Biochemical analysis of Hyalomma dromedarii salivary glands and gut tissues using SR-FTIR micro-spectroscopy.

Ticks are obligatory voracious blood feeders infesting diverse vertebrate hosts, that have a crucial role in the transmission of diverse pathogens that threaten human and animal health. The continuous emergence of tick-borne diseases due to combined worldwide climatic changes, human activities, and acaricide-resistant tick strains, necessitates the development of novel ameliorative tick control strategies such as vaccines. The synchrotron-based Fourier transform infrared micro-spectroscopy (SR-FTIR) is a bioanalytical microprobe capable of exploring the molecular chemistry within microstructures at a cellular or subcellular level and is considered as a nondestructive analytical approach for biological specimens. In this study, SR-FTIR analysis was able to explore a qualitative and semi-quantitative biochemical composition of gut and salivary glands of Hyalomma dromedarii (H. dromedarii) tick detecting differences in the biochemical composition of both tissues. A notable observation regarding Amide I secondary structure protein profile was the higher ratio of aggregated strands in salivary gland and beta turns in gut tissues. Regarding the lipid profile, there was a higher intensity of lipid regions in gut tissue when compared to salivary glands. This detailed information on the biochemical compositions of tick tissues could assist in selecting vaccine and/or control candidates. Altogether, these findings confirmed SR-FTIR spectroscopy as a tool for detecting differences in the biochemical composition of H. dromedarii salivary glands and gut tissues. This approach could potentially be extended to the analysis of other ticks that are vectors of important diseases such as babesiosis and theileriosis.

Emergent and Neglected Equine Filariosis in Egypt: Species Diversity and Host Immune Response.

Equine filariosis (EF) is a neglected vector-borne disease caused by nematode species belonging to the Onchocercidae and Setariidae families. Aside from their zoonotic potential, some species are responsible for serious health problems in equids worldwide, leading to significant economic difficulties. Here, we molecularly investigated equine blood samples (320 horses and 109 donkeys from Egypt) and four adult worms isolated from the peritoneal cavity of 5 out of the 94 slaughtered donkeys. In addition, quantitative enzyme-linked immunoassays (ELISAs) targeting circulating cytokines were used to identify whether the immunological profile of the infected animals is a Th1 (i.e., INF-gamma as indicator) or Th2 (i.e., IL-5 and IL-10 as indicators) response type. Overall, 13.8% and 0.3% of the donkeys and horses, respectively, were scored as positive for filaroid DNA. The 18S phylogeny revealed the occurrence of three different filaroid species, identified here as Mansonella (Tetrapetalonema) sp., Setaria digitata and Dirofilaria repens. Th1 (INF-gamma and IL-5) and Th2 (IL-10) immune response types were identified in equines infected with S. digitata and Mansonella (T.) sp., respectively. These results provide new data on the species diversity of EF in Egypt and extend knowledge of the downregulation of the protective immune response by the potentially zoonotic Mansonella (T) sp. There is an urgent need to implement control measures to preserve equine health and limit the propagation of these vector-borne filaroids in Egypt.

Fundamental Tick Vaccinomic Approach to Evade Host Autoimmune Reaction.

Ticks are obligate hematophagous ectoparasites that infect domestic animals, humans, and wildlife. Ticks can transmit a wide range of pathogens (viruses, rickettsia, bacteria, parasites, etc.), and some of those are of zoonotic importance. Tick-borne diseases have a negative economic impact in several tropical and subtropical countries. With climate change, tick distribution and tick-associated pathogens have increased. Currently, tick control procedures have more environmental drawbacks and there are pitfalls in vaccination process. Since vaccinations have helped to prevent several diseases and infections, several vaccination trials are ongoing to control ticks and tick-borne pathogens. However, autoimmune reactions to vaccinations are reported as an adverse reaction since vaccines were used to protect against disease in humans and animals. The antibodies against the vaccine antigen might harm similar antigen in the host. Therefore, in this chapter, we attempt to shed light on the importance of raising awareness of possible adverse events associated with vaccinations and the methods that should be used to address this problem. In silico and lab work should be performed ahead of the vaccination process to evaluate the vaccine candidates and avoid the vaccination opposing consequences.

Copro-microscopical and immunological diagnosis of cryptosporidiosis in Egyptian buffalo-calves with special reference to their cytokine profiles.

Cryptosporidiosis is considered to be one of the most devasting gastrointestinal diseases in calves. The aim of this study was to investigate Cryptosporidium parvum infection (C. parvum) in buffalo-calves with both copro-microscopic examination and enzyme linked immunosorbent assay (ELISA) using two C. parvum prepared antigens with regards to their cytokines profile; interferon- γ (IFN-γ), interleukin (IL)-12 and IL-14 to achieve a proper diagnosis. All collected buffalo- calves' fecal samples were examined by modified Ziehl-Neelsen staining technique. ELISA was performed to evaluate the diagnostic accuracy of the two C. parvum prepared antigens; crude whole oocyst (CWO) and crude sonicated oocyst (CSO) in detection of anti-C. parvum IgG in buffalo-calves' sera. As well, concentrations of INF-γ, IL-12 and IL-14 in the buffalo-calves' serum samples were estimated. The results revealed that the overall parasitological incidence of cryptosporidiosis was 40%. However, the serological diagnosis by ELISA assay showed 53.75% and 27.5% when using CWO and CSO antigen, respectively. Also, the diagnostic efficacy parameters of both antigens; CWO and CSO showed a significant high specificity (83.3%) achieved by CSO antigen and a high sensitivity (71.8%) by CWO antigen. The levels of INF-γ, IL-12 and IL-14 were significantly increased in positive Cryptosporidium infected group by both coprological and serological assays followed by the group which was positive for cryptosporidiosis by copro-microscopic examination only. The present study concluded that a combination of coprological and serological examination with reference to the cytokines profile is needed for proper diagnosis of cryptosporidiosis in buffalo-calves.

Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4).

BACKGROUND: The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. METHODS: BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). RESULTS: Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. CONCLUSIONS: The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.

Cell-mediated and humoral immune profile to hydatidosis among naturally infected farm animals.

BACKGROUND AND AIM: Cystic echinococcosis (CE) is a widespread parasitic disease caused by Echinococcus granulosus tapeworm infect different intermediate hosts including sheep, cattle, and camels. The intermediate host's immune response to the hydatid cyst is still conflict and complex. The current study was designed to evaluate the immune response in sera of hydatid naturally infected sheep, cattle, and camels in the form of features of inflammatory cell infiltrations, levels of Th1 and Th2 cytokines, besides the humoral specific immunoglobulin G (IgG) responses. MATERIALS AND METHODS: Thirty-nine sheep, 74 cattle, and 79 camels' sera were collected and considered as CE naturally infected and ten samples from each species were graded as non-infected. Lung specimens were collected for histopathological examination. The quantitative concentrations of tumor necrosis factor-α, interleukin (IL)-6, IL-4, and IL-10 were determined. Different antigens were prepared from hydatid cyst; hydatid cyst fluid of lung origin hydatid cyst fluid of liver origin, hydatid cyst protoscoleces of lung origin (HCP-g), hydatid cyst protoscoleces of liver origin, hydatid cyst germinal layer of lung origin, and hydatid cyst germinal layer of liver origin; and characterized by gel electrophoresis and Western blotting analysis. The total specific IgG level against E. granulosus infection was measured using an indirect enzyme-linked immunosorbent assay. RESULTS: The results indicated that the cellular immune response in the infected tissues was characterized by inflammatory cell penetration. The pro-inflammatory Th1 cytokine profile was predominant in infected animals in comparison with non-infected ones. However, the humoral immune response was seen as a high level of IgG in infected animals. The presented data approved that the HCP-g antigen could be considered as a delegate antigen for all other prepared antigens with an immunoreactive band at molecular weights 32 kDa. CONCLUSION: This study provides a fundamental insight into the events that manipulate cellular and humoral immune profiles in an intermediate host; sheep, cattle, and camel that naturally infected with CE. Hence, it was concluded that CE is a constant disease and confirm the reactivity Th1 in combating hydatid cyst. Besides, it could lead to the activation of the humoral immune response in the form of a high level of IgG.

Camel hydatidosis diagnostic kit: optimization of turnip and horseradish peroxidase conjugates using glutaraldehyde method.

Echinococcosis/hydatidosis is one of the most important parasitic zoonotic diseases in the world. Cystic echinococcosis increases public health and socio-economic concern due to considerable morbidity rates that give rise to elevated economic losses both in the public health part and in the farm animal field. The enzyme linked immunosorbent assay (ELISA) is consider the more accurate tool for diagnosis of hydatidosis in camels. In the present study, affinity purified Echinococcus granulosus (E. granulosus) antigens (APA) were purified from crude hydatide E. granulosus germinal layer proteins for detection of E. granulosus antibodies in infected camels, using affinity matrix (camel IgGs coupled to CNBr-activated Sepharose). The electrophoretic profile of the APA showed that it was separated into two bands; one major band of 130 kDa and one minor band at 55 kDa. These antigens were used successfully as specific coating antigenic proteins in detection of echinococcosis in camel. In a trial to prepare an anti-camel IgGs peroxidase conjugate; peroxidase enzyme was purified from turnip roots (TPOD) using ammonium sulfate precipitation and affinity chromatography on phenyl Sepharose CL-4B. The purified TPOD showed a major band at 35 kDa. Rabbit anti-camel IgG antibodies (AC IgGs) were prepared then purified using affinity chromatography on Protein G-Sepharose. The TPOD, and commercial HRP for comparison, enzymes were conjugated to AC IgGs using 1%, 5% and 10% glutaraldehyde. The results revealed that the HRP was much better than TPOD in conjugation with AC-IgG antibodies and the 10% glutaraldehyde concentration was the most efficient concentration with ELISA titer 1:50.

Immunity to gastrointestinal nematodes in ruminants: effector cell mechanisms and cytokines.

Gastrointestinal nematodes (GINs) of ruminants are prevalent and have major economic impacts worldwide. The insight studies of immune responses triggered against GINs are of great concern to understand interaction between host's immune system and parasite. T-helper 2 cytokines drive the effector cell mechanisms which include eosinophils and mast cells. The immune responses are controlled by Th2 secreted interleukins (IL); IL3, IL-4, IL-5, IL-9, IL-10 and IL-13. B-Cell immune response is incorporated in defense mechanisms developed against GINs specially immunoglobulins (Ig); IgA, IgE and IgG. The immune resistance of the infected host is presented by failure of larval establishment or hypobiosis, low worm burden and decreased female fecundity. The host-parasite interaction is a complex series that affected by host's genetic constitution, nutrition, age and physiological status. The GINs have different immune evasion mechanisms to improve their survival within the host. Also, management of the host influences GINs parasitism. Thus, the aim of this review is to highlight the hallmarks of immune responses that endorse GINs parasitism. The insights studies of the triggered immune responses developed against GINs will improve the appropriate protective immune strategy.

Efficacy of glutathione-S-transferase purified antigen of the gastro-intestinal nematode Haemonchus contortus in diagnosis of sheep haemonchosis.

Haemonchus contortus is one of the most pathogenic and economically important parasites of sheep. Different H. contortus antigens; crude somatic antigen (CSA), excretory/secretory antigen (ESA), crude larval antigen (CLA), glutathione-S-transferase antigen (GST) and recombinant protein (rhcp 26/23) were prepared and characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The antigens were immunologically evaluated through indirect enzyme linked immunosorbent assay (ELISA) for diagnosis of haemonchosis in experimentally and naturally infected sheep. Analysis of the resultant bands of SDS-PAGE demonstrated that 13, 6, 11, 2 and 1 protein bands from CSA, ESA, CLA, GST and rhcp 26/23, respectively and analysis of the resultant bands of western blot showed that 13, 6, 4 and 1 reactive bands detected from CSA, ESA, CLA and GST, respectively. The results of ELISA of different antigens revealed that sero-prevelance of CSA, ESA, CLA, GST and rhcp 26/23 were 78.51, 82.34, 85.319, 45.319 and 90.8% respectively, sensitivity were 100, 90, 100, 96.66 and 90%, respectively and specificity were 0, 70, 10, 70 and 6.66%, respectively with diagnostic potency were 50, 80, 55, 83.33 and 48.33%, respectively. Statistical analysis using Chi square test found that GST is the best one that can be used. The cross reactivity of GST antigen, crude Fasciola antigen and crude Moniezia antigen tested versus their homologous hyper immune sera at different dilutions using ELISA. The current study reported that GST antigen could be considered as a promising antigen for diagnosis of haemonchosis.

Evaluation of crude larval protein and recombinant somatic protein 26/23 (rHcp26/23) immunization against Haemonchuscontortus in sheep.

AIM: The aim of this study was to evaluate the potential possibility of crude larval and recombinant (rHcp26/23) antigens of Haemonchus contortus for immunization to control sheep hemonchosis. MATERIALS AND METHODS: A total of 21 lambs were divided into five groups. Lambs were immunized with larval and recombinant (rHcp26/23) proteins at day 0 and day 14 and after that challenged with 5000 infective larvae of H. contortus on day 42. An unvaccinated positive control group was challenged with L3 in the meantime. An unvaccinated negative control group was not challenged. RESULTS: Fecal egg count reduction taking after challenge for rHcp26/23 and larval antigens was 92.2% and 38.2%, respectively, compared with the positive control group. Vaccine incited protection in rHcp26/23 and larval immunization was reflected in significant (p<0.05) decreases in worm burden; 59.9% and 40.1%, respectively. CONCLUSION: Recombinant rHcp26/23 vaccine induced a partial immune response and had immune-protective effect against sheep hemonchosis.

Evaluation of different Haemonchus contortus antigens for diagnosis of sheep haemonchosis by ELISA and their cross reactivity with other helminthes.

Haemonchus contortus (H. contortus) remains important nematode that infecting sheep all over the world. Truthful diagnosis of haemonchosis needs reliable Enzyme linked immune sorbent assay test as well as the immuno-reactive protein profile of different prepared H. contortus antigens; larval (L), excretory secretory product (ESP) and adult somatic H. contortus (AS). The current study fulfilled that L antigen is the talented antigen for such serological diagnosis. Immunodominant band at molecular weight 57 kDa were answerable for highest specificity and accuracy of positive predictive value of this antigen. Moreover, the highest apparent prevalence value was 92 and 75% obtained by L and ESP antigens, respectively. The results of cross reactivity among AS, Monezia expansa (M. expansa) and Fasciola spp. revealed that AS antigen appeared major cross reactivity with other cestode and trematode. Best dilution of serum was (1:800) to rise above this phenomenon.

Th1/Th2 balance and humoral immune response to potential antigens as early diagnostic method of equine Strongylus nematode infection.

AIM: The aim of this study was to investigate the early diagnosis of strongyle infection based on early changes in Th1 and Th2 cytokines beside the diagnostic accuracy values and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting profiles using prepared strongyles antigens. MATERIALS AND METHODS: A total of 73 donkeys had a mean age of 4-32 years old were parasitologically examined for strongyle infection. The early changes in Th1 and Th2 cytokines were determined, and the diagnostic accuracy values and SDS-PAGE and western blotting profiles were performed using prepared strongyles antigens; crude somatic Strongylusvulgaris (CSS), excretory-secretory S. vulgaris (ESS), crude somatic Cyathostomins (CSC), and excretory-secretory Cyathostomins (ESC). RESULTS: The results revealed highest 437.04% and lowest 37.81% immunoglobulin G (IgG) in high and low egg shedder groups when using ESC and CSS antigens, respectively. Antibodies index for ESS and CSC were significantly higher in moderate egg shedder group while that for ESS and CSC, ESC was significantly higher in high egg shedder group. Tumor necrosis factor alpha (TNF-α)/interleukin-4 (IL-4) balance in S. vulgaris infected donkeys was approximately equal in apparently healthy, low and high egg shedder groups while TNF-α < IL-4 in moderate egg shedder. In Cyathostomins infected animals, TNF-α/IL-4 balance was approximately equal in apparently healthy group while it was low in moderate and high egg shedder groups. The diagnostic accuracy showed that the higher specificity (46.6%) and prevalence (95.40%) were recorded by CSS and ESC antigens, respectively. However, SDS-PAGE and western blotting profiling proved that the band at molecular weight 25 kDa is exhibited by CSS antigen. CONCLUSION: Combination of detecting level of TNF-α/IL-4 balance, CSS antigen and IgG concentration is good tool for appropriate diagnosis of such infection. More advancement research must be done concerning Th1/Th2 balance and cross-reactivity of S. vulgaris and Cyathostomins spp. at the base of serological and molecular investigation.

Serological and molecular diagnostic surveys combined with examining hematological profiles suggests increased levels of infection and hematological response of cattle to babesiosis infections compared to native buffaloes in Egypt.

BACKGROUND: Babesiosis threatens the development of the cattle and buffaloes industries in Egypt and improved control is needed. The main objectives of this study are surveying the presence of bovine babesiosis in distinct selected bovine and buffalo populations in Egypt using novel molecular and previously validated serological methods, while also comparing the occurrence of hematological alterations among Babesia infected cattle and buffalos. METHODS: A total of 253 and 81 blood samples from apparently healthy cattle and buffaloes, respectively, were randomly collected from diverse locations in Egypt. All samples were tested for Babesia bovis and B. bigemina infection using blood film examination, competitive ELISA (cELISA) and PCR. Novel semi-nested and nested PCR assays for the detection of B. bovis and B. bigemina respectively, were developed and used to analyze DNA extracted from bovine and buffalo samples. Hematological profiles were studied using a hematological analyzer. RESULTS: Blood films examination revealed 13.8% and 7.4% Babesia infection rates in cattle and buffaloes, respectively. However, in cattle, the cELISA detected 32.8%, 21.3% and 10.7% infection rates with B. bigemina, B. bovis and mixed infection, respectively. In addition, cELISA identified 22.2%, 22.2% and 6.2% infection rates with B. bigemina, B. bovis and mixed infection, respectively in buffaloes. The semi-nested PCR assay showed that 15% of the tested samples were positive for B. bovis in cattle, but just 3% in buffaloes. Infections with B. bigemina were also found in cattle (32.4%), but not in buffaloes upon nested PCR analysis. Sequencing analysis confirmed the identity of the PCR amplicons and showed that Egyptian genotypes of B. bigemina and B. bovis highly resemble sequences previously deposited in GenBank. Hemograms performed on the sampled animals revealed macrocytic hypochromic anemia associated with reduced platelet counts in infected cattle with babesiosis. In addition, marked increases in total leukocyte and granulocytic counts and decreases in lymphocytic counts were found in infected cattle. In contrast, no such hematological anomalies were found in presumably Babesia-infected buffaloes. CONCLUSIONS: Frequent occurrence of babesiosis among apparently healthy bovines in Egypt, suggests the need for appropriately designed prevalence studies in this country. Infected bovine, but not buffalo, populations often present hematological disorders compatible with intravascular hemolysis and thrombocytopenia.