MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica

During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11 wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4–7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3–6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33–66 adults), while it was for F. gigantica infection (burden of 17–69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.     

Immunological features of LPS from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica

The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium in sheep with fasciolosis was reported previously, resulting in lower fecal egg counts and fluke burden. In the current study, we analyzed its immunological effects in two groups of sheep, treated (T) and controls (C). Fasciolosis induces a T helper (Th) type-2 response, characterized by IL-4 and IL-10 production; however, at the beginning of the infection, the IFN-γ production predominates (Th type-1 response). Although we did not find differences in IL-4 production or in the expression level of this gene in the hepatic lymph nodes, the expression level of IL-10 was higher (P < 0.05) in the T group at 4 wpi. The IFN-γ production was higher (P < 0.01) at 12 wpi as well as its level of expression at 4 wpi (P < 0.05) in the T group. We found a higher expression level of TGF-β at 4 wpi in the T group (P < 0.05), associated with the previous report of thicker fibrous tracks in a treated group. Immunoglobulin G1, related with a Th type-2 response, was higher (P < 0.01) in the T group at 4 and 12 wpi. In conclusion, the effects of LPS from O. intermedium could have resulted from a predominant Th type-2 immune response.     

Anthelmintic activity of artesunate against Fasciola hepatica in naturally infected sheep

In light of rapidly spreading triclabendazole resistance alternative fasciocidal drugs are urgently needed. Following up on promising results obtained with artemether in Fasciola hepatica infected sheep, we here report the efficacy and safety of artesunate in sheep with a natural F. hepatica infection. Artesunate was administered intravenously and intramuscularly, adverse events were monitored and drug efficacy was elucidated by means of faecal egg and worm burden reductions. A single 40 mg/kg intravenous dose of artesunate induced an egg count reduction of 68.9% and a worm burden reduction of 77.4%. Intramuscular artesunate at 40 mg/kg reduced faecal egg count and worm burden by 97.6% and 91.9%, respectively; whereas at 60 mg/kg it caused 93.2% and 87.1% reduction in faecal egg count and worm burden, respectively. Three sheep died 24-72 h post-treatment with a double dose of 40 mg/kg intramuscular artesunate, showing lethargy, sialorrhoea, reduced rumination and tremors. Egg and worm burden reductions of 93.3% and 83.9%, respectively, were calculated in the three surviving sheep. In conclusion, the interesting fasciocidal properties of artesunate in sheep warrant further investigations with an emphasis on toxicity studies.     

Effects of synbiotic-based Bifidobacterium animalis in female rats experimentally infected with Toxoplasma gondii

The aim of this study was to assess the effects of a synbiotic composed of Bifidobacterium animalis and fructooligosaccharides on female rats infected with Toxoplasma gondii. Female Wistar rats, treated or not with dexamethasone, were daily supplemented with synbiotics for 21 days. After 15 days of supplementation, the rats were orally infected with 10⁴T. gondii bradyzoites. Blood samples were collected to measure the levels of IFN-γ, IL-10 and T. gondii antibodies. All synbiotic-supplemented rats survived until the end of the experiment; however, non-supplemented dexamethasone-treated rats died between the fifth and the eighth days after T. gondii infection. Dexamethasone-treated rats supplemented with synbiotics (P < 0.05) were capable of synthesizing IFN-γ, and this immunological response was essential to ensure their survival. In addition, brain cysts were found in one rat not supplemented with synbiotics. Results suggest that the synbiotic composed of B. animalis and fructooligosaccharides may be beneficial to toxoplasmosis control.     

Molecular detection of Theileria and Babesia infections in cattle

This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.     

Comparison of nested PCR and real-time PCR for the detection of Toxoplasma gondii in biological samples from naturally infected cats

There are different protocols of molecular diagnosis methods available including DNA extraction methods to diagnose of Toxoplasma gondii, being necessary to perform comparative studies in biological samples. The aim of this study is to compare real-time PCR (rtPCR) and nested PCR (nPCR) to evaluate the detection of T. gondii in naturally infected cats. Biological samples of Toxoplasma-seropositive cats were assayed for detection of T. gondii DNA - extracted by both the lysis buffer and proteinase K (LB proteinase K) method and the acid guanidinium thiocyanate (GuSCN) method - using rtPCR and nPCR. T. gondii DNA was detected by nPCR in 43.6% and 40.8% of the samples from which it was extracted by the LB proteinase K and the GuSCN method, respectively. With rtPCR these figures fell significantly to 33.8% and 14.1%. Despite of nPCR showed higher sensitivity, the agreement observed between two PCRs was good; this agreement, however, was affected by the DNA extraction method used, LB proteinase K method showed better results.     

Effect of dietary supplementation with flaxseed oil or vitamin E on sheep experimentally infected with Fasciola hepatica

The effects of two diets supplemented with flaxseed oil (FO) or vitamin E (VE) were studied in sheep infected with Fasciola hepatica. The control group (CO), not supplemented, had higher weight gain and faecal egg count (P < 0.05) at the end of the study. At necropsy, the number (71.2 ± 26.5) and size of flukes were lower in the FO group (P < 0.01), probably due to the higher levels of white blood cells and lymphocytes (P < 0.01). This group also had higher red blood cell and haematocrit values (P < 0.01). The CO group had more severe hypoproteinaemia and hypoalbuminaemia, due to having the highest fluke burden (123.0 ± 35.2), and also the highest IgG1 titre (P < 0.01). The diet did not affect production or gene expression levels of IFN-γ and IL-4. On the other hand, the supplementation with VE led to a reduction in adult fluke burden (97.7 ± 39.9) and lower lipid oxidation in the liver (P < 0.001).     

Infections with Anaplasma phagocytophilum in dogs in Germany

The main objectives of this prospective study were to establish prevalence of Anaplasma phagocytophilum infections in dogs from Northeast Germany; and to evaluate the hematological parameters of sero- or real-time PCR-positive clinically healthy dogs. The mean prevalence of A. phagocytophilum seropositivity of 522 dogs (258 suspected to have anaplasmosis, 264 healthy) was 43%. There was no difference between sick (46.9%) and healthy dogs (39.8%) (p = 0.100). The PCR test was positive in 30 dogs (20 sick, 10 healthy); morulae were found in 12 of them. Twenty-six of 30 dogs tested PCR-positive between May and September (p < 0.05). There was no difference with regard to abnormal CBC parameters between seropositive and seronegative clinically healthy dogs. The CBC was within reference range in 10 PCR-positive clinically healthy dogs suggesting a routine examination of blood donors for A. phagocytophilum in endemic areas to minimize the risk of transmission.     

Seasonal differences in the incidence of infection with Fasciola gigantica in cambodian cattle

Farmer's cattle were treated with triclabendazole and used as tracer animals to detect new infections with Fasciola gigantica in three villages located on the bank of the Bassac River (a major tributary of the Mekong River) and in a fourth village located on farmland away from the river, from April 1999 until January 2001. The month of infection was estimated by subtracting 4 months from the date when eggs of F. gigantica were detected in faeces. Farmers were interviewed each month to record the nature of the agricultural and animal husbandry activities that occurred during the previous month, especially events that might have exposed cattle to infection with F. gigantica. Results support the conclusions that infection of cattle in riverbank villages acquired from about August or September until November originated from herbage and water in irrigation canals and dams on the riverbank, and that the progressively increasing monthly incidence from December until April (up to 87% per month in April 2000) was derived from herbage and water in recently harvested rice fields and lakes adjacent to the riverbank. The abrupt cessation of new infection in riverbank villages in May coincided with flooding of low-lying land, the movement of cattle to land above flood height on the riverbank, and a change of diet to dry-land crop residues, stored dry rice stalks, and herbage and water that were unlikely to contain metacercariae. It was concluded that snails in dams and canals on the riverbank became infected with F. gigantica after cattle were moved to the riverbank in May, and cercariae shed from these snails provided the new infections that occurred in cattle in August and September. In the village located away from the river, infection of cattle between September and March coincided with the rice harvest, supporting the conclusion that feeding of fresh rice stalks and stubble after the rice was harvested was the main source of infection. The low monthly incidence observed (up to 6.4% per month) was consistent with the hypothesis that snails did not survive in the dry rice fields between crops and that few snails would have been available from the small number of aquatic refuges that persisted through the dry season to recolonize rice fields during the wet season. Between April and August there was no opportunity for new infection because cattle were fed forage from around houses and headlands, and on dry-land crop residues and stored dry rice stalks. Control of fasciolosis was proposed using a single treatment of cattle with triclabendazole in riverbank villages in May when cattle were moved to the riverbank, and after harvest of the last rice field in villages located away from the river.     

Detection and identification of Toxocara canis DNA in bronchoalveolar lavage of infected mice using a novel real-time PCR

Toxocarosis is a zoonosis with worldwide distribution caused by Toxocara spp. of dogs and cats. In humans, diagnosis relies mainly on detection of parasite-specific antibodies. Although serological assays in current use have defined sensitivity and specificity, the problem of cross-reactivity still remains, particularly in areas of endemic polyparasitism. Microscopic detection of the parasite in tissue biopsies is not recommended for diagnosis because larvae can be difficult to locate, and finding the parasite eggs in faeces is not applicable since the larvae do not develop to the adult stage in the human host. In this study we describe a novel real-time PCR (‘Nemo-PCR’) that, in combination with DNA sequencing, allows the detection and identification of Toxocara canis and other nematodes in the Superfamily Ascaridoidea. Results indicate that this approach can detect Toxocara spp. DNA in bronchoalveolar lavage (BAL) of experimentally-infected mice. For diagnostic purposes further studies are necessary to evaluate this assay including testing human BAL fluid. The availability of such a direct assay would improve diagnosis of toxocarosis particularly for patients with pulmonary signs and symptoms.     

Expression of regulatory dendritic cell-related cytokines in cattle experimentally infected with Trypanosoma evansi

Trypanosoma evansi causes wasting disease in many livestock. T. evansi infection gives rise to inflammatory immune responses, which contribute to the development of inflammation-associated tissue injury. We previously reported that regulatory dendritic cells (DCs), which act as potential regulators of inflammation, were activated in infected mice and transfer of regulatory DCs to infected mice prolonged their survival. However, the kinetics of regulatory DCs in cattle, which are natural hosts of T. evansi, remained unclear. In this study, we report that the expressions of CCL8 and IL-10, which promote the development of regulatory DCs, were up-regulated in cattle experimentally infected with T. evansi. This finding is potentially useful for studying the control strategy of T. evansi infection in cattle.     

Evidence for a relationship between Leishmania load and clinical manifestations

Visceral leishmaniasis (VL) is a life-threatening disease of medical, social and economic importance in endemic areas. Dogs are the main reservoir of Leishmaniainfantum. In this study, the authors investigated a group of 56 natural infected dogs to establish the relationship between parasite load and various clinical forms of leishmaniasis.The sick dogs were monitored at the beginning from clinical and physiological point of view. Leishmania load was measured by real-time PCR assay on whole blood samples and lymph node aspirates, collected at the time of diagnosis.Our results indicate that a higher quantity of Leishmania DNA was found in the lymph nodes of dogs characterized by maximum clinical score. This interesting finding indicates the presence of a positive relationship between Leishmania load and clinical manifestations in dogs showing a severe clinical form of leishmaniasis.     

Host differences in response to trickle infection with Fasciola gigantica in buffalo, Ongole and Bali calves

Progressive weight gain, faecal egg counts, packed cell volume, percent eosinophils in blood, serum antibody and serum levels of glutamate dehydrogenase and gamma-glutamyl transpeptidase were recorded in seven swamp buffalo (Bubalis bubalis), 7 Ongole (Bos indicus) and four Bali calves (Bos sundiacus) which were infected orally with 15 metacercariae of Fasciola gigantica twice weekly for 32 weeks. Similar observations were made on four buffalo, 4 Ongole calves and 3 Bali calves maintained fluke-free as controls. Flukes were counted at slaughter 36 weeks after initial infection. Mean daily weight gains of infected Bali (228 ± 100 (SD) g/day) and infected Ongole calves (328 ± 57 (SD) g/day) were lower (p = 0.026 and 0.067, respectively) than those of control calves (405 ± 107 (SD) g/day), but infected buffalo calves (379 ± 78 (SD) g/day) had similar weight gains to those of the controls (p = 0.57). Throughout the trial, faecal Fasciola egg counts in buffaloes were about one-fifth of counts of Ongole calves, and counts in Bali calves were intermediate. Ongole calves had three times the number of flukes at slaughter in their liver compared to buffalo and Bali calves, which had similar numbers. However, there was evidence that Bali calves had acquired a degree of resistance about 24 weeks after infection commenced and may have lost adult flukes as a consequence.     

A zoospore inhibition technique to evaluate the activity of antifungal compounds against Batrachochytrium dendrobatidis and unsuccessful treatment of experimentally infected green tree frogs (Litoria caerulea) by fluconazole and benzalkonium chloride

Effective and safe treatments of chytridiomycosis in amphibians, caused by Batrachochytrium dendrobatidis, are needed to help prevent mortality in captive programs for threatened species, to reduce the risk of spread, and to better manage the disease in threatened populations. We describe a simple method to determine minimum inhibitory concentrations (MICs) of antifungal agents that involves adding zoospores to various drug concentrations in 96 well plates and microscopic observation after four days. We report results from testing 10 commercially available antifungal compounds: benzalkonium chloride (<0.78 μg/ml), povidone iodine (312.5 μg/ml), amphotericin B (3.125 μg/ml), fluconazole (<1.56 μg/ml), itraconazole (<1.56 μg/ml), enilconazole (<1.56 μg/ml), mercurochrome (6.25 μg/ml), sodium chloride (12.5 mg/ml), methylene blue (<1.56 μg/ml) and Virkon (3.125 μg/ml). For treatment trials of juvenile Litoria caerulea, baths of benzalkonium chloride at 1 mg/L and fluconazole at 25 mg/L were used on 18 experimentally infected frogs per treatment. Although these treatments resulted in longer survival times (mean 43.7 ± 11.3 days) than in the untreated controls (37.9 ± 9.3 days), the mortality rate was still 100%. Higher doses of fluconazole are suggested for further animal trials.     

Prevalence and antibiotic resistance profiles of Enterococcus species in chicken at slaughter level; absence of vanA and vanB genes in E. faecalis and E. faecium

The prevalence of enterococci in neck skin samples of poultry from Ankara region in Turkey was investigated and their antibiotic resistance patterns were determined. In the study, 83 of 106 analyzed neck skin samples were positive for Enterococcus, with E. faecium as the most prevalent species (48%) followed by E. durans (23%) and E. faecalis (19%). Lower numbers were detected for E. gallinarum, E. hirae, E. mundtii and E. casseliflavus. Using the disc diffusion method, it was established that over 90% of E. faecium and E. faecalis isolates were high-level resistant against erythromycin and tetracycline. Four E. faecium isolates were additionally resistant to chloramphenicol, gentamicin and streptomycin, though they were susceptible to penicillin G. The most frequently observed multiple resistance in E. faecium (25%) was against erythromycin, tetracycline, chloramphenicol and streptomycin. Of the E. faecalis isolates, 44% were multiple resistant to erythromycin, tetracycline and streptomycin. Vancomycin resistance could not be demonstrated phenotypically and vanA or vanB genes were not detected by multiplex PCR in any of the isolates. Nevertheless, the observed resistance patterns are of concern for public health.     

Gene markers of generic Escherichia coli associated with colonization and persistence of Escherichia coli O157 in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR = 16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR = 0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR = 0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC O157.     

Cessation of clinical disease and spirochete shedding after tiamulin treatment in pigs experimentally infected with “Brachyspira hampsonii”

With the emergence of “Brachyspira hampsonii” associated with swine dysentery in North America, identification of effective treatments and interventions is a pressing need. Denagard® (tiamulin hydrogen fumarate) Liquid Concentrate 12.5% is approved in the United States for treatment of dysentery caused by Brachyspira hyodysenteriae at 0.006% in the water. In this study, the effectiveness of tiamulin in resolving clinical disease, eliminating viable spirochete shedding, and reducing neutrophilic colitis following infection with either “B. hampsonii” or B. hyodysenteriae was evaluated. Seventy-eight 7-week-old crossbred pigs were divided into three groups [sham-inoculated (n = 18), “B. hampsonii”-inoculated (n = 30), and B. hyodysenteriae-inoculated (n = 30)]. Each inoculum group was divided into three subgroups which received either 0.006% tiamulin, 0.018% tiamulin, or no medication. Both levels of tiamulin resolved clinical disease within 24 h of treatment initiation, eliminated spirochete shedding within 72 h of treatment initiation, and resolved and/or prevented histologic lesions in pigs infected with either Brachyspira spp.     

First report on Parafilaria bovicola (Nematoda: Filaroidea) in Germany

From September 2008 to April 2009 several cases of spontaneous bleeding and nodules from the back and withers area of cattle otherwise considered as healthy have been reported from several farms in south-western and southern Germany. Successive investigation of these cases gave strong evidence of Parafilaria (P.) bovicola infections due to their seasonal occurrence and specific pathological lesions. There was no history of animal import from endemic countries for the affected cattle herds. In one case, a nodule containing nematode fragments and an intact female nematode were recovered from a slaughtered White Galloway bull. This sample was identified as P. bovicola and is the first report of bovine parafilariosis in Germany.