An ELISA for the diagnosis of Babesia ovis infection utilizing a synthetic, Babesia bovis-derived antigen

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of Babesia ovis antibodies is described. In an initial study, a crude Babesia bovis antigen and a synthetic B. bovis-derived antigen (designated 11C5) were used to screen 46 B. ovis-positive and 55 negative sheep sera. A 95% correlation between the two antigenic preparations was found with the positive sera; no negative sera gave positive reactions. The synthetic antigen was then used in the screening of 1466 sera collected from sheep from 18 regions of Turkey. A high incidence of B. ovis-positive reactions was found from all regions (60-80%) in sheep over 1 year old, while from two smaller samples the incidence in young sheep was much less (28 and 52%). This test is superior to existing ones because the synthetic antigen can be produced in a highly reproducible state, is specific and is stable over extended periods of time.     

A novel 57-kDa merozoite protein of Babesia gibsoni is a prospective antigen for diagnosis and serosurvey of canine babesiosis by enzyme-linked immunosorbent assay

We isolated a novel single copy gene encoding a 57-kDa merozoite protein of Babesia gibsoni (BgP57). The nucleotide sequence of the cDNA was 2387 bp with an open reading frame (ORF) of 1644 bp encoding a 57-kDa predicted polypeptide having 547 amino acid residues. The recombinant BgP57 (rBgP57) without a predicted signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein. Western blotting showed that the corresponding native protein was 57-kDa, consistent with molecular weight of predicted mature polypeptide. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP57 detected specific antibodies in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with antibodies to B. canis sub-species and closely related apicomplexan parasites indicating that the rBgP57 was a specific antigen for B. gibsoni antibodies. The diagnostic performance of ELISA based on rBgP57 using 107 sera from B. gibsoni-naturally infected dogs was the same as the previously identified rBgP32 but performed better than the previously studied rBgP50. Although, seminested PCR detected higher proportions (82%) of positive samples than the ELISAs, the Mcnemar's chi-square test showed that there was no significant difference in relative effectiveness of rBgP57-ELISA and seminested PCR (chi(2)=2.70; P=0.1003) in identifying positive samples. The rBgP57-ELISA when used in combination with rBgP32-ELISA and rBgP50-ELISA appeared to improve sensitivity of the rBgP57-ELISA for detection of B. gibsoni antibodies. Overall, the rBgP57-ELISA and seminested PCR when used in combination, could improve epidemiological surveys and clinical diagnosis of B. gibsoni infection.     

Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-γ, IL-2, IL-4, and IL-10, as well as the percentage of CD4(+) cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice.     

Purification of a 28 kDa Babesia (Theileria) equi antigen and a 29 kDa spurious erythrocyte antigen from in vitro culture through ion exchange chromatography.

An extract of in vitro cultivated Babesia equi was fractionated using a MonoQ anion exchange column. Separation of a 28 kDa B. equi antigen from a 29 kDa spurious erythrocyte antigen, both of which were intensely immunoreactive, was achieved by chromatography of the infected erythrocyte proteins. Using tricine-SDS-PAGE, the 28 kDa antigen of B. equi showed multiple band resolution, while the 29 kDa antigen was consistently resolved as a single band. The 29 kDa antigen was identified in both infected and non-infected erythrocyte extracts. Moreover, B. equi antiserum recognised this antigen in the non-infected erythrocyte extract, and conversely serum from horses not infected with babesia detected the antigen in infected erythrocyte extract. This 29 kDa antigen could represent a horse erythrocyte isoantigen. The purified 28 kDa antigen is specifically recognised by B. equi antisera and therefore could be useful for the production of the recombinant replica and to employ these in further test systems.     

Antibodies to foot-and-mouth disease virus infection associated (VIA) antigen: use of a bioengineered VIA protein as antigen in an ELISA

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to foot-and-mouth disease (FMD) virus infection associated (VIA) antigen (viral RNA polymerase) in cattle sera, was developed using a bioengineered VIA (BioVIA) protein antigen. Compared with the classical immunodiffusion test, with viral RNA polymerase purified from infected cell cultures as antigen, this ELISA was more sensitive. However, depending on the cattle population examined, sera with antibodies to viral RNA polymerase, probably due to infection with other picornaviruses, were detected. Despite these observations, the ELISA using BioVIA provided a rapid answer as to whether or not FMD virus circulated in a given herd of cattle. The main advantage of this ELISA is its absolute safety, since in no step of the antigen production was infectious or uninfectious FMD virus involved. The test can therefore be performed under normal laboratory conditions and no isolation units are needed as they are for the immunodiffusion test.     

Use of MSG1 protein in a novel blocking ELISA for the detection of Mycoplasma suis infection

A blocking ELISA based on the monoclonal antibody (MAb) 1A7 was developed to detect antibodies against Mycoplasma suis. The MAb was produced by immunising BALB/c mice with recombinant MSG1 protein (rMSG1) from M. suis expressed in E. coli. Following identification by Western blotting, the MAb was purified and labelled with horseradish peroxidase. The parameters of the ELISA were optimised, and the cut-off value determined as 36.35% by analysing the percentage inhibition of M. suis negative serum. The sensitivity and specificity of the ELISA were 92% and 100%, respectively. In repeatability tests, the intra- and inter-batch variation coefficients were <10%. The results suggest this blocking ELISA is specific, sensitive and reproducible, and will be a valuable tool in the serodiagnosis of M. suis infection in pigs.     

Detection of Bartonella sp. and a novel spotted fever group Rickettsia sp. in Neotropical fleas of wild rodents (Cricetidae) from Southern Brazil

The Neotropical region shows a great diversity of fleas, comprising more than 50 genera. The importance of the study of fleas is linked to their potential role as disease vectors. The aim of this study is to investigate the presence of Rickettsia spp. and Bartonella spp. in Neotropical fleas collected from wild rodents in Southern Brazil. From 350 rodents captured, 30 were parasitized by fleas. A total of 61 fleas belonging to two genera and six different species were collected (Craneopsylla minerva minerva, Polygenis occidentalis occidentalis, Polygenis platensis, Polygenis pradoi, Polygenis rimatus, and Polygenis roberti roberti). In 13 % of fleas of three different species (C. minerva, P. platensis, and P. pradoi) Rickettsia sp. DNA was found. Phylogenetic analysis of concatenated sequences of gltA, htrA, and ompA genes showed that Rickettsia sp. found in rodent fleas (referred as strain Taim) grouped together with Spotted Fever Group Rickettsia. In reference to Bartonella spp., five genotypes were identified in seven fleas of two species (C. minerva and P. platensis) and in five rodent spleens. Also, 207 frozen samples of wild rodents were screened for these pathogens: while none was positive for Rickettsia spp.; five rodent spleens were PCR-positive for Bartonella spp.. Herein, we show the detection of potential novel variants of Bartonella sp. and Rickettsia sp. in fleas collected of wild rodents from Southern Brazil. Further studies are needed to fully characterize these microorganisms, as well as to improve the knowledge on the potential role of Neotropical flea species as diseases vectors.     

Furosemide loading test in a case of homozygous solute carrier family 12, member 1 (SLC12A1) mutation (g.62382825G>A,p.Pro372Leu) in Japanese Black cattle

Hydrallantois is the excessive accumulation of fluid in the allantoic cavity in a pregnant animal and is associated with fetal death. We recently identified a recessive missense mutation in the solute carrier family 12, member 1 (SLC 12A1 ) gene (g.62382825G>A, p.Pro372Leu) that is associated with hydrallantois in Japanese Black cattle. Unexpectedly, we found a case of the homozygous risk‐allele for SLC 12A1 in a calf, using a PCR ‐based direct DNA sequencing test. The homozygote was outwardly healthy up to 3 months of age and the mother did not exhibit any clinical symptoms of hydrallantois. In order to validate these observations, we performed confirmation tests for the genotype and a diuretic loading test using furosemide, which inhibits the transporter activity of the SLC 12A1 protein. The results showed that the calf was really homozygous for the risk‐allele. In the homozygous calf, administration of furosemide did not alter urinary Na⁺ or Cl^? levels, in contrast to the heterozygote and wild‐type calves in which these were significantly increased. These results demonstrate that the SLC 12A1 (g.62382825G>A, p.Pro372Leu) is a hypomorphic or loss‐of‐function mutation and the hydrallantois with this mutation shows incomplete penetrance in Japanese Black cattle.     

Evaluation of the recombinant Heat shock protein B (HspB) of Coxiella burnetii as a potential antigen for immunodiagnostic of Q fever in goats

Coxiella burnetii is an intracellular bacterium that causes a worldwide zoonosis, the Q fever. Currently, to diagnose the infection in ruminants, whole cell antigens-based ELISAs are used. In this study a heat shock protein, the HspB, was evaluated for its ability to be recognized by the goat immune system and its capacity to sign a stage of infection. The htpB gene of C. burnetii was cloned and sequenced. A high identity (>90%) was observed among the htpB genes of four ruminant strains tested. A recombinant protein was expressed in a prokaryotic expression system. The rHspB protein was used to determine the IgG reactivity by ELISA. Sera from experimentally and naturally infected goats were tested. The rHspB is recognized early during the infection course, at 18 days post-infection. Moreover, 80-90% of the animals tested were positive at 39-60dpi. In addition, animals presenting a reactivation of the infection displayed a higher reactivity, statistically significant (p<0.05), than that of the animals in latent infection. These findings suggest that the rHspB could be a good candidate for the development of an ELISA test making possible the detection of recent C. burnetii infection in goats as well as reactivation in those with latent infection.     

Afrotropical Culicoides: C. (Avaritia) loxodontis sp. nov., a new member of the Imicola group (Diptera: Ceratopogonidae) associated with the African elephant in the Kruger National Park, South Africa.

Culicoides (Avaritia) loxodontis sp. nov., is described and illustrated from both sexes collected in South Africa. It is the 5th species of the Imicola group of the subgenus Avaritia to be described from the Afrotropical Region, and is presently known only from the Kruger National Park where it has been collected in light-traps and reared from the dung of the African elephant (Loxodonta africana) on various occasions. A number of character states, and statistical analyses of antennal and palpal measurements, are used to separate the new species from its taxonomic congeners C. imicola Kieffer, 1913, C. pseudopallidipennis Clastrier, 1958, C. bolitinos Meiswinkel, 1989 and C. miombo Meiswinkel, 1991. It is suggested that the occurrence of the African elephant is the primary factor that determines the distribution of Culicoides loxodontis sp. nov., and that this close association, coupled with the fact that C. loxodontis sp. nov. can be locally abundant, may result in the cycling of certain arboviruses between this biting midge and the elephant.     

A rapid slide agglutination test for the serodiagnosis of Brucella canis infection that employs a variant (M-) organism as antigen

An improved antigen is described for use in the serodiagnosis of canine brucellosis. The novel antigen employs a less mucoid (M-) variant of B. canis. The replacement of Brucella ovis with B. canis (M-) cells as antigen in slide agglutination tests would increase accuracy by reducing the rate of false positive results from ca. 50% to ca. 10%. Rose bengal stained B. canis (M-) cells are slightly more sensitive than the commercially available Brucella ovis as slide test antigens. Both test procedures require brief (less than 1 min) reaction with 2-mercaptoethanol in order to reduce the rate of false positive reactions.     

High mobility group box 1 (HMGB1) protein is present in the cerebrospinal fluid of dogs with encephalitis

Many cases of encephalitic disease with unknown etiologies have been reported in specific breeds of small dogs. High mobility group box 1 (HMGB1) in neuronal cells was recently found to be a novel cytokine-like mediator that is a marker of neuronal necrosis and inflammation. The aim of this study was to determine whether HMGB1 levels are elevated in the cerebrospinal fluid (CSF) of dogs suspected of having encephalitis. CSF was obtained from 31 dogs that were diagnosed with an encephalitic disease by clinical examinations and magnetic resonance image (MRI) scanning. The CSF samples were analyzed via western blotting (WB) and an enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody against HMGB1. The mean HMGB1 concentration was significantly higher in the encephalitic dogs than that in the healthy controls. The concentrations of HMGB1 were correlated with the cell counts and total protein concentrations, which are known CSF indicators of the neuronal inflammation associated with encephalitis. These results suggest that HMGB1 protein in CSF confirms the presence of necrosis and inflammation in most cases of canine encephalitis and that HMGB1 will be a new indicator of encephalitis.     

Biological potentials for a family of disintegrin and metalloproteinase (ADAMDEC)-1 in mouse normal pregnancy

Our previous research has indicated local expression of ADAMDEC-1, a family of disintegrin and metalloproteinase, was confirmed in the mouse placentas and enhancement was found in the sites for spontaneous abortion. Present study was aimed to identify biological effects of ADAMDEC-1 in pregnancy process. Syngeneic pairs of C57BL/6J mice and heterogenic mating pairs of CBA/J and DBA/2 mice were used. Pregnant mice were treated with recombinant ADAMDEC-1 protein. Vasculogenesis effects was evaluated using the Matrigel plugs including vascular endothelial growth factor singularity or combination with ADAMDEC-1. ADAMDEC-1 single effects were evaluated by tubal formation and proliferation assays using HuEht-1 endothelial cells. Expression of ADAMDEC-1 was not exactly corresponded with the time periods for miscarriage initiation. ADAMDEC-1 was distributed in normal placentas and fetuses, especially at extraembryonic ectoderm, decidua cells, uterine natural killer (uNK) cells in decidua, trophoblasts in labyrinthine zone, and hematopoietic cells in umbilical blood and fetal liver. ADAMDEC-1 treatment did not affect reproductive performances, while it elevated uNK cell recruitment in placenta and enlarged lumen sizes of the intraplacental vessels. In vitro analysis also indicated ADAMDEC-1 promoting effect on tubal formation and cell length of HuEht-1. qPCR analysis showed that ADAMDEC-1 modified placental gene expression especially for linkage of actin filament rearrangement. Our findings suggested that ADAMDEC-1 is correlated on cell shape, stability, and movement via modification of actin cytoskeleton. ADMADEC-1 suspected to regulate cellular activity of endothelial cells, trophoblasts, and uNK cells and may support normal developing of mouse placentas.     

Sarcocystis infection in wild reindeer (Rangifer tarandus) from Hardangervidda in southern Norway: with a description of the cysts of Sarcocystis hardangeri n. sp

Fresh preparations of micro-isolated sarcosysts from skeletal muscle of 5 wild reindeer were examined by light microscopy. Slender, spindelshaped cysts measuring 821 × 60 µm, and having short knob-like cyst wall protrusions were found in all animals. In 1 animal cysts different in structure from the cysts of the 4 previously known Sarcocystis spp. of reindeer were found, These cysts are considered to be cysts of a new Sarcocystis sp. of reindeer, for which the name Sarcocystis hardangeri has been proposed.S. hardangeri n. sp. had macroscopic, ovoid to cylindrical cysts measuring 1667 (900–2570) × 819 (450–1575) µm. The cysts were surrounded by a 8–10 µm thick layer of fibrillar material. After removal of this layer, relatively few and irregularly spaced, slanting protrusions became visible. The 20–30 µm long protrusions were tongue-like, and were lying close to the surface of the cyst.Cysts of S. grueneri, S. rangiferi and S. tarandi were not demonstrated in the 5 wild reindeer examined.     

Pathological findings in horses dying during an outbreak of the paralytic form of Equid herpesvirus type 1 (EHV-1) infection.

In 1988 an outbreak of the paralytic form of Equid herpesvirus type 1 (EHV-1) infection occurred on a stud farm and several animals died. This provided an opportunity to perform detailed pathological investigations to gain insights into the pathogenesis of this spontaneous disease. Two paretic mares, three foals, an aborted foetus and its non-paretic dam were examined. The endotheliotropism of the virus was clearly demonstrated by the use of an indirect immunoperoxidase (IP) stain. At autopsy, evidence of viral infection was widespread in the foetus and foals, but limited or absent in the mares, probably reflecting differences in their immune status. Vascular lesions were present in the central nervous system (CNS) of the foals as well as the adults; they resulted in minimal neural lesions in the foals. Severe changes in the upper and lower respiratory tracts were a particular feature in the foals, two of which exhibited extensive vasculitis and thrombosis in the lungs. The IP technique was of great value in locating antigen-containing cells in the CNS of one mare when virus isolation was negative. It also revealed the presence of virus in less well documented sites such as the pancreas, gut, thyroid, uveal tract and the skin of the nares.     

Bovine neonatal pancytopenia (BNP): novel insights into the incidence,vaccination-associated epidemiological factors and a potential genetic predisposition for clinical and subclinical cases

Bovine neonatal pancytopenia (BNP) is a haemorrhagic disease of newborn calves elicited by colostrum from specific cows. Two studies have indicated that BNP-inducing colostrum might be associated with alloantibodies directed against MHC class I in response to vaccination with a distinct inactivated viral vaccine. However, the proportion of alloantibody-producing individuals by far exceeds the proportion of clinical BNP cases in the vaccinated population. This raises the question about the incidence of subclinical, unrecognised cases and also suggests further factors involved in BNP pathogenesis, e.g., genetic predisposition. Our results on neonatal calves from a closely monitored resource population confirmed the hypothesis of a genetic predisposition for clinical BNP and suggest that the predisposition is also involved in subclinical BNP-cases. No indication was obtained for a higher frequency of subclinical BNP-cases compared with clinical cases. Neither time point nor frequency of vaccination was a relevant factor for BNP in our resource population.     

亚洲璃眼蜱唾液腺新基因GP15的表达和鉴定

将GP15的阅读框序列插入pET32a( )制成重组质粒,通过BL21成功表达出相对分子质量为36 000的蛋白.该蛋白可被His·Bind从细菌裂解物纯化出来,也可为抗融合蛋白抗体所识别.表达形式分析显示,融合蛋白以包涵体形式在大肠肝菌内表达.     

Molecular characterization of a 13-amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D^pol) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D^pol coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp₂₆→Glu substitution in a beta sheet located within a small groove of the 3D^pol protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.