Constantin Constantinoiu

Real-time polymerase chain reaction (PCR) assays for the specific detection and quantification of seven Eimeria species that cause coccidiosis in chickens

Coccidiosis of chickens is an economically important disease caused by infection with species of Eimeria. The oocysts of some of the seven recognized species are difficult to distinguish morphologically and for this reason diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria. The real-time PCR provides both sensitivity and speed for the analysis of DNA samples, and the approach has the capability of quantifying DNA. Together with a protocol for the extraction of DNA directly from faecal samples, real-time PCR assays have been established for the detection and quantification of seven species of Eimeria that infect chickens in Australia. The assays target one genetic marker, the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2), use TaqMan MGB technology with species-specific probes, and can be multiplexed in pairs such that the seven species of Eimeria can be screened in four reaction tubes. A test screen of commercial flocks identified more Eimeria-infected chickens than were detected by coproscopic examination for oocysts. These molecular assays can also be used for the quality control of mixed-species vaccines. The ability to multiplex the assays makes them particularly practical for screening samples from chickens with mixed-species infections where the relative abundance of each Eimeria species present is required.

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3(+), CD4(+), CD8(+) and gammadelta T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for gammadelta T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite life cycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.

Host resistance in cattle to infestation with the cattle tick Rhipicephalus microplus

Resistance to Rhipicephalus microplus infestation in cattle has many effector mechanisms, each of which is likely to be modulated by complex, interacting factors. Some of the mechanisms of host resistance and their modulating factors have been identified and quantified, although much remains to be explained. The variation in resistance to tick infestation is most marked between Bos taurus and Bos indicus cattle, taurine cattle given the same exposure carrying between five and 10 times as many ticks as indicine cattle. Tick resistance is mostly manifest against attaching larvae, which attempt to feed often and without success, death occurring mostly within 24 h of finding a host. There is evidence of innate and adaptive immune response to tick infestation, and it appears that the relative importance of each differs between indicine and taurine cattle. There is conflicting information regarding the role of humoral immunity in tick resistance, and recent studies indicate that strong IgG responses to tick antigens are not protective. A strong T‐cell‐mediated response directed against larval stages, as mounted by indicine cattle, seems to be protective. Variation in the extracellular matrix of skin (epidermal growth factors, collagens and other matrix components such as lumican) also contributes to variation in host resistance.

Characterization of monoclonal antibodies that recognize the Eimeria tenella Microneme Protein MIC2

Abstract The apicomplexan pathogens of Eimeria tenella cause coccidiosis, an intestinal disease of chickens that has a major economic impact on the poultry industry. Members of Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes, and dense granules) that mediate invasion of host cells and the formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from E. tenella (EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. In an immunofluorescence assay, all mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina, and E. maxima. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2-related proteins in Eimeria species.

Prolonged Subcutaneous Administration of Oxytocin Accelerates Angiotensin II-Induced Hypertension and Renal Damage in Male Rats.

Oxytocin and its receptor are synthesised in the heart and blood vessels but effects of chronic activation of this peripheral oxytocinergic system on cardiovascular function are not known. In acute studies, systemic administration of low dose oxytocin exerted a protective, preconditioning effect in experimental models of myocardial ischemia and infarction. In this study, we investigated the effects of chronic administration of low dose oxytocin following angiotensin II-induced hypertension, cardiac hypertrophy and renal damage. Angiotensin II (40 μg/Kg/h) only, oxytocin only (20 or 100 ng/Kg/h), or angiotensin II combined with oxytocin (20 or 100 ng/Kg/h) were infused subcutaneously in adult male Sprague-Dawley rats for 28 days. At day 7, oxytocin or angiotensin-II only did not change hemodynamic parameters, but animals that received a combination of oxytocin and angiotensin-II had significantly elevated systolic, diastolic and mean arterial pressure compared to controls (P < 0.01). Hemodynamic changes were accompanied by significant left ventricular cardiac hypertrophy and renal damage at day 28 in animals treated with angiotensin II (P < 0.05) or both oxytocin and angiotensin II, compared to controls (P < 0.01). Prolonged oxytocin administration did not affect plasma concentrations of renin and atrial natriuretic peptide, but was associated with the activation of calcium-dependent protein phosphatase calcineurin, a canonical signalling mechanism in pressure overload-induced cardiovascular disease. These data demonstrate that oxytocin accelerated angiotensin-II induced hypertension and end-organ renal damage, suggesting caution should be exercised in the chronic use of oxytocin in individuals with hypertension.

Tracking transparent monogenean parasites on fish from infection to maturity

The infection dynamics and distribution of the ectoparasitic fish monogenean Neobenedenia sp. (Monogenea: Capsalidae) throughout its development was examined on barramundi, Lates calcarifer (Bloch) (Latidae), by labelling transparent, ciliated larvae (oncomiracidia) with a fluorescent dye. Replicate fish were each exposed to approximately 50 fluorescent oncomiracidia and then examined for parasites using an epifluorescence stereomicroscope at 10 time intervals post-exposure (15, 30, 60, 120 min, 24, 48 h, four, eight, 12, and 16 days). Fluorescent labelling revealed that parasites attached underneath and on the surface of the scales of host fish. Parasite infection success was 20% within 15 min, and peaked at 93% two days post-exposure, before gradually declining between four and sixteen days. Differences in parasite distribution on L. calcarifer over time provided strong evidence that Neobenedenia sp. larvae settled opportunistically and then migrated to specific microhabitats. Parasites initially attached (<24 h) in greater mean numbers on the body surface (13 ± 1.5) compared to the fins (4 ± 0.42) and head region (2 ± 0.41). Once larvae recruitment had ceased (48 h), there were significantly higher mean post-larvae counts on the head (5 ± 3.4) and fins (12 ± 3) compared to previous time intervals. Neobenedenia sp. aggregated on the eyes, fins, and dorsal and ventral extremities on the main body. As parasites neared sexual maturity, there was a marked aggregation on the fins (22 ± 2.35) compared to the head (4 ± 0.97) and body (9 ± 1.33), indicating that Neobenedenia sp. may form mating aggregations.

Natural plant extracts and prebiotic compounds as alternatives to antibiotics in broiler chicken diets in a necrotic enteritis challenge model

An experiment was conducted to determine the effects of two different water-soluble carbohydrate extracts (renga renga lily extract and Acacia extract), and two commercially available prebiotic compounds, Fibregum and Raftifeed-IPE, on the performance of broiler chickens subjected to a necrotic enteritis (NE) challenge model. These treatments were compared with negative control and a positive (Zn-bacitracin) control treatments. An overall 8.8% NE-related mortality was recorded, with mean jejunal and ileal lesion scores in dead birds ranging from 3.03 to 3.90 in all challenged groups except the positive control groups. NE-specific deaths or clinical abnormalities were not observed with unchallenged control and positive control groups. At 7 days post-challenge, the concentration of specific IgY antibodies against the α-toxin of Clostridium perfringens in the serum was lower (P < 0.05) in birds fed the positive control and Fibregum-supplemented diets than in the negative control group. However, birds fed Fibregum had increased (P < 0.05) IgM concentration compared with those fed Acacia extract and lily extract. The Fibregum-fed group also had higher (P < 0.05) IgA concentrations in serum than did the positive-control and lily extract-supplemented groups at 14 days but this effect did not persist to 21 days. The results from the present study demonstrated that supplementation with water-soluble carbohydrates from two plant sources was not effective in controlling NE. However, the prebiotic compound Fibregum was found to be having some immunomodulatory effects. Addition of Zn-bacitracin and monensin was highly effective in counteracting the negative effects of the disease challenge.

Characterization of the antibody response in birds following infection with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix

Live vaccines containing attenuated parasite strains are increasingly used to control chicken coccidiosis. In this paper antibody responses elicited by infections with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix were characterized by immunoblotting and ELISA with homologous and heterologous antisera. Few differences between antisera from birds infected with wild and attenuated strains of E. tenella were evident in immunoblots conducted with merozoite antigen preparations from both E. tenella strains, however the reactivity of sera raised in birds infected with the wild-type strain was noticeably more intense. In ELISAs conducted with merozoite antigen preparations, antisera from birds infected with the wild-type strains of E. tenella and E. necatrix consistently produced a significantly higher (P<0.05) antibody response than antisera from birds infected with the attenuated strains. Likewise, avidity ELISAs conducted with the E. tenella strains demonstrated that antibodies in birds infected with the wild-type strain were of significantly higher avidity (P<0.05) than antibodies in birds infected with the attenuated strain. The differences in the antibody responses are probably due to changes in the attenuated strain as a result of selection for precocious development and the less severe tissue damage and inflammation of the intestine resulting from infection with the attenuated strain.

Revisiting the Ancylostoma Caninum Secretome Provides New Information on Hookworm–Host Interactions

Hookworm infection is a major tropical parasitic disease affecting almost 500 million people worldwide. These soil‐transmitted helminths can survive for many years in the intestine of the host, where they feed on blood, causing iron deficiency anemia and other complications. These parasites release a variety of molecules known as excretory/secretory products (ESPs) that are involved in many different biological processes that govern parasite survival. Using a combination of separation techniques such as SDS‐PAGE and OFFGEL electrophoresis, in combination with state‐of‐the‐art mass spectrometry we have reanalyzed the dog hookworm, Ancylostoma caninum, ESPs (AcESP). We identified 315 proteins present in the AcESP, compared with just 105 identified in previous studies. The most highly represented family of proteins is the SCP/TAPs (110 of the 315 proteins), and the most abundant constituents of AcESP are homologues of the tissue inhibitors of metalloproteases (TIMP) family. Interestingly, we identified new homologs of well‐known vaccine candidates and immunomodulatory proteins. This study provides novel information about the proteins secreted by A. caninum, and constitutes a comprehensive dataset to study the proteins involved in host–hookworm interactions.