Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n = 53), 29% of foxes (n = 48), 29% of stone martens (n = 21) and in 25–50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.     

Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

Effect of isometamidium on infections by Trypanosoma vivax and T. Evansi in experimentally-infected animals

Assays dealing with the therapeutic and prophylactic activity of isometamidium on experimental infections by Trypanosoma vivax and T. evansi were carried out. The drug was found to be highly effective against T. vivax infection in sheep and cattle in which periods of protection ranging from 118 to 195 days were achieved. No complete effects against infection by T. evansi were observed. The drug was well tolerated in sheep and cattle while side-effects were noted in treated mares. It was concluded that isometamidium could be used to prevent damage and economical losses caused by T. vivax in Venezuela.     

Mycobacterium bovis infection in the brush-tailed possum (Trichosurus vulpecula): I. Preliminary observations on experimental infection

An Australian cattle strain of Mycobacterium bovis was injected intramuscularly into 10 brush-tailed possums (Trichosurus vulpecula) which were shown to be highly sensitive to experimental infection with this organism. The possums were killed and examined throughout the course of infection. At necropsy, gross and microscopic lesions were recorded and several tissues cultured for recovery of M. bovis. Infection spread rapidly via the lymphatic system from the injection site to the lumbar lymph nodes, then to the spleen. There was a bacteraemia after 2 week and by 6 weeks lesions were present in spleen, lymph nodes, lungs and kidneys; M. bovis proliferated rapidly and host response was minimal. Few organisms were detected in the liver where miliary lesions were found. M. bovis was excreted in large numbers in urine, faeces and discharging sinuses from subcutaneous abscesses. In two possums that died early in the infection, stress had rendered then more susceptible; infection spread more rapidly than in other possums and liver involvement was more severe. Although aerosol transmission was considered to be a possible means of spreading M. bovis, three in-contact possums did not acquire infection.     

Leishmania infantum secreted iron superoxide dismutase purification and its application to the diagnosis of canine Leishmaniasis

Leishmania spp. are digenetic parasites whose infection occurs inside the mononuclear phagocitary system. The iron superoxide dismutase secreted (Fe-SODe) by promastigotes of Leishmania spp. seems to plays an important role in the defense to environmental detoxification and neutralization of oxidative stress damage caused by reactive oxygen species (ROS) produced by macrophages during the infection. Parasites Fe-SODe is involved in establishing the infection and manifestation of Leishmaniasis. Its high immunogenicity makes it a useful molecular marker in diagnosing trypanosomatids infections. The aim of this study is demonstrate that purified Fe-SODe from Leishmania infantum is much more sensitive than un-purified Fe-SODe for diagnosis canine Leishmaniasis. We have purified a Fe-SODe of L. infantum using an ion exchange and a molecular sieve chromatographies and its application in diagnosis of canine Leishmaniasis was tested. One hundred and forty-five dogs’ sera from Andalusia Autonomous Community, Spain were tested by ELISA and Western blot and the antigen Fe-SODe purified is compared with two different antigens: the total parasites soluble lysate and the unpurified Fe-SODe. To validate the results obtained using the Fe-SODe purified we tasted 10 L. infantum infected dogs’ sera from Lombardy, Italy as positive control.     

Prevalence of Anaplasma phagocytophilum in fallow deer (Dama dama) and feeding ticks from an Italy preserve

Up to date, information concerning the Anaplasma phagocytophilum infection in fallow deer is scant, therefore, to verify its prevalence in these ungulates serological and PCR screenings were performed on blood of 72 fallow deer hunted in a Central-Northern Italian preserve. Molecular analyses were also performed on 90 ticks removed from the animals.A. phagocytophilum infection in fallow deer was confirmed in 20 out 72 by IFA assay and in 11 out 72 by PCR. The sequence obtained revealed a complete genetic homology among the blood samples and strong degrees of homology with other European isolates. Considering the 90 ticks collected we found that 7.3% of Ixodes ricinus harboured A. phagocytophilum specific DNA. The data obtained confirmed that fallow deer can be a competent host for A. phagocytophilum and, therefore, that may represent a biological reservoir playing an important role in the epidemiological scenarios of the infection, in the geographical areas where is widespread.     

A comparison of two laboratory methods of maintaining field-collected Lymnaea tomentosa for the production of Fasciola hepatica metacercariae

Four replicates of 50 field-collected Lymnaea tomentosa were maintained in two laboratory culture systems and their efficiency and practicability for the production of Fasciola hepatica metacercariae were assessed. There was no significant difference in the total number of metacercariae produced (56 552 from 64 snails in the closed container system and 54 073 from 82 snails in the shallow aquarium system).There was no significant difference between culture systems as assessed by snail survival, the number of infected snails or the number of infected snails harvested for the recovery of metacercariae. Within both systems there was a significant effect of snail size on the number of metacercariae produced. Optimum survival and production of metacercariae was achieved in young adult snails of 5–8 mm shell length at the time of infection.The collection of L. tomentosa from the field habitat and their maintenance in either culture system was shown to be a practical alternative to the breeding of laboratory adapted strains of L. tomentosa for the regular production of large numbers of F. hepatica metacercariae.     

Experimental infections with Campylobacter fetus in bulls of different ages

Factors affecting the establishment of a carrier state of Campylobactor fetus subsp. fetus (venerealis) in preputial cavities of bulls were investigated by following infection in bulls of two different age groups until slaughter, 9–18 weeks post infection. Each of the four older bulls (66–74 months at infection) and three of the four younger bulls (41–49 months at infection) were culturally positive until slaughter, indicating no increased susceptibility with age. Relative proportions of immunoglobulin classes and albumin in preputial fluids were generally similar to those determined prior to infection, although protein concentrations decreased and ratios of IgG/IgA and IgG₁/IgG₂ increased in most of the bulls. An unexplained diminution of sample volumes occurred with progressive sampling. Changes in concentrations of proteins and in sample volumes bore no apparent relationship to infection of bulls with C. fetus. Low levels of C. fetus agglutinins were detected in sample obtained both before and after infection, but no appreciable rise in antibody titers occurred following infection. Alterations in superficial antigens of C. fetus isolates obtained during the course of infection were demonstrated in the majority of animals. The capacity of the organism to undergo antigenic variation and to provoke a minimal immune response may contribute to its prolonged survival in the preputial cavity.     

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.     

Equine ehrlichiosis: A comparison between E. equi and other pathogenic species of Ehrlichia

Ehrlichia equi, etiologic agent of equine ehrlichiosis, is a rickettsia which morphologically closely resembles the agents of bovine petechial fever, tick-borne fever, and canine ehrlichiosis (tropical canine pancytopenia). Natural infections of E. equi have been reported only in horses; however, the experimental host range of E. equi has been broadened to include burros, sheep, goats, dogs, cats, monkeys and baboons. Infection of primates indicates that E. equi may be a zoonotic agent. An indirect fluorescent antibody test employing E. equi-infected granulocytes as antigen has been developed and used to show that infected horses develop a prolonged antibody response to E. equi. Cell-mediated immune responses measured by the leukocyte migration inhibition test were also detected in horses recovered from acute illness. Protective immunity in horses, monkeys and baboons to reinfection is long-lasting. In contrast to the blood of dogs recovered from clinical E. canis infection, the blood of horses and dogs recovered from clinical infection with E. equi is not infectious for susceptible animals. Infection of dogs with E. equi does not provide protection against subsequent infection with E. canis.     

Factors affecting seroprevalence of Toxoplasma gondii in the endangered Iberian lynx (Lynx pardinus)

Wild felids are considered important in maintaining the sylvatic cycle of Toxoplasma gondii. Although, T. gondii antibodies have been reported in several species of wild felids, little is known of the epidemiology and risk factors associated with T. gondii infection in wild cats. The Iberian lynx (Lynx pardinus) is the most endangered felid species in the world. In the present study, seroprevalence and associated risk factors for T. gondii infection in a large population of Iberian lynx in Spain were determined. Serum samples from 129 Iberian lynx collected from 2005 to 2009 and 85 wild rabbits (Oryctolagus cuniculus), sharing the habitat with the Iberian lynx, were tested for antibodies to T. gondii by the modified agglutination test (MAT) using a cut-off value of 1:25. Antibodies to T. gondii were found in 81 of 129 (62.8%) Iberian lynx. Seroprevalence to T. gondii in Iberian lynx significantly increased with age (P < 0.001). T. gondii seroprevalences were similar in free-ranging (66.7% of 93) and wild-caught captive lynx (69% of 84) but significantly lower in captive-born lynx (22.5% of 40). Seroprevalence was higher in lynx with concurrent Cytauxzoonfelis (88% of 25) but not with concurrent Feline Leukemia Virus (FeLV) infection (53.8% of 13). There were no significant differences in seroprevalence between sexes, geographic region and year of sample collection (2005–2009). Oocysts of T. gondii were not detected microscopically in fecal samples from 58 lynx. Wild rabbits are considered the most important food for the lynx. Antibodies to T. gondii were found in 14 (11.9%) of 85 rabbits tested. The present results indicate that T. gondii infection is widespread in the two areas where Iberian lynx survive in Spain. The fact that four captive-born lynx seroconverted was indication of contact with T. gondii also in the Captive Breeding Centers, hence, control measures to prevent T. gondii infection would be necessary in these centers.     

In vitro effects of Tabernaemontana citrifolia extracts on Haemonchus contortus

Tabernaemontana citrifolia (Apocynaceae) is traditionally used as an anthelmintic preparation for ruminants in Guadeloupe (French West Indies). This study was carried out to evaluate the in vitro effect of this plant against the parasitic nematode of small ruminants Haemonchus contortus. Three extracts (aqueous, methanolic and dichloromethane) of T. citrifolia fruit, leaf and root were tested on four developmental stages of the parasite, using egg hatch assay (EHA), larval development assay (LDA), L3 migration inhibition assay (LMI), and adult worm motility assay (AWM). Compared to the negative control, significant effects were observed for the different parts of T. citrifolia but with differences depending on the parasitic stage; efficacies on the larval development of H. contortus from 88.9% to 99.8% for fruit, from 72.1% to 83.8% for root and from 33.5% to 85% for leaf with dose-dependent effect for the methanolic extract. The root gave the best result on EHA (22.7% efficacy for dichloromethane extract) and AWM (56% efficacy, with dose-dependent effect for dichloromethane extract) and the leaf on LMI (49.4% efficacy). These results suggest that T. citrifolia possess anthelmintic activity against H. contortus. The active ingredients responsible for the activity could be the alkaloid compounds present in the plant parts of the plant.     

An immunofluorescence technique for the detection of Toxocara canis antibodies

An immunofluorescent (IF) test for the serodiagnosis of Toxocara canis infections in puppies is described. Frozen sections of male adult T. canis worms were used as antigen.A group of seven puppies, 6 weeks of age, was infected orally with 10 000 embryonated T. canis eggs each. In the sera of all animals IF antibodies could be detected from approximately 4 weeks after infection onwards. Titers were detectable until the end of the observation period (22 weeks).Two puppies of the same age were infected with 30 000 or 50 000 embryonated T. canis eggs respectively. Positive IF results were also obtained in the sera of these pups from week 4 post infection (p.i.) onwards. No correlation between titer and initial number of egges administered was observed. Furthermore, no correlation was noticed between titer and number of adult worms recovered from the dogs. For comparison all sera were tested with the complement fixation (CF) test, using cuticle material of adult worms as antigen. Complement fixing antibodies could be detected in none of the serum samples.     

Pigeons (Columba livia) are a suitable experimental model for Neospora caninum infection in birds

Neospora caninum infections in chickens have been recently described by epidemiological and experimental approaches, and these birds may be considered natural intermediate hosts of the parasite. It has been postulated that other bird species might perform this role in wildlife as well. To better understand the sylvatic life cycle of N. caninum, further studies are required. In that sense, this work aimed to observe infection kinetics in pigeons experimentally infected with N. caninum. Experimental infections were conducted in parallel with a related protozoan, Toxoplasma gondii, which has been already described as able to infect pigeons in nature. Our results demonstrated that N. caninum disseminated through various tissues of this host and induced parasite-specific IgG seroconversion. Infection parameters were similar to that observed in the T. gondii infected group, although N. caninum-infected pigeons presented lower IgG titers during acute phase. The results herein described demonstrate that pigeons are a suitable model for N. caninum infection, considering that these data are in agreement with those observed in chickens experimentally infected with this parasite. As pigeons may be revealed as important reservoirs for N. caninum infection in nature, future studies are necessary to determine the real prevalence of this parasite in this and other birds in wildlife.     

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.     

In vitro effects of Musa x paradisiaca extracts on four developmental stages of Haemonchus contortus

This study was carried out to evaluate the in vitro effect of Musa x paradisiaca stem and leaf against the parasitic nematode of small ruminants Haemonchus contortus. Three extracts (aqueous, methanolic and/or dichloromethane) of Musa x paradisiaca stem and leaf were tested in vitro on four developmental stages of H. contortus using egg hatch assay (EHA), larval development assay (LDA), L3 migration inhibition assay (LMI) and adult worm motility assay (AWM). The highly significant (P < 0.0001) ability to stop larval development (inhibition >67% for each extract) and the negative effect of the dichloromethane extract of leaf on adult worm motility (43% of inhibition of motility after 24 h of incubation) compared to the negative controls, suggest anthelmintic properties of Musa x paradisiaca stem and leaf against H. contortus. The active principles responsible for the activity could be secondary metabolites such as terpenoid and flavonoid compounds present in the leaf and stem of the plant.     

New multiplex PCR method for the simultaneous diagnosis of the three known species of equine tapeworm

Although several techniques exist for the detection of equine tapeworms in serum and feces, the differential diagnosis of tapeworm infection is usually based on postmortem findings and the morphological identification of eggs in feces. In this study, a multiplex polymerase chain reaction (PCR)-based method for the simultaneuos detection of Anoplocephala magna, Anoplocephala perfoliata and Anoplocephaloides mamillana has been developed and validated. The method simultaneously amplifies hypervariable SSUrRNA gene regions in the three tapeworm species in a single reaction using three pairs of primers, which exclusively amplify the internal transcribed spacer 2 (ITS-2) in each target gene. The method was tested on three types of sample: (a) 1/10, 1/100, 1/500, 1/1000, 1/2000 and 1/5000 dilutions of 70 ng of genomic DNA of the three tapeworm species, (b) DNA extracted from negative aliquots of sediments of negative control fecal samples spiked with 500, 200, 100, 50 and 10 eggs (only for A. magna and A. perfoliata; no A. mamillana eggs available) and (c) DNA extracted from 80, 50, 40, 30, 10 and 1 egg per 2 μl of PCR reaction mix (only for A. magna and A. perfoliata; no A. mamillana eggs available). No amplification was observed against the DNA of Gasterophilus intestinalis, Parascaris equorum and Strongylus vulgaris. The multiplex PCR method emerged as specific for the three tapeworms and was able to identify as few as 50 eggs per fecal sample and as little as 0.7 ng of control genomic DNA obtained from the three species. The method proposed is able to differentiate infections caused by the two most frequent species A. magna or A. perfoliata when the eggs are present in feces and is also able to detect mixed infections by the three cestode species.     

Growth hormones therapy in immune response against Trypanosoma cruzi

Growth hormone (GH) is an important hypophyseal hormone that is primarily involved in body growth and metabolism. In mammals, control of Trypanosoma cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. To explore the possibility that GH might be effective in the treatment of Chagas’ disease, we investigated its effects on the course of T. cruzi infection in rats, focusing our analyses on its influences on parasitemia, NO, TNF-α and IFN-γ concentration and on histopathological alterations and parasite burden in heart tissue. T. cruzi-infected male Wistar rats were intraperitoneally treated with 5 ng/10 g body weight/day of GH. Animals treated with GH showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection compared with untreated animals (P < 0.05). For all experimental days (7, 14 and 21 post infection) of the acute phase, infected and GH treated animals reached higher concentrations of TNF-α, IFN-γ and nitric oxide as compared to untreated and infected counterparts (P < 0.05) Histopathological observations of heart tissue revealed that GH administration also resulted in fewer and smaller amastigote burdens, and less inflammatory infiltrate and tissue disorganization, indicating a reduced parasitism of this tissue. These results show that GH can be considered as an immunomodulator substance for controlling parasite replication and combined with the current drug used may represent in the future a new therapeutic tool to reduce the harmful effects of Chagas’ disease.     

Occurrence of Mycobacterium avium subspecies paratuberculosis and Neospora caninum in Alberta cow-calf operations

Mycobacterium avium subsp. paratuberculosis (MAP) and Neospora caninum (NC) are two pathogens causing important production limiting diseases in the cattle industry. Significant impacts of MAP and NC have been reported on dairy cattle herds, but little is known about the importance, risk factors and transmission patterns in western Canadian cow-calf herds. In this cross-sectional study, the prevalence of MAP and NC infection in southwest Alberta cow-calf herds was estimated, risk factors for NC were identified, and the reproductive impacts of the two pathogens were assessed.     

Mycoplasma synoviae cell invasion: Elucidation of the Mycoplasma pathogenesis in chicken

Fluorochrome-labelled cells of two field isolates and Mycoplasma synoviae (Ms) were inoculated onto monolayer cultures of fluorochrome-labelled HEp-2 cells and monitored by confocal laser scanning microscopy (CLSM). Ms was detected initially adhered to and subsequently inside the host cells. Between 24 and 48 h of infection, Ms was detected in the perinuclear region, and after 72 h of infection was confirmed by gentamicin invasion assay. High and low passage Ms strains showed no differences in adherence or invasion. The morphology and the actin filaments of the infected HEp-2 cells were preserved throughout the study period. The observed invasion by Ms is consistent with the biology of Mollicutes, and could explain the difficulties in recovering field isolates of the mycoplasma and in controlling the infection in birds even after long-term antibiotic treatment.