Dogs as victims of their own worms: Serodiagnosis of canine alveolar echinococcosis.

BACKGROUND: Besides acting as definitive hosts for Echinococcus multilocularis, dogs can become infected by the larval form of this parasite and thereby develop life-threatening alveolar echinococcosis (AE). Although AE is a zoonotic disease, most therapeutic and diagnostic approaches have been developed for human patients. In dogs, AE is typically diagnosed in the advanced stage of the disease when the parasitic mass has already caused abdominal distension. At that stage, complete resection of the parasitic mass is often impossible, leaving a guarded prognosis for the affected dogs. For humans, sensitive and specific diagnostic protocols relying on serology have been validated and are now widely used. In contrast, sensitive and specific laboratory diagnostic tools that would enable early diagnosis of canine AE are still lacking. The aim of the current study was to establish a serological protocol specifically adapted to dogs. METHODS: We tested several native and recombinant antigens (EmVF, Em2, recEm95, recEm18) in in-house ELISA, an in-house Western blot (WB), as well as a commercially available WB developed for serodiagnosing human AE (Anti-Echinococcus EUROLINE-WB®), using a panel of known status dog sera. RESULTS: RecEm95-antigen was revealed to be the most promising antigen for use in ELISA, demonstrating 100% (95% CI: 72-100%) sensitivity and 100% (95% CI: 93-100%) specificity in our study. The in-house WB using EmVF antigen performed as well as the recEm95-ELISA. The commercial WB also correctly identified all infected dogs, coupled with a specificity of 98% (95% CI: 91-100%). CONCLUSION: The recEm95-ELISA alone or in combination with either the in-house WB or the Anti-Echinococcus EUROLINE-WB® (IgG) with a minor modification should be considered as the best current approach for the serological diagnosis of dogs infected with the larval stage of E. multilocularis. However, larger studies with a focus on potentially cross-reacting sera should be undertaken to verify these findings.

Identification and partial characterization of a Salp15 homolog from Ixodes ricinus.

The immunomodulatory molecule Salp15 is originally described in Ixodes scapularis and has been shown to inhibit CD4 T cell activation. Many Salp15 homologs have been described from Ixodes species, and all were well conserved at C-terminal residues that seem to be essential for the function of the protein. In this study, a gene sequence was amplified from cDNA isolated from engorged female I. ricinus ticks, which was predicted to generate a protein of 12.3 kDa. The protein displayed distinct amino acid differences from previously described I. ricinus Salp15 homologs, with amino acid identity ranging between 46.6% and 93.9%. It was referred to as I. ricinus Salp15-like protein. The protein showed 48.1% sequence identity to I. scapularis Salp15. We analyzed the effect of the recombinant I. ricinus Salp15-like protein on the production of cytokines from human peripheral blood mononuclear cells stimulated with LPS. The recombinant protein exerted no effect on the production of TNF-α and IL-6, but the production of IL-10 was dose-dependently reduced. It can be concluded that I. ricinus Salp15-like protein exerts an immunomodulatory effect on the host. The inhibition of IL-10 production may possibly lead to a retardation of B cell activity.

Can Anaplasma ovis in small ruminants be neglected any longer?

Anaplasma species are obligate intracellular rickettsial pathogens transmitted by ticks with an impact on human and animal health. Anaplasma ovis infects sheep and goats in many regions of the world, and it can be diagnosed by different methods like Giemsa staining, PCR or competitive ELISA. In this study, a PCR based on the gene coding for major surface protein 4 (MSP-4) was used to examine field samples collected from sheep in different countries. Altogether, 1161 blood samples from Turkey (n = 830), Iraq (n = 195), Sudan (n = 96) and Portugal (n = 40) were examined, of which 31.4%, 66.6% 41.6% and 82.5%, respectively, were positive. This indicates high prevalence of A. ovis in the countries under investigation, and it can be assumed that the situation in other areas of the world might be similar. Thus, A. ovis should be considered as an important constraint of livestock production, and further efforts are needed to better understand the epidemiology and to implement suitable control measures.

Coinfection of sheep with Anaplasma, Theileria and Babesia species in the Kurdistan Region, Iraq.

Infections of small ruminants with Anaplasma, Theileria and Babesia species are widely distributed in the old world and are of great economic impact. In Iraq, data on disease occurrence in sheep caused by above-mentioned infectious agents are scarce. This study provides information on various haemoparasitic agents infecting sheep in the Kurdistan Region, Iraq, using molecular diagnostic tools. Altogether, 195 samples originating from three governorates in the Kurdistan Region, namely Duhok, Erbil and Sulaimaniya, were analysed. The following pathogens were identified: Anaplasma ovis (62.6%), Theileria ovis (14.35%), T. lestoquardi (7.7%), T. uilenbergi (5.6%) and Babesia ovis (1.5%). T. uilenbergi is detected for the first time in Iraq. Coinfection of sheep with different pathogens could be observed in this study, and it was found that 45 of 195 (23%) of the samples contained more than one pathogen. Even triple-positive samples were identified in 3% of the investigated animals. In conclusion, we confirm the coinfection of sheep with various haemoparasitic pathogen species in the Kurdistan Region of Iraq. Further investigations are needed to reveal the epidemiology of the diseases, the respective tick vectors, and, in the case of coinfection, pathogens' interaction and possible cross-protection.

Whole antigenic lysates of Ixodes ricinus, but not Der-p2 allergen-like protein, are potent inducers of basophil activation in previously tick-exposed human hosts.

The clinical suspicion of tick anaphylaxis is based on a history of the bite and occurs often during the warm season. Further arguments are the presence of natural hosts in the immediate environment and, eventually, the identification of the tick. The diagnosis is confirmed when immediate-type sensitization is shown by positive skin prick tests performed with specific tick extracts or the demonstration of specific IgE in vitro. In the current study, we hypothesize that hard tick-derived material contains potent inducers being able to promote basophil stimulation, which correlates with a sensitization immunological response following tick bites. To this end, biological material from two hard tick cell lines (IRE11 and IDE8 - derived from Ixodes ricinus and I. scapularis, respectively) as well as I. ricinus salivary gland and body lysates were used in a human basophil activation test (BAT) to analyse binding and cross-linking capacity of membrane-bound IgE, because basophils are one of the main effector cells of allergic reactions. Additionally, Der-p2 allergen-like gene from I. ricinus was recombinantly expressed as a 15-kDa histidine-tagged fusion protein, purified and included as a stimulus within the setup. Blood was drawn and submitted to BAT screening from a pool of 36 individuals, both bitten and who served solely as negative controls. We have found that seven subjects (19%), all of whom were at least two times tick-bitten, positively reacted to the aforementioned stimuli, whereas the reactivity level of the ones bearing single bites proved to be within the normal range. Moreover, no significant upregulation of the assessed basophil activation marker was detected in the case of Der-p2, except a faint reaction at high dosages. We conclude that at least two tick bites of the human host must occur in order to induce significant basophil activation.

Development of a competitive ELISA for detection of Theileria annulata infection.

In previous studies, Theileria annulata surface protein (TaSP) was identified as an immunodominant antigen and successfully used to develop and validate a recombinant-protein-based ELISA for the detection of circulating antibodies in serum of T. annulata-infected animals. In this study, the same antigen was used to develop a competitive ELISA (cELISA) using a monoclonal antibody that was found to bind to TaSP. The cELISA accurately differentiated T. annulata-infected from uninfected animals and demonstrated a satisfactory performance with a calculated sensitivity and specificity of 77.4% and 100%, respectively. Thus the test proved its suitability for the diagnosis of tropical theileriosis and has application for use in serological surveys to monitor the prevalence of the disease or identify carrier animals with high specificity.