Carlos Gutiérrez

Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study

Summary Background A new betacoronavirus—Middle East respiratory syndrome coronavirus (MERS-CoV)—has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. Methods We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. Findings 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralising antibody titres varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralisation by bovine coronavirus antibodies. Interpretation MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection. Funding European Union, European Centre For Disease Prevention and Control, Deutsche Forschungsgemeinschaft.

Camel trypanosomosis in the Canary Islands: assessment of seroprevalence and infection rates using the card agglutination test (CATT/T. evansi) and parasite detection tests.

Trypanosomosis due to Trypanosoma evansi (surra) is a major enzootic disease of the dromedary camel. Thus, the purpose of the present study was to assess seroprevalence and infection rates in the Canary Islands using antibody(-card agglutination test-CATT/T. evansi) and parasite detection tests (micro-Haematocrit Centrifugation technique, Giemsa stained blood smears, microscopic examination of lymph node aspirates and mouse inoculation). PCV was also determined. 745 dromedary camels (483 females and 262 males) were examined. Trypanosomes were detected in seven animals. 36 animals yielded CATT positive results while 709 animals were negative. All parasitologically positive animals were also CATT positive. Results showed a good correlation between CATT positive and low PCV and a higher seroprevalence in older animals. Trypanocidal drugs have not been registered in Spain and, consequently, if vigilance is not exercised the prevalence could be increased in the future.

Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

Trypanosoma evansi: recent outbreaks in Europe.

Here, two recent outbreaks of Trypanosoma evansi infection in mainland France and Spain associated with the importation of dromedary camels from the Canary Islands, are reported. The disease is well-known on the Archipelago since 1997 and many efforts have been made towards control and eventual eradication, but some areas still remain affected. Both mainland outbreaks were controlled by means of massive treatments and monthly serological, parasitological and molecular (PCR) evaluations carried out by Valencian Regional Animal Health laboratory and by CIRAD, Montpellier, respectively. Possible causes for the persistence of the parasite in a small area of the Canaries are discussed. T. evansi must be included among the animal health conditions for international trade within the European Union as well as many other countries. Moreover, procedures including diagnosis, curative or preventive treatment and quarantine should be established to insure the status of the animals moving from a country to another.

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. KD of CA52–DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs’ potentialities to target, to purify, and to modulate the function of cellular markers.

Trypanosomosis in Goats

Abstract:  Trypanosomosis is a major constraint on ruminant livestock production in Africa, Asia, and South America. The principal host species affected varies geographically, but buffalo, cattle, camels, and horses are particularly sensitive. Natural infections with Trypanosoma congolense, T. vivax, T. brucei, and T. evansi have been described in goats. Trypanosomosis in goats produces acute, subacute, chronic, or subclinical forms, being T. vivax, T. congolense, and T. evansi, the most invasive trypanosomes for goats. However, the role of goats in the epidemiology of trypanosomosis is largely discussed and not well understood. Thus, it has commonly been assumed that trypanosomosis presents a subclinical course and that goats do not play an important role in the epidemiology of the disease. This can partially be due to parasitemia caused by trypanosomes which has been considered low in goats. However, this assumption is currently undergoing a critical reappraisal because of goats may also serve as a reservoir of trypanosome infection for other species, including the human beings in the case of T. brucei rhodesiense. The present article describes the current status of trypanosomosis in goats in Africa, Asia, and South America. Pathogenesis, clinical features, diagnosis, and treatment of the different trypanosomes are also described. The possible role in the epidemiology of the disease in the different areas is also discussed.

Structure of Cyclin G-Associated Kinase (Gak) Trapped in Different Conformations Using Nanobodies.

GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent ‘off-target’ of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK.

A transversal study on antibodies against selected pathogens in dromedary camels in the Canary Islands, Spain.

The Canary Islands contain the most important dromedary camel (Camelus dromedarius) population in the European Union and are the main export point of dromedaries to continental Europe and Latin America. We investigated the presence of antibodies against relevant disease agents in 100 Canarian camel sera. Selected blood samples of the same animals were also tested by PCR. Sera were tested for antibodies against Bluetongue virus (BTV; 0%), Bovine Viral Diarrhoea virus (BVDV; 0%), Camelpox virus (CPV; 8% by serum neutralization, 16% by ELISA), Peste des Petits Ruminants virus (PPRV, 0%), Rift Valley Fever virus (RVFV; 0%) and West Nile Fever virus (WNV; 3%), the bacterial pathogens Anaplasma sp. (3%), Brucella sp. (1%), Coxiella burnetii (19%), Mycobacterium avium paratuberculosis (MAP; 22%), Mycobacterium tuberculosis complex (MTC; 10%) and Rickettsia sp. (83%), and the parasites Toxoplasma gondii (36%) and Neospora caninum (86%). The most remarkable findings were the detection of antibodies against CPV and the high antibody prevalence against C. burnetii, Rickettsia sp., T. gondii and N. caninum. By PCR, we found no C. burnetii, N. caninum and Anaplasma sp. DNA in the tested samples. However, Rickettsia sp. DNA was detected in six antibody positive tested samples. These results should be taken into consideration in order to implement adequate control measures and avoid a potential dissemination of infections to other territories.

Presence of Trypanosoma theileri in Spanish Cattle

Trypanosoma theileri (Laveran, 1902) has been diagnosed in many countries and is commonly considered as a nonpathogenic hemoparasite, although some authors have described clinical signs in cattle infected with T. theileri. In April and May, 2005, 12 blood samples were received at the Exopol Diagnostic Laboratory (Zaragoza, Spain) from a Spanish bull‐fighting farm located at Seville province. Clinical exploration of the animals revealed fever, progressive weight loss, anemia, and frequent recumbent position. Microscopic examination showed Theileria spp. in all cases (12), and in four of them, T. theileri was also observed. The clinical picture observed in the animals could be compatible with T. theileria infection. However, the contribution of T. theileri to the clinical signs seen at least in four cases is unknown. Further studies are necessary to determine the pathogenicity of T. theileri in the different animal species. To our knowledge, this is the first isolation of T. theileri in Spain.

Clinical, Hematological, and Biochemical Findings in an Outbreak of Abortion and Neonatal Mortality Associated with Trypanosoma evansi Infection in Dromedary Camels

Abstract:  This article presents the clinical and laboratorial findings in an outbreak of abortions and high neonatal mortality attributable to Trypanosoma evansi infection in camels. A total of 16 females were diagnosed, 2 of which showed moderate signs of chronic form, particularly hyporexia and intolerance to exercise. The main laboratorial findings were regenerative anemia (hemolytic anemia), lymphocytic and monocytic leukocytosis, hyperproteinemia, hyperglobulinemia, hypoglycemia, serum urea increased, and serum iron decreased. The most characteristic finding in the examined females would be the uremia, probably due to the higher protein metabolism.