The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells

Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future

Seroprevalence of Encephalitozoon cuniculi in Humans and Rabbits in China

Background: Encephalitozoon cuniculi is a microsporidian parasite commonly found in rabbits that can infect humans, causing encephalitozoonosis. Our objective in this study was to evaluate the seroprevalence of this parasite in rabbits and humans in China. Methods: Overall, 300 serum samples each from clinically healthy rabbit and hu-man were collected from three regions of China (Sichuan Province, Chongqing Municipality and Jilin Province) from January to September 2013 and tested for anti- E. Cuniculi antibodies using an ELISA. Results: An overall seroprevalence of E. cuniculi was recorded as 56/300 (18.76%) and 29/300 (9.76%) in rabbit and human sera, respectively. The seropositivity of rabbit samples collected from Jilin province was 41%, which was significantly higher (P<0.01) than Sichuan Province (9%) and Chongqing Municipality (6%). Three breeds of rabbit were used in the present study and antibody detection in Rex Rabbit was significantly (P<0.01) higher than Japanese White and New Zea-land Rabbit. In human, Jilin province was more prevalent (18%) followed by Si-chuan Province (6%) and Chongqing Municipality (5%). Conclusions The E. cuniculi was present and widespread among healthy rabbits and humans in China

Babesiosis: Current status and future perspectives in Pakistan and chemotherapy used in livestock and pet animals

Babesiosis is a protozoal disease affect livestock and pet animals such as cattle, buffaloes, sheep, goats, horses, donkeys, mules, dogs, and cats. It causes severe economic losses in livestock as well as in pet animals. A large number of dairy animals are imported in order to fulfill the demands of milk, milk, meat and its products. In addition, different pet animals are transported from Pakistan to various parts of the world, therefore, it is important to identify the current status and distribution of babesiosis throughout Pakistan in order to control the disease and draw attention for future research, diagnosis, treatment and control of this diseases. No work has been done on a complete review on up-to-date on blood protozoal disease burden in Pakistan. This article will provide about the complete background of babesiosis in ruminants, equines and pet animals, its current status, distribution, vectors in Pakistan and allopathic and ethnoveterinary treatments used against babesiosis. Babesiosis may be subclinical (apparently normal) and may be clinical with acute to chronic disease and sometimes fatal. Babesia is found and develops inside the erythrocytes (red blood cells). Clinically, it causes fever, fatigue, lethargy, pallor mucus membranes, malaise, cachexia, respiratory distress, jaundice, icterus, hemolytic anemia, hemoglobinuria, lymphadenopathy, chollangocytitis, hepatomegaly, and splenomegaly. Chemotherapy for babesiosis includes Imidocarb dipropionate, Diaminazine aceturate Atovaquone and Bupravaquone, Azithromycin, Quinuronium sulfate and Amicarbalidesio-thionate are most widely used. Supportive therapy includes multivitamins, fluid therapy, antipyretics intravenous fluids, and blood transfusions are used if necessary. In addition, there are certain ethnoveterinary (homeopathic) ingredients which having anti-babesial activity. As the resistance against these drugs is developing every day. New more specific long-lasting drugs should be developed for the treatment of Babesiosis. Further studies should be done on disease genome of different species of Babesia for vaccine development like malarial parasites

Prevalence and molecular detection of Babesia bigemina in water buffalo from southeastern region of Pakistan

Abstract Babesia spp. are tick-transmitted apicomplexan parasites that infect erythrocytes of a wide range of vertebrates including domestic animals, and can be associated with a considerable economic loss due to introduction of prophylactic measures, decreased production and rates of morbidity and mortality. An efficient detection assay needs to be set up for accurate diagnosis and prevention of disease. The present work is aimed at verifying the occurrence of Babesia bigemina infection in water buffaloes in the southeastern region of Pakistan by using both traditional blood smear and molecular techniques. A total of 100 blood samples were collected from tick-free and tick-infested animals (50 animals each group). The overall prevalence was recorded as 20% and 17% by using thin blood smear and PCR methods, respectively. Moreover, the analysis of infection in tick-infested and tick-free animals showed the infection rates of 36% and 4% by using blood smear method, and 30% and 4% with that of PCR method, respectively. Despite the higher prevalence rate recorded by blood smear method, which may be the result of non-specific identification of Babesia species, these results indicate that the PCR assay used in this study provides a useful tool for accurate diagnosis of the Babesia bigemina infection in bovines. Key words: Babesia bigemina, diagnosis, PCR, prevalence, blood smear techniqu

Characterization of Haemonchus contortus Excretory/Secretory Antigen (ES-15) and Its Modulatory Functions on Goat Immune Cells In Vitro

Small size excretory/secretory (ES) antigens of the Haemonchus contortus parasite have intense interest among researchers for understanding the molecular basis of helminths immune regulation in term of control strategies. Immunomodulatory roles of H. contortus ES-15 kDa (HcES-15) on host immune cells during host&ndash;parasite interactions are unknown. In this study, the HcES-15 gene was cloned and expression of recombinant protein (rHcES-15) was induced by isopropyl-&szlig;-d-thiogalactopyranoside (IPTG). Binding activity of rHcES-15 to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay (IFA) and immunohistochemical analysis showed that H. contortus 15 kDa protein localized in the outer and inner structure of the adult worm, clearly indicated as the parasite&rsquo;s ES antigen. The immunoregulatory role on cytokines production, cell proliferation, cell migration, nitric oxide (NO) production, apoptosis, and phagocytosis were observed by co-incubation of rHcES-15 with goat PBMCs. The results showed that cytokines IL-4, IL-10, IL-17, the production of nitric oxide (NO), PBMCs apoptosis, and monocytes phagocytosis were all elevated after cells incubated with rHcES-15 at differential protein concentrations. We also found that IFN-&gamma;, TGF-&beta;1, cells proliferation and migration were significantly suppressed with the interaction of rHcES-15 protein. Our findings indicated that low molecular ES antigens of H. contortus possessed discrete immunoregulatory roles, which will help to understand the mechanisms involved in immune evasion by the parasite during host&ndash;parasite interactions

Proteomic Analysis of the Excretory and Secretory Proteins of <i>Haemonchus contortus</i> (HcESP) Binding to Goat PBMCs <i>In Vivo</i> Revealed Stage-Specific Binding Profiles

<div><p><i>Haemonchus contortus</i> is a parasitic gastrointestinal nematode, and its excretory and secretory products (HcESPs) interact extensively with the host cells. In this study, we report the interaction of proteins from HcESPs at different developmental stages to goat peripheral blood mononuclear cells (PBMCs) <i>in vivo</i> using liquid chromatography-tandem mass spectrometry. A total of 407 HcESPs that interacted with goat PBMCs at different time points were identified from a <i>H</i>. <i>contortus</i> protein database using SEQUEST searches. The L₄ and L₅ stages of <i>H</i>. <i>contortus</i> represented a higher proportion of the identified proteins compared with the early and late adult stages. Both stage-specific interacting proteins and proteins that were common to multiple stages were identified. Forty-seven interacting proteins were shared among all stages. The gene ontology (GO) distributions of the identified goat PBMC-interacting proteins were nearly identical among all developmental stages, with high representation of binding and catalytic activity. Cellular, metabolic and single-organism processes were also annotated as major biological processes, but interestingly, more proteins were annotated as localization processes at the L₅ stage than at the L₄ and adult stages. Based on the clustering of homologous proteins, we improved the functional annotations of un-annotated proteins identified at different developmental stages. Some unnamed <i>H</i>. <i>contortus</i> ATP-binding cassette proteins, including ADP-ribosylation factor and P-glycoprotein-9, were identified by STRING protein clustering analysis.</p></div

SDS PAGE and western blot analysis of the Co-IP of <i>in vivo</i> interacting HcESPs with goat PBMCs at different developmental stages.

<p>Marker (M), Immunoprecipitates (IP) and normal rat serum Control (C). (A) L₄, (B) L5, (C) early adult and (D) late adult.</p