Prevalence of Toxoplasma gondii oocysts through Copro-PCR in cats at Pet Center (UVAS), Lahore, Pakistan.

Toxoplasmosis is a major zoonotic disease of warm-blooded animals caused by Toxoplasma gondii. Cats are the only definitive host and they excrete environmentally resistant T. gondii oocysts in their faeces. Coproscopy was used to detect oocysts of enteric coccidians and then Copro-PCR was employed to test specifically for T. gondii in 470 cat samples. The prevalence of T. gondii oocysts was 2.3% (11/470) based on PCR. We observed 15 (3.2%) of 470 samples positive for coccidian oocysts by microscopy. The presence of Copro-DNA of T. gondii was found significantly higher (p<0.05) in males than females. We tested 11 samples of T. gondii oocysts in which 9 samples were from coccidian oocysts positive samples and 2 samples from negative faecal samples. Our results showed that PCR is the reliable method for the detection of faecal oocysts of T. gondii in cats as compared to microscopy. As per our knowledge, ours is first study for Copro-PCR prevalence of cats' T. gondii oocysts excretion in Pakistan.

Induction of specific humoral immune response in mice immunized with ROP18 nanospheres from Toxoplasma 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 ß-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.

FIRST REPORT OF TRYPANOSOMA EVANSI INFECTION (SURRA) IN A PUMA (FELIS CONCOLOR) OF LAHORE ZOO, PAKISTAN.

The blood protozoan Trypanosoma evansi, which is transmitted by biting flies, is frequently neglected due to subclinical infections. This report describes a case of trypanosomiasis due to T. evansi in a 9-yr-old male puma (Felis concolor) housed at the Lahore Zoo in Pakistan. Early in January 2015, this male puma presented with chronic lethargy, weight loss, incoordination, hyperthermia, anorexia, sunken eyes, and unthriftiness. Microscopic examination of Giemsa-stained blood smears showed numerous Trypanosoma parasites. The puma was treated with diminazene aceturate subcutaneously twice. A few days later, a blood smear examination showed absence of trypanosomes. Five months later the cat presented with acute epistaxis and died. Postmortem examination showed emaciation, pale liver and kidneys, and hemorrhages on the spleen. Examination of a blood smear taken at the time of death showed numerous Trypanosoma parasites. PCR testing confirmed the presence of Trypanosoma DNA. DNA sequencing of two amplicons confirmed the presence of Trypanosoma in the blood smears with a 98-99% identity with the previously identified GenBank sequences. A phylogenetic tree was then constructed. Further studies are needed to improve our knowledge about the epidemiology and pathogenesis of T. evansi infection in wild animal species.

Epidemiology of Trypanosomiasis in Wildlife-Implications for Humans at the Wildlife Interface in Africa.

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.

Population demographic history and population structure for Pakistani Nili-Ravi breeding bulls based on SNP genotyping to identify genomic regions associated with male effects for milk yield and body weight.

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.

Economic Significance of Tropical Theileriosis on a Holstein Friesian Dairy Farm 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.

Induction of Th1 type-oriented humoral response through intranasal immunization of mice with SAG1-Toxoplasma gondii polymeric nanospheres.

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 ß-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.

DNA Amplification Techniques for the Detection of Toxoplasma gondii Tissue Cysts in Meat Producing Animals: A Narrative Review Article.

BACKGROUND: Toxoplasma gondii is an intracellular parasite, which infects one-third population of world. Humans and animals acquire infection by ingesting oocytes from feces of cats or by meat of other animals having cysts that may lead to congenital, ocular or cephalic toxoplasmosis. Either it is important to detect T. gondii from meat of food animals from retail shops or directly at slaughterhouses, which is meant for export. METHODS: The current research was done without time limitation using such terms as follows: "Toxoplasma gondii", "Meat", "Tissue cyst", "PCR", "LAMP", "Screening" and "Immunological assay" alone or in combination, in English language. The used electronic databases for searching included as follows: Pub-Med, Scopus, Google Scholar, Web of Science and Science Direct. The searches were limited to the published papers to English language. RESULTS: Sensitivity of different molecular techniques for diagnosis of Toxoplasma is real-time PCR > LAMP > conventional PCR. In addition to these DNA analysis tools, bioassay in mice and cats is considered as "gold standard" to detect T. gondii. CONCLUSION: This review article will help the readers for grasping advantages and limitations of different diagnostic tools for screening meat samples for T. gondii. This review also makes bibliography about the type of meat sample to be processed for diagnosis and different primers or sequences to be targeted for T. gondii by number of researches for its detection from meat or tissue sample using DNA amplification techniques.