Control Of Neosporosis Research Articles

Pathological characteristics and congenital immunological responses of pigeons-infected with Neospora caninum

Pigeons are natural intermediate host of Neospora caninum (N. caninum). In comparison to ruminants, N. caninum causes milder clinical symptoms and less financial loss to pigeons. Natural infectious rates and high prevalence of N. caninum in pigeons, and death cases of N. caninum-infected pigeons under experimental conditions have been reported, but the detailed pathological characteristics and congenital immunological responses of pigeons-infected with N. caninum remain not well described. In this study, pigeons were infected intraperitoneally with 107 N. caninum tachyzoites. N. caninum in tissues was detected by qPCR. Pathological changes of tissues were examined by hematoxylin-eosin staining. Blood smears were prepared for counting eosinophils changes in blood. Heterophil extracellular traps (HETs) in vivo and in vitro were quantified by Pico Green. N. caninum-induced HETs structures were observed by immunofluorescence staining. The model of pigeons-infected with N. caninum was successfully established. Lung and duodenum were the main target organs of pigeons-infected with N. caninum. N. caninum caused hemorrhage, edema and inflammatory cell infiltration in liver, pulmonary congestion and hemorrhage, organizational destruction in lung, and shorter villi or even disappear in duodenum. N. caninum also increased the number of eosinophils in blood of pigeons. Moreover, N. caninum-induced HETs release in the congenital immunological system of pigeons were first demonstrated, and the HETs structures were consisted of DNA as the skeleton and modified with citH3 and elastase. N. caninum-induced HETs release was related with NADPH oxidase, TLR 2 and 4, ERK1/2 and p38 MAPK signaling pathways, and glycolysis. In summary, it is the first report on the detailed pathological characteristics and congenital immunological responses of pigeons-infected with N. caninum, which may provide theoretical basis for the prevention and control of Neosporosis in pigeons.

Involvement of chemokine receptor CXCR3 in the defense mechanism against Neospora caninum infection in C57BL/6 mice.

C-X-C motif chemokine receptor 3 (CXCR3) is an important receptor controlling the migration of leukocytes, although there is no report regarding its role in Neospora caninum infection. Herein, we investigated the relevance of CXCR3 in the resistance mechanism to N. caninum infection in mice. Wild-type (WT) C57BL/6 mice and CXCR3-knockout (CXCR3KO) mice were used in all experiments. WT mice displayed a high survival rate (100%), while 80% of CXCR3KO mice succumbed to N. caninum infection within 50 days. Compared with WT mice, CXCR3KO mice exhibited significantly lower body weights and higher clinical scores at the subacute stage of infection. Flow cytometric analysis revealed CXCR3KO mice as having significantly increased proportions and numbers of CD11c-positive cells compared with WT mice at 5 days post infection (dpi). However, levels of interleukin-6 and interferon-γ in serum and ascites were similar in all groups at 5 dpi. Furthermore, no differences in parasite load were detected in brain, spleen, lungs or liver tissue of CXCR3KO and WT mice at 5 and 21 dpi. mRNA analysis of brain tissue collected from infected mice at 30 dpi revealed no changes in expression levels of inflammatory response genes. Nevertheless, the brain tissue of infected CXCR3KO mice displayed significant necrosis and microglial activation compared with that of WT mice at 21 dpi. Interestingly, the brain tissue of CXCR3KO mice displayed significantly lower numbers of FoxP3+ cells compared with the brain tissue of WT mice at 30 dpi. Accordingly, our study suggests that the lack of active regulatory T cells in brain tissue of infected CXCR3KO mice is the main cause of these mice having severe necrosis and lower survival compared with WT mice. Thus, CXCR3+ regulatory T cells may play a crucial role in control of neosporosis.

Control measures to reduce Neospora caninum abortions in dairy farms: a pilot study in Iran.

Neospora caninum plays an important role in abortion and economic losses in dairy industry worldwide. The main target of this work was to detect the infection rate of N. caninum in various hosts in dairy farms for identifying the risk factors and applying appropriate control programs to reduce the number of abortions. The study was conducted in dairy farms with high incidence of abortion in Hamedan province, West of Iran. After the primary assessment, we conducted a controlling program for reducing the Neospora‑infection rate and associated abortions. Before implementing the control program, the seropositivity was 24.8% in cows (N=476 distributed in 10 farms) and 8.6% (N=185) in dogs. Abortion occurred in 3.57% of pregnant cows. 94.1% of aborted cows were positive for Neospora‑infection. Based on molecular technique, the infection rate was detected in 7.3% of cats (N=41), in 25% of pigeons (N=19)and in 11.8% of rodents (N=51). After the implementation of neosporosis control programs in the farms, the seropositivity of N. caninum decreased to 8.2% in cows and 2.9% in dogs. After the one‑year follow‑up, no cases of abortions were reported in the farms. This was the first parallel evaluation of Neospora‑infection and controlling programs in Iranian dairy farms. Regular control of rodents, retesting of seronegative animals and farm biosecurity measures are recommended for reducing the abortion incidence. The access of dogs to the herd and to aborted materials should be restricted.

Recombinant actin-depolymerizing factor of the apicomplexan Neospora caninum (NcADF) is susceptible to oxidation.

Neospora caninum is a member of Apicomplexa Phylum and the causative agent of neosporosis, a disease responsible for abortions in cattle. Apicomplexan parasites have a limited set of actin-binding proteins conducting the regulation of the dynamics of nonconventional actin. The parasite actin-based motility is implicated in the parasite invasion process in the host cell. Once no commercial strategy for the neosporosis control is available, the interference in the parasite actin function may result in novel drug targets. Actin-depolymerization factor (ADF) is a member of the ADF/cofilin family, primarily known for its function in actin severing and depolymerization. ADF/cofilins are versatile proteins modulated by different mechanisms, including reduction and oxidation. In apicomplexan parasites, the mechanisms involved in the modulation of ADF function are barely explored and the effects of oxidation in the protein are unknown so far. In this study, we used the oxidants N-chlorotaurine (NCT) and H2O2 to investigate the susceptibility of the recombinant N. caninum ADF (NcADF) to oxidation. After exposing the protein to either NCT or H2O2, the dimerization status and cysteine residue oxidation were determined. Also, the interference of NcADF oxidation in the interaction with actin was assessed. The treatment of the recombinant protein with oxidants reversibly induced the production of dimers, indicating that disulfide bonds between NcADF cysteine residues were formed. In addition, the exposure of NcADF to NCT resulted in more efficient oxidation of the cysteine residues compared to H2O2. Finally, the oxidation of NcADF by NCT reduced the ability of actin-binding and altered the function of NcADF in actin polymerization. Altogether, our results clearly show that recombinant NcADF is sensitive to redox conditions, indicating that the function of this protein in cellular processes involving actin dynamics may be modulated by oxidation.

Extracellular H2O2, peroxiredoxin, and glutathione reductase alter Neospora caninum invasion and proliferation in Vero cells

Neospora caninum is a protozoan member of the Apicomplexa phylum and is closely connected with abortion in cattle. The development of the parasite in host cells is characterized by the active secretion of proteins, allied to the tight control of the redox status. In this sense, elucidating the mechanisms related to the role of the redox agents and enzymes during the invasion and proliferation of N. caninum may contribute to developing novel forms of neosporosis control. In this study we verified the effects of the recombinant forms of N. caninum glutathione reductase (rNcGR) and thioredoxin-dependent peroxide reductase (rNcPrx), as well as H2O2 in the tachyzoite invasion and proliferation. rNcPrx interfered in the N. caninum invasion in a redox state manner. Oxidized rNcPrx inhibited the N. caninum invasion and proliferation with no toxic effects observed in Vero cells. In contrast, lower concentrations of H2O2 (10 μM) stimulated the N. caninum invasion, which was reverted in higher doses (>100 μM). H2O2 inhibited the parasite proliferation in lower concentrations than cytotoxicity in host cells, resulting in a positive selectivity index (1.8). Besides, rNcPrx (reduced and non-reduced) and rNcGR inhibited the parasite proliferation without affecting the host cell. Our results indicate the connection between the N. caninum development and the redox state, contributing to the elucidation of parasite propagation and control mechanisms.

Pathohistological changes in aborted foetuses of cows due to neosporosis: Evidence from Ukraine

Neosporosis is a parasitic disease characterized by abortions and the birth of weak offspring in cows. The causative agent of Neospora caninum is an obligate, protozoan parasite that belongs to the type Apicomplexa. The relevance of the study is conditioned upon the adverse impact of neosporosis on the economy of Ukraine (loss of productivity, veterinary and diagnostic costs). Furthermore, the issue of neosporosis is understudied. In this regard, the purpose of this study was to establish pathohistological changes in aborted foetuses and the foetal part of placentas and to confirm the involvement of the parasite (Neospora caninum) in cases of abortions recorded in different regions of the country. Two methods were used to investigate this problem: histological and real-time polymerase chain reaction. In aborted foetuses positive for N. caninum, the following pathohistological changes were most often detected: focal gliosis and perivascular mononuclear infiltrates in the brain; focal or diffuse mononuclear infiltration in the heart and skeletal muscles; periportal mononuclear infiltrates in the liver; focal necrosis of the mucous membrane and mononuclear infiltration in the foetal part of the placenta. Changes were less often detected in the lungs – mononuclear infiltration of the interstitium and diffuse lymphocytic alveolitis, and in the kidneys – diffuse interstitial mononuclear infiltration. No changes were found in the spleen. Neospore-like cysts were found in one out of twelve foetuses. Lesions established of foetal organs and placentas are inherent in neosporosis. The results of histological studies substantially complement the data of other authors, confirm the involvement of N. caninum in the occurrence of abortions in cows in certain regions of Ukraine, and also represent practical value for the diagnosis and control of neosporosis in cattle

The Protective Role of TLR2 Mediates Impaired Autophagic Flux by Activating the mTOR Pathway During Neospora caninum Infection in Mice

Autophagy has been shown to play an essential role in defending against intracellular bacteria, viruses, and parasites. Mounting evidence suggests that autophagy plays different roles in the infection process of different pathogens. Until now, there has been no conclusive evidence regarding whether host autophagy is involved in Neospora caninum infection. In the current study, we first monitored the activation of autophagy by N. caninum, which occurred mainly in the early stages of infection, and examined the role of host autophagy in N. caninum infection. Here, we presented evidence that N. caninum induced an increase in autophagic vesicles with double-membrane structures in macrophages at the early stage of infection. LC3-II expression peaked and decreased as infection continued. However, the expression of P62/SQSTM1 showed significant accumulation within 12 h of infection, indicating that autophagic flux was blocked. A tandem fluorescence protein mCherry-GFP-LC3 construct was used to corroborate the impaired autophagic flux. Subsequently, we found that N. caninum infection induced the activation of the TLR2–AKT–mTOR pathways. Further investigation revealed that TLR2–mTOR, accompanied by the blockade of autophagic flux, was responsible for impaired autophagy but was not associated with AKT. In vitro and in vivo, N. caninum replication was strongly blocked by the kinase inhibitor 3-methyladenine (3-MA, autophagy inhibitor). In contrast, rapamycin (Rapa, an autophagy inducer) was able to promote intracellular proliferation and reduce the survival rate of N. caninum-infected mice. On the other hand, the accumulation of autophagosomes facilitated the proliferation of N. caninum. Collectively, our findings suggest that activation of host autophagy facilitates N. caninum replication and may counteract the innate immune response of the host. In short, inhibition of the early stages of autophagy could potentially be a strategy for neosporosis control.

Antibody seropositivity for Neospora caninum in Blanco Orejinegro cattle in Colombia and factors associated with the infection.

Neosporosis is a parasitic disease that causes abortions and economic losses in bovine production systems, but no studies have been found concerning its effect on the Creole cattle breed, Blanco Orejinegro (BON). The aim of this research was to establish the serological status of Colombian BON cattle against Neospora caninum and to determine the factors associated with seropositivity. Blood samples were taken from 363 animals from 13 herds in six states of Colombia, and analyzed by indirect ELISA using a commercial test. Information on sex, herd, generation group, and state was recorded. A survey was carried out with 26 questions related to productive, reproductive, and health factors per herd. A logistic regression analysis was performed and the ORs for significantly associated variables were estimated using the R software. General seropositivity of 73.5% (95% CI 68.6-77.9%) was obtained, and sex, age group, and herd were the variables significantly associated with seropositivity (p < 0.05). For the sex variable, seroprevalence levels of 79.6% (95% CI 74.3-84.1%) were recorded for females and 54.5% (95% CI 43.6-65.1%) for males. Herd seroprevalence varied between 58.3 and 95.8%, and the last generation showed the lowest positivity (51.2%, 95% CI 42.1-60.2%). The inadequate disposal of fetuses was a risk factor, while carrying out serological tests to new animals that enter the herd, the use of new gloves and palpation utensils for each animal, supplementation, and stabling were stated as protective factors. No effect of positivity was found in the last calving interval. The implementation of bovine neosporosis control programs to support breeding and conservation programs of the BON breed in Colombia is recommended.

Risk factor analysis associated with Neospora caninum in dairy cattle in Western Brazilian Amazon.

Neospora caninum is considered to be one of the main causes of abortion among cattle. The present survey was conducted in the municipality of Rolim de Moura, Rondônia State, Brazil. A questionnaire that investigates the epidemiological aspects of neosporosis was used in the analysis of risk factors associated with the animal-level and herd-level prevalence in dairy cattle. A total of 416 bovine blood samples were collected from 30 farms, and N. caninum antibody levels were measured by Indirect Fluorescent Antibody Test (IFAT). Analysis of dairy cattle serum samples revealed the presence of anti-N. caninum antibodies to be 47.36% (n = 197). Risk factors associated with N. caninum infection were the management system and access locations of dogs. The results of the present survey indicated that infection of dairy cattle with N. caninum is widespread in the studied region of Western Amazon, which has implications for prevention and control of neosporosis in this region. Therefore, integrated control strategies and measures are recommended to prevent and control N. caninum infection in dairy cattle. In addition, direct contact between dairy cattle, dogs and wild animals, which can influence the epidemiology of neosporosis, should be investigated further.

Risk factors of canine neosporosis in Iran: a meta-analysis study

Neospora caninum is an obligate intracellular protozoan that causes neurological disorders in dogs. This meta-analysis based review aimed to evaluate the risk factors associated with N. caninum infection in Iran.The relevant articles, published up to January 2021, have been searched from five English databases, including PubMed, Google Scholar, ScienceDirect, Scopus, and Web of Science and five Persian databases namely, Element, Civilica, Magiran, Irandoc, and Scientific Information Database (SID). The odds ratio (OR) and the corresponding 95% confidence intervals (CI)) were measured using random-effects model. A total of 549 articles were identified by the databases without setting any time limit, of which 28 papers met the evaluation criteria for this study. Age, gender, breed, presence of an intermediate host, habitat, and diet were the risk factors reported in Iranian studies. Dogs equal or below 1 year of age had higher infection compared to that of more than 2 years of age and the difference was statistically significant. In relation to types of habitats, household dogs had lower infection in comparison to stray and farm dogs and the difference was statistically significant. Dogs are important source of N. caninum infection. Therefore, the systematic analysis of the risk factors associated with N. caninum may help in prevention and control of neosporosis among the dog population.

Cloning and expression of Neospora caninum rhoptry 2 and its potential as an immunobiological for neosporosis control

ABSTRACT: The protozoan Neospora caninum is known worldwide as one of the main causes of abortion in cattle. During infection, rhoptry proteins present in the apical complex of the parasite play important roles in adhesion and parasitophorous vacuole formation. The use of N. caninum ROP2 in experimental vaccines has shown promising protective results. In our study we performed cloning and expression in Escherichia coli of an antigenic portion of N. caninum ROP2. The recombinant protein (rROP2) was obtained in insoluble form, and the purified protein showed a size of approximately 18kDa. Even being a small truncate NcROP2 region, it was possible to conserve the antigenic epitopes which were recognized by bovine serum naturally infected with N. caninum. Vaccination with rROP2 on aluminum hydroxide adjuvant induced high levels of rROP2-specific IgG antibodies capable of recognizing native protein in tachyzoite lysates. In conclusion, our approaches were effective in obtaining the rROP2 protein, which induced specific mouse immune response and was also recognized by sera from N. caninum naturally infected cattle. These results suggest that it is a promising antigen for the development of neosporosis subunit vaccines as well as a suitable antigen for use in immunodiagnosis.

Inhibitory action of phenothiazinium dyes against Neospora caninum

Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9–Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019–1.83 μM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9–Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9–Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control.

Evaluation of abortions spontaneously induced by Neospora caninum and risk factors in dairy cattle from Lima, Peru.

Our objective was to identify the direct and indirect presence of Neospora caninum in dairy cattle and their aborted fetuses from Lima, Peru. A total 219 blood samples obtained from dairy cattle with records of spontaneous abortion were collected to detect antibodies against N. caninum in serum with indirect ELISA and search for risk-factor associations. 68 fetal aborted tissue samples of these cows were analyzed by PCR, indirect ELISA and histopathology assay to detect N. caninum presence. The prevalence ratio (PR) and 95% confidence intervals (CI) were estimated. Univariate analysis was performed using the chi-squared test. Among the 68 aborted fetuses collected, 10 (15%) were positive in at least two diagnostic tests. Among 219 serum samples, 46.6% (95% CI: 40.0%-53.3%) were positive. Cows with 4 years or older (PR: 7.10; 95% CI: 4.89-10.67) and multiparous (PR: 1.76; 95% CI: 1.11-2.80) were found to be more likely to possess N. caninum antibodies. This study detects presence of N. caninum in dairy cattle and their aborted fetus from Lima valley, suggesting biosecurity management improve to neosporosis control.

Safety and efficacy of the bumped kinase inhibitor BKI-1553 in pregnant sheep experimentally infected with Neospora caninum tachyzoites

Neospora caninum is one of the main causes of abortion in cattle, and recent studies have highlighted its relevance as an abortifacient in small ruminants. Vaccines or drugs for the control of neosporosis are lacking. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. We here present the pharmacokinetics, safety and efficacy of BKI-1553 in pregnant ewes and foetuses using a pregnant sheep model of N. caninum infection. BKI-1553 showed exposure in pregnant ewes with trough concentrations of approximately 4 μM, and of 1 μM in foetuses. Subcutaneous BKI-1553 administration increased rectal temperatures shortly after treatment, and resulted in dermal nodules triggering a slight monocytosis after repeated doses at short intervals. BKI-1553 treatment decreased fever in infected pregnant ewes already after two applications, resulted in a 37–50% reduction in foetal mortality, and modulated immune responses; IFNγ levels were increased early after infection and IgG levels were reduced subsequently. N. caninum was abundantly found in placental tissues; however, parasite detection in foetal brain tissue decreased from 94% in the infected/untreated group to 69–71% in the treated groups. In summary, BKI-1553 confers partial protection against abortion in a ruminant experimental model of N. caninum infection during pregnancy. In addition, reduced parasite detection, parasite load and lesions in foetal brains were observed.

A new microneme protein of Neospora caninum, NcMIC8 is involved in host cell invasion

Microneme proteins play an important role in the invasion process of Apicomplexan parasites through adhesion to host cells. We discovered a new N. caninum protein, NcMIC8, which is highly identical to TgMIC8. The NcMIC8 sequence has 2049 bp and no intron in the open reading fragment. It has a molecular weight of 73.8 kDa and contains a signal peptide, a transmembrane region, a low complexity region and 10 epidermal growth factor (EGF) domains. Immuno-fluorescence assay showed that NcMIC8 is located in the microneme. NcMIC8 was secreted to culture medium under stimulation of 1% ethanol, and cleaved to form the mature body of 40 kDa before transporting to microneme or during secretion. Blocking NcMIC8 using anti-NcMIC8 serum effectively inhibited host cell invasion by tachyzoites in vitro. NcMIC8 in the form of mature body interacts with NcMIC3, and the two microneme proteins form a complex probably during transportation. NcMIC8 is a new microneme protein of N. caninum and could be an attractive target for the control of neosporosis.

Evaluation of methylene blue, pyrimethamine and its combination on an in vitro Neospora caninum model.

Neospora caninum is an apicomplexan parasite strongly related to reproductive problems in cattle. The neosporosis control is not well established and several fronts are under development, predominantly based on immune protection, immunomodulation and chemotherapy. The use of anti-malarial drugs as therapeutic sources has, in theory, considerable potential for any apicomplexan. Drugs such as methylene blue (MB) and pyrimethamine (Pyr) represent therapeutic options for malaria; thus, their use for neosporosis should be assessed. In this work, we tested the effects of MB and Pyr on N. caninum proliferation and clearance, using LacZ-tagged tachyzoites. The drugs inhibited at nanomolar dosages and its combination demonstrated an antagonistic interaction in proliferation assays, according to the Chou and Talalay method for drug combination index. However, the drug combination significantly improved the parasite in vitro clearance. The repositioning of well-established drugs opens a short-term strategy to obtain low-cost therapeutics approaches against neosporosis.

Seroprevalence and risk factors associated with exposure of water buffalo (Bubalus bubalis) to Neospora caninum in northeast Thailand

Water buffalo are important draft animals for agriculture in resource-restricted areas worldwide. Water buffalo were shown to be experimentally susceptible to infection with Neospora caninum, potentially affected by neosporosis, and naturally exposed to the parasite in Asia. Although enzootic to Thailand, the distribution of N. caninum among Thai water buffalo is unclear. The objectives of this study were to determine the seroprevalence of N. caninum among water buffalo of northeast Thailand and to identify risk factors associated with their exposure to N. caninum. Sera from 628 water buffalo from 288 farms were tested with an indirect fluorescent antibody test (IFAT). A total of 57 samples from 48 herds contained antibodies to N. caninum, indicating overall seroprevalence of 9.1% and 16.7% among individual animals and herds, respectively. The overall seroprevalence was highest in provinces located in the Khorat Basin in the southern part of the region tested. Host age was also associated with seroprevalence, with the greatest seroprevalence (16.1%) among buffalo over 10 years of age, followed by 5–10 years of age (13.4%), 3–5 years (9.2%), and less than 3 years (1.2%). These results collectively suggested that horizontal transmission from canine definitive hosts was an important route of water buffalo exposure to N. caninum. These results also verified the importance of risk factor analysis for effective bovine neosporosis control strategies at the local level.

Seroprevalence of Neospora caninum infection in dairy cows in Northern provinces, Thailand.

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis, recognized as a major cause of bovine abortion around the world. Thailand is a developing agricultural country located in Southeast Asia. Livestock developments particularly in dairy cows of this country have been hampered by low productivity including milk and slow growth rate due to the impact of many pathogens including N. caninum. Currently, there is no effective method for control of neosporosis since there is less information regarding current status of infections. The objective of this study was to investigate the seroprevalence of neosporosis in dairy cows of the northern part of Thailand. During 2006-2007, the sera of 642 cows from 42 small farm holders with the top three highest consensus of dairy farms in the northern provinces, such as Chiang Rai, Chiang Mai and Lumpang were collected and performed tests. Antibodies to N. caninum were assayed by enzyme-linked immunosorbent assay (ELISA) with recombinant N. caninum surface antigen 1 (NcSAG1) and indirect fluorescent antibody test (IFAT). The overall prevalence of N. caninum infection in this study was 46.9% (301/642) by ELISA and 34.3% (220/642) by IFAT.

Serological diagnosis of bovine neosporosis: A comparative study of commercially available ELISA tests

Bovine neosporosis control programs are currently based on herd management and serodiagnosis because effective treatments and vaccines are unavailable. Although a wide variety of serological tools have been developed, enzyme-linked immunosorbent assays (ELISAs) are the most commonly commercialized tests. Partial comparative studies have been performed in the past, and the panel of available ELISAs has notably changed in the last few years. Therefore, diagnostic laboratories are requesting updated information about the performance of these tests.Accordingly, the aim of this study was to compare all of the commercially available ELISAs (n=10) by evaluating their performance and to re-standardize them based on TG-ROC analyses when necessary. For this purpose, a well-characterized serum panel from experimentally and naturally infected bovines and non-infected bovines (n=458) was used. Two different definitions of gold standard were considered: (i) the result of the majority of tests and (ii) pre-test information based on epidemiological, clinical and serological data. Most of the tests displayed high sensitivity (Se) and specificity (Sp) values when both gold standard criteria were considered. Furthermore, all the tests showed near perfect agreement, with the exception of the pair-wise comparisons that included the VMRD and SVANOVIR. The best-adjusted ELISAs were the HIPRA-CIVTEST, IDVET, BIOVET and IDEXX Rum (Se and Sp>95%). After the TG-ROC analyses, higher Se and Sp values were obtained for the BIO-X, LSI Bov, LSI Rum and IDEXX Bov, though the increases were more significant for the SVANOVIR and VMRD. The Kappa values also increased with the new adjusted cut-offs. This is the first study that offers updated performance evaluations of commercially available ELISAs. Such analyses are essential for diagnostic laboratories and are valuable to the companies that develop and distribute these tests.

Oligomannose-coated liposome-entrapped dense granule protein 7 induces protective immune response to Neospora caninum in cattle

Neospora caninum is an intracellular protozoan parasite that causes abortion in cows. Vaccination is an important strategy for control of neosporosis, and a safe and effective vaccine suitable for cattle is required. Dense granule protein 7 of N. caninum (NcGRA7) is a secretory protein with high antigenicity in hosts. We demonstrated previously that NcGRA7 entrapped in liposomes coated with mannotriose (M3-NcGRA7) could induce a parasite-specific T-helper type 1 immune response and produce humoral antibodies that resulted in increased offspring survival and decreased infection in the brains of mice dams. In the present study, the efficacy of M3-NcGRA7 as a vaccine candidate against N. caninum has been evaluated in cattle (n=12). Cattle were immunized with M3-NcGRA7 containing 50μg (n=4) or 200μg NcGRA7 (n=4) subcutaneously twice with a 4-week interval and all cattle including the non-immunized controls (n=4) were inoculated with 107 tachyzoites of Nc-1 strain 27 days after the second immunization and euthanized at 85–87 days post infection (dpi). In immunized cattle, NcGRA7-specific antibody production and IFN-γ production in PBMC was induced before challenge. At 3dpi, body temperature and concentration of serum IFN-γ tended to be higher in control cattle than in the immunized cattle. Furthermore, the parasite load in the brain significantly decreased in cattle immunized with 50μg M3-NcGRA7 compared with controls. These results suggest that M3-NcGRA7 can induce protective immune responses to N. caninum tachyzoites in cattle, which could lead to practical application of safe and effective subunit vaccines.