b. gambiense, and within Reduviid bugs for T. cruzi. T. evansi is mechanically transmitted, and T. vixax is also commonly transmitted by biting flies including tsetse. Wildlife animal species serve as long-term reservoirs of infection, but the delicate acquired balance between trypanotolerance and trypanosome challenge can be disrupted by an increase in challenge and/or the introduction of new more virulent species into the ecosystem. There is a need to protect wildlife, animal, and human populations from the infectious consequences of encroachment to preserve and protect these populations. In this review, we explore the ecology and epidemiology of Trypanosoma spp. in wildlife.

Bovine anaplasmosis is endemic in Pakistan where it reduces livestock productivity and leads to high mortality, especially in young animals. This study was aimed to identify the potential risk factors responsible for the occurrence and spread of anaplasmosis in cattle and buffaloes for the first time in Pakistan. A total of 900 (cattle = 479, buffalo = 421) blood samples were collected irrespective of age and sex from three distinct zones of Khyber Pakhtunkhhwa (KP) province of Pakistan. Polymerase chain reaction (PCR) technique was used for the molecular detection of anaplasmosis. Data collected on a piloted questionnaire including 11 predicting variables which were analyzed using R-statistical software, and association between the dependent and independent variables was assessed using univariable analysis. Automated and manual approaches were exercised, producing comparable models. Key risk factors identified in all the approaches included species of the animal, breed of animal, sex of animal, tick infestation status, previous tick history, tick control status, and acaricides used (odds ratio > 1). The 611 bp DNA fragment specific for 16S rRNA gene of Anaplasma spp. was produced from 165 samples. The samples were confirmed for anaplasmosis through sequencing and BLAST queries. The findings of the current study conclude that by enhancing the protective measures to control the identified risk factors can reduce the spread of anaplasmosis in Pakistan.

The dairy industry in Pakistan is booming, and investors are anxious to fund dairy farms that are using high-milk-producing (exotic) cattle breeds such as Holstein Friesians that are not native to the country. Unfortunately, the benefits of increased milk production do not provide resistance to pathogens present in regions where the exotic breeds are introduced. Therefore, the current study was conducted to evaluate the economic impact of Theileria annulata on a commercial Holstein Friesian dairy farm in the District of Ranjanpur, in the Province of Punjab, Pakistan. The economic impact of T. annulata infection was calculated for cattle with subclinical and clinical theileriosis. Losses were estimated based on milk production, morbidity, mortality, and tick control costs (organophosphate sprays). Animals were classified into groups after screening for mastitis, teat abnormality, abnormal parturition, intestinal parasites, and hemoparasites ( T. annulata, Babesia spp., and Anaplasma spp.). Microscopy was done for hemoparasites and intestinal parasites. PCR was used to confirm microscopic identification of T. annulata. Animals were classified into 3 groups: group A (normal), group B (subclinical theileriosis), and group C (acute theileriosis). Hemoparasites were observed microscopically in 28.7% of cows. Theileria annulata was found in 8%, and the herd incidence (new cases) of T. annulata was 2.8%. Milk production, animal rectal temperature, and body condition scores between group A and groups B and C were significantly different ( P < 0.05). But the enlargement of sub-scapular lymph node and interval of body condition score of the 3 groups were not significant ( P > 0.05). The total expenditure incurred due to theileriosis was US $74.98 per animal and 13.83% of total farm costs. Hence theileriosis caused significant economic loss of US $18,743.76 (0.02 million) on this Holstein Friesian dairy.

About one-third of the world population is prone to have infection with T. gondii, which can cause toxoplasmosis in the developing fetus and in people whose immune system is compromised through disease or chemotherapy. Surface antigen-1 (SAG1) is the candidate of vaccine against toxoplasmosis. Recent advances in biotechnology and nano-pharmaceuticals have made possible to formulate nanospheres of recombinant protein, which are suitable for sub-unit vaccine delivery. In current study, the local strain was obtained from cat feces as toxoplasma oocysts. Amplified 957 bp of SAG1 was cloned into pGEM-T and further sub-cloned into pET28-SAG1. BL21 bacteria were induced at different concentrations of isopropyl β-d-1-thiogalactopyranoside for the expression of rSAG1 protein. An immunoblot was developed for the confirmation of recombinant protein expression at 35 kDa that was actually recognized by anti-HIS antibodies and sera were collected from infected mice. PLGA encapsulated nanospheres of recombinant SAG1 were characterized through scanning electron microscopy. Experimental mice were intraperitoneally immunized with rSAG1 protein and intra-nasally immunized with nanosphere. The immune response was evaluated by indirect ELISA. In results intra-nasally administered rSAG1 in nanospheres appeared to elicit elevated responses of specific IgA and IgG2a than in control. Nanospheres of rSAG1 are found to be a bio-compatible candidate for the development of vaccine against T. gondii.

Toxoplasmosis is one of the most common zoonotic protozoal diseases. Recent advances in biotechnology have produced recombinant protein, which are immunogenic, and progress in nano-pharmaceutics has generated encapsulated protein in nanospheres, which are suitable for vaccine delivery. DNA was extracted from Toxoplasma gondii oocysts and was confirmed through nested PCR and sequencing. The 1665 bp of ROP18 was cloned into the easy vector system: pGEM-T by the T-A cloning method. DH5α bacteria were transfected with pGEM-ROP18. ROP18 was subcloned from pGEM-ROP18 into pET28-ROP18. BL21 bacteria were transfected with pET28-ROP18. Thus, rROP18 protein was expressed in BL21 bacteria by induction at different concentrations of isopropyl β-D-1-thiogalactopyranoside. Protein expression was confirmed through SDS-PAGE and Western blotting. The immunoblot of rROP18 was recognized by anti-HIS antibodies and sera from infected mice at 67 kDa. Recombinant ROP18 protein was encapsulated in nanoparticles with PLGA and was characterized through scanning electron microscopy. Intraperitoneal immunizations with rROP18 protein and intranasal immunization of nanospheres were carried out in mice, and the immune response was detected by ELISA. Results showed that rROP18 in nanospheres administered intra-nasally elicited elevated responses of specific IgA and IgG2a as compared to groups inoculated intra-nasally with rROP18 alone, or injected subcutaneously with rROP18 in montanide adjuvant. It was concluded that nanospheres of ROP18 would be a non-invasive approach to develop vaccination against T. gondii. Further experiments are needed to determine the cellular response to these nanospheres in a mouse model for chronic toxoplasmosis.